TARGETED RADIOPHARMACEUTICALS FOR THE DIAGNOSIS AND TREATMENT OF

PROSTATE CANCER

The present invention relates to chelators of general formula (I) and targeted radiopharmaceuticals prepared thereform as described and defined herein, methods of preparing said conjugates, intermediate compounds useful for preparing said compounds, pharmaceutical compositions and combinations comprising said compounds, and the use of said compounds for manufacturing pharmaceutical compositions for the imaging, treatment or prophylaxis of diseases, in particular prostate cancer, as a sole agent or in combination with other active ingredients.

BACKGROUND

Specific cell killing can be essential for the successful treatment of a variety of diseases in mammalian subjects. Typical examples of this are in the treatment of malignant diseases such as sarcomas and carcinomas. However the selective elimination of certain cell types can also play a key role in the treatment of other diseases, especially hyperplastic and neoplastic diseases.

The most common methods of selective treatment are currently surgery, chemotherapy and external beam irradiation. Targeted radionuclide therapy is, however, a promising and developing area with the potential to deliver highly cytotoxic radiation specifically to cell types associated with disease. The most common forms of radiopharmaceuticals currently authorised for use in humans employ beta-emitting and/or gamma-emitting radionuclides. There has, however, been some interest in the use of alpha-emitting radionuclides in therapy because of their potential for more specific cell killing.

The radiation range of typical alpha emitters in physiological surroundings is generally less than 100 micrometers, the equivalent of only a few cell diameters. This makes these sources well suited for the treatment of tumours, including micrometastases, because they have the range to reach neighbouring cells within a tumour but if they are well targeted then little of the radiated energy will pass beyond the target cells. Thus, not every cell need be targeted but damage to surrounding healthy tissue may be minimised (see Feinendegen et al. , Radiat Res 148: 195-201 (1997)). In contrast, a beta particle has a range of 1 mm or more in water (see Wilbur, Antibody Immunocon Radiopharm 4: 85-96 (1991)).

The energy of alpha-particle radiation is high in comparison with that carried by beta particles, gamma rays and X-rays, typically being 5-8 MeV, or 5 to 10 times that of a beta particle and 20 or more times the energy of a gamma ray. Thus, this deposition of a large amount of energy over a very short distance gives a-radiation an exceptionally high linear energy transfer (LET), high relative biological efficacy (RBE) and low oxygen enhancement ratio (OER) compared to gamma and beta radiation (see Hall, "Radiobiology for the radiologist", Fifth edition, Lippincott Williams & Wilkins, Philadelphia PA, USA, 2000). This explains the exceptional cytotoxicity of alpha emitting radionuclides and also imposes stringent demands on the biological targeting of such isotopes and upon the level of control and study of alpha emitting radionuclide distribution which is necessary in order to avoid unacceptable side effects. Table 1 below shows the physical decay properties of the alpha emitters so far broadly proposed in the literature as possibly having therapeutic efficacy. Table 1

* Half life So far, with regards to the application in radioimmunotherapy the main attention has been focused on ²¹¹ At, ²¹³ Bi and ²²⁵ Ac and these three nuclides have been explored in clinical immunotherapy trials. Several of the radionuclides which have been proposed are short-lived, i.e. have half-lives of less than 12 hours. Such a short half-life makes it difficult to produce and distribute radiopharmaceuticals based upon these radionuclides in a commercial manner. Administration of a short-lived nuclide also increases the proportion of the radiation dose which will be emitted in the body before the target site is reached. The recoil energy from alpha-emission will in many cases cause the release of daughter nuclides from the position of decay of the parent. This recoil energy is sufficient to break many daughter nuclei out from the chemical environment which may have held the parent, e.g. where the parent was complexed by a ligand such as a chelating agent. This will occur even where the daughter is chemically compatible with, i.e. complexable by, the same ligand. Equally, where the daughter  

nuclide is a gas, particularly a noble gas such as radon, or is chemically incompatible with the ligand, this release effect will be even greater. When daughter nuclides have half-lives of more than a few seconds, they can diffuse away into the blood system, unrestrained by the complexant which held the parent. These free radioactive daughters can then cause undesired systemic toxicity. The use of Thorium-227 (T _1/2 = 18.7 days) under conditions where control of the ²²³ Ra daughter isotope is maintained was proposed a few years ago (see WO 01/60417 and WO 02/05859).

This was in situations where a carrier system is used which allows the daughter nuclides to be retained by a closed environment. In one case, the radionuclide is disposed within a liposome and the substantial size of the liposome (as compared to recoil distance) helps retain daughter nuclides within the liposome. In the second case, bone-seeking complexes of the radionuclide are used which incorporate into the bone matrix and therefore restrict release of the daughter nuclides. These are potentially highly advantageous methods, but the administration of liposomes is not desirable in some circumstances and there are many diseases of soft tissue in which the radionuclides cannot be surrounded by a mineralised matrix so as to retain the daughter isotopes. More recently, it was established that the toxicity of the ²²³ Ra daughter nuclei released upon decay of ²²⁷ Th could be tolerated in the mammalian body to a much greater extent than would be predicted from prior tests on comparable nuclei. In the absence of the specific means of retaining the radium daughters of thorium-227 discussed above, the publicly available information regarding radium toxicity made it clear that it was not possible to use thorium-227 as a therapeutic agent since the dosages required to achieve a therapeutic effect from thorium-227 decay would result in a highly toxic and possibly lethal dosage of radiation from the decay of the radium daughters, i.e. there is no therapeutic window. WO 04/091668 describes the unexpected finding that a therapeutic treatment window does exist in which a therapeutically effective amount of a targeted thorium-227 radionuclide can be administered to a subject (typically a mammal) without generating an amount of radium-223 sufficient to cause unacceptable myelotoxicity. This can therefore be used for treatment and prophylaxis of all types of diseases at both bony and soft-tissue sites. In view of the above developments, it is now possible to employ alpha-emitting thorium-227 nuclei in endoradionuclide therapy without lethal myelotoxicity resulting from the generated ²²³ Ra. Nonetheless, the therapeutic window remains relatively narrow and it is in all cases desirable to administer no more alpha-emitting radioisotope to a subject than absolutely

 

necessary. Useful exploitation of this new therapeutic window would therefore be greatly enhanced if the alpha-emitting thorium-227 nuclei could be complexed and targeted with a high degree of reliability. Because radionuclides are constantly decaying, the time spent handling the material between isolation and administration to the subject is of great importance. It would also be of considerable value if the alpha-emitting thorium nuclei could be complexed, targeted and/or administered in a form which was quick and convenient to prepare, preferably requiring few steps, short incubation periods and/or temperatures not irreversibly affecting the properties of the targeting entity.

Furthermore, processes which can be conducted in solvents that do not need removal before administration (essentially in aqueous solution) have the considerable advantage of avoiding a solvent evaporation or dialysis step. It would also be considered of significant value if a thorium labelled drug product formulation could be developed which demonstrated significantly enhanced stability. This is critical to ensure that robust product quality standards are adhered to while at the same time enabling a logistical path to delivering patient doses. Thus formulations with minimal radiolysis over a period of 1-4 days are preferred. Octadentate chelating agents containing hydroxypyridinone groups have previously been shown to be suitable for coordinating the alpha emitter thorium-277, for subsequent attachment to a targeting moiety (WO2011098611). Octadentate chelators were described, containing four 3,2- hydroxypyridinone groups joined by linker groups to an amine-based scaffold, having a separate reactive group used for conjugation to a targeting molecule. Preferred structures of the previous invention contained 3,2-hydroxypyridinone groups and employed the isothiocyanate moiety as the preferred coupling chemistry to the antibody component as shown in compound ALG-DD- NCS. The isothiocyanate is widely used to attach a label to proteins via amine groups. The isothiocyanate group reacts with amino terminal and primary amines in proteins and has been used for the labelling of many proteins including antibodies. Although the thiourea bond formed in these conjugates is reasonably stable, it has been reported that antibody conjugates prepared from fluorescent isothiocyanates deteriorate over time. [Banks PR, Paquette DM., Bioconjug Chem (1995) 6:447-458]. The thiourea formed by the reaction of fluorescein isothiocyanate with amines is also susceptible to conversion to a guanidine under basic conditions [Dubey I, Pratviel G, Meunier BJournal: Bioconjug Chem (1998) 9:627-632]. Due to the long decay half-life of thorium-227 (18.7 days) coupled to the long biological half-life of a monoclonal antibody it is desirable to use more stable linking moieties so as to generate conjugates which are more chemically stable both in vivo and to storage.

 

Work on conjugation of hydroxypyridinone ligands was published in WO2013/167754 and discloses ligands possessing a water solubilising moiety comprising a hydroxyalkyl functionality.

Due to the reactivity of the hydroxyl groups of this chelate class activation as an activated ester is not possible as multiple competing reactions ensue leading to a complex mixture of products through esterification reactions. The ligands of WO2013/167754 must therefore be coupled to the tissue-targeting protein via alternative chemistries such as the isothiocyanate giving a less stable thiourea conjugate as described above. In addition WO2013167755 and WO2013167756 discloses the hydroxyalkyl/ isothiocyanate conjugates applied to CD33 and CD22 targeted antibodies respectively. Furthermore, although the introduction of the alcohol groups do contribute to water solubilization by increasing the hydrophilicity of the conjugates they are still hydrophobic enough to effect the pharmacokinetics of the labeled conjugate with significant liver uptake observed in some cases particularly in applications utilizing small molecules such as peptides. WO2013/022797 discloses a PSMA-targeting peptide and linking structure for use with beta- emitting radionuclides. The application discloses a number of specific chelators suitable for use with the peptide, but does not suggest the use of alpha-emitters, nor the use of HOPO chelators. WO2015/055318 discloses an alternative PSMA-targeting peptide (PSMA-617) and linking structure for use with beta-emitting radionuclides. The application discloses a number of specific chelators suitable for use with the peptide, but does not suggest the use of alpha-emitters, nor the use of HOPO chelators. WO2016/096843 discloses 3,2-HOPO chelators radiolabeled with thorium and attached to a variety of tissue-targeted moieties. However, while such chelators are effective in the therapeutic setting, when labeled with a zirconium ion for imaging purposes, such chelators can experience p-p stacking which can lead to unwanted agglomeration. However, the state of the art does not describe the chelators of general formula (I) of the present invention as described and defined herein.

It has now been found, and this constitutes the basis of the present invention, that the compounds of the present invention have surprising and advantageous properties.

In particular, the compounds of the present invention have surprisingly been found to be suitable for use with tissue targeting moieties, such as peptides and monoclonal antibodies and antibody fragments with reduced dimerization or agglomeration. Without being bound by one particular theory, the inventors belive the reduced dimerization or agglomeration is the result of reduction of p-p stacking.  

Further, the compounds of the present invention have surprisingly been found to effectively address several biological targets associated with tumor growth.

Further, the compounds of the present invention have surprisingly been found to effectively target PSMA and may therefore be used for the treatment or prophylaxis of oncologic disorders, such as prostate cancer, for example.

DESCRIPTION of the INVENTION In accordance with a first aspect, the present invention covers compounds of general formula (I):

wherein:

n is 1, 2 or 3;

R1, R2, R3 and R4, independently represent OH or Q; and

Q represents a tissue-targeting moeity selected from the group consisting of poly- and oligo-peptides, proteins, DNA and RNA fragments, aptamers polyclonal or monoclonal antibodies, and a mixture of proteins or fragments or constructs of protein

or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In one embodiment of the first aspect of the invention, Q represents the following structure:

 

wherein X represents aryl, heteroaryl, butylurea, fluoro-substituted phenyl, butyl- substituted phenyl, quinolinyl and 2-napthyl;

wherein Y represents aryl, heteroaryl, aralkyl, hetaralkyl,C ₃ -C ₈ -cycloalkyl, or pyridine; wherein G represents CH ₂ N*H or N*H with * being the attachment point to the remainder of compound (I);

and wherein R ₅ represents azole,–SO ₂ H, - SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H ₂ , -PO ₃ H, - PO ₄ H ₂ , -CO ₂ H, -C(O)R; wherein

R represents–H, -OH, -(C ₁ -C ₁₀ )alkyl, -O(C ₁ -C ₁₀ )alkyl, -NHR ₆ , or NR ₆ R ₇ ;

R ₆ , R ₇ and R ₈ each independently represent H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, BR, I, C(O), C(S), -C(S)-NH-benzyl-, -C(O)-NH-benzyl-, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ )v-NR ₈ ,

-(CH ₂ )p-NH-C(O)-(CH ₂ )p-, -(CH ₂ -CH ₂ )t-NH-C(O)-(CH ₂ )p-, -(CH ₂ )p-COR, -(CH ₂ )p- C(O)NH-C[(CH ₂ )p-COR] ₃ , -C[(CH ₂ )p-COR] ₃ , or–(CH ₂ )p-(C ₅ -C ₁₄ )heteroaryl;

wherein v, p and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment of the first aspect of the invention, Q represents a monoclonal antibody with binding affinity for targets selected from the list consisting of FAP, HER2, and PSMA.

In accordance with a second aspect, the present invention covers compounds wherein compound (I) is radiolabled with a radionuclide A selected from the group consisting of of ⁴³ Sc, 4 ⁴ Sc, ⁴⁷ Sc, ⁸⁹ Zr, ⁹⁰ Y, ¹¹¹ In, ¹⁴⁹ Tb, ¹⁵² Tb, ¹⁵⁵ Tb, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac, 2 ²⁷ Th, and ²³² Th. In one embodiment, the radionuclide A is chlated according to the general structure :

 

wherein:

n is 1, 2 or 3;

R1, R2, R3 and R4, independently represent OH or Q; and

Q represents a tissue-targeting moeity selected from the group consisting of poly- and oligo-peptides, proteins, DNA and RNA fragments, aptamers polyclonal or monoclonal antibodies, and a mixture of proteins or fragments or constructs of protein

or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In one embodiment of the second aspect of the present invention, n Q represents the following structure:

wherein X represents aryl, heteroaryl, butylurea, fluoro-substituted phenyl, butyl- substituted phenyl, quinolinyl and 2-napthyl;

  wherein Y represents aryl, heteroaryl, aralkyl, hetaralkyl,C ₃ -C ₈ -cycloalkyl, or pyridine;

wherein G represents CH ₂ N*H or N*H with * being the attachment point to the remainder of compound (I);

and wherein R ₅ represents azole,–SO ₂ H, - SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H ₂ , -PO ₃ H, - PO ₄ H ₂ , -CO ₂ H, -C(O)R; wherein

R represents–H, -OH, -(C ₁ -C ₁₀ )alkyl, -O(C ₁ -C ₁₀ )alkyl, -NHR ₆ , or NR ₆ R ₇ ;

R ₆ , R ₇ and R ₈ each independently represent H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, BR, I,

C(O), C(S), -C(S)-NH-benzyl-, -C(O)-NH-benzyl-, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ )v-NR ₈ ,

-(CH ₂ )p-NH-C(O)-(CH ₂ )p-, -(CH ₂ -CH ₂ )t-NH-C(O)-(CH ₂ )p-, -(CH ₂ )p-COR, -(CH ₂ )p- C(O)NH-C[(CH ₂ )p-COR] ₃ , -C[(CH ₂ )p-COR] ₃ , or–(CH ₂ )p-(C ₅ -C ₁₄ )heteroaryl;

wherein v, p and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In accordance with an embodiment of the third aspect, the present invention covers compounds wherein compound (I) is radiolabled with a radionuclide A selected from the group consisting of of ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ⁸⁹ Zr, ⁹⁰ Y, ¹¹¹ In, ¹⁴⁹ Tb, ¹⁵² Tb, ¹⁵⁵ Tb, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac, ²²⁷ Th, and ²³² Th. In one embodiment, the radionuclide A is chlated according to the general structure :

wherein Q represents a monoclonal antibody with binding affinity for targets selected from the list consisting of FAP, HER2, and PSMA.

DEFINITIONS  

The term“substituted” means that one or more hydrogen atoms on the designated atom or group are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded. Combinations of substituents and/or variables are permissible.

The term“optionally substituted” means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen atom.

Commonly, it is possible for the number of optional substituents, when present, to be 1, 2, 3, 4 or 5, in particular 1, 2 or 3.

As used herein, the term“one or more”, e.g. in the definition of the substituents of the compounds of general formula (I) of the present invention, means“1, 2, 3, 4 or 5, particularly 1, 2, 3 or 4, more particularly 1, 2 or 3, even more particularly 1 or 2”.

As used herein, an oxo substituent represents an oxygen atom, which is bound to a carbon atom or to a sulfur atom via a double bond.

The term“ring substituent” means a substituent attached to an aromatic or nonaromatic ring which replaces an available hydrogen atom on the ring.

The term“comprising” when used in the specification includes“consisting of”.

If within the present text any item is referred to as“as mentioned herein”, it means that it may be mentioned anywhere in the present text.

The terms as mentioned in the present text have the following meanings:

The term“halogen atom” means a fluorine, chlorine, bromine or iodine atom, particularly a fluorine, chlorine or bromine atom.

The term“C ₁ -C ₆ -alkyl” means a linear or branched, saturated, monovalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, e.g. a methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neo-pentyl, 1,1-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2,3-dimethylbutyl, 1,2-dimethylbutyl or 1,3-dimethylbutyl group, or an isomer thereof. Particularly, said group has 1, 2, 3 or 4 carbon atoms (“C ₁ -C ₄ -alkyl”), e.g. a methyl, ethyl, propyl, isopropyl, butyl, sec-butyl isobutyl, or tert-butyl group, more particularly 1, 2 or 3 carbon atoms (“C ₁ -C ₃ -alkyl”), e.g. a methyl, ethyl, n-propyl or isopropyl group.

The term“C ₁ -C ₆ -hydroxyalkyl” means a linear or branched, saturated, monovalent hydrocarbon group in which the term“C ₁ -C ₆ -alkyl” is defined supra, and in which 1, 2 or 3 hydrogen atoms are replaced with a hydroxy group, e.g. a hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl,  

1,2-dihydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, 1-hydroxypropyl, 1-hydroxypropan-2-yl, 2-hydroxypropan-2-yl, 2,3-dihydroxypropyl, 1,3-dihydroxypropan-2-yl, 3-hydroxy-2-methyl-propyl, 2-hydroxy-2-methyl-propyl, 1-hydroxy-2-methyl-propyl group.

The term“C ₁ -C ₆ -alkylsulfanyl” means a linear or branched, saturated, monovalent group of formula (C ₁ -C ₆ -alkyl)-S-, in which the term “C ₁ -C ₆ -alkyl” is as defined supra, e.g. a methylsulfanyl, ethylsulfanyl, propylsulfanyl, isopropylsulfanyl, butylsulfanyl, sec-butylsulfanyl, isobutylsulfanyl, tert-butylsulfanyl, pentylsulfanyl, isopentylsulfanyl, hexylsulfanyl group.

The term“C ₁ -C ₆ -haloalkyl” means a linear or branched, saturated, monovalent hydrocarbon group in which the term“C ₁ -C ₆ -alkyl” is as defined supra, and in which one or more of the hydrogen atoms are replaced, identically or differently, with a halogen atom. Particularly, said halogen atom is a fluorine atom. Said C ₁ -C ₆ -haloalkyl group is, for example, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 3,3,3-trifluoropropyl or 1,3-difluoropropan-2-yl.

The term“C ₁ -C ₆ -alkoxy” means a linear or branched, saturated, monovalent group of formula (C ₁ -C ₆ -alkyl)-O-, in which the term“C ₁ -C ₆ -alkyl” is as defined supra, e.g. a methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy or n-hexyloxy group, or an isomer thereof.

The term“C ₁ -C ₆ -haloalkoxy” means a linear or branched, saturated, monovalent C ₁ -C ₆ -alkoxy group, as defined supra, in which one or more of the hydrogen atoms is replaced, identically or differently, with a halogen atom. Particularly, said halogen atom is a fluorine atom. Said C ₁ -C ₆ -haloalkoxy group is, for example, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy or pentafluoroethoxy.

The term“C ₂ -C ₆ -alkenyl” means a linear or branched, monovalent hydrocarbon group, which contains one or two double bonds, and which has 2, 3, 4, 5 or 6 carbon atoms, particularly 2 or 3 carbon atoms (“C ₂ -C ₃ -alkenyl”), it being understood that in the case in which said alkenyl group contains more than one double bond, then it is possible for said double bonds to be isolated from, or conjugated with, each other. Said alkenyl group is, for example, an ethenyl (or“vinyl”), prop-2-en-1-yl (or “allyl”), prop-1-en-1-yl, but-3-enyl, but-2-enyl, but-1-enyl, pent-4-enyl, pent-3-enyl, pent-2-enyl, pent-1-enyl, hex-5-enyl, hex-4-enyl, hex-3-enyl, hex-2-enyl, hex-1-enyl, prop-1-en-2-yl (or “isopropenyl”), 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, 1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, 2-methylbut-2-enyl, 1-methylbut-2-enyl, 3-methylbut-1-enyl, 2-methylbut-1-enyl, 1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl, 4-methylpent-4-enyl, 3-methylpent-4-enyl, 2-methylpent-4-enyl, 1-methylpent-4-enyl, 4-methylpent-3-enyl, 3-methylpent-3-enyl, 2-methylpent-3-enyl, 1-methylpent-3-enyl, 4-methylpent-2-enyl, 3-methylpent-2-enyl, 2-methylpent-2-enyl, 1-methylpent-2-enyl, 4-methylpent-1-enyl, 3-methylpent-1-enyl,  

2-methylpent-1-enyl, 1-methylpent-1-enyl, 3-ethylbut-3-enyl, 2-ethylbut-3-enyl, 1-ethylbut-3-enyl, 3-ethylbut-2-enyl, 2-ethylbut-2-enyl, 1-ethylbut-2-enyl, 3-ethylbut-1-enyl, 2-ethylbut-1-enyl, 1-ethylbut-1-enyl, 2-propylprop-2-enyl, 1-propylprop-2-enyl, 2-isopropylprop-2-enyl, 1-isopropylprop-2-enyl, 2-propylprop-1-enyl, 1-propylprop-1-enyl, 2-isopropylprop-1-enyl, 1-isopropylprop-1-enyl, 3,3-dimethylprop-1-enyl, 1-(1,1-dimethylethyl)ethenyl, buta-1,3-dienyl, penta-1,4-dienyl or hexa-1,5-dienyl group.

Particularly, said group is vinyl or allyl.

The term“C ₂ -C ₆ -alkynyl” means a linear or branched, monovalent hydrocarbon group which contains one triple bond, and which contains 2, 3, 4, 5 or 6 carbon atoms, particularly 2 or 3 carbon atoms (“C ₂ -C ₃ -alkynyl”). Said C ₂ -C ₆ -alkynyl group is, for example, ethynyl, prop-1-ynyl, prop-2-ynyl (or “propargyl”), but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl, hex-5-ynyl, 1-methylprop-2-ynyl, 2-methylbut-3-ynyl, 1-methylbut-3-ynyl, 1-methylbut-2-ynyl, 3-methylbut-1-ynyl, 1-ethylprop-2-ynyl, 3-methylpent-4-ynyl, 2-methylpent-4-ynyl, 1-methyl- pent-4-ynyl, 2-methylpent-3-ynyl, 1-methylpent-3-ynyl, 4-methylpent-2-ynyl, 1-methyl- pent-2-ynyl, 4-methylpent-1-ynyl, 3-methylpent-1-ynyl, 2-ethylbut-3-ynyl, 1-ethylbut-3-ynyl, 1-ethylbut-2-ynyl, 1-propylprop-2-ynyl, 1-isopropylprop-2-ynyl, 2,2-dimethylbut-3-ynyl, 1,1-dimethylbut-3-ynyl, 1,1-dimethylbut-2-ynyl or 3,3-dimethylbut-1-ynyl group. Particularly, said alkynyl group is ethynyl, prop-1-ynyl or prop-2-ynyl.

The term“C ₃ -C ₈ -cycloalkyl” means a saturated, monovalent, mono- or bicyclic hydrocarbon ring which contains 3, 4, 5, 6, 7 or 8 carbon atoms (“C ₃ -C ₈ -cycloalkyl”). Said C ₃ -C ₈ -cycloalkyl group is for example, a monocyclic hydrocarbon ring, e.g. a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl group, or a bicyclic hydrocarbon ring, e.g. a bicyclo[4.2.0]octyl or octahydropentalenyl.

The term“C ₄ -C ₈ -cycloalkenyl” means a monovalent, mono- or bicyclic hydrocarbon ring which contains 4, 5, 6, 7 or 8 carbon atoms and one double bond. Particularly, said ring contains 4, 5 or 6 carbon atoms (“C ₄ -C ₆ -cycloalkenyl”). Said C ₄ -C ₈ -cycloalkenyl group is for example, a monocyclic hydrocarbon ring, e.g. a cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl or cyclooctenyl group, or a bicyclic hydrocarbon ring, e.g. a bicyclo[2.2.1]hept-2-enyl or bicyclo[2.2.2]oct-2-enyl.

The term“C ₃ -C ₈ -cycloalkoxy” means a saturated, monovalent, mono- or bicyclic group of formula (C ₃ -C ₈ -cycloalkyl)-O-, which contains 3, 4, 5, 6, 7 or 8 carbon atoms, in which the term “C ₃ -C ₈ -cycloalkyl” is defined supra, e.g. a cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy or cyclooctyloxy group.

The term "spirocycloalkyl" means a saturated, monovalent bicyclic hydrocarbon group in which the two rings share one common ring carbon atom, and wherein said bicyclic hydrocarbon group contains 5, 6, 7, 8, 9, 10 or 11 carbon atoms, it being possible for said spirocycloalkyl group to  

be attached to the rest of the molecule via any one of the carbon atoms except the spiro carbon atom. Said spirocycloalkyl group is, for example, spiro[2.2]pentyl, spiro[2.3]hexyl, spiro[2.4]heptyl, spiro[2.5]octyl, spiro[2.6]nonyl, spiro[3.3]heptyl, spiro[3.4]octyl, spiro[3.5]nonyl, spiro[3.6]decyl, spiro[4.4]nonyl, spiro[4.5]decyl, spiro[4.6]undecyl or spiro[5.5]undecyl.

The terms“4- to 7-membered heterocycloalkyl” and“4- to 6-membered heterocycloalkyl” mean a monocyclic, saturated heterocycle with 4, 5, 6 or 7 or, respectively, 4, 5 or 6 ring atoms in total, which contains one or two identical or different ring heteroatoms from the series N, O and S, it being possible for said heterocycloalkyl group to be attached to the rest of the molecule via any one of the carbon atoms or, if present, a nitrogen atom.

Said heterocycloalkyl group, without being limited thereto, can be a 4-membered ring, such as azetidinyl, oxetanyl or thietanyl, for example; or a 5-membered ring, such as tetrahydrofuranyl, 1,3-dioxolanyl, thiolanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, 1,1-dioxidothiolanyl, 1,2-oxazolidinyl, 1,3-oxazolidinyl or 1,3-thiazolidinyl, for example; or a 6-membered ring, such as tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, 1,3-dioxanyl, 1,4-dioxanyl or 1,2-oxazinanyl, for example, or a 7-membered ring, such as azepanyl, 1,4-diazepanyl or 1,4-oxazepanyl, for example.

Particularly,“4- to 6-membered heterocycloalkyl” means a 4- to 6-membered heterocycloalkyl as defined supra containing one ring nitrogen atom and optionally one further ring heteroatom from the series: N, O, S. More particularly,“5- or 6-membered heterocycloalkyl” means a monocyclic, saturated heterocycle with 5 or 6 ring atoms in total, containing one ring nitrogen atom and optionally one further ring heteroatom from the series: N, O.

The term“5- to 8-membered heterocycloalkenyl” means a monocyclic, unsaturated, non- aromatic heterocycle with 5, 6, 7 or 8 ring atoms in total, which contains one or two double bonds and one or two identical or different ring heteroatoms from the series: N, O, S; it being possible for said heterocycloalkenyl group to be attached to the rest of the molecule via any one of the carbon atoms or, if present, a nitrogen atom.

Said heterocycloalkenyl group is, for example, 4H-pyranyl, 2H-pyranyl, 2,5-dihydro-1H-pyrrolyl,

[1,3]dioxolyl, 4H-[1,3,4]thiadiazinyl, 2,5-dihydrofuranyl, 2,3-dihydrofuranyl, 2,5-dihydrothio- phenyl, 2,3-dihydrothiophenyl, 4,5-dihydrooxazolyl or 4H-[1,4]thiazinyl.

The term“heterospirocycloalkyl” means a bicyclic, saturated heterocycle with 6, 7, 8, 9, 10 or 11 ring atoms in total, in which the two rings share one common ring carbon atom, which “heterospirocycloalkyl” contains one or two identical or different ring heteroatoms from the series: N, O, S; it being possible for said heterospirocycloalkyl group to be attached to the rest of the molecule via any one of the carbon atoms, except the spiro carbon atom, or, if present, a nitrogen atom.

 

Said heterospirocycloalkyl group is, for example, azaspiro[2.3]hexyl, azaspiro[3.3]heptyl, oxaazaspiro[3.3]heptyl, thiaazaspiro[3.3]heptyl, oxaspiro[3.3]heptyl, oxazaspiro[5.3]nonyl, oxazaspiro[4.3]octyl, azaspiro[4,5]decyl, oxazaspiro [5.5]undecyl, diazaspiro[3.3]heptyl, thiazaspiro[3.3]heptyl, thiazaspiro[4.3]octyl, azaspiro[5.5]undecyl, or one of the further homologous scaffolds such as spiro[3.4]-, spiro[4.4]-, spiro[2.4]-, spiro[2.5]-, spiro[2.6]-, spiro[3.5]-, spiro[3.6]-, spiro[4.5]- and spiro[4.6]-.

The term“fused heterocycloalkyl” means a bicyclic, saturated heterocycle with 6, 7, 8, 9 or 10 ring atoms in total, in which the two rings share two adjacent ring atoms, which“fused heterocycloalkyl” contains one or two identical or different ring heteroatoms from the series: N, O, S; it being possible for said fused heterocycloalkyl group to be attached to the rest of the molecule via any one of the carbon atoms or, if present, a nitrogen atom.

Said fused heterocycloalkyl group is, for example, azabicyclo[3.3.0]octyl, azabicyclo[4.3.0]nonyl, diazabicyclo[4.3.0]nonyl, oxazabicyclo[4.3.0]nonyl, thiazabicyclo[4.3.0]nonyl or azabicyclo[4.4.0]decyl.

The term“bridged heterocycloalkyl” means a bicyclic, saturated heterocycle with 7, 8, 9 or 10 ring atoms in total, in which the two rings share two common ring atoms which are not adjacent, which“bridged heterocycloalkyl” contains one or two identical or different ring heteroatoms from the series: N, O, S; it being possible for said bridged heterocycloalkyl group to be attached to the rest of the molecule via any one of the carbon atoms, except the spiro carbon atom, or, if present, a nitrogen atom.

Said bridged heterocycloalkyl group is, for example, azabicyclo[2.2.1]heptyl, oxazabicyclo[2.2.1]heptyl, thiazabicyclo[2.2.1]heptyl, diazabicyclo[2.2.1]heptyl, azabicyclo- [2.2.2]octyl, diazabicyclo[2.2.2]octyl, oxazabicyclo[2.2.2]octyl, thiazabicyclo[2.2.2]octyl, azabi- cyclo[3.2.1]octyl, diazabicyclo[3.2.1]octyl, oxazabicyclo[3.2.1]octyl, thiazabicyclo[3.2.1]octyl, azabicyclo[3.3.1]nonyl, diazabicyclo[3.3.1]nonyl, oxazabicyclo[3.3.1]nonyl, thiazabicyclo[3.3.1]- nonyl, azabicyclo[4.2.1]nonyl, diazabicyclo[4.2.1]nonyl, oxazabicyclo[4.2.1]nonyl, thiaza- bicyclo[4.2.1]nonyl, azabicyclo[3.3.2]decyl, diazabicyclo[3.3.2]decyl, oxazabicyclo[3.3.2]decyl, thiazabicyclo[3.3.2]decyl or azabicyclo[4.2.2]decyl.

The term“heteroaryl” means a monovalent, monocyclic, bicyclic or tricyclic aromatic ring having 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms (a“5- to 14-membered heteroaryl” group), particularly 5, 6, 9 or 10 ring atoms, which contains at least one ring heteroatom and optionally one, two or three further ring heteroatoms from the series: N, O and/or S, and which is bound via a ring carbon atom or optionally via a ring nitrogen atom (if allowed by valency).

Said heteroaryl group can be a 5-membered heteroaryl group, such as, for example, thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl or tetrazolyl; or a 6-membered heteroaryl group, such as, for example,  

pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl; or a tricyclic heteroaryl group, such as, for example, carbazolyl, acridinyl or phenazinyl; or a 9-membered heteroaryl group, such as, for example, benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzothiazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, indolizinyl or purinyl; or a 10- membered heteroaryl group, such as, for example, quinolinyl, quinazolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinoxalinyl or pteridinyl.

In general, and unless otherwise mentioned, the heteroaryl or heteroarylene groups include all possible isomeric forms thereof, e.g.: tautomers and positional isomers with respect to the point of linkage to the rest of the molecule. Thus, for some illustrative non-restricting examples, the term pyridinyl includes pyridin-2-yl, pyridin-3-yl and pyridin-4-yl; or the term thienyl includes thien-2-yl and thien-3-yl.

Particularly, the heteroaryl group is a quinolinyl group.

The term“azole” includes imidazoles, pyrazoles, triazoles and tetrazoles.

The term“C ₁ -C ₆ ”, as used in the present text, e.g. in the context of the definition of“C ₁ -C ₆ -alkyl”, “C ₁ -C ₆ -haloalkyl”,“C ₁ -C ₆ -hydroxyalkyl”,“C ₁ -C ₆ -alkoxy” or“C ₁ -C ₆ -haloalkoxy” means an alkyl group having a finite number of carbon atoms of 1 to 6, i.e.1, 2, 3, 4, 5 or 6 carbon atoms.

Further, as used herein, the term“C ₃ -C ₈ ”, as used in the present text, e.g. in the context of the definition of“C ₃ -C ₈ -cycloalkyl”, means a cycloalkyl group having a finite number of carbon atoms of 3 to 8, i.e.3, 4, 5, 6, 7 or 8 carbon atoms.

When a range of values is given, said range encompasses each value and sub-range within said range.

For example:

"C ₁ -C ₆ " encompasses C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₁ -C ₆ , C ₁ -C ₅ , C ₁ -C ₄ , C ₁ -C ₃ , C ₁ -C ₂ , C ₂ -C ₆ , C ₂ -C ₅ , C ₂ - C ₄ , C ₂ -C ₃ , C ₃ -C ₆ , C ₃ -C ₅ , C ₃ -C ₄ , C ₄ -C ₆ , C ₄ -C ₅ , and C ₅ -C ₆ ;

"C ₂ -C ₆ " encompasses C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₂ -C ₆ , C ₂ -C ₅ , C ₂ -C ₄ , C ₂ -C ₃ , C ₃ -C ₆ , C ₃ -C ₅ , C ₃ -C ₄ , C ₄ -C ₆ , C ₄ -C ₅ , and C ₅ -C ₆ ;

"C ₃ -C ₁₀ " encompasses C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₃ -C ₁₀ , C ₃ -C ₉ , C ₃ -C ₈ , C ₃ -C ₇ , C ₃ -C ₆ , C ₃ -C ₅ , C ₃ -C ₄ , C ₄ -C ₁₀ , C ₄ -C ₉ , C ₄ -C ₈ , C ₄ -C ₇ , C ₄ -C ₆ , C ₄ -C ₅ , C ₅ -C ₁₀ , C ₅ -C ₉ , C ₅ -C ₈ , C ₅ -C ₇ , C ₅ -C ₆ , C ₆ -C ₁₀ , C ₆ -C ₉ , C ₆ -C ₈ , C ₆ -C ₇ , C ₇ -C ₁₀ , C ₇ -C ₉ , C ₇ -C ₈ , C ₈ -C ₁₀ , C ₈ -C ₉ and C ₉ -C ₁₀ ;

"C ₃ -C ₈ " encompasses C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₃ -C ₈ , C ₃ -C ₇ , C ₃ -C ₆ , C ₃ -C ₅ , C ₃ -C ₄ , C ₄ -C ₈ , C ₄ -C ₇ , C ₄ - C ₆ , C ₄ -C ₅ , C ₅ -C ₈ , C ₅ -C ₇ , C ₅ -C ₆ , C ₆ -C ₈ , C ₆ -C ₇ and C ₇ -C ₈ ;

"C ₃ -C ₆ " encompasses C ₃ , C ₄ , C ₅ , C ₆ , C ₃ -C ₆ , C ₃ -C ₅ , C ₃ -C ₄ , C ₄ -C ₆ , C ₄ -C ₅ , and C ₅ -C ₆ ;

 

"C ₄ -C ₈ " encompasses C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₄ -C ₈ , C ₄ -C ₇ , C ₄ -C ₆ , C ₄ -C ₅ , C ₅ -C ₈ , C ₅ -C ₇ , C ₅ -C ₆ , C ₆ -C ₈ , C ₆ -C ₇ and C ₇ -C ₈ ;

"C ₄ -C ₇ " encompasses C ₄ , C ₅ , C ₆ , C ₇ , C ₄ -C ₇ , C ₄ -C ₆ , C ₄ -C ₅ , C ₅ -C ₇ , C ₅ -C ₆ and C ₆ -C ₇ ;

"C ₄ -C ₆ " encompasses C ₄ , C ₅ , C ₆ , C ₄ -C ₆ , C ₄ -C ₅ and C ₅ -C ₆ ;

"C ₅ -C ₁₀ " encompasses C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₅ -C ₁₀ , C ₅ -C ₉ , C ₅ -C ₈ , C ₅ -C ₇ , C ₅ -C ₆ , C ₆ -C ₁₀ , C ₆ -C ₉ , C ₆ -C ₈ , C ₆ -C ₇ , C ₇ -C ₁₀ , C ₇ -C ₉ , C ₇ -C ₈ , C ₈ -C ₁₀ , C ₈ -C ₉ and C ₉ -C ₁₀ ;

"C ₆ -C ₁₀ " encompasses C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₆ -C ₁₀ , C ₆ -C ₉ , C ₆ -C ₈ , C ₆ -C ₇ , C ₇ -C ₁₀ , C ₇ -C ₉ , C ₇ -C ₈ , C ₈ - C ₁₀ , C ₈ -C ₉ and C ₉ -C ₁₀ .

As used herein, the term“leaving group” means an atom or a group of atoms that is displaced in a chemical reaction as stable species taking with it the bonding electrons. In particular, such a leaving group is selected from the group comprising: halide, in particular fluoride, chloride, bromide or iodide, (methylsulfonyl)oxy, [(trifluoromethyl)sulfonyl]oxy, [(nonafluorobutyl)- sulfonyl]oxy, (phenylsulfonyl)oxy, [(4-methylphenyl)sulfonyl]oxy, [(4-bromophenyl)sulfonyl]oxy,

[(4-nitrophenyl)sulfonyl]oxy, [(2-nitrophenyl)sulfonyl]oxy, [(4-isopropylphenyl)sulfonyl]oxy,

[(2,4,6-triisopropylphenyl)sulfonyl]oxy, [(2,4,6-trimethylphenyl)sulfonyl]oxy, [(4-tert-butyl- phenyl)sulfonyl]oxy and [(4-methoxyphenyl)sulfonyl]oxy.

The schemes and procedures described below illustrate synthetic routes to the compounds of formula (I) of the invention and are not intended to be limiting. It is obvious to the person skilled in the art that the order of transformations as exemplified in the Schemes can be modified in various ways. The order of transformations exemplified in the Schemes is therefore not intended to be limiting. In addition, interconversion of any of the substituentscan be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W.

Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999).

Specific examples are described in the subsequent paragraphs.

It is possible for the compounds of general formula (I) to exist as isotopic variants. The invention therefore includes one or more isotopic variant(s) of the compounds of general formula (I), particularly deuterium-containing compounds of general formula (I).

 

The term“Isotopic variant” of a compound or a reagent is defined as a compound exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound.

The term“Isotopic variant of the compound of general formula (I)” is defined as a compound of general formula (I) exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound.

The expression“unnatural proportion” means a proportion of such isotope which is higher than its natural abundance. The natural abundances of isotopes to be applied in this context are described in“Isotopic Compositions of the Elements 1997”, Pure Appl. Chem., 70(1), 217-235, 1998.

Examples of such isotopes include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as ² H

(deuterium), ³ H (tritium), ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³² P, ³³ P, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ¹⁸ F, ³⁶ Cl, ⁸² Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁹ I and ¹³¹ I, respectively.

With respect to the treatment and/or prophylaxis of the disorders specified herein the isotopic variant(s) of the compounds of general formula (I) preferably contain deuterium (“deuterium- containing compounds of general formula (I)”). Isotopic variants of the compounds of general formula (I) in which one or more radioactive isotopes, such as ³ H or ¹⁴ C, are incorporated are useful e.g. in drug and/or substrate tissue distribution studies. These isotopes are particularly preferred for the ease of their incorporation and detectability. Positron emitting isotopes such as ¹⁸ F or ¹¹ C may be incorporated into a compound of general formula (I). These isotopic variants of the compounds of general formula (I) are useful for in vivo imaging applications. Deuterium- containing and ¹³ C-containing compounds of general formula (I) can be used in mass spectrometry analyses (H. J. Leis et al., Curr. Org. Chem., 1998, 2, 131) in the context of preclinical or clinical studies.

Isotopic variants of the compounds of general formula (I) can generally be prepared by methods known to a person skilled in the art, such as those described in the schemes and/or examples herein, by substituting a reagent for an isotopic variant of said reagent, preferably for a deuterium-containing reagent. Depending on the desired sites of deuteration, in some cases deuterium from D ₂ O can be incorporated either directly into the compounds or into reagents that are useful for synthesizing such compounds (Esaki et al., Tetrahedron, 2006, 62, 10954; Esaki et al., Chem. Eur. J., 2007, 13, 4052). Deuterium gas is also a useful reagent for incorporating deuterium into molecules. Catalytic deuteration of olefinic bonds (H. J. Leis et al., Curr. Org.

Chem., 1998, 2, 131; J. R. Morandi et al., J. Org. Chem., 1969, 34 (6), 1889) and acetylenic bonds (N. H. Khan, J. Am. Chem. Soc., 1952, 74 (12), 3018; S. Chandrasekhar et al., Tetrahedron Letters, 2011, 52, 3865) is a rapid route for incorporation of deuterium. Metal catalysts (i.e. Pd, Pt, and Rh) in the presence of deuterium gas can be used to directly exchange deuterium for hydrogen in functional groups containing hydrocarbons (J. G. Atkinson et al., US  

Patent 3966781). A variety of deuterated reagents and synthetic building blocks are commercially available from companies such as for example C/D/N Isotopes, Quebec, Canada;

Cambridge Isotope Laboratories Inc., Andover, MA, USA; and CombiPhos Catalysts, Inc., Princeton, NJ, USA. Further information on the state of the art with respect to deuterium- hydrogen exchange is given for example in Hanzlik et al., J. Org. Chem.55, 3992-3997, 1990;

R. P. Hanzlik et al., Biochem. Biophys. Res. Commun.160, 844, 1989; P. J. Reider et al., J. Org.

Chem.52, 3326-3334, 1987; M. Jarman et al., Carcinogenesis 16(4), 683-688, 1995; J. Atzrodt et al., Angew. Chem., Int. Ed. 2007, 46, 7744; K. Matoishi et al., Chem. Commun. 2000, 1519-1520; K. Kassahun et al., WO2012/112363.

The term“deuterium-containing compound of general formula (I)” is defined as a compound of general formula (I), in which one or more hydrogen atom(s) is/are replaced by one or more deuterium atom(s) and in which the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than the natural abundance of deuterium, which is about 0.015%. Particularly, in a deuterium-containing compound of general formula (I) the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, preferably higher than 90%, 95%, 96% or 97%, even more preferably higher than 98% or 99% at said position(s). It is understood that the abundance of deuterium at each deuterated position is independent of the abundance of deuterium at other deuterated position(s).

The selective incorporation of one or more deuterium atom(s) into a compound of general formula (I) may alter the physicochemical properties (such as for example acidity [C. L. Perrin, et al., J. Am. Chem. Soc., 2007, 129, 4490; A. Streitwieser et al., J. Am. Chem. Soc., 1963, 85, 2759;], basicity [C. L. Perrin et al., J. Am. Chem. Soc., 2005, 127, 9641; C. L. Perrin, et al., J.

Am. Chem. Soc., 2003, 125, 15008; C. L. Perrin in Advances in Physical Organic Chemistry, 44, 144], lipophilicity [B. Testa et al., Int. J. Pharm., 1984, 19(3), 271]) and/or the metabolic profile of the molecule and may result in changes in the ratio of parent compound to metabolites or in the amounts of metabolites formed. Such changes may result in certain therapeutic advantages and hence may be preferred in some circumstances. Reduced rates of metabolism and metabolic switching, where the ratio of metabolites is changed, have been reported (A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102; D. J. Kushner et al., Can. J. Physiol.

Pharmacol., 1999, 77, 79). These changes in the exposure to parent drug and metabolites can have important consequences with respect to the pharmacodynamics, tolerability and efficacy of a deuterium-containing compound of general formula (I). In some cases deuterium substitution reduces or eliminates the formation of an undesired or toxic metabolite and enhances the formation of a desired metabolite (e.g. Nevirapine: A. M. Sharma et al., Chem.

Res. Toxicol., 2013, 26, 410; Efavirenz: A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). In other cases the major effect of deuteration is to reduce the rate of systemic clearance.

 

As a result, the biological half-life of the compound is increased. The potential clinical benefits would include the ability to maintain similar systemic exposure with decreased peak levels and increased trough levels. This could result in lower side effects and enhanced efficacy, depending on the particular compound’s pharmacokinetic/ pharmacodynamic relationship. ML-337 (C. J.

Wenthur et al., J. Med. Chem., 2013, 56, 5208) and Odanacatib (K. Kassahun et al., WO2012/112363) are examples for this deuterium effect. Still other cases have been reported in which reduced rates of metabolism result in an increase in exposure of the drug without changing the rate of systemic clearance (e.g. Rofecoxib: F. Schneider et al., Arzneim. Forsch. / Drug. Res., 2006, 56, 295; Telaprevir: F. Maltais et al., J. Med. Chem., 2009, 52, 7993).

Deuterated drugs showing this effect may have reduced dosing requirements (e.g. lower number of doses or lower dosage to achieve the desired effect) and/or may produce lower metabolite loads.

A compound of general formula (I) may have multiple potential sites of attack for metabolism.

To optimize the above-described effects on physicochemical properties and metabolic profile, deuterium-containing compounds of general formula (I) having a certain pattern of one or more deuterium-hydrogen exchange(s) can be selected. Particularly, the deuterium atom(s) of deuterium-containing compound(s) of general formula (I) is/are attached to a carbon atom and/or is/are located at those positions of the compound of general formula (I), which are sites of attack for metabolizing enzymes such as e.g. cytochrome P ₄₅₀ .

Where the plural form of the word compounds, salts, polymorphs, hydrates, solvates and the like, is used herein, this is taken to mean also a single compound, salt, polymorph, isomer, hydrate, solvate or the like.

By "stable compound' or "stable structure" is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

The compounds of the present invention optionally contain one or more asymmetric centres, depending upon the location and nature of the various substituents desired. It is possible that one or more asymmetric carbon atoms are present in the (R) or (S) configuration, which can result in racemic mixtures in the case of a single asymmetric centre, and in diastereomeric mixtures in the case of multiple asymmetric centres. In certain instances, it is possible that asymmetry also be present due to restricted rotation about a given bond, for example, the central bond adjoining two substituted aromatic rings of the specified compounds.

Preferred compounds are those which produce the more desirable biological activity. Separated, pure or partially purified isomers and stereoisomers or racemic or diastereomeric mixtures of the compounds of the present invention are also included within the scope of the present invention.

The purification and the separation of such materials can be accomplished by standard techniques known in the art.  

The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by the formation of diastereoisomeric salts using an optically active acid or base or formation of covalent diastereomers. Examples of appropriate acids are tartaric, diacetyltartaric, ditoluoyltartaric and camphorsulfonic acid. Mixtures of diastereoisomers can be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known in the art, for example, by chromatography or fractional crystallisation. The optically active bases or acids are then liberated from the separated diastereomeric salts. A different process for separation of optical isomers involves the use of chiral chromatography (e.g., HPLC columns using a chiral phase), with or without conventional derivatisation, optimally chosen to maximise the separation of the enantiomers. Suitable HPLC columns using a chiral phase are commercially available, such as those manufactured by Daicel, e.g., Chiracel OD and Chiracel OJ, for example, among many others, which are all routinely selectable. Enzymatic separations, with or without derivatisation, are also useful. The optically active compounds of the present invention can likewise be obtained by chiral syntheses utilizing optically active starting materials.

In order to distinguish different types of isomers from each other reference is made to IUPAC

Rules Section E (Pure Appl Chem 45, 11-30, 1976).

The present invention includes all possible stereoisomers of the compounds of the present invention as single stereoisomers, or as any mixture of said stereoisomers, e.g. (R)- or (S)- isomers, in any ratio. Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of the present invention is achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example.

Further, it is possible for the compounds of the present invention to exist as tautomers. For example, any compound of the present invention which contains an imidazopyridine moiety as a heteroaryl group for example can exist as a 1H tautomer, or a 3H tautomer, or even a mixture in any amount of the two tautomers, namely :

1 ^{H tautomer 3H tautomer}

The present invention includes all possible tautomers of the compounds of the present invention as single tautomers, or as any mixture of said tautomers, in any ratio.

Further, the compounds of the present invention can exist as N-oxides, which are defined in that at least one nitrogen of the compounds of the present invention is oxidised. The present invention includes all such possible N-oxides.

 

The present invention also covers useful forms of the compounds of the present invention, such as metabolites, hydrates, solvates, prodrugs, salts, in particular pharmaceutically acceptable salts, and/or co-precipitates.

The compounds of the present invention can exist as a hydrate, or as a solvate, wherein the compounds of the present invention contain polar solvents, in particular water, methanol or ethanol for example, as structural element of the crystal lattice of the compounds. It is possible for the amount of polar solvents, in particular water, to exist in a stoichiometric or non- stoichiometric ratio. In the case of stoichiometric solvates, e.g. a hydrate, hemi-, (semi-), mono- , sesqui-, di-, tri-, tetra-, penta- etc. solvates or hydrates, respectively, are possible. The present invention includes all such hydrates or solvates.

Further, it is possible for the compounds of the present invention to exist in free form, e.g. as a free base, or as a free acid, or as a zwitterion, or to exist in the form of a salt. Said salt may be any salt, either an organic or inorganic addition salt, particularly any pharmaceutically acceptable organic or inorganic addition salt, which is customarily used in pharmacy, or which is used, for example, for isolating or purifying the compounds of the present invention.

The term“pharmaceutically acceptable salt" refers to an inorganic or organic acid addition salt of a compound of the present invention. For example, see S. M. Berge, et al.“Pharmaceutical Salts,” J. Pharm. Sci.1977, 66, 1-19.

A suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, or“mineral acid”, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic, 3- phenylpropionic, pivalic, 2-hydroxyethanesulfonic, itaconic, trifluoromethanesulfonic, dodecylsulfuric, ethanesulfonic, benzenesulfonic, para-toluenesulfonic, methanesulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, or thiocyanic acid, for example.

Further, another suitably pharmaceutically acceptable salt of a compound of the present invention which is sufficiently acidic, is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium, magnesium or strontium salt, or an aluminium or a zinc salt, or an ammonium salt derived from ammonia or from an organic primary, secondary or tertiary amine having 1 to 20 carbon atoms, such as ethylamine, diethylamine,  

triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, 1,2-ethylenediamine, N-methylpiperidine, N-methyl-glucamine, N,N-dimethyl-glucamine, N-ethyl-glucamine, 1,6-hexanediamine, glucosamine, sarcosine, serinol, 2-amino-1,3- propanediol, 3-amino-1,2-propanediol, 4-amino-1,2,3-butanetriol, or a salt with a quarternary ammonium ion having 1 to 20 carbon atoms, such as tetramethylammonium, tetraethylammonium, tetra(n-propyl)ammonium, tetra(n-butyl)ammonium, N-benzyl-N,N,N- trimethylammonium, choline or benzalkonium.

Those skilled in the art will further recognise that it is possible for acid addition salts of the claimed compounds to be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts of acidic compounds of the present invention are prepared by reacting the compounds of the present invention with the appropriate base via a variety of known methods.

The present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.

In the present text, in particular in the Experimental Section, for the synthesis of intermediates and of examples of the present invention, when a compound is mentioned as a salt form with the corresponding base or acid, the exact stoichiometric composition of said salt form, as obtained by the respective preparation and/or purification process, is, in most cases, unknown.

Unless specified otherwise, suffixes to chemical names or structural formulae relating to salts, such as "hydrochloride", "trifluoroacetate", "sodium salt", or "x HCl", "x CF ₃ COOH", "x Na ⁺ ", for example, mean a salt form, the stoichiometry of which salt form not being specified.

This applies analogously to cases in which synthesis intermediates or example compounds or salts thereof have been obtained, by the preparation and/or purification processes described, as solvates, such as hydrates, with (if defined) unknown stoichiometric composition.

Furthermore, the present invention includes all possible crystalline forms, or polymorphs, of the compounds of the present invention, either as single polymorph, or as a mixture of more than one polymorph, in any ratio.

Moreover, the present invention also includes prodrugs of the compounds according to the invention. The term “prodrugs” here designates compounds which themselves can be biologically active or inactive, but are converted (for example metabolically or hydrolytically) into compounds according to the invention during their residence time in the body.

Suitable targeting moieties include poly- and oligo-peptides, proteins, DNA and RNA fragments, aptamers etc, preferably a protein, e.g. avidin, strepatavidin, a polyclonal or monoclonal antibody (including IgG and IgM type antibodies), or a mixture of proteins or fragments or constructs of   protein. Antibodies, antibody constructs, fragments of antibodies (e.g. Fab fragments or any fragment comprising at least one antigen binding region(s)), constructs of fragments (e.g. single chain antibodies) or a mixture thereof are particularly preferred. Suitable fragments particularly include Fab, F(ab') ₂ , Fab' and/or scFv. Antibody constructs may be of any antibody or fragment indicated herein. In accordance with an embodiment of the first aspect, the present invention covers compounds of general formula (I), supra, in wherein Q represents the following structure:

wherein X represents aryl, heteroaryl, butylurea, fluoro-substituted phenyl, butyl- substituted phenyl, quinolinyl and 2-napthyl;

wherein Y represents aryl, heteroaryl, aralkyl, hetaralkyl,C ₃ -C ₈ -cycloalkyl, or pyridine;

wherein G represents CH ₂ N*H or N*H with * being the attachment point to the remainder of compound (I);

and wherein R ₅ represents tetrazole,–SO ₂ H, - SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H ₂ , -PO ₃ H, - PO ₄ H ₂ , -CO ₂ H, -C(O)R; wherein

R represents–H, -OH, -(C ₁ -C ₁₀ )alkyl, -O(C ₁ -C ₁₀ )alkyl, -NHR ₆ , or NR ₆ R ₇ ;

R ₆ , R ₇ and R ₈ each independently represent H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, BR, I,

C(O), C(S), -C(S)-NH-benzyl-, -C(O)-NH-benzyl-, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ )v-NR ₈ ,

-(CH ₂ )p-NH-C(O)-(CH ₂ )p-, -(CH ₂ -CH ₂ )t-NH-C(O)-(CH ₂ )p-, -(CH ₂ )p-COR, -(CH ₂ )p- C(O)NH-C[(CH ₂ )p-COR] ₃ , -C[(CH ₂ )p-COR] ₃ , or–(CH ₂ )p-(C ₅ -C ₁₄ )heteroaryl;

wherein v, p and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In accordance with an alternate embodiment of the second aspect, the present invention covers compounds of general formula (IA), supra, in which:

  wherein compound (I) is radiolabled with a radionuclide A selected from the group consisting of ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ⁸⁹ Zr, ⁹⁰ Y, ¹¹¹ In, ¹⁴⁹ Tb, ¹⁵² Tb, ¹⁵⁵ Tb, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac, ²²⁷ Th, and ²³² Th :

wherein X represents aryl, heteroaryl, butylurea, fluoro-substituted phenyl, butyl- substituted phenyl, quinoline and 2-napthyl;

wherein Y represents aryl, heteroaryl, aralkyl, hetaralkyl,C ₃ -C ₈ -cycloalkyl, or pyridine;

wherein G represents CH ₂ N*H or N*H with * being the attachment point to the remainder of compound (I);

and wherein R5 represents R ₅ represents–SO ₂ H, - SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H ₂ , - PO ₃ H, -PO ₄ H ₂ , -CO ₂ H, -C(O)R; wherein  

R represents–H, -OH, -(C ₁ -C ₁₀ )alkyl, -O(C ₁ -C ₁₀ )alkyl, -NHR ₆ , or NR ₆ R ₇ ;

R ₆ , R ₇ and R ₈ each independently represent H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, BR, I,

C(O), C(S), -C(S)-NH-benzyl-, -C(O)-NH-benzyl-, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ )v-NR ₈ ,

-(CH ₂ )p-NH-C(O)-(CH ₂ )p-, -(CH ₂ -CH ₂ )t-NH-C(O)-(CH ₂ )p-, -(CH ₂ )p-COR, -(CH ₂ )p- C(O)NH-C[(CH ₂ )p-COR] ₃ , -C[(CH ₂ )p-COR] ₃ , or–(CH ₂ )p-(C ₅ -C ₁₄ )heteroaryl;

wherein v, p and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In accordance with an alternate embodiment of the second aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

two of R1, R2, R3 and R4 represent OH and two of R1, R2, R3 and R4 represent Q,;or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same. It should be noted in this embodiment, both constitutional isomers (1,1 and 1,2) can both be present.

In accordance with an alternate embodiment of the second aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

three of R1, R2, R3 and R4 represent OH and one of R1, R2, R3 and R4 represents Q,or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In accordance with an alternate embodiment of the second aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

one of R1, R2, R3 and R4 represents OH and three of R1, R2, R3 and R4 represent Q, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In accordance with an alternate embodiment of the second aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

all of R1, R2, R3 and R4 represent Q,

or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same

In accordance with a further embodiment of the second aspect, the present invention covers compounds of general formula (I), supra, in which:

X represents 2-quinolinyl, and Y represents pyridine,   and stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same.

In a particular further embodiment of the first aspect, the present invention covers combinations of two or more of the above mentioned embodiments under the heading“further embodiments of the second aspect of the present invention”.

The present invention covers any sub-combination within any embodiment or aspect of the present invention of compounds of general formula (I), supra.

In accordance with an embodiment of the third aspect, the present invention covers compounds of general formula (IA) wherein compound (I) is radiolabled with a radionuclide A selected from the group consisting of of ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ⁸⁹ Zr, ⁹⁰ Y, ¹¹¹ In, ¹⁴⁹ Tb, ¹⁵² Tb, ¹⁵⁵ Tb, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac, ²²⁷ Th, and ²³² Th

wherein Q represents a monoclonal antibody with binding affinity for targets selected from the list consisting of FAP, , HER2, and PSMA.

In accordance with an alternate embodiment of the third aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

 

two of R1, R2, R3 and R4 represent OH and two of R1, R2, R3 and R4 represent Q,;or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same. It should be noted in this embodiment, both constitutional isomers (1,1 and 1,2) can both be present.

In accordance with an alternate embodiment of the third aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

three of R1, R2, R3 and R4 represent OH and one of R1, R2, R3 and R4 represents Q,or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In accordance with an alternate embodiment of the third aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

one of R1, R2, R3 and R4 represents OH and three of R1, R2, R3 and R4 represent Q, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

In accordance with an alternate embodiment of the third aspect, the present invention covers compounds of general formula (I), supra, in which:

n is 1;

all of R1, R2, R3 and R4 represent Q,

or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same

The present invention covers any sub-combination within any embodiment or aspect of the present invention of intermediate compounds of general formula (IA).

The present invention covers the compounds of general formula (I) which are disclosed in the Example Section of this text, infra.

The compounds of general formula (I) of the present invention can be converted to any salt, preferably pharmaceutically acceptable salts, as described herein, by any method which is known to the person skilled in the art. Similarly, any salt of a compound of general formula (I) of the present invention can be converted into the free compound, by any method which is known to the person skilled in the art.

Compounds of general formula (I) of the present invention demonstrate a valuable pharmacological spectrum of action and pharmacokinetic profile, both of which could not have been predicted. Compounds of the present invention have surprisingly been found to effectively

 

target tumors and it is possible therefore that said compounds be used for the treatment or prophylaxis of diseases, preferably soft tissue disorders in humans and animals.

Compounds of the present invention can be utilized to inhibit, block, reduce, decrease, etc., cell proliferation and/or cell division, and/or produce apoptosis. This method comprises administering to a mammal in need thereof, including a human, an amount of a compound of general formula (I) of the present invention, or a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof, which is effective to treat the disorder.

Hyperproliferative disorders include, but are not limited to, for example : psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), solid tumours, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. Those disorders also include lymphomas, sarcomas, and leukaemias.

Examples of breast cancers include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are not limited to, small-cell and non- small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to, brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumour.

Tumours of the male reproductive organs include, but are not limited to, prostate and testicular cancer.

Tumours of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.

Tumours of the digestive tract include, but are not limited to, anal, colon, colorectal, oesophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.

Tumours of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, urethral and human papillary renal cancers.

Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi’s sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

 

Head-and-neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oral cavity cancer and squamous cell.

Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin’s lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin’s disease, and lymphoma of the central nervous system.

Sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

The present invention also provides methods of treating angiogenic disorders including diseases associated with excessive and/or abnormal angiogenesis.

Inappropriate and ectopic expression of angiogenesis can be deleterious to an organism. A number of pathological conditions are associated with the growth of extraneous blood vessels.

These include, for example, diabetic retinopathy, ischemic retinal-vein occlusion, and retinopathy of prematurity [Aiello et al., New Engl. J. Med., 1994, 331, 1480 ; Peer et al., Lab.

Invest., 1995, 72, 638], age-related macular degeneration (AMD) [Lopez et al., Invest.

Opththalmol. Vis. Sci., 1996, 37, 855], neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma, inflammation, rheumatoid arthritis (RA), restenosis, in-stent restenosis, vascular graft restenosis, etc. In addition, the increased blood supply associated with cancerous and neoplastic tissue, encourages growth, leading to rapid tumour enlargement and metastasis.

Moreover, the growth of new blood and lymph vessels in a tumour provides an escape route for renegade cells, encouraging metastasis and the consequence spread of the cancer. Thus, compounds of general formula (I) of the present invention can be utilized to treat and/or prevent any of the aforementioned angiogenesis disorders, for example by inhibiting and/or reducing blood vessel formation; by inhibiting, blocking, reducing, decreasing, etc. endothelial cell proliferation, or other types involved in angiogenesis, as well as causing cell death or apoptosis of such cell types.

These disorders have been well characterized in humans, but also exist with a similar etiology in other mammals, and can be treated by administering pharmaceutical compositions of the present invention.

The term“treating” or“treatment” as stated throughout this document is used conventionally, for example the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of a disease or disorder, such as a carcinoma.

The compounds of the present invention can be used in particular in therapy and prevention, i.e.

prophylaxis, of tumour growth and metastases, especially in solid tumours of all indications and stages with or without pre-treatment of the tumour growth.  

Generally, the use of chemotherapeutic agents and/or anti-cancer agents in combination with a compound or pharmaceutical composition of the present invention will serve to:

1. yield better efficacy in reducing the growth of a tumour or even eliminate the tumour as

compared to administration of either agent alone,

2. provide for the administration of lesser amounts of the administered chemotherapeutic

agents,

3. provide for a chemotherapeutic treatment that is well tolerated in the patient with fewer

deleterious pharmacological complications than observed with single agent chemotherapies and certain other combined therapies,

4. provide for treating a broader spectrum of different cancer types in mammals, especially

humans,

5. provide for a higher response rate among treated patients,

6. provide for a longer survival time among treated patients compared to standard

chemotherapy treatments,

7. provide a longer time for tumour progression, and/or

8. yield efficacy and tolerability results at least as good as those of the agents used alone,

compared to known instances where other cancer agent combinations produce antagonistic effects.

In addition, the compounds of general formula (I) of the present invention can also be used in combination with radiotherapy and/or surgical intervention.

In a further embodiment of the present invention, the compounds of general formula (I) of the present invention may be used to sensitize a cell to radiation, i.e. treatment of a cell with a compound of the present invention prior to radiation treatment of the cell renders the cell more susceptible to DNA damage and cell death than the cell would be in the absence of any treatment with a compound of the present invention. In one aspect, the cell is treated with at least one compound of general formula (I) of the present invention.

Thus, the present invention also provides a method of killing a cell, wherein a cell is administered one or more compounds of the present invention in combination with conventional radiation therapy.

The present invention also provides a method of rendering a cell more susceptible to cell death, wherein the cell is treated with one or more compounds of general formula (I) of the present invention prior to the treatment of the cell to cause or induce cell death. In one aspect, after the cell is treated with one or more compounds of general formula (I) of the present invention, the cell is treated with at least one compound, or at least one method, or a combination thereof, in  

order to cause DNA damage for the purpose of inhibiting the function of the normal cell or killing the cell.

In other embodiments of the present invention, a cell is killed by treating the cell with at least one DNA damaging agent, i.e. after treating a cell with one or more compounds of general formula (I) of the present invention to sensitize the cell to cell death, the cell is treated with at least one DNA damaging agent to kill the cell. DNA damaging agents useful in the present invention include, but are not limited to, chemotherapeutic agents (e.g. cis platin), ionizing radiation (X-rays, ultraviolet radiation), carcinogenic agents, and mutagenic agents.

In other embodiments, a cell is killed by treating the cell with at least one method to cause or induce DNA damage. Such methods include, but are not limited to, activation of a cell signalling pathway that results in DNA damage when the pathway is activated, inhibiting of a cell signalling pathway that results in DNA damage when the pathway is inhibited, and inducing a biochemical change in a cell, wherein the change results in DNA damage. By way of a non-limiting example, a DNA repair pathway in a cell can be inhibited, thereby preventing the repair of DNA damage and resulting in an abnormal accumulation of DNA damage in a cell.

In one aspect of the invention, a compound of general formula (I) of the present invention is administered to a cell prior to the radiation or other induction of DNA damage in the cell. In another aspect of the invention, a compound of general formula (I) of the present invention is administered to a cell concomitantly with the radiation or other induction of DNA damage in the cell. In yet another aspect of the invention, a compound of general formula (I) of the present invention is administered to a cell immediately after radiation or other induction of DNA damage in the cell has begun.

In another aspect, the cell is in vitro. In another embodiment, the cell is in vivo.

In accordance with a further aspect, the present invention covers compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for use in the diagnosis, treatment, or prophylaxis of diseases, in particular soft tissue disorders.

In accordance with a further aspect, the present invention covers the use of compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for the diagnosis, treatment, or prophylaxis of diseases, in particular soft tissue disorders, particularly prostate cancer.

In accordance with a further aspect, the present invention covers the use of a compound of formula (I), described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, for

 

the diagnosis, prophylaxis, or treatment of diseases, in particular soft tissue disorders, particularly prostate cancer.

In accordance with a further aspect, the present invention covers the use of compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, in a method of diagnosis, treatment or prophylaxis of diseases, in particular soft tissue disorders, particularly prostate cancer.

In accordance with a further aspect, the present invention covers use of a compound of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for the preparation of a pharmaceutical composition, preferably a medicament, for the diagnosis, prophylaxis, or treatment of diseases, in particular soft tissue disorders, particularly prostate cancer.

In accordance with a further aspect, the present invention covers a method of diagnosis, treatment, or prophylaxis of diseases, in particular soft tissue disorders, particularly prostate cancer, using an effective amount of a compound of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same.

In a preferred embodiment, compounds of general formula (I) can be radiolabled with an appropriate radionuclide and used for the imaging of an internal organ of a mammal according to conventional methods.

In accordance with a further aspect, the present invention covers pharmaceutical compositions, in particular a medicament, comprising a compound of general formula (I), as described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, a salt thereof, particularly a pharmaceutically acceptable salt, or a mixture of same, and one or more excipients), in particular one or more pharmaceutically acceptable excipient(s). Conventional procedures for preparing such pharmaceutical compositions in appropriate dosage forms can be utilized.

The present invention furthermore covers pharmaceutical compositions, in particular medicaments, which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipients, and to their use for the above mentioned purposes.

It is possible for the compounds according to the invention to have systemic and/or local activity.

For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent.

 

For these administration routes, it is possible for the compounds according to the invention to be administered in suitable administration forms.

For oral administration, it is possible to formulate the compounds according to the invention to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally- disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the invention in crystalline and/or amorphised and/or dissolved form into said dosage forms.

Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal).

Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders.

Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays;

tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents.

The compounds according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, inter alia,

● fillers and carriers (for example cellulose, microcrystalline cellulose (such as, for example, Avicel ^® ), lactose, mannitol, starch, calcium phosphate (such as, for example, Di-Cafos ^® )), ● ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols),

● bases for suppositories (for example polyethylene glycols, cacao butter, hard fat),

● solvents (for example water, ethanol, isopropanol, glycerol, propylene glycol, medium

chain-length triglycerides fatty oils, liquid polyethylene glycols, paraffins),

 

● surfactants, emulsifiers, dispersants or wetters (for example sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols (such as, for example, Lanette ^® ), sorbitan fatty acid esters (such as, for example, Span ^® ), polyoxyethylene sorbitan fatty acid esters (such as, for example, Tween ^® ), polyoxyethylene fatty acid glycerides (such as, for example, Cremophor ^® ), polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers (such as, for example, Pluronic ^® ), ● buffers, acids and bases (for example phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine),

● isotonicity agents (for example glucose, sodium chloride),

● adsorbents (for example highly-disperse silicas),

● viscosity-increasing agents, gel formers, thickeners and/or binders (for example polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropyl- cellulose, carboxymethylcellulose-sodium, starch, carbomers, polyacrylic acids (such as, for example, Carbopol ^® ); alginates, gelatine), ● disintegrants (for example modified starch, carboxymethylcellulose-sodium, sodium

starch glycolate (such as, for example, Explotab ^® ), cross- linked polyvinylpyrrolidone, croscarmellose-sodium (such as, for example, AcDiSol ^® )), ● flow regulators, lubricants, glidants and mould release agents (for example magnesium

stearate, stearic acid, talc, highly-disperse silicas (such as, for example, Aerosil ^® )), ● coating materials (for example sugar, shellac) and film formers for films or diffusion membranes which dissolve rapidly or in a modified manner (for example polyvinylpyrrolidones (such as, for example, Kollidon ^® ), polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropyl- methylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates such as, for example, Eudragit ^® )), ● capsule materials (for example gelatine, hydroxypropylmethylcellulose),

● synthetic polymers (for example polylactides, polyglycolides, polyacrylates, polymethacrylates (such as, for example, Eudragit ^® ), polyvinylpyrrolidones (such as, for example, Kollidon ^® ), polyvinyl alcohols, polyvinyl acetates, polyethylene oxides, polyethylene glycols and their copolymers and blockcopolymers),

  ● plasticizers (for example polyethylene glycols, propylene glycol, glycerol, triacetine, triacetyl citrate, dibutyl phthalate),

● penetration enhancers,

● stabilisers (for example antioxidants such as, for example, ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate), ● preservatives (for example parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate),

● colourants (for example inorganic pigments such as, for example, iron oxides, titanium

dioxide),

● flavourings, sweeteners, flavour- and/or odour-masking agents.

The present invention furthermore relates to a pharmaceutical composition which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention.

In accordance with another aspect, the present invention covers pharmaceutical combinations, in particular medicaments, comprising at least one compound of general formula (I) of the present invention and at least one or more further active ingredients, in particular for the diagnosis, treatment, and/or prophylaxis of prostate cancer.

Particularly, the present invention covers a pharmaceutical combination, which comprises:

● one or more first active ingredients, in particular compounds of general formula (I) as

defined supra, and

● one or more further active ingredients, in particular prostate cancer

The term“combination” in the present invention is used as known to persons skilled in the art, it being possible for said combination to be a fixed combination, a non-fixed combination or a kit- of-parts.

A“fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein, for example, a first active ingredient, such as one or more compounds of general formula (I) of the present invention, and a further active ingredient are present together in one unit dosage or in one single entity. One example of a“fixed combination” is a pharmaceutical composition wherein a first active ingredient and a further active ingredient are present in admixture for simultaneous administration, such as in a formulation. Another example of a“fixed combination” is a pharmaceutical combination wherein a first active ingredient and a further active ingredient are present in one unit without being in admixture.

A non-fixed combination or“kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein a first active ingredient and a further  

active ingredient are present in more than one unit. One example of a non-fixed combination or kit-of-parts is a combination wherein the first active ingredient and the further active ingredient are present separately. It is possible for the components of the non-fixed combination or kit-of- parts to be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

The compounds of the present invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutically active ingredients where the combination causes no unacceptable adverse effects. The present invention also covers such pharmaceutical combinations. For example, the compounds of the present invention can be combined with known anti-cancer agents.

Examples of anti-cancer agents include:

131I-chTNT, abarelix, abemaciclib, abiraterone, acalabrutinib, aclarubicin, adalimumab, ado- trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine, angiotensin II, antithrombin III, apalutamide, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, atezolizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, basiliximab, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, bosutinib, buserelin, brentuximab vedotin, brigatinib, busulfan, cabazitaxel, cabozantinib, calcitonine, calcium folinate, calcium levofolinate, capecitabine, capromab, carbamazepine carboplatin, carboquone, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, durvalumab, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, enasidenib, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinylestradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds,  

lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, inotuzumab ozogamicin, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, Iasocholine, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, lutetium Lu 177 dotatate, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, midostaurin, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, mvasi, nabilone, nabiximols, nafarelin, naloxone + pentazocine, naltrexone, nartograstim, necitumumab, nedaplatin, nelarabine, neratinib, neridronic acid, netupitant/palonosetron, nivolumab, pentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, niraparib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palbociclib, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG- epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pembrolizumab, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, ribociclib, risedronic acid, rhenium-186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, rucaparib, samarium (153Sm) lexidronam, sargramostim, sarilumab, satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogene laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium

(99mTc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tisagenlecleucel, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine + tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine,  

vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.

Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of soft tissue disease, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known active ingredients or medicaments that are used to treat these conditions, the effective dosage of the compounds of the present invention can readily be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.

In all aspects of the present invention, the tissue-targeting radiopharmaceutical preferably comprises Th-227. The radiopharmaceutical is preferably administered at a dosage level of thorium- 227 dosage of 500 kBq/kg to 2 MBq/kg bodyweight, preferably 1.5 MBq/kg.

Correspondingly, a single dosage until may comprise around any of these ranges multiplied by a suitable bodyweight, such as 30 to 150 Kg, preferably 40 to 100 Kg Of course the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests. General Procedures The compounds according to the invention can be prepared according to the following schemes 1 through 15.

The schemes and procedures described below illustrate synthetic routes to the compounds of formula (I) of the invention and are not intended to be limiting. It is obvious to the person skilled in the art that the order of transformations as exemplified in the Schemes can be modified in various ways. The order of transformations exemplified in the Schemes is therefore not intended to be limiting. In addition, interconversion of any of the substituents, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, V, W, Y, Z and Het can be achieved before and/or after the  

exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs.

 

Scheme 1: Route for the preparation of compounds of formula (I) wherein n, R1, R2, R3, R4, R5, X, and Y have the meaning as given for general formula (I) supra. PG ₁ as a carboxylic acid protecting group. PG-R1, PG-R2, PG-R3, and PG-R4 means protected R1, R2, R3, and R4 or OH.

HOPO chelator A is reacted with a suitably protected amine Q-PG-NH2 under amide coupling conditions known to those skilled in the art to give amide PG-I. Possible reaction conditions include but are not limited to amide coupling reagents like HATU (1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridi nium 3-oxide hexafluorophosphate) or PyAOP ((3-hydroxy-3H-1,2,3-triazolo[4,5-b]pyridinato-O)tri-1- pyrrolidinyl-phosphorus hexafluorophosphate). In this reaction in theory 0–4 amidation reactions with Q-PG-NH2 can occur at HOPO chelator A yielding different multimers: monomer for one reaction, dimer for two reactions, trimer for three reactions, tetramer for four reactions at the HOPO chelator A.

In the next step the protect compound PG-I is deprotected by conditions known to those skilled in the art to give compound I. For example the removal of Boc-amine protecting groups or tert- butyl esters can be achieved by TFA (trifluoroacetic acid) or hydrochloric acid.

 

Scheme 2: Route for the preparation of compounds of formula (I) wherein n, R1, R2, R3, and R4 have the meaning as given for general formula (I) supra.

(I-223Th)

Scheme 3: Route for the preparation of compounds of formula (I) wherein n, R1, R2, R3, and R4 have the meaning as given for general formula (I) supra.

 

'

Scheme 4: Route for the preparation of HOPO chelator (A) wherein n, have the meaning as given for general formula (I) supra. PG ₁ is a carboxylic acid protecting group tert-butyl, PG ₂ is a phenol protecting group like benzyl.

Suitably protected hydroxypyridone A-HOPO is coupled to tetraamine A-amine under amide coupling conditions known to those skilled in the art. Possible reaction conditions include but are not limited to amide coupling reagents like HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate). In the following step the protecting groups are removed by conditions known to those skilled in the art for the respective protecting groups. Possible reaction conditions include but are not limited to cleavage by hydrochloric acid, hydrobromic acid, hydrogen bromide in acetic acid or trifluoroacetic acid.

 

Scheme 5: Route for the preparation of HOPO chelator (A) wherein n, have the meaning as given for general formula (I) supra and LG is a leaving group. PG ₁ is a carboxylic acid protecting group methyl or tert-butyl, PG ₂ is a phenol protecting group like benzyl. PG ₃ is a carboxylic acid protecting group like methyl or ethyl which can be cleaved orthogonally to PG ₁ .

PG ₃ -protected oxalacetate sodium salt is reacted with chloroacetone and ammonia under suitable conditions to give protected hydroxypyridone A-HOPO-1. These reaction conditions include but are not limited to heating, elevated pressure or the use of a Lewis acid like aluminium trichloride. A-HOPO-1 is then protected at the phenol position by reaction with PG ₂ -X to give A- HOPO-2. After that A-HOPO-2 is reacted with an activated protected acetic acid equivalent like tert-butyl bromoacetate to give PG-HOPO-3. Finally PG ₃ is cleaved selectively by conditions known to those skilled in the art like for example lithium hydroxide for the cleavage of ethyl or methyl esters to give A-HOPO.

The order of the second and third step in this synthesis can be exchanged meaning to alkylate the pyridine NH before protection of the phenol.

 

Scheme 6: Route for the preparation of A-amine wherein n has the meaning as given for general formula (I) supra and LG is a leaving group.

Bis-reactive A-amine-1 is reacted with an appropriate azide like sodium azide under conditions for alkylic nucleophilic displacement known to those skilled in the art to give bis azide A-amine- 2. This is then further reacted with an appropriate bis-reactive alkane like 1,3-dibromopropoane under conditions for alkylic nucleophilic displacement known to those skilled in the art to give tetraazide A-amine-3. Tetraazide A-amine-3 is the reduced to tetraamien A-amine under conditions typical for the reduced of azides to the corresponding amine like catalytic hydrogenation with palladium on charcoal or with triphenyl phosphine.

Scheme 7: Alternative route for the preparation of A-amine wherein n has the meaning as given for general formula (I) supra, PG ₄ is an amine protecting group and LG is a leaving group.

Trisamine A-amine-4 is protected at the terminal primary amines with a suitable protecting group like Boc, Fmoc, Cbz, or trityl to give bis-protected trisamine A-amine-5. This is then further reacted with an appropriate bis-reactive alkane like 1,3-dibromopropoane under conditions for alkylic nucleophilic displacement known to those skilled in the art to give tetrakis-protected

  hexaamine A-amine-6. A-amine-6 is then deprotected under conditions known to those skilled in the art to give A-amine.

'

Scheme 8: Route for the preparation of amine 1-4 wherein R ⁵ has the meaning as given for general formula (I) supra, PG ₁ is a carboxylic acid protecting group, PG ₄ is an amine protecting group and LG is a leaving group.

Protected lysine 1-1 is coupled with the a-amino group of protected amino acid 1-2 via an appropriate carbonic acid equivalent to give urea 1-3. This carbonic acid equivalent can be but is not limited to N,N’-carbonyl diimidazole, phosgene, diphosgene, triphosgene, or p- nitrophenylchloroformate and is reacted first with 1-1 and then treated with 1-2 together with a suitable base like for example N,N-diisopropylethylamine or triethylamine or reacted first with 1- 2 and then treated with 1-1 together with a suitable base like for example N,N- diisopropylethylamine or triethylamine. In the next step 1-3 is deprotected at the e-amino group of the lysine moiety under conditions known to those skilled in the art to give amine 1-4.

 

Scheme 9: Route for the preparation of amines Q-PG and Q-NH2 wherein X, Y and R ⁵ have the meaning as given for general formula (I) supra, PG ₁ is a carboxylic acid protecting group and PG ₄ is an amine protecting group.

Amine 1-4 is coupled to N-protected amino acid AA1 by typical peptide coupling conditions known to those skilled in the art to give protected peptide 1-5. Typical reaction conditions include but are to limited to the use of coupling reagents like HATU, PyAOP, or DMTMM. 1-5 is then deprotected at the amine position by conditions known to those skilled in the art to give amine 1-6. These conditions include but are not limited to the use of trifluoroacetic acid for Boc protecting groups, catalytic hydrogenation for Cbz protecting groups or piperidine for Fmoc protecting groups. Amine 1-6 is then coupling with N-protected amino acid AA2 by typical peptide coupling conditions known to those skilled in the art to give protected peptide 1-7. 1-7 is then

 

deprotected at the amine position by conditions known to those skilled in the art to give amine Q-PG. Q-PG is then preferably conjugated via amide bond formation using standard acid activating coupling reagents to the a carboxylate group on the HOPO chelators described in this invention. The use of stable tetrafluorophenol esters of compound (I) are preferred. Alternatively Q-PG can be deprotected at the carboxylic acid groups by conditions known to those skilled in the art to give Q-NH2. These conditions include but are not limited to trifluoro acetic acid or hydrochloric acid. The chelator moiety may then be coupled through amide bond formation using preformed active esters or other suitably activated carboxylate groups on the chelator.

These syntheses can also be performed on solid phase by conditions known to those skilled in the art by attaching amine AA2 or amine 1-4 to a solid phase like e.g. 2-chlorotrityl resin and cleaving it off the resin by suitable reagents like trifluoroacetic acid.

 

Scheme 10: Route for the preparation of PEGylated HOPO conjugate 1-11 wherein X, Y, R5 and have the meaning as given for general formula (I) supra, PG ₁ is a carboxylic acid protecting group, PG ₄ is an amine protecting group and m is from 2 to 16, preferably 10.   Q-PG is coupled to N-protected PEG carboxylic acid PG-PEG by standard amide coupling conditions known to those skilled in the art to give 1-8. These conditions include but are not limited to the use of coupling reagents like HATU or PyAOP.1-8 is then deprotected at the amino group by by conditions known to those skilled in the art to give amine 1-9. Amine 1-9 is the coupled to HOPO chelator glycolic acid HOPO1 by amide coupling conditions known to those skilled in the art to give 1-10. These conditions include but are not limited to the use of coupling reagents like HATU or PyAOP. HOPO1 can be synthesized by reacting the free aniline of HOPO1 (described in WO2013167756) with diglycolic acid anhydride with a suitable base like 2,4,6-trimethylpyridine. 1-10 is then deprotected at the carboxylic acid groups by conditions known to those skilled in the art to give conjugate 1-11. These conditions include but are not limited to trifluoro acetic acid or hydrochloric acid.

  Scheme 11: Route for the preparation of thiourea HOPO conjugate 1-12 wherein X, Y, R5 and m have the meaning as given above.

Amine Q-NH2 is reacted with HOPO isothiocyanate HOPO2 (described in WO 2013167756) in an appropriate buffer like borate buffer to give thiourea 1-12.

Scheme 12: Route for the preparation of DOTA conjugate 1-14 wherein X, Y and R5 have the meaning as given for general formula (I) supra and PG ₁ is a carboxylic acid protecting group.

Amine Q-PG is coupled to tris-protected DOTA derivative (DOTA) by amide coupling conditions known to those skilled in the art to give 1-13. These conditions include but are not limited to the use of coupling reagents like HATU or PyAOP. 1-13 is then deprotected at the carboxylic acid groups by conditions known to those skilled in the art to give conjugate 1-14. These conditions include but are not limited to trifluoro acetic acid or hydrochloric acid.

 

Scheme 13: Route for the preparation of tritiated DOTA conjugate T-1-14 wherein X, Y and R5 have the meaning as given for general formula (I) supra and PG ₁ is a carboxylic acid protecting group.

  Amine Q-PG is brominated at residue X by conditions known to those skilled in the art to give Br-Q-PG. These conditions include but are not limited to the use of N-bromosuccinimide or bromine. Br-Q-PG is then coupled to tris-protected DOTA derivative (DOTA) by amide coupling conditions known to those skilled in the art to give Br-1-13. These conditions include but are not limited to the use of coupling reagents like HATU or PyAOP. Br-1-13 is then tritiated via catalytic tritiation to give T-1-13. T-1-13 is then deprotected at the carboxylic acid groups by conditions known to those skilled in the art to give conjugate T-1-14. These conditions include but are not limited to trifluoro acetic acid or hydrochloric acid.

Scheme 14: Route for the preparation of amino acid AA2 derivative AAX2 wherein PG ₄ is an amine protecting group and X is an aromatic carbocycle or heterocycle.

Amino acids of type AAX2 can be prepared according to R. Ramón et al., ChemBioChem 2011, 12, 625-632. Benzylic aldehyde 1-15 is reacted with phosphonate 1-16 in a Wittig-type reaction to give aminoacrylic ester 1-17.1-17 is then reduced to protected amino acid 1-18. To yield the racemic amino acid 1-18 the reduction can be performed using achiral catalysts like palladium on charcoal under a hydrogen atmosphere. For stereoselective reduction yielding the enantiomerically pure or enriched amino acids 1-18 and AAX2 the reduction can be performed using a chiral catalyst like (R)-[Rh(COD)(MaxPhos)]BF ₄ under a hydrogen atmosphere.

The benzylic aldehydes 1-15 can be prepared for example via oxidation of the corresponding benzyl alcohol using Dess-Martin periodinane or Swern conditions or by reduction of the corresponding benzoic acid using DIBAL-H. The corresponding benzyl alcohol can be synthesized by bromination of the methyl group of the corresponding toluoyl derivative followed by hydrolysis or substitution with acetate and subsequent ester hydrolysis or by reduction of the corresponding methyl ester with lithium aluminium hydride. Moreover 1-15 can be prepared by treating the corresponding arylbromide with n-butyllithium and N,N-dimethylformamide.

 

Scheme 15: Route for the preparation of amino acid (S)-AA2 derivative AAX2 wherein PG ₄ is an amine protecting group, LG is a leaving group and X is an aromatic carbocycle or heterocycle.

Another possibility to synthesize enantiomerically enriched or pure AAX2 is described in D. A.

Evans et al., J. Am. Chem. Soc.1987, 22, 6881 and starts with activation of the corresponding 3-aryl-propanoic acid 1-19 by for example oxalyl chloride or thionyl chloride to yield 1-20. This is then reacted with (4S)-4-benzyl-1,3-oxazolidin-2-one which was deprotonated with N- butyllithium to yield chiral imide 1-21. This is then deprotonated with a lithium bis(trimethylsilyl)amide and reacted with 2,4,6-tri(propan-2-yl)benzene-1-sulfonyl azide to give chirakl azido acid 1-23. Reduction of the a-azido group by triphenyl phosphine yield chiral amino acid 1-24 which is protected at the amino group to give (S)-AAX2.

EXPERIMENTAL SECTION Abbreviations The following table lists the abbreviations used in this paragraph and in the Intermediates and Examples section as far as they are not explained within the text body. Table 1: Abbreviations

 

 

 

Amino acid Abbreviations Ala = Alanine

Arg = Arginine

Asn = Asparagine  

Asp = Aspartic acid

Cys = Cysteine

Glu = Glutamic acid

Gln = Glutamine

Gly = Glycine

His = Histidine

Ile = Isoleucine

Leu = Leucine

Lys = Lysine

Met = Methionine

Phe = Phenylalanine

Pro = Proline

Ser = Serine

Thr = Threonine

Trp = Tryptophan

Tyr = Tyrosine

Other abbreviations not specified herein have their meanings customary to the skilled person.

The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit in any way the full scope of the invention as described herein.

Specific Experimental Descriptions NMR peak forms in the following specific experimental descriptions are stated as they appear in the spectra, possible higher order effects have not been considered. Chemical shifts (d) are given in ppm. The chemical shifts were corrected by setting the DMSO signal to 2.50 ppm unless otherwise stated. The ¹ H-NMR data of selected compounds are listed in the form of ¹ H-NMR peaklists. Therein, for each signal peak the d value in ppm is given, followed by the signal intensity, reported in round brackets. The d value-signal intensity pairs from different peaks are separated by  

commas. Therefore, a peaklist is described by the general form: d ₁ (intensity ₁ ), d ₂ (intensity ₂ ), ...

, d _i (intensity _i ), ... , d _n (intensity _n ). The intensity of a sharp signal correlates with the height (in cm) of the signal in a printed NMR spectrum. When compared with other signals, this data can be correlated to the real ratios of the signal intensities. In the case of broad signals, more than one peak, or the center of the signal along with their relative intensity, compared to the most intense signal displayed in the spectrum, are shown. A ¹ H-NMR peaklist is similar to a classical ¹ H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation. Moreover, similar to classical ¹ H- NMR printouts, peaklists can show solvent signals, signals derived from stereoisomers of the particular target compound, peaks of impurities, ¹³ C satellite peaks, and/or spinning sidebands.

The peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compound (e.g., with a purity of >90%). Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify a reproduction of the manufacturing process on the basis of "by-product fingerprints". An expert who calculates the peaks of the target compound by known methods (MestReC, ACD simulation, or by use of empirically evaluated expectation values), can isolate the peaks of the target compound as required, optionally using additional intensity filters. Such an operation would be similar to peak-picking in classical ¹ H-NMR interpretation. A detailed description of the reporting of NMR data in the form of peaklists can be found in the publication "Citation of NMR Peaklist Data within Patent Applications" (cf.

http://www.researchdisclosure.com/searching-disclosures, Research Disclosure Database Number 605005, 2014, 01 Aug 2014). In the peak picking routine, as described in the Research Disclosure Database Number 605005, the parameter "MinimumHeight" can be adjusted between 1% and 4%. However, depending on the chemical structure and/or depending on the concentration of the measured compound it may be reasonable to set the parameter "MinimumHeight" <1%. Reactions employing microwave irradiation may be run with a Biotage Initator ^{^} microwave oven optionally equipped with a robotic unit. The reported reaction times employing microwave heating are intended to be understood as fixed reaction times after reaching the indicated reaction temperature. The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. from Separtis such as Isolute ^® Flash silica gel (“SiO ₂ ”) or Isolute ^® Flash NH ₂ silica gel (“amine-coated SiO ₂ ”) in  

combination with a Isolera autopurifier (Biotage) and eluents such as gradients of e.g. hexane/ ethyl acetate or dichloromethane/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia. In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc) of a compound of the present invention as isolated as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity. The purities of the intermediates and examples are ³95% as judged by LC/MS or ¹ H-NMR spectra if not stated otherwise. The percentage yields reported in the following examples are based on the starting component that was used in the lowest molar amount. Air and moisture sensitive liquids and solutions were transferred via syringe or cannula, and introduced into reaction vessels through rubber septa.

Commercial grade reagents and solvents were used without further purification. The term “concentrated under reduced pressure” refers to use of a Buchi rotary evaporator at a minimum pressure of approximately 15 mm of Hg. All temperatures are reported uncorrected in degrees Celsius (°C). In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety.

Generation of ²²⁷ Th ²²⁷ Th was selectively isolated from an ²²⁷ Ac mixture, which had been growing in daughters for two weeks, by adding 0.25 ml of 7M HNO ₃ to the actinium mixture (which had been evaporated to dryness) and eluting the solution through an anion exchange column. The column had an  

inner diameter of 2 mm and a length of 30 mm containing approximately 70 mg of AG-1×8 anion exchange resin (Biorad Laboratories, Hercules, Calif., USA) (nitrate form). The column was washed with 2-4 ml of 7M HNO ₃ to remove ²²⁷ Ac, ²²³ Ra and Ra daughters while retaining ²²⁷ Th.

Subsequently ²²⁷ Th was stripped from the column with a few ml of 12M HCl. Finally the HCl was evaporated to dryness and the ²²⁷ Th re-dissolved in 0.05 M HCl.

Antibodies

The FAP-targeting antibody was prepared according to WO2017/211809.

The PSMA-targeting antibody is BAY 2315497 and is prepared according to Example 9, specifically Examples 9a and 9b of WO 2016/096843.

The HER2-targeting antibody is prepared according to Example 5, specifically Examples 5a and 5b of WO 2016/096843.

Analytical LC-MS and HPLC conditions LC-MS-data given in the subsequent specific experimental descriptions refer (unless otherwise noted) to the following conditions:

Method 1:

Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50x2.1mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; DAD scan: 210-400 nm.

Method 2:

Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50x2.1mm; eluent A: water + 0.2 vol % aqueous ammonia (32%), eluent B: acetonitrile; gradient:

0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; DAD scan: 210- 400 nm.

Method 3:

Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50x2.1mm; eluent A: water + 0.1 vol-% formic acid (99%), eluent B: acetonitrile; gradient: 0-1.7 min 1-45% B, 1.7-1.72 min 45-99% B, 1.72-2.0 min 99% B; flow 0.8 ml/min; temperatur: 60 °C;

ELSD.

Method A:

Instrument: SHIMADZU LCMS-2020 SingleQuad; Column: Chromolith@Flash RP-18E 25-2 MM; eluent A: water + 0.0375 vol % trifluoroacetic acid, eluent B: acetonitrile + 0.01875 vol %  

trifluoroacetic acid; gradient: 0-0.8 min 0-60% B, 0.8-1.2 min 60% B; flow 1.5 ml/min;

temperature: 50 °C; PDA: 220 nm & 254 nm. Method B:

Instrument: Agilent 1100\G1956A SingleQuad; Column: Kinetex@ 5um EVO C18 30*2.1 mm;

eluent A: water + 0.0375 vol % trifluoroacetic acid, eluent B: acetonitrile + 0.01875 vol % trifluoroacetic acid; gradient: 0-0.8 min 0-60% B, 0.8-1.2 min 60% B; flow 1.5 ml/min;

temperature: 50 °C; PDA: 220 nm & 254 nm. Method C:

Instrument: SHIMADZU LCMS-2020 SingleQuad; Column: Chromolith@Flash RP-18E 25-2 MM; eluent A: water + 0.0375 vol % trifluoroacetic acid, eluent B: acetonitrile + 0.01875 vol % trifluoroacetic acid; gradient: 0-0.8 min, 5-95% B, 0.8-1.2 min 95% B; flow 1.5 ml/min;

temperature: 50 °C; PDA: 220 nm & 254 nm. Method D:

Instrument: Agilent 1100\G1956A SingleQuad; Column: Kinetex@ 5 mm EVO C18 30*2.1 mm;

eluent A: water + 0.0375 vol % trifluoroacetic acid, eluent B: acetonitrile + 0.01875 vol % trifluoroacetic acid; gradient: 0-0.8 min 5-95% B, 0.8-1.2 min 95% B; flow 1.5 ml/min;

temperature: 50 °C; PDA: 220 nm & 254 nm. Method E:

Instrument: Agilent 1200\G6110A SingleQuad; Column: XBridge C182.1*50 mm, 5 mm; eluent A: water + 0.025 vol % ammonium hydroxide, eluent B: acetonitrile; gradient: 0-1.2 min 10-80%

B, 1.2-1.6 min 80% B; flow 1.2 ml/min; temperature: 40 °C; DAD: 220 nm & 254 nm. Method F:

Instrument: Agilent 1200\G6110A SingleQuad; Column: XBridge C182.1*50 mm, 5 mm; eluent A: water + 0.025 vol % ammonium hydroxide, eluent B: acetonitrile; gradient: 0-1.2 min 0-60%

B, 1.2-1.6 min 60% B; flow 1.0 ml/min; temperature: 40 °C; DAD: 220 nm & 254 nm. Method G:

Instrument: SHIMADZU LCMS-2020 SingleQuad; Column: Kinetex EVO C182.1*30 mm, 5 mm;

eluent A: water + 0.025 vol % ammonium hydroxide, eluent B: acetonitrile; gradient: 0-0.8 min, 5-95% B, 0.8-1.2 min 95% B; flow 1.5 ml/min; temperature: 40 °C; PDA: 220 nm & 254 nm. Method H:

 

Instrument: SHIMADZU LCMS-2020 SingleQuad; Column: Kinetex EVO C182.1*30 mm, 5 mm;

eluent A: water + 0.025 vol % ammonium hydroxide, eluent B: acetonitrile; gradient: 0-1.2 min, 0-60% B, 1.2-1.6 min, 60% B; flow 1.0 ml/min; temperature: 40 °C; PDA: 220 nm & 254 nm. Method I:

Instrument: SHIMADZU LCMS-2020 SingleQuad; Column: Kinetex EVO C182.1*30 mm, 5um;

eluent A: water + 0.025 vol % ammonium hydroxide, eluent B: acetonitrile; gradient: 0-0.8 min, 5-95% B, 0.8-1.2 min, 95% B; flow 1.5 ml/min; temperature: 40 °C; PDA: 220 nm & 254 nm. Method J:

Instrument: SHIMADZU LCMS-2020 SingleQuad; Column: Kinetex EVO C182.1*30 mm, 5 mm;

eluent A: water + 0.025 vol % ammonium hydroxide, eluent B: acetonitrile; gradient: 0-0.8 min, 0-60% B, 0.8-1.2 min, 60% B; flow 1.5 ml/min; temperature: 40 °C; PDA: 220 nm & 254 nm. Method K:

Instrument: Waters Acquity/QTOF; Column: Phenomenex Kinetex 1.7 µm C18, 100 Å, 30 x 2.1 mm; eluent A: Water/0.1% TFA; eluent B: ACN/0.1% TFA; flow: 0.5 mL/min; temperature:

ambient; Detection: PDA

Method L:

Column: Agilent Kinetex EVO C18 30*2.1 mm, 5 um. MPA: 0.0375% TFA in water. MPB:

0.0188% TFA in acetonitrile. Gradient: 0.00 min 5% B ^ 0.80 min 95% B ^1.20 min 95% B

^1.21 min 5% B ^1.50 min 5% B; flow rate: 1.5 mL/min; Column Temperature: 50 °C; UV detection: 220 nm & 254 nm.

Method M

Equipment type MS: Waters TOF instrument; Equipment type UPLC: Waters Acquity I-CLASS;

Column: Waters, HSST3, 2.1 x 50 mm, C181.8 µm; eluent A: 1 L water + 0.01% formic acid;

eluent B: 1 L acetonitrile + 0.01% formic acid; gradient: 0.0 min 2% B ® 0.5 min 2% B ® 7.5 min 95% B ® 10.0 min 95% B; oven: 50°C; flow rate: 1.00 mL/min; UV-detection: 210 nm

Method N

Equipment type MS: ThermoFisherScientific LTQ-Orbitrap-XL; Equipment type HPLC: Agilent

1200SL; Column: Agilent, POROSHELL 120, 3 x 150 mm, SB– C182.7 µm; eluent A: 1 L

water + 0.1% trifluoroacetic acid; eluent B: 1 L acetonitrile + 0.1% trifluoroacetic acid; gradient:  

0.0 min 2% B ® 1.5 min 2% B ® 15.5 min 98% B ® 18.0 min 98% B; oven: 40°C; flow rate:

0.75 mL/min; UV-detection: 210 nm Methode o (preparative RP-chromatography)

Instrument: Knauer Blue Shadow (Pump 40P, Azura Detector UVD 2.1S) with Merck/Hitachi D- 2500 Chromato-Integrator; Column: Nucleodur C18 5 µm Gravity, 250x21 mm; eluent A:

acetonitrile, eluent B: water + 0.2% trifluoroacetic acid; gradient: 0-25 min 55% A, 25-30 min 55- 90% A, 30-45 min 90% A; flow 5.0 mL/min; temperature: r.t.; UV scan: 226 nm.

Methode P

Instrument: Agilent 6550 Series iFunnel Q-TOF; Instrument HPLC: Agilent 1290 Infinity Series;

Column: Kinetix C181.7 µm, 100x1.7mm; eluent A: water + 0.1 vol % formic acid (99%),

eluent B: acetonitrile + 0.1 vol % formic acid (99%); gradient: 0-2 min 5% B, 2.0-3.0 min 5-90%

B; flow 1.0 mL/min; temperature: 40°C; UV scan: 230 nm. Methode Q (analytical RP-chromatography)

Instrument: Agilent HP 1200 (UV) and Raytest Ramona® 2000 (radioactivity signal), the radioactivity signal was digitized by Agilent A/D-converter 35900E and evaluated by the work station; Column: Aquasil C185 µm 125 A, 150x4.6 mm; eluent A: acetonitrile, eluent B: water + 0.2% trifluoroacetic acid; gradient: 0-30 min 10-90% A, 30-40 min 90% A; flow 1.3 mL/min;

temperature: r.t.; UV scan: 254 nm.

Methode R (preparative RP-chromatography)

Instrument: Knauer Blue Shadow (Pump 40P, Azura Detector UVD 2.1S) with Merck/Hitachi D- 2500 Chromato-Integrator; Column: Nucleodur C18 5 µm Gravity, 250x21 mm; eluent A:

acetonitrile, eluent B: water + 0.2 vol % trifluoroacetic acid; gradient: 0-30 min 10-50% A, 30-35 min 50-90% A, 35-40 min 90% A; flow 5.0 mL/min; temperature: r.t.; UV scan: 226 nm.

Methode S

Instrument: Agilent 6200 Series TOF; Instrument HPLC: Agilent 1290 Infinity Series; Column:

Kinetix C181.7 µm, 100x1.7mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B:

acetonitrile + 0.1 vol % formic acid (99%); gradient: 0-2 min 5% B, 2.0-3.0 min 5-90% B; flow

1.0 mL/min; temperature: 40°C; UV scan: 210 nm.

 

Methode T (analytical RP-chromatography)

Instrument: Agilent HP 1260 Infinity (UV) and Raytest Ramona Star® (radioactivity signal), the radioactivity signal was digitized by Agilent A/D-converter 35900E and evaluated by the work station; Column: Aquasil C185 µm 125 A, 150x4.6 mm; eluent A: acetonitrile, eluent B: water + 0.2% trifluoroacetic acid; gradient: 0-30 min 10-90% A, 30-40 min 90% A; flow 1.3 mL/min;

temperature: r.t.; UV scan: 254 nm.

Flash column chromatography conditions “Purification by (flash) column chromatography” as stated in the subsequent specific experimental descriptions refers to the use of a Biotage Isolera purification system. For technical specifications see“Biotage product catalogue” on www.biotage.com.

Synthetic procedures Carboxy-HOPO chelator Intermediate a

Ethyl 3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate

To a solution of sodium diethyloxylacetate (42.1 g, 200 mmol) in dry THF (500 mL) in a 1-liter 3- neck round bottom flask was added chloroacetone (16 mL, 200 mmol). After 10 min NH3 gas was bubbled trough the reaction followed by careful addition of AlCl3 (2.67 g, 20 mmol). The reaction was stirred under ambient temperature for 5 days. The resulting orange solid was filtered and taken up in 1 M HCl (500 mL) so that pH was below 3. The resulting suspension was stirred for 30 min and the precipitate filtered, washed with water and dried to give 25.5 g (65%) of the target compound as a pale yellow solid. Intermediate b

3-(benzyloxy)-6-methyl-2-oxo-1,2-dihydropyridine-4-carbox ylate  

1,8-Diazabicyclo[5.4.0]undec-7-ene (30.0 g, 197 mmol) was added to a solution of ethyl 3- hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate (25.5 g, 129 mmol) in isopropanol (300 mL). The reaction mixture was refluxed at 83 °C under N ₂ before adding benzyl bromide (24 mL, 202 mmol) slowly. Refluxing was maintained for 4 hours, and then the solvent was evaporated. The resulting dark brown oil was dissolved in dichloromethane (100 mL), washed with aqueous 3 M HCl (2 x 100 mL) and water (3 x 100 mL). The organic layer was dried (Na2SO4), filtered and concentrated. Diisopropyl ether (500 mL) was added to the oily residue and the after spinning on the rotary evaporator the solid was filtered and dried to give 25 grams (67%) of the target compound. as a light grey solid. Intermediate c

Ethyl 3-(benzyloxy)-1-(2-(tert-butoxy)-2-oxoethyl)-6-methyl-2-oxo- 1,2-dihydropyridine-4- carboxylate

To a solution of tert-butylbromoacetate (25.0 g, 0.128 mol) in acetone (130 mL) was added sodium iodide (25.6 g, 0.17 mol). The reaction mixture was heated at reflux for 9 hours, cooled to room temperature, filtered and concentrated. The crude product was used without further purification. To a 1 L round bottom flask was added 3-(benzyloxy)-6-methyl-2-oxo-1,2-dihydropyridine-4- carboxylate (20.9 g, 72.8 mmol) followed by potassium fluoride on alumina (40 g). These reagents were purged under nitrogen for 15 minutes after which dimethoxyethane (320 mL) was added. To this solution was added tert-butyliodoacetate (35 g, 144 mmol) and the reaction was left to stir overnight, filtered and the filter cake washed thoroughly with THF. The filtrate was  

concentrated and the residue purified by flash chromatography (heptane– heptane/EtOAc 90:10 – 80:20– 70:30– 50:50) to afford 25.5 gram (87%) of the target compound. Intermediate d

3-(Benzyloxy)-1-(2-(tert-butoxy)-2-oxoethyl)-6-methyl-2-o xo-1,2-dihydropyridine-4- carboxylic acid

To a solution of ethyl 3-(benzyloxy)-1-(2-(tert-butoxy)-2-oxoethyl)-6-methyl-2-oxo- 1,2- dihydropyridine-4-carboxylate (25.5 g, 63.5 mmol) in 1:1 THF/H2O (500 mL) was added aqueous LiOH (76 mL 1M, 76.2 mmol) and the reaction mixture stirred overnight at ambient temperature. Aqueous citric acid was added to neutral pH and the aqueous phase was extracted with EtOAc (x3). The combined organic extract was dried (Na2SO4), filtered and concentrated.

The residue was triturated with heptane/EtOAc 90:10 and the precipitated material filtered and dried to afford 21.9 g (92%) of the target compound as a colorless solid.

MS (ESIpos): m/z = 374.1 [M+H] ⁺ Intermediate e

Bis(2-azidoethyl)amine To a stirred solution of sodium azide (56.0 g, 0.86 mol) in water (500 mL) was added bis(2- chloroethyl)amine hydrochloride (76.8 g, 0.43 mol). After stirring for 2 hours at 90 °C, another portion of sodium azide (56.0 g, 0.86 g) was added, and the reaction mixture stirred for 48 hours at 90 °C. After cooling to room temperature, the pH was adjusted to around 10 with aqueous NaOH (10 M). The aqueous solution was extracted with diethyl ether (x5). The combined organic extract was dried (Na2SO4), filtered and concentrated. Purification by chromatography (heptane /EtOAc gradient) afforded 45.5 g (68%) of the target compound as a yellow oil. Intermediate f

 

N1,N1,N3,N3-tetrakis(2-azidoethyl)propane-1,3-diamine

To a solution of 1,3-dibromopropane (15 mL, 147 mmol), potassium carbonate (101 g, 730 mmol) and potassium iodide (48 g, 280 mmol) in acetonitrile (500 mL) was added slowly bis(2- azidoethyl)amine dissolved in acetonitrile and the reaction mixture heated at 80 °C for 48 hours.

The solid was removed by filtration and the filtrate concentrated in vacuo. The residue was purified by column chromatography using heptane/EtOAc (0-50% EtOAc) to afford 38 grams (74%) of the target compound as a yellow oil. Intermediate g

N1,N1'-(propane-1,3-diyl)bis(N1-(2-aminoethyl)ethane-1,2- diamine)

A solution of N1,N1,N3,N3-tetrakis(2-azidoethyl)propane-1,3-diamine (5.20 g) in EtOH (100 mL) was hydrogenated overnight at 5 bar in the presence of Pd (2 g). Filtration and concentration afforded the target compound as a yellow oil used without further purification.

MS (ESIpos): m/z = 247.2 [M+H] ⁺ Intermediate h

Tetra-tert-butyl 2,2',2'',2'''-((5,9-bis(2-formamidoethyl)-2,5,9,12-tetraazat ridecanedioyl)- tetrakis-(3-(benzyloxy)-6-methyl-2-oxopyridine-4,1(2H)-diyl) )tetraacetate

 

To a solution of N1,N1'-(propane-1,3-diyl)bis(N1-(2-aminoethyl)ethane-1,2-dia mine) (246 mg, 1 mmol) and 3-(benzyloxy)-1-(2-(tert-butoxy)-2-oxoethyl)-6-methyl-2-oxo- 1,2-dihydropyridine-4- carboxylic acid (1.50 g, 4 mmol) in DMF (15 mL) was added DIPEA (2 mL, 12 mmol), followed by HATU (3.0 g, 8 mmol). The reaction mixture was stirred at room temperature overnight, poured into water and extracted three times with EtOAc. The combined organic extract was washed with brine, dried (Na ₂ SO ₄ ), filtered and concentrated. Flash chromatography (DCM/EtOAc 50:50– DCM/EtOAc/MeOH 47.5:47.5:5– DCM/EtOAc/MeOH 45:45:10) afforded 0.45 g (27%) of the target compound as a yellow solid. Different batches were combined and purified by flash chromatography to afford 2.70 g of the target compound as a yellow solid used in the final step.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 3.01 min; MS (ESIpos): m/z = 1667.7  

[M+H] ⁺  

1H NMR ((400 MHz, CDCl3) d 8.13 (s, 3H), 7.39– 7.25 (m, 18H), 6.37 (s, 4H), 5.27 (s, 8H), 4.68 (s, 8H), 3.39– 3.03 (m, 9H), 2.72 (d, J = 44.6, 8H), 2.10 (d, J = 63.3, 15H), 1.50– 1.34 (m, 40H)), and 13C NMR ((101 MHz, CDCl3) d 166.8, 144.1, 130.1, 129.2, 129.3, 128.5, 83.2, 74.9, 55.9, 46.9, 38.8, 28.2, 20.2). Example A

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl- 2-oxopyridine-4,1(2H)-diyl)]})tetraacetic acid

 

Tetra-tert-butyl 2,2',2'',2'''-((5,9-bis(2-formamidoethyl)-2,5,9,12-tetraazat ridecanedioyl)-tetrakis- (3-(benzyloxy)-6-methyl-2-oxopyridine-4,1(2H)-diyl))tetraace tate (2.70 grams) was treated with concentrated hydrochloric acid (100 mL) at room temperature for 3 hours and concentrated to dryness by evaporation in vacuo. The residue was purified by reverse phase flash chromatography (0-50% ACN in water) to give 2.2 grams of the target compund. This material was again purified using preparative HPLC (column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 50 mm; mobile phase: water/0.1% TFA; ACN; gradient: 0-30% B over 40 min; flow: 50 mL/min;

detection: UV 280/335 nm) to afford 400 mg of the target compound.

LC-MS (Method K, gradient: 5-30% B over 3 min): R _t = 1.68 min; MS (ESIpos): m/z = 1083.5

[M+H] ⁺  

1H NMR ((400 MHz, DMSO) d 11.03 (4H), 8.62 (s, 4H), 6.39 (s, 4H), 4.74 (s, 8H), 4.16– 2.85 (m, 26H), 2.19 (s, 12H)), and 13C NMR ((101 MHz, DMSO) d 169.3, 165.5, 158.9, 144.3, 134.3, 117.5, 102.9, 51.7, 46.1, 34.6, 19.1)

Alternative synthesis route Intermediate i

Ethyl 3-hydroxy-6-methyl-2-oxo-1H-pyridine-4-carboxylate

Compound diethyl oxalacetate sodium salt (250 g, 1.19 mol) was partitioned between aq. HCl (1 M, 1.7 L) and DCM (2 L). The layers were stirred at rt for 3 hours until all the solid was dissolved and the pH of the aqueous phase < 3. The reaction was parallel performed for 2 batches and worked together. The organic phase (2 batches) was separated and the aqueous phase was extracted with DCM (400 mL x 2). The combined organic phase was dried over  

MgSO ₄ , filtered and concentrated under reduced pressure to give diethyl 2-hydroxybut-2- enedioate (460 g, crude) as a yellow oil.

To a solution of diethyl 2-hydroxybut-2-enedioate (460 g, the above yellow oil) in THF (4.5 L) was added 1-chloropropan-2-one (285 g, 3.08 mol) at rt. Then NH ₃ (g) was bubbled through the reaction mixture for 1.5 hours at -20 °C - 0 °C. The resulting mixture was stirred at 85 °C in an autoclave (30 Psi) for 4 hours. The reaction mixture was cooled to rt and filtered. The filter cake was washed with MTBE (300 mL). A yellow solid (440 g) was collected by filtration. The crude product (440 g) was combined with other 2 batches crude product (430 g from 500 g of diethyl oxalacetate sodium salt; 450 g from 500 g of diethyl oxalacetate sodium salt). All the crude product was slurried with aq. HCl (1 M, 9 L). The mixture was filtered. The filter cake was washed with H ₂ O (500 mL) and dried in high vacuum to give ethyl 3-hydroxy-6-methyl-2-oxo-1H-pyridine- 4-carboxylate (612 g, 43.5% yield) as a red solid.

1H NMR: (DMSO-d ₆ 400MHz) 11.95 (brs, 1H), 9.77 (brs, 1H), 6.08 (s, 1H), 4.29 (q, 2H), 2.10 (s, 3H), 1.27 (t, 3H).

Note, containing ~0.5 eq NH ⁺

4 Intermediate j

Ethyl 1-(2-tert-butoxy-2-oxo-ethyl)-3-hydroxy-6-methyl-2-oxo-pyrid ine-4-carboxylate

A mixture of ethyl 3-hydroxy-6-methyl-2-oxo-1H-pyridine-4-carboxylate (50 g, 253.57 mmol), KOtBu (31.50 g, 280.72 mmol) and ditert-butoxymagnesium (95.50 g, 560.01 mmol) in THF (1.2 L) was stirred at rt under N ₂ for 1 hour. Tert-butyl 2-bromoacetate (50 mL, 338.37 mmol) in THF

(300 mL) was added to the reaction mixture drop-wise at rt. The mixture was stirred at rt for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EA (1 L) and cold sat. aq. citric acid (500 mL). Two phases were separated. The organic phase was washed with H ₂ O (200 mL x 2) and brine (200 mL), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The reaction was parallel performed for 6 batches and all of the residues were combined. The combined crude product was diluted with DCM (2 L) and filtered. The filtrate was concentrated under reduced pressure. The residue was slurried with MTBE (1.5 L) to give ethyl 1-(2-tert-butoxy-2-oxo-ethyl)-3-hydroxy-6-methyl-2-oxo-pyrid ine-4- carboxylate (314 g, 66.3% yield) as an off-white solid.

 

¹ H NMR: (CDCl ₃ 400MHz) 10.30 (brs, 1H), 6.34 (s, 1H), 4.74 (s, 2H), 4.40 (q, 2H), 2.24 (s, 3H), 1.47 (s, 9H), 1.41 (t, 3H). Intermediate k

Ethyl 3-benzyloxy-1-(2-tert-butoxy-2-oxo-ethyl)-6-methyl-2-oxo-pyr idine-4-carboxylate

A mixture of ethyl 1-(2-tert-butoxy-2-oxo-ethyl)-3-hydroxy-6-methyl-2-oxo-pyrid ine-4- carboxylate (183 g, 587.80 mmol), K ₂ CO ₃ (122 g, 882.74 mmol) and BnBr (84 mL, 707.23 mmol) in CH ₃ CN (1.9 L) was stirred at reflux under N ₂ for 3 hours. The reaction mixture was cooled to rt and filtered. The reaction was parallel performed for 2 batches. The combined filtrate was concentrated under reduced pressure to give 500 g of crude product. The crude product was purified with another batch crude product (220 g, 150 g of ethyl 1-(2-tert-butoxy-2-oxo-ethyl)-3- hydroxy-6-methyl-2-oxo-pyridine-4-carboxylate was used in this batch from EW99-2305) together by silica gel chromatography (PE: EA = 10: 1 to 3: 1) to give ethyl 3-benzyloxy-1-(2- tert-butoxy-2-oxo-ethyl)-6-methyl-2-oxo-pyridine-4-carboxyla te (615 g, 92.5% yield) as a red oil.

1H NMR: (CDCl ₃ 400MHz) 7.51-7.49 (m, 2H), 7.35-7.30 (m, 3H), 6.21 (d, 1H), 5.25 (s, 2H), 4.75 (s, 2H), 4.28 (q, 2H), 2.27 (s, 3H), 1.49 (s, 9H), 1.28 (t, 3H). Intermediate l

3-benzyloxy-1-(2-tert-butoxy-2-oxo-ethyl)-6-methyl-2-oxo- pyridine-4-carboxylic acid

To a solution of ethyl 3-benzyloxy-1-(2-tert-butoxy-2-oxo-ethyl)-6-methyl-2-oxo-pyr idine-4- carboxylate (102.5 g, 255.32 mmol) in THF (1 L) and H ₂ O (1 L) was added LiOH.H ₂ O (12.86 g, 306.39 mmol) in H ₂ O (300 mL) at rt. The mixture was stirred at rt for 5 hours and then adjusted pH to 3 with sat. aq. citric acid. The resulting mixture was diluted with EA (1.5 L). Two phases were separated. The organic phase was washed with H ₂ O (500 mL) and brine (500 mL), dried  

over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The reaction was parallel performed for 6 batches. All of the crude products were slurried with a mixed solvent (1.8 L, PE:

EA = 10: 1) to give 3-benzyloxy-1-(2-tert-butoxy-2-oxo-ethyl)-6-methyl-2-oxo-pyr idine-4- carboxylic acid (452 g, 79.0% yield) as a white solid.

1H NMR: (CDCl ₃ 400MHz) 7.44-7.42 (m, 2H), 7.39-7.37 (m, 3H), 6.58 (d, 1H), 5.57 (s, 2H), 4.75 (s, 2H), 2.29 (s, 3H), 1.51 (s, 9H).

LC-MS: (method L) t = 0.789 min, m/z = 374.2 (M+H) ⁺ . Intermediate m

N ¹ -trityl-N ² -(2-(tritylamino)ethyl)ethane-1,2-diamine

The reactions were performed as 4 batches in parallel: to a solution of N'-(2-aminoethyl)ethane- 1,2-diamine (50 g, 484.66 mmol) in i-PrOH (2 L) was added diethylamine (110 mL, 1.07 mol), followed by TrtCl (270 g, 968.52 mmol) in portions at 0 °C. The mixture was stirred at room temperature for 16 hours. TLC (DCM: MeOH = 20: 1) indicated the reaction completed. NaOH

(aq., 5 M, 300 mL for each batch) was added and the mixture was stirred at room temperature for 0.5 h. The suspension of 4 batches were combined and filtered. The cake was washed with water (1 L x 6) and dried under air. The crude product was stirred in refluxing MeOH (2.4 L) for 20 min and then filtered while hot. The cake was washed with MeOH (200 mL) and dried in vacuum. The resulting solid was slurried in MeCN (3.0 L) to afford N ¹ -trityl-N ² -(2- (tritylamino)ethyl)ethane-1,2-diamine (741 g, 90% purity) as a white solid.

1H NMR: (CDCl ₃ 400MHz) 7.52-7.50 (m 12H), 7.28-7.17 (m, 18H), 2.68 (t, 4H), 2.28 (t, 4H). Intermediate n

N ¹ ,N ¹ '-(propane-1,3-diyl)bis(N ² -trityl-N ¹ -(2-(tritylamino)ethyl)ethane-1,2-diamine)

 

The reactions were performed as 3 batches in parallel: to a suspension of N ¹ -trityl-N ² -(2- (tritylamino)ethyl)ethane-1,2-diamine (240 g, 367.48 mmol, 90% purity) and 1,3-diiodopropane (60.38 g, 204.06 mmol) in MeCN (1.65 L) was added K ₂ CO ₃ (240 g, 1.74 mol), followed by DMF

(3.2 mL, 41.59 mmol). The mixture was stirred at reflux for 3 days under N ₂ . The mixture was filtered. The cake was washed with water (3.5 L x 3) and MeCN (1.5 L) and then stirred in MeCN

(3 L) at 70 °C for 0.5 h. The suspension was filtered while hot, and the cake was washed with MeCN (2.5 L). The resulting solid was stirred in MeCN (2 L) at 70 °C for 20 hours and then filtered while hot. The cake was washed with MeCN (2.5 L) and water (4 L) and then dried in vacuum to afford N ¹ ,N ¹ '-(propane-1,3-diyl)bis(N ² -trityl-N ¹ -(2-(tritylamino)ethyl)ethane-1,2- diamine) (540 g, 444.21 mmol) as a white solid.

1H NMR: (CDCl ₃ 400MHz) 7.46-7.44 (m 24H), 7.22-7.14 (m, 36H), 2.33 (t, 8H), 2.15 (t, 8H), 1.95 (t, 4H), 1.85 (t, 4H), 1.19 (s, 2H). Intermediate o

N ¹ ,N ¹ '-(propane-1,3-diyl)bis(N ¹ -(2-aminoethyl)ethane-1,2-diamine)

The reactions were performed as 3 batches in parallel: a suspension of N ¹ ,N ¹ '-(propane-1,3- diyl)bis(N ² -trityl-N ¹ -(2-(tritylamino)ethyl)ethane-1,2-diamine) (177 g, 145.60 mmol) in HCl (aq., 6 M, 900 mL) was stirred at reflux for 2 hours. MTBE (1 L) was added and the mixture was refluxed for further 1 hour. The organic layer was removed. The aqueous phase was washed with MTBE

(1.5 L x 5) and lyophilized. The resulting solid was combined with two batches of product to give N ¹ ,N ¹ '-(propane-1,3-diyl)bis(N ¹ -(2-aminoethyl)ethane-1,2-diamine) hydrochloride (234 g, 503.05 mmol, 6HCl), which was dissolved in H ₂ O (2 L). NaOH (126 g, 3.15 mol) was added in portions.  

The mixture was stirred at room temperature for 10 min and then lyophilized. The solid obtained was stirred in DCM (2.5 L) for 10 min and then filtered. The filtrate was concentrated in vacuum.

The residue was dissolved in water (300 mL) and then lyophilized to give N ¹ ,N ¹ '-(propane-1,3- diyl)bis(N ¹ -(2-aminoethyl)ethane-1,2-diamine) (107 g, 86.33% yield) as a yellow oil.

1H NMR: (D ₂ O 400MHz) 2.71-2.67 (m 8H), 2.54-2.50 (m, 8H), 2.43 (t, 4H), 1.62-1.56 (m, 2H).

IC: Average Cl- content: 3.1%. Intermediate p

tetra-tert-butyl 2,2',2'',2'''-[propane-1,3-diylbis(nitrilobis{(ethane-2,1-di yl)carbamoyl[3- (benzyloxy)-6-methyl-2-oxopyridine-4,1(2H)-diyl]})]tetraacet ate

Under argon 24.2 g (64.9 mmol) of 3-(benzyloxy)-1-(2-tert-butoxy-2-oxoethyl)-6-methyl-2-oxo- 1,2-dihydropyridine-4-carboxylic acid were dissolved in 80 mL of N,N-dimethylacetamide.8.4 g (64.9 mmol) of N,N-diisopropylethylamine and 24.7 g (64.9 mmol) of HATU (CAS-RN:[148893- 10-1]) were added at room temperature. The reaction mixture is stirred 30 min at this temperature. Then 4.00 g (16.2 mmol) of N ¹ ,N ¹ ,N ³ ,N ³ -tetrakis(2-aminoethyl)propane-1,3- diamine dissolved in 20 mL of N,N-dimethylacetamide were added dropwise. The reaction mixture was stirred 2 h at room temperature. Water was added and the formed viscous solid was separated from the liquid phase. The remaining solid residue was dissolved in 400 mL ethyl acetate and extracted with saturated aqueous NaHCO ₃ -solution. The organic phase is washed twice with water and dried over MgSO ₄ . The solvent was evaporated under reduced pressure.

The residue is purified by using column chromatography A followed by a second column chromatography B (A: Biotage autopurifier system (Isolera LS®), 340 g Biotage SNAP cartridge KP-Sil® ultra column, dichloromethane/ethyl acetate to ethyl acetate/methanol: 0-100% ethyl acetate to 5-10% methanol. B: Biotage autopurifier system (Isolera LS®), 375 g Biotage SNAP  

cartridge KP-NH®, dichloromethane/ethyl acetate: 0-50% ethyl acetate). Yield: 72%, 19.5 g (11.7 mmol).

1H NMR (400 MHz, DMSO-d ₆ ) d ppm 1.43 (br s, 38 H) 2.21 (s, 12 H) 2.31 - 2.38 (m, 4 H) 2.42 (br t, J=6.72 Hz, 8 H) 3.13 - 3.26 (m, 8 H) 4.73 (s, 8 H) 5.13 (s, 8 H) 6.10 - 6.29 (m, 4 H) 7.23 - 7.33 (m, 12 H) 7.35 - 7.40 (m, 6 H) 8.19 (t, J=5.45 Hz, 4 H)

LC-MS: R _t = 1.68 min; MS (ESIpos): m/z = 1668 [M+H] ⁺ Instrument: Waters Acquity UPLCMS SingleQuad; column: XBridge C18 5 µm, 150x4.6mm;

eluent A: water + 0.2 vol % aqueous ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; DAD scan: 210-400 nm. Example A1

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl- 2-oxopyridine-4,1(2H)-diyl)]})tetraacetic acid—hydrogen chloride (1/2)

23.2 g (13.9 mmol) of tetra-tert-butyl 2,2',2'',2'''-[propane-1,3-diylbis(nitrilobis{(ethane-2,1- diyl)carbamoyl[3-(benzyloxy)-6-methyl-2-oxopyridine-4,1(2H)- diyl]})]tetraacetate were suspended in 115 mL aqueous hydrochloric acid (37%). The mixture was stirred 16 h at room temperature and then 2 h at 50 °C. Afterwards the mixture was stirred further 48 h at room temperature.500 mL of 2-methyltetrahydrofuran were added at room temperature and the yellow precipitate was collected by filtration. After washing with a small amount of ethanol the residue was titurated in 50 mL of ethanol at 45 °C. Yield: 96%, 15.5 g (13.4 mmol).

 

¹ H NMR (400 MHz, DMSO-d ₆ ) d ppm 2.03 - 2.33 (m, 14 H) 3.66 - 3.86 (br m, 12 H) 4.74 (s, 8 H) 6.46 (s, 4 H) 8.64 - 8.82 (br m, 4 H) 10.49 - 10.60 (br s, 2 H) 11.09 - 11.22 (br s, 2 H) 12.89 - 13.51 (br s, 4 H)

LC-MS: R _t = 0.50 min; MS (ESIpos): m/z = 1083 [M+H] ⁺ Instrument: Waters Acquity UPLCMS SingleQuad; Column: XBridge C18 5 µm, 150x4.6mm;

Eluent A: water + 0.1 Vol-% formic acid (99%), Eluent B: acetonitrile; Gradient: 0-1.7 min 1-45%

B, 1.7-1.72 min 45-99% B, 1.72-2.0 min 99% B; Flow 0.8 ml/min; Temperature: 60 °C; ELSD. Small molecule Conugates targeting PSMA Intermediate 1

di-tert-butyl N-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tert-butoxy-1-oxoh exan-2- yl]carbamoyl}-L-glutamate 

tert-butyl N6-[(benzyloxy)carbonyl]-L-lysinate—hydrogen chloride (1/1) (7.27 g, 19.5 mmol;

CAS-RN:[5978-22-3]) was solubilised in DCM (100 ml), cooled to 0°C under argon, and N,N- diisopropylethylamine (14 ml, 78 mmol) was added dropwise. The mixture was stirred for 5min at 0°C and 30min at rt.4-nitrophenyl carbonochloridate (3.59 g, 17.8 mmol; CAS-RN:[7693-46- 1]) and di-tert-butyl L-glutamate—hydrogen chloride (1/1) (5.00 g, 16.9 mmol; CAS-RN:[32677- 01-3]) were added and N,N-diisopropylethylamine (14 ml, 78 mmol) was added dropwise to the mixture. It was stirred overnight at rt. The mixture was concentrated under reduced pressure, diluted with DCM, washed 2 times with sodium hydroxide (0.5 M) and once with brine. The organic layer was dried and evaporated. The residue was purified by flash chromatography (SiO2, hexane/EtOAc gradient 0%-50%) to give 8.40 g (96 % purity, 67 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.48 min; MS (ESIpos): m/z = 623 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.154 (0.66), 1.172 (1.40), 1.190 (0.71), 1.384 (16.00), 1.392 (7.49), 1.987 (2.48), 3.331 (2.46), 4.017 (0.64), 4.035 (0.68), 4.992 (1.08), 7.318 (0.43), 7.334 (1.05), 7.342 (0.98), 7.360 (0.43). 

 

Intermediate 2

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate 

di-tert-butyl N-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tert-butoxy-1-oxoh exan-2-yl]carbamoyl}- L-glutamate (8.48 g, 13.6 mmol) was solubilised in MeOH (42 ml), palladium on carbon (1.45 g, 10 % purity, 1.36 mmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 4h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was diluted with DCM, washed 3 times with sat. sodium hydrogen carbonate solution and once with brine. The organic layer was dried and evaporated.

The residue was purified by flash chromatography (SiO2, DCM/Ethanol gradient 0%-20%) to give 4.06 g (97 % purity, 59 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.97 min; MS (ESIpos): m/z = 489 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.053 (0.43), 1.288 (0.42), 1.297 (0.48), 1.302 (0.48), 1.386 (16.00), 1.390 (13.68), 1.396 (13.08), 2.477 (0.52), 3.165 (1.50), 3.330 (0.58), 6.239 (0.40).  Intermediate 3

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(naphthalen-2-yl)methyl] -3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (1.00 g, 70 % purity, 1.44 mmol) and (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(naphth alen-2- yl)propanoic acid (940 mg, 2.15 mmol; CAS-RN:[112883-43-9]) were solubilised in DMF (11 ml), 4-methylmorpholine (470 µl, 4.3 mmol, CAS-RN: 109-02-4) and HATU (819 mg, 2.15 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 1.11 g (93 % purity, 79 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.67 min; MS (ESIpos): m/z = 908 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.379 (16.00), 2.297 (2.06), 4.101 (0.61), 4.107 (0.70), 7.164 (0.59), 7.181 (0.62), 7.230 (0.56), 7.249 (0.74), 7.363 (0.42), 7.457 (0.50), 7.758 (0.43), 7.844 (0.68), 7.863 (0.60).  Intermediate 4

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(naphthalen-1-yl)methyl] -3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (1.11 g, 93 % purity, 1.14 mmol) was solubilised in DMF (26 ml), piperidine (2.3 ml, 23 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 663 mg (85 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.22 min; MS (ESIpos): m/z = 686 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.382 (13.90), 1.387 (16.00), 2.297 (3.11), 7.162 (0.66), 7.180 (0.78), 7.230 (0.67), 7.249 (0.71), 7.455 (0.48), 7.679 (0.55), 7.806 (0.81), 7.828 (0.77), 7.845 (0.55).  Intermediate 5

 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (116 mg, 70 % purity, 119 µmol) and (1R,4R)- 4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohe xane-1-carboxylic acid (67.5 mg, 178 µmol) were solubilised in DMF (910 µl), 4-methylmorpholine (39 µl, 360 µmol, CAS-RN:

109-02-4) and HATU (67.6 mg, 178 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 134 mg (80 % purity, 86 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.66 min; MS (ESIpos): m/z = 1047 [M+H] ⁺

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.231 (0.44), 1.378 (13.79), 1.385 (16.00), 2.298 (0.55), 2.518 (1.25), 2.522 (0.78), 4.279 (0.64), 4.296 (0.45), 7.306 (0.61), 7.324 (0.42), 7.381 (0.59), 7.400 (0.75), 7.418 (0.40), 7.448 (0.45), 7.668 (0.63), 7.685 (0.77), 7.789 (0.50), 7.874 (0.67), 7.893 (0.61), 7.947 (0.40). Intermediate 6

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-1-{(1R,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (730 mg, 98 % purity, 684 µmol) was solubilised in DMF (18 ml), piperidine (1.4 ml, 14 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 381 mg (98 % purity, 66 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.20 min; MS (ESIpos): m/z = 825 [M+H] ⁺

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.381 (14.66), 1.387 (16.00), 2.518 (1.24), 2.523 (0.84), 3.342 (0.66), 7.451 (0.53), 7.678 (0.48), 7.785 (0.58), 8.419 (0.62). Example 1-A

(3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(na phthalen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (25.0 mg, 98 % purity, 29.7 µmol) was solubilised in DCM (1.0 ml), TFA (1.0 ml, 13 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC

(C18, acetonitrile/water with 0.1% formic acid) to give 12.0 mg (95 % purity, 58 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.70 min; MS (ESIpos): m/z = 657 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (1.18), 1.904 (1.25), 2.327 (3.80), 2.331 (2.75), 2.336 (1.25), 2.518 (16.00), 2.523 (10.10), 2.558 (0.52), 2.577 (0.52), 2.669 (3.87), 2.673 (2.75), 2.678 (1.25), 3.119 (0.46), 7.452 (0.59), 7.693 (0.59), 7.783 (0.72), 7.804 (0.59).

The amino group of Intermediate 6 can then used in amide bond forming reactions with the carboxylate containing chelators of this invention to form chelator conjugates containing monomeric, dimeric, trimeric or tetrameric pharmacophore moieties. The protcting groups are

 

removed using standard conditions. The conjugates can also be prepared by activating the chelator moiety followed by addition of the amine-containing pharmacophore (Example 1A).  Intermediate 7

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[(1 r,4S)-4-({2- [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

4

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (289 mg, 505 µmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N-dimethylmetha niminium hexafluoridophosphate(1-) (189 mg, 498 µmol; CAS-RN:94790-37-1) and N,N- diisopropylethylamine (88 µl, 500 µmol) were stirred in DMF (3.5 ml) for 15min at rt. tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (104 mg, 126 µmol) was added and the mixture was stirred at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 97.0 mg (99 % purity, 55 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.42 min; MS (ESIpos): m/z = 1379 [M+H] ⁺  

 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.381 (15.02), 1.387 (16.00), 1.407 (4.24), 1.412 (2.61), 1.479 (2.90), 2.332 (0.43), 2.518 (2.40), 2.523 (1.70), 2.673 (0.44), 2.917 (0.45), 3.513 (0.42), 7.449 (0.44).  Example 1-B, PSMA-617

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1- [(1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[(1 r,4S)-4-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate (96.0 mg, 98 % purity, 68.2 µmol) was solubilised in DCM (9.6 ml), TFA (79 µl, 1.0 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 48.0 mg (95 % purity, 64 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.72 min; MS (ESIneg): m/z = 1041 [M-H]- 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.793 (0.86), 0.822 (1.00), 1.093 (0.43), 1.124 (0.43), 1.245 (1.64), 1.313 (1.36), 1.328 (1.64), 1.463 (1.14), 1.572 (1.14), 1.613 (1.14), 1.656 (1.07), 1.699 (0.79), 1.717 (0.64), 1.734 (0.71), 1.751 (0.79), 1.860 (0.57), 1.876 (0.57), 1.907 (1.71), 2.049 (0.79), 2.075 (1.50), 2.214 (1.29), 2.236 (2.00), 2.252 (1.00), 2.337 (1.43), 2.518 (16.00),  

2.523 (11.00), 2.540 (10.79), 2.632 (1.93), 2.674 (3.29), 2.679 (1.64), 2.909 (2.14), 2.966 (5.29), 3.074 (2.86), 3.099 (2.00), 3.121 (1.57), 3.134 (1.50), 3.427 (8.07), 3.983 (0.86), 3.994 (0.93), 4.066 (0.86), 4.082 (0.86), 4.463 (0.50), 4.484 (0.79), 4.498 (0.79), 4.521 (0.43), 6.304 (1.71), 6.325 (1.57), 7.389 (1.50), 7.411 (1.64), 7.432 (1.57), 7.435 (1.43), 7.452 (2.43), 7.466 (1.36), 7.469 (1.36), 7.483 (0.57), 7.693 (2.71), 7.785 (3.71), 7.806 (3.29), 7.834 (1.50), 7.839 (1.50), 7.857 (1.43), 7.923 (1.00), 8.068 (1.14), 8.083 (1.21), 8.106 (0.71), 8.142 (1.00).  Example 1-B- ²³² Th

{3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-{4-[(2-{4,7, 10-tris[(carboxy-kappaO)methyl]- 1,4,7,10-tetraazacyclododecan-1-yl-kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]cyclohexyl}- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylato(4-)}tho rium(3+) 

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[(1 r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid (28.6 mg, 27.4 µmol) was solubilised in ammonium acetate (29 ml, 1.0 M, 29 mmol, prepared with ultrafiltered and autoclaved water), thorium solution (6.6 ml, 27 µmol, 1µg/µl in HNO ₃ [2%]) was added and the mixture was stirred for 3.5h at 90°C. The mixture was evaporated and diluted with DMF. The resulting suspension was filtered, washed with DMF and the filtrate was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 1.20 mg (75 % purity, 3 % yield) of the target compound.

 

¹ H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.44), 1.232 (1.27), 1.352 (0.89), 1.452 (0.57), 1.656 (0.57), 2.235 (0.44), 2.322 (2.98), 2.327 (3.87), 2.331 (2.92), 2.336 (1.52), 2.518 (16.00), 2.522 (9.40), 2.539 (1.08), 2.549 (2.29), 2.664 (2.98), 2.669 (3.94), 2.673 (2.98), 2.678 (1.59), 2.727 (0.83), 2.888 (0.83), 2.938 (0.51), 3.088 (0.57), 3.131 (0.51), 3.505 (1.02), 7.085 (0.76), 7.384 (0.51), 7.404 (0.51), 7.428 (0.38), 7.443 (0.89), 7.461 (1.40), 7.476 (0.89), 7.493 (0.44), 7.698 (0.76), 7.801 (1.21), 7.821 (1.02), 7.842 (0.95), 7.863 (0.89). Example 1-B- ²²⁷ Th

[ ²²⁷ Th]Thorium (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-{(1 r,4S)-4-[(2- {4,7,10-tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclod odecan-1-yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]cyclohexyl}-2,5,11,13-tetraazahexade cane- 10,14,16-tricarboxylate 

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[(1 r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid dissolved in 400 mM sodium acetate buffer (pH 5.6) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl at 0.3 MBq/nmol specific activity and RAC of 7.2 MBq/mL at 95 °C for 40 min. The labelling efficiency was determined to be 92% by iTLC. Intermediate 8

{4-[(2-{(3-{bis[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11 -bis(tert-butoxycarbonyl)-2,2- dimethyl-19-(naphthalen-2-yl)-4,9,17-trioxo-3-oxa-8,10,16-tr iazanonadecan-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl]amino}propyl)[2-({1-[ 2-({[(1S,4r)-4- {[(7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19 -(naphthalen-2-yl)-4,9,17- trioxo-3-oxa-8,10,16-triazanonadecan-18-yl]carbamoyl}cyclohe xyl]methyl}amino)-2- oxoethyl]-3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4-

 

carbonyl}amino)ethyl]amino}ethyl)carbamoyl]-3-hydroxy-6-m ethyl-2-oxopyridin-1(2H)- yl}acetic acid 

'

' c

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5 mg, 4.6 µmol) and tri-tert-butyl (3S,10S,14S)-1- [(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(naphthalen-2-yl)meth yl]-1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (15.2 mg, 18.5 µmol) are dissolved in NMP (1.0 mL), DIPEA (8.0 µL) is added, then PyAOP (9.6 mg, 18.5 µmol) in NMP (960 µL) is added.

Reaction mix is diluted with 50% ACN/water/0.1% TFA (6 mL) and the products purified by preparative HPLC (Äkta pure system, column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 50-100% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR: 33 min) affording 2.5 mg of the target compound.

LC-MS (Method K, gradient: 50-100% B over 3 min): R _t = 2.02 min; MS (ESIpos): m/z = 1750.5  

[M+2H] ²⁺   Example 1-C monomer

(3S,10S,14S)-1-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carb oxymethyl)-3-hydroxy-6-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl} (2-{[1-(carboxymethyl)-3- hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-hydroxy-6-meth yl-2-oxopyridin-1(2H)- yl]acetamido}methyl)cyclohexyl]-3-[(naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid 

 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.70 mg, 5.26 µmol) in 570 µL NMP and PyAOP (2.7 mg, 5.26 µmol) in 270 µL NMP are mixed in a vial. DIPEA (4.6 µL, 26 µmol) is added.

(3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(na phthalen-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid (5.18 mg, 7.89 µmol) in 520 µL NMP is added. Reaction mix is diluted with 10% ACN/water/0.1% TFA (7 mL) and products purified by preparative HPLC (RP-HPLC using Äkta pure system (ALG-106) Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 10- 50% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm tR product: 31 min tR by-product:

37 min) affording 4.6 mg of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.75 min; MS (ESIpos): m/z = 1720.4  

[M+H] ⁺   Example 1-C monomer- ²³² Th

(3S,10S,14S)-1-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carb oxymethyl)-3-(hydroxy-kappaO)-6- methyl-2-(oxo-kappaO)-1,2-dihydropyridine-4-carbonyl]amino}e thyl)amino]propyl}(2-{[1- (carboxymethyl)-3-(hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)-1 ,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2-(oxo- kappaO)pyridin-1(2H)-yl]acetamido}methyl)cyclohexyl]-3-[(nap hthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylato(4-)( ²³² Th)thorium 

 

(3S,10S,14S)-1-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carb oxymethyl)-3-hydroxy-6-methyl-2-oxo- 1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{ [1-(carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6- methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)cyclohexyl]-3- [(naphthalen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid (1.00 mg, 0.581 µmol)  is dissolved in 0.5 M carbonate buffer (0.5 mL), Th-232 solution (1 mg/mL in 2% HNO3, 200 µg, 0.87 µmol) is added. Reaction mixture is lyophilised affording 1.10 mg (95 % purity, 92 % yield) of the target compound. 

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 2.22 min; MS (ESIpos): m/z = 1948.7  

[M+H] ⁺   Example 1-D monomer- ²²⁷ Th

(3S,10S,14S)-1-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carb oxymethyl)-3-(hydroxy-kappaO)-6- methyl-2-(oxo-kappaO)-1,2-dihydropyridine-4-carbonyl]amino}e thyl)amino]propyl}(2-{[1- (carboxymethyl)-3-(hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)-1 ,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2-(oxo- kappaO)pyridin-1(2H)-yl]acetamido}methyl)cyclohexyl]-3-[(nap hthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylato(4-)( ²²⁷ Th)thorium 

(3S,10S,14S)-1-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxy methyl)-3-hydroxy-6-methyl-2-oxo- 1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{ [1-(carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6- methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)cyclohexyl]-3- [(naphthalen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid (5.4 µg) dissolved in 186 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 2.9 MBq/mL. 0.1M carbonate buffer pH 9 (140 µL) was added and mixture incubated for 60 min. The labelling efficiency was determined to be 99% by iTLC.  Example 1-E dimer

 

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)az anediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis{3-[( naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid} 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.70 mg, 5.26 µmol) in 570 µL NMP and PyAOP (2.7 mg, 5.26 µmol) in 270 µL NMP are mixed in a vial. DIPEA (4.6 µL, 26 µmol) is added.

(3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(na phthalen-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid (5.18 mg, 7.89 µmol) in 520 µL NMP is added. Reaction mix is diluted with 10% ACN/water/0.1% TFA (7 mL) and products purified by preparative HPLC (RP-HPLC using Äkta pure system (ALG-106) Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 10- 50% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm tR product: 31 min tR by-product:

37 min) affording 3.60 mg (95 % purity, 28 % yield) of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 2.27 min; MS (ESIpos): m/z = 1179.3  

[M+2H] ²⁺   Example 1-F dimer- ²²⁷ Th

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-(hydroxy- kappaO)-6-methyl-2-(oxo-kappaO)-1,2-dihydropyridine-4- carbonyl]amino}ethyl)azanediyl]ethane-2,1-diylcarbamoyl[3-(h ydroxy-kappaO)-6-methyl- 2-(oxo-kappaO)pyridine-4,1(2H)-diyl](1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis{3-[( naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylato}(4-)( ²²⁷ Th)thorium 

 

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)azan ediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis{3-[( naphthalen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid} (8 µg) dissolved in 20 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 5.9 MBq/mL. 0.1M carbonate buffer pH 9 (150 µL) was added and mixture incubated for 60 min. The labelling efficiency was determined to be 99% by iTLC.  Intermediate 7, trimer

{4-[(2-{(3-{bis[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11 -bis(tert-butoxycarbonyl)-2,2- dimethyl-19-(naphthalen-2-yl)-4,9,17-trioxo-3-oxa-8,10,16-tr iazanonadecan-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl]amino}propyl)[2-({1-[ 2-({[(1S,4r)-4- {[(7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19 -(naphthalen-2-yl)-4,9,17- trioxo-3-oxa-8,10,16-triazanonadecan-18-yl]carbamoyl}cyclohe xyl]methyl}amino)-2- oxoethyl]-3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl}amino)ethyl]amino}ethyl)carbamoyl]-3-hydroxy-6-meth yl-2-oxopyridin-1(2H)- yl}acetic acid 

c

C

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5 mg, 4.6 µmol) and tri-tert-butyl (3S,10S,14S)-1- [(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(naphthalen-2-yl)meth yl]-1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (15.2 mg, 18.5 µmol) are dissolved in NMP (1.0 mL), DIPEA (8.0 µL) is added, then PyAOP (9.6 mg, 18.5 µmol) in NMP (960 µL) is added.  

Reaction mix is diluted with 50% ACN/water/0.1% TFA (6 mL) and the products purified by preparative HPLC (Äkta pure system, column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 50-100% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR: 33 min) affording 2.5 mg of the target compound.

LC-MS (Method K, gradient: 50-100% B over 3 min): R _t = 2.02 min; MS (ESIpos): m/z = 1750.5  

[M+2H] ²⁺  

Example 1-G trimer

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{[(3-{[2-({1-[2-({[(1S,4r )-4-{[(2S)-1-{[(5S)-5-carboxy-5- ({[(1S)-1,3-dicarboxypropyl]carbamoyl}amino)pentyl]amino}-3- (naphthalen-2-yl)-1- oxopropan-2-yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl ]-3-hydroxy-6-methyl-2- oxopyridine-4(2H)-carbonyl}amino)ethyl](2-{[1-(carboxymethyl )-3-hydroxy-6-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino}propyl) azanediyl]bis[(ethane- 2,1-diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)- diyl)(1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl]}bis{3-[( naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid} 

H

{4-[(2-{(3-{bis[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11 -bis(tert-butoxycarbonyl)-2,2-dimethyl- 19-(naphthalen-2-yl)-4,9,17-trioxo-3-oxa-8,10,16-triazanonad ecan-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl]amino}propyl)[2-({1-[ 2-({[(1S,4r)-4-{[(7S,11S,18S)- 7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19-(naphthalen-2- yl)-4,9,17-trioxo-3-oxa-8,10,16- triazanonadecan-18-yl]carbamoyl}cyclohexyl]methyl}amino)-2-o xoethyl]-3-hydroxy-6-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl}amino)ethyl]amino}ethyl)c arbamoyl]-3-hydroxy-6-methyl-2-  

oxopyridin-1(2H)-yl}acetic acid (2.50 mg, 0.714 µmol) is treated with 90% TFA in water (0.5 mL) for 2 hrs. Water (18 mL) is addedamd reaction mixture is lyophilised affording 2.10 mg (95 % purity, 93 % yield) of the target compound. 

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 1.84 min; MS (ESIpos): m/z = 1498.1  

[M+2H] ²⁺   Example 1-H trimer- ²²⁷ Th

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{[(3-{[2-({1-[2-({[(1S,4r )-4-{[(2S)-1-{[(5S)-5-carboxy-5- ({[(1S)-1,3-dicarboxypropyl]carbamoyl}amino)pentyl]amino}-3- (naphthalen-2-yl)-1- oxopropan-2-yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl ]-3-(hydroxy-kappaO)-6- methyl-2-oxopyridine-4(2H)-carbonyl}amino)ethyl](2-{[1-(carb oxymethyl)-3-(hydroxy- kappaO)-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino}propyl)azanediyl]bis[(ethane-2,1- diyl)carbamoyl[3- (hydroxy-kappaO)-6-methyl-2-oxopyridine-4,1(2H)-diyl](1-oxoe thane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl]}bis{3-[( naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylato}(4-)( ²²⁷ Th)thorium 

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{[(3-{[2-({1-[2-({[(1S,4r )-4-{[(2S)-1-{[(5S)-5-carboxy-5-({[(1S)- 1,3-dicarboxypropyl]carbamoyl}amino)pentyl]amino}-3-(naphtha len-2-yl)-1-oxopropan-2- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxopyridine-4(2H)- carbonyl}amino)ethyl](2-{[1-(carboxymethyl)-3-hydroxy-6-meth yl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino}propyl)azanediyl]bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)(1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl]}bis{3-[( naphthalen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid} (8 µg) dissolved in 159 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl  

(2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.6 MBq/mL. 0.1M carbonate buffer pH 9 (16 µL) was added and mixture incubated for 60 min. The labelling efficiency was determined to be 97% by iTLC.  Example 1-I tetramer

(2S)-2-[[(1S)-5-[[(2S)-2-[[4-[[[2-[4-[2-[3-[bis[2-[[1-[2- [[4-[[(1S)-2-[[(5S)-5-carboxy-5-[[(1S)- 1,3-dicarboxypropyl]carbamoylamino]pentyl]amino]-1-(2-naphth ylmethyl)-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo- pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[[4-[ [(1S)-2-[[(5S)-5-carboxy-5- [[(1S)-1,3-dicarboxypropyl]carbamoylamino]pentyl]amino]-1-(2 -naphthylmethyl)-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo- pyridine-4-carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydr oxy-6-methyl-2-oxo-1- pyridyl]acetyl]amino]methyl]cyclohexanecarbonyl]amino]-3-(2- naphthyl)propanoyl]amino]-1-carboxy-pentyl]carbamoylamino]pe ntanedioic acid 

H

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (7.40 mg, 6.83 µmol) and PyAOP (14.3 mg, 27.3 µmol) are dissolved in NMP (1 mL), DIPEA (11.9 µL, 68 µmol) is added. (3S,10S,14S)-1- [(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(naphthalen-2-yl)meth yl]-1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid (31.4 mg, 47.8 µmol) is added. Reaction mix is quenched with 20% ACN/water (8 mL) and the product purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 20-60% B over 40 min Flow: 10 mL/min Detection: UV

280/335 nm tR: 34 min) affording 2.50 mg (95 % purity, 10 % yield) of the target compound. 

 

LC-MS (Method K, gradient: 20-60% B over 3 min): R _t = 1.89 min; MS (ESIpos): m/z = 1816.8  

[M+2H] ²⁺   Example 1-J tetramer- ²²⁷ Th

(2S)-2-[[(1S)-5-[[(2S)-2-[[4-[[[2-[4-[2-[3-[bis[2-[[1-[2- [[4-[[(1S)-2-[[(5S)-5-carboxy-5-[[(1S)- 1,3-dicarboxypropyl]carbamoylamino]pentyl]amino]-1-(2-naphth ylmethyl)-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-(hydr oxy -kappaO)-6-methyl-2- oxo-pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[ [4-[[(1S)-2-[[(5S)-5-carboxy- 5-[[(1S)-1,3-dicarboxypropyl]carbamoylamino]pentyl]amino]-1- (2-naphthylmethyl)-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-(hydr oxy-kappaO)-6-methyl-2- oxo-pyridine-4-carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3- (hydroxyl-kappaO)-6- methyl-2-oxo-1-pyridyl]acetyl]amino]methyl]cyclohexanecarbon yl]amino]-3-(2- naphthyl)propanoyl]amino]-1-carboxylato-pentyl]carbamoylamin o]pentanedioate(4- )( ²²⁷ Th)thorium  

(2S)-2-[[(1S)-5-[[(2S)-2-[[4-[[[2-[4-[2-[3-[bis[2-[[1-[2- [[4-[[(1S)-2-[[(5S)-5-carboxy-5-[[(1S)-1,3- dicarboxypropyl]carbamoylamino]pentyl]amino]-1-(2-naphthylme thyl)-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[[4-[[(1S)-2-[[( 5S)-5-carboxy-5-[[(1S)-1,3- dicarboxypropyl]carbamoylamino]pentyl]amino]-1-(2-naphthylme thyl)-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydroxy-6-methy l-2-oxo-1- pyridyl]acetyl]amino]methyl]cyclohexanecarbonyl]amino]-3-(2- naphthyl)propanoyl]amino]-1- carboxy-pentyl]carbamoylamino]pentanedioic acid 

(7 µg) dissolved in 108 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 5.7 MBq/mL.  

0.1M carbonate buffer pH 9 (11 µL) was added and mixture incubated for 2 hrs. The labelling efficiency was determined to be 98% by iTLC. Example 1-K

(3S,10S,14S)-1-{(1r,4S)-4-[(2-{4-[(7,10-bis{3-[(2-{[1-(ca rboxymethyl)-3-hydroxy-6-methyl- 2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]-3-ox opropyl}-18-[1- (carboxymethyl)-3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridin- 4-yl]-4,13,18-trioxo- 3,7,10,14,17-pentaazaoctadecan-1-yl)carbamoyl]-3-hydroxy-6-m ethyl-2-oxopyridin-1(2H)- yl}acetamido)methyl]cyclohexyl}-3-[(naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid 

)

{4-[(7,10-bis{3-[(2-{[1-(carboxymethyl)-3-hydroxy-6-methy l-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]-3-oxopropyl}-18-[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo- 1,2-dihydropyridin-4-yl]-4,13,18-trioxo-3,7,10,14,17-pentaaz aoctadecan-1-yl)carbamoyl]-3- hydroxy-6-methyl-2-oxopyridin-1(2H)-yl}acetic acid (2.10 mg, 1.55 µmol) in 210 µL NMP, 2,4,6- trimethylpyridine (2.1 µL) and TSTU (0.5 mg in 50 µL NMP) are mixed in a vial. (3S,10S,14S)- 1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(naphthalen-2-yl)me thyl]-1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid (1.53 mg, 2.33 µmol) is added. Reaction mix is diluted with water/0.1% TFA (4 mL) and the product purified by preparative HPLC (RP-HPLC using Äkta pure system (ALG-106) Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 10-50% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 32 min) affording 2.00 mg (95 % purity, 61 % yield) of the target compound. 

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.78 min; MS (ESIpos): m/z = 1990.9  

[M+H] ⁺   Example 1-L- ²²⁷ Th

 

(3S,10S,14S)-1-{(1r,4S)-4-[(2-{4-[(7,10-bis{3-[(2-{[1-(ca rboxymethyl)-3-(hydroxy-kappaO)- 6-methyl-2-(oxo-kappaO)-1,2-dihydropyridine-4-carbonyl]amino }ethyl)amino]-3- oxopropyl}-18-[1-(carboxymethyl)-3-(hydroxy-kappaO)-6-methyl -2-(oxo-kappaO)-1,2- dihydropyridin-4-yl]-4,13,18-trioxo-3,7,10,14,17-pentaazaoct adecan-1-yl)carbamoyl]-3- (hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)pyridin-1(2H)- yl}acetamido)methyl]cyclohexyl}-3-[(naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylato(4-)( ²²⁷ Th)thorium 

(3S,10S,14S)-1-{(1r,4S)-4-[(2-{4-[(7,10-bis{3-[(2-{[1-(carbo xymethyl)-3-hydroxy-6-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]-3-oxop ropyl}-18-[1-(carboxymethyl)-3- hydroxy-6-methyl-2-oxo-1,2-dihydropyridin-4-yl]-4,13,18-trio xo-3,7,10,14,17- pentaazaoctadecan-1-yl)carbamoyl]-3-hydroxy-6-methyl-2-oxopy ridin-1(2H)- yl}acetamido)methyl]cyclohexyl}-3-[(naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid (4.9 µg) dissolved in 149 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.375 MBq/nmol specific activity and RAC of 5.6 MBq/mL.1M HEPES buffer pH 7.5 (15 µL) was added and mixture incubated for 30 min. The labelling efficiency was determined to be 65% by iTLC. Intermediate 8

(3S,10S,14S)-1-[(1r,4S)-4-(47,47-dimethyl-3,7,45-trioxo- 5,11,14,17,20,23,26,29,32,35,38,41,46-tridecaoxa-2,8,44-tria zaoctatetracontan-1- yl)cyclohexyl]-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5 ,11,13-tetraazahexadecane- 10,14,16-tricarboxylic acid 

 

2,2-dimethyl-4,42-dioxo-3,8,11,14,17,20,23,26,29,32,35,38,44 -tridecaoxa-5,41- diazahexatetracontan-46-oic acid (5.00 mg, 6.57 µmol) (5 mg in 500 µL NMP) and PyAOP (3.4 mg, 6.6 µmol) in 340 µL NMP are mixed in a vial. N,N-diisopropylethylamine (2.3 µL) is added.

pH strip indicates neutral reaction mixture. N,N-diisopropylethylamine (1.0 µL, 19 µmol) is added. (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylic acid (4.31 mg, 6.57 µmol)  in 430 µL NMP is added. Purification by RP-HPLC (Äkta pure system Column:

Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN

Gradient: 20-50% B over 40 min Flow: 10 mL/min Detection: UV 280/276 nm tR product = 40 min afforded 4.5 mg (49%) of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 2.29 min; MS (ESIpos): m/z = 1398.7  

[M+H] ⁺   Intermediate 9

(3S,10S,14S)-1-[(1r,4S)-4-(43-amino-3,7-dioxo-5,11,14,17, 20,23,26,29,32,35,38,41- dodecaoxa-2,8-diazatritetracontan-1-yl)cyclohexyl]-3-[(napht halen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

(3S,10S,14S)-1-[(1r,4S)-4-(47,47-dimethyl-3,7,45-trioxo- 5,11,14,17,20,23,26,29,32,35,38,41,46-tridecaoxa-2,8,44-tria zaoctatetracontan-1-  

yl)cyclohexyl]-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylic acid (9.00 mg, 6.43 µmol) is treated with 95% TFA/water. TFA is removed by evaporation, water (10 mL) is added to the residue followed by lyophilisation affording 8.4 mg (100% yield) of the target compoubd.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.8 min; MS (ESIpos): m/z = 1298.7  

[M+H] ⁺

Intermediate 10a

{2-[4-(3-[bis(2-{[3-hydroxy-1-(2-hydroxyethyl)-2-oxo-1,2- dihydropyridine-4- carbonyl]amino}ethyl)amino]-2-{[bis(2-{[3-hydroxy-1-(2-hydro xyethyl)-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]methyl}propyl)a nilino]-2- oxoethoxy}acetic acid

To a solution of N,N',N'',N'''-({2-[(4-aminophenyl)methyl]propane-1,3-diyl}bi s[nitrilodi(ethane- 2,1-diyl)])tetrakis[3-hydroxy-1-(2-hydroxyethyl)-2-oxo-1,2-d ihydropyridine-4-carboxamide] (100 mg, 80 % purity, 74.3 µmol) in NMP ()1 mL) was added at r.t.1,4-dioxane-2,6-dione (32.9 mg, 97 % purity, 275 µmol) and 2,4,6-trimethylpiperidine (48 µl, 360 µmol). After stirring for 15min at r.t. the mixture was diluted with 0.1% aqueous formic acid (3.5 mL), adjusted to pH 14 with 5N aq. NaOH and then adjusted to pH with TFA. The mixture was purified by preparative

LC-MS (Method 1): R ⁺

t = 0.62 min; MS (ESIpos): m/z = 1193 [M+H]

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.45), 1.907 (0.45), 2.074 (0.90), 2.195 (0.65), 2.332 (2.65), 2.336 (1.16), 2.518 (16.00), 2.522 (10.97), 2.669 (3.68), 2.673 (2.71), 2.678 (1.23),

 

3.608 (4.06), 3.622 (8.58), 3.635 (4.77), 3.918 (3.87), 3.932 (6.58), 3.945 (3.29), 4.119 (4.52), 4.162 (3.68), 6.481 (5.16), 6.499 (5.16), 6.989 (1.55), 7.011 (1.68), 7.051 (6.77), 7.069 (6.13), 7.407 (2.26), 7.428 (1.87), 8.133 (2.00), 8.523 (1.35).

  Example 1-PEG

(3S,10S,14S)-1-[(1r,4S)-4-{49-[4-(3-[bis(2-{[3-hydroxy-1- (2-hydroxyethyl)-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]-2-{[bis(2-{[3- hydroxy-1-(2-hydroxyethyl)- 2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]methy l}propyl)anilino]- 3,7,45,49-tetraoxo-5,11,14,17,20,23,26,29,32,35,38,41,47-tri decaoxa-2,8,44- triazanonatetracontan-1-yl}cyclohexyl]-3-[(naphthalen-2-yl)m ethyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

{2-[4-(3-[bis(2-{[3-hydroxy-1-(2-hydroxyethyl)-2-oxo-1,2- dihydropyridine-4- carbonyl]amino}ethyl)amino]-2-{[bis(2-{[3-hydroxy-1-(2-hydro xyethyl)-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]methyl}propyl)a nilino]-2-oxoethoxy}acetic acid (5.00 mg, 4.19 µmol) in 500 µL NMP and PyAOP (2.2 mg, 4.19 µmol) in 220 µL NMP are mixed in a vial.2,4,6-Trimethylpyridine (2.8 µL, 42 µmol) is added, then (3S,10S,14S)-1-[(1r,4S)-4-(43- amino-3,7-dioxo-5,11,14,17,20,23,26,29,32,35,38,41-dodecaoxa -2,8-diazatritetracontan-1- yl)cyclohexyl]-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5 ,11,13-tetraazahexadecane- 10,14,16-tricarboxylic acid (5.99 mg, 4.61 µmol) in 600 µL NMP is added. Reaction mixture is diluted with water/0.1% TFA (3.5 mL) and the product purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 10-50% B over 40 min Flow: 10 mL/min Detection: UV

280/335 nm, tR product = 32 min) affording 2.00 mg (93 % purity, 18 % yield)

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.69 min; MS (ESIpos): m/z = 1236.6  

[M+H] ⁺   Example 1-PEG- ²²⁷ Th  

(3S,10S,14S)-1-[(1r,4S)-4-{49-[4-(3-[bis(2-{[3-(hydroxy-k appaO)-1-(2-hydroxyethyl)-2- (oxo-kappaO)-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]-2-{[bis(2-{[3-(hydroxy- kappaO)-1-(2-hydroxyethyl)-2-(oxo-kappaO)-1,2-dihydropyridin e-4- carbonyl]amino}ethyl)amino]methyl}propyl)anilino]-3,7,45,49- tetraoxo- 5,11,14,17,20,23,26,29,32,35,38,41,47-tridecaoxa-2,8,44-tria zanonatetracontan-1- yl}cyclohexyl]-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5 ,11,13-tetraazahexadecane- 10,14,16-tricarboxylato(4-)( ²²⁷ Th)thorium 

(3S,10S,14S)-1-[(1r,4S)-4-{49-[4-(3-[bis(2-{[3-hydroxy-1- (2-hydroxyethyl)-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]-2-{[bis(2-{[3- hydroxy-1-(2-hydroxyethyl)-2-oxo- 1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]methyl}prop yl)anilino]-3,7,45,49-tetraoxo- 5,11,14,17,20,23,26,29,32,35,38,41,47-tridecaoxa-2,8,44-tria zanonatetracontan-1- yl}cyclohexyl]-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5 ,11,13-tetraazahexadecane- 10,14,16-tricarboxylic acid (6.9 µg) dissolved in 167 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.375 MBq/nmol specific activity and RAC of 4.8 MBq/mL. Ethanol (50 µL) was added and mixture incubated for 60 min.

The labelling efficiency was determined to be 96% by iTLC.  Example 1-F

(3S,10S,14S)-1-{(1r,4S)-4-[({[4-(3-[bis(2-{[3-hydroxy-1-( 2-hydroxyethyl)-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]-2-{[bis(2-{[3- hydroxy-1-(2-hydroxyethyl)- 2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]methyl}propyl)phenyl]carbamothioy l}amino)methyl]cycloh exyl}-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-te traazahexadecane-10,14,16- tricarboxylic acid 

 

N,N',N'',N'''-({2-[(4-isothiocyanatophenyl)methyl]propane-1, 3-diyl}bis[nitrilodi(ethane-2,1- diyl)])tetrakis[3-hydroxy-1-(2-hydroxyethyl)-2-oxo-1,2-dihyd ropyridine-4-carboxamide] (9.0 mg, 8.0 µmol) and (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid (3.0 mg, 4.6 µmol) are dissolved in borate buffer (1 mL) and the mixture heated at 50 °C. Product is purified using preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-40% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR = 26 min) affording 3.0 mg (37 % yield) of the target compound. 

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.88 min; MS (ESIpos): m/z = 1773.7  

[M+H] ⁺   Example 1-F- ²²⁷ Th

(3S,10S,14S)-1-{(1r,4S)-4-[({[4-(3-[bis(2-{[3-(hydroxy-ka ppaO)-1-(2-hydroxyethyl)-2-(oxo- kappaO)-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]-2- {[bis(2-{[3-(hydroxy- kappaO)-1-(2-hydroxyethyl)-2-(oxo-kappaO)-1,2-dihydropyridin e-4- carbonyl]amino}ethyl)amino]methyl}propyl)phenyl]carbamothioy l}amino)methyl]cycloh exyl}-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-te traazahexadecane-10,14,16- tricarboxylato(4-)( ²²⁷ Th)thorium 

 

(3S,10S,14S)-1-{(1r,4S)-4-[({[4-(3-[bis(2-{[3-hydroxy-1-(2-h ydroxyethyl)-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]-2-{[bis(2-{[3- hydroxy-1-(2-hydroxyethyl)-2-oxo- 1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]methyl}propyl)phenyl]carbamothioy l}amino)methyl]cyclohexyl}-3- [(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahe xadecane-10,14,16-tricarboxylic acid (6.9 µg) dissolved in 230 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 3.7 MBq/mL and the mixture incubated for 60 min. The labelling efficiency was determined to be 97% by iTLC.  Example 1-M

(3S,10S,14S)-1-[(1r,4S)-4-(acetamidomethyl)cyclohexyl]-3- [(naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(acetamidomethyl)cyclohexyl]-3-[(n aphthalen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (20.0 mg, 98 % purity, 22.6 µmol) was solubilised in DCM (1.0 ml), TFA (1.0 ml, 13 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 8.00 mg (96 % purity, 49 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 699 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.755 (0.43), 0.786 (0.72), 0.818 (0.58), 1.009 (0.43), 1.178 (0.65), 1.231 (1.52), 1.300 (0.87), 1.319 (0.87), 1.352 (0.43), 1.461 (0.87), 1.482 (0.58), 1.577 (0.80), 1.612 (0.87), 1.772 (13.39), 1.907 (0.80), 2.062 (0.58), 2.217 (0.80), 2.235 (1.52), 2.254 (0.65), 2.332 (2.97), 2.336 (1.30), 2.518 (16.00), 2.523 (10.79), 2.673 (3.04), 2.678 (1.30), 2.810 (1.01), 2.826 (1.59), 2.841 (0.94), 2.887 (0.51), 2.910 (0.51), 2.921 (0.58), 2.945 (0.65), 2.984 (0.51), 2.999 (0.58), 3.017 (0.51), 3.034 (0.58), 3.074 (0.58), 3.088 (0.65), 3.109 (0.51), 3.122 (0.43), 3.998 (0.58), 4.011 (0.58), 4.070 (0.58), 4.086 (0.58), 4.513 (0.58), 4.526 (0.58), 6.280 (0.65), 6.298 (1.09), 6.317 (0.65), 7.381 (1.01), 7.385 (1.01), 7.402 (1.09), 7.406 (1.16), 7.419 (0.43), 7.433 (1.09), 7.437 (0.94), 7.447 (1.16), 7.453 (1.52), 7.456 (1.30), 7.467 (0.94), 7.471 (1.09), 7.484 (0.43), 7.681 (1.88), 7.726 (0.51), 7.741 (0.94), 7.755 (0.43), 7.772 (1.09), 7.783 (1.74), 7.789 (1.09), 7.804 (1.45), 7.839 (1.01), 7.857 (0.94), 7.943 (1.23), 7.955 (1.23), 7.965 (1.09).  Intermediate 10

di-tert-butyl N-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tert-butoxy-1-oxoh exan-2- yl]carbamoyl}-D-glutamate 

 

di-tert-butyl D-glutamate—hydrogen chloride (1/1) (4.97 g, 16.8 mmol) and 4-nitrophenyl carbonochloridate (3.57 g, 17.7 mmol) were solubilised in DCM (51 ml), cooled to 0°C under argon, and N,N-diisopropylethylamine (6.7 ml, 39 mmol) was added dropwise. The mixture was stirred for 5min at 0°C and 30min at rt. tert-butyl N6-[(benzyloxy)carbonyl]-L-lysinate—hydrogen chloride (1/1) (7.22 g, 19.4 mmol) was added and N,N-diisopropylethylamine (6.7 ml, 39 mmol) was added dropwise to the mixture. It was stirred 1h at rt. The mixture was washed 3 times with sat. sodium hydrogen carbonate, once with sodium hydroxide (1.0 M) and once with brine. The organic layer was dried and concentrated under reduced pressure to give 13.3 g (78 % purity, 99 % yield) of the target compound, which was used without further purification.

LC-MS (Method 1): R _t = 1.47 min; MS (ESIpos): m/z = 623 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.409 (0.86), 1.419 (0.83), 1.447 (16.00), 1.737 (0.56), 1.754 (0.54), 2.518 (0.43), 2.983 (0.68), 2.997 (0.69), 3.836 (0.75), 4.995 (2.79), 7.270 (0.50), 7.307 (0.75), 7.321 (0.90), 7.324 (0.93), 7.327 (0.91), 7.337 (2.40), 7.348 (2.00), 7.361 (0.84), 7.365 (1.00), 7.368 (0.64), 8.345 (0.80).  Intermediate 11

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-D- glutamate 

di-tert-butyl N-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tert-butoxy-1-oxoh exan-2-yl]carbamoyl}- D-glutamate (2.37 g, 70 % purity, 2.67 mmol) was solubilised in MeOH (40 ml), palladium on carbon (60.0 mg, 10 % purity) was added and the mixture was purged with hydrogen. The mixture was stirred for 4.5h at rt under hydrogen atmosphere. The mixture was filtered over

 

Celite, washed with MeOH and concentrated under reduced pressure to give 1.80 g (70 % purity, 97 % yield) of the target compound, which was used without further purification.

LC-MS (Method 1): R _t = 0.98 min; MS (ESIpos): m/z = 489 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.389 (16.00), 1.397 (9.42), 2.478 (0.42), 2.728 (1.77), 2.888 (2.10), 6.319 (0.43).  Intermediate 12

tri-tert-butyl (5S,12S,16R)-1-(9H-fluoren-9-yl)-5-[(naphthalen-2-yl)methyl] -3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-D- glutamate (4.00 g, 85 % purity, 6.97 mmol) and (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(naphth alen- 2-yl)propanoic acid (4.58 g, 10.5 mmol; CAS-RN:[112883-43-9]) were solubilised in DMF (54 ml), 4-methylmorpholine (2.3 ml, 21 mmol, CAS-RN: 109-02-4) and HATU (3.98 g, 10.5 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated, diluted with water and extracted 3 times with DCM. The combined organic layers were washed with brine, dried and evaporated. The residue was purified by flash chromatography (SiO2, hexane/EtOAc gradient 0%-25%) to give 6.07 g (96 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.65 min; MS (ESIpos): m/z = 908 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.317 (0.43), 1.368 (9.34), 1.382 (7.41), 1.389 (16.00), 2.518 (1.06), 2.522 (0.73), 3.096 (0.40), 4.070 (1.47), 4.089 (1.18), 4.097 (1.55), 4.106 (0.87), 6.310 (0.42), 7.121 (0.44), 7.138 (0.44), 7.155 (0.44), 7.213 (0.60), 7.232 (0.68), 7.250 (0.42), 7.336 (0.76), 7.354 (1.06), 7.372 (0.76), 7.381 (0.57), 7.423 (0.60), 7.434 (0.92), 7.440 (1.20), 7.449 (1.40), 7.459 (1.25), 7.475 (0.62), 7.524 (0.71), 7.545 (0.63), 7.564 (0.66), 7.583 (0.65), 7.591 (0.65), 7.609 (0.57), 7.667 (0.55), 7.688 (0.84), 7.750 (0.85), 7.760 (0.79), 7.780 (0.83), 7.800 (1.13), 7.821 (1.86), 7.837 (1.77), 7.853 (1.11), 7.861 (0.91). 

 

Intermediate 13

di-tert-butyl (2R)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16R)-1-(9H-fluoren-9-yl)-5-[(naphthalen-2-yl)methyl] -3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (940 mg, 95 % purity, 984 µmol) was solubilised in DMF (26 ml), piperidine (1.9 ml, 20 mmol) was added and the mixture was stirred under argon over the weekend at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.2% ammonia) to give 305 mg (98 % purity, 44 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.21 min; MS (ESIpos): m/z = 686 [M+H] ⁺   Intermediate 14

tri-tert-butyl (3S,10S,14R)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� � )

di-tert-butyl (2R)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (305 mg, 445 µmol) and (1r,4r)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-car boxylic acid (253 mg, 668 µmol) were solubilised in DMF (3.4 ml), 4-methylmorpholine (150 µl, 1.3 mmol, CAS-RN: 109-02-4)  

and HATU (254 mg, 668 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 160 mg (96 % purity, 33 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.65 min; MS (ESIpos): m/z = 1047 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.214 (0.59), 1.230 (0.78), 1.373 (16.00), 1.379 (4.03), 1.387 (15.09), 1.390 (15.72), 1.643 (0.40), 2.297 (0.85), 2.522 (0.43), 2.781 (0.46), 4.280 (0.88), 4.297 (0.62), 6.312 (0.46), 6.333 (0.42), 7.245 (0.48), 7.289 (0.41), 7.306 (0.86), 7.324 (0.55), 7.382 (0.77), 7.400 (0.95), 7.418 (0.53), 7.448 (0.61), 7.668 (0.

87), 7.686 (1.10), 7.788 (0.65), 7.808 (0.53), 7.873 (0.95), 7.892 (0.84), 7.944 (0.51).  Intermediate 15

tri-tert-butyl (3S,10S,14R)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

tri-tert-butyl (3S,10S,14R)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (160 mg, 96 % purity, 147 µmol) was solubilised in DMF (3.9 ml), piperidine (290 µl, 2.9 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 95.0 mg (100 % purity, 79 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.18 min; MS (ESIpos): m/z = 825 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.195 (0.43), 1.232 (0.47), 1.377 (16.00), 1.388 (13.40), 1.394 (14.23), 1.677 (0.40), 2.332 (0.64), 2.518 (3.55), 2.523 (2.55), 2.673 (0.66), 6.332 (0.42), 7.452 (0.74), 7.678 (0.66), 7.784 (0.71), 7.804 (0.53), 7.960 (0.42), 7.981 (0.64), 8.411 (1.22).  Example 1-A-D

 

(3S,10S,14R)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(na phthalen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3S,10S,14R)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (21.9 mg, 98 % purity, 26.0 µmol) was solubilised in DCM (1.6 ml), TFA (1.6 ml, 21 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (95 % purity, 33 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 657 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.886 (1.25), 0.912 (1.33), 1.067 (0.86), 1.094 (0.86), 1.145 (0.62), 1.251 (2.42), 1.314 (2.50), 1.330 (2.58), 1.522 (2.73), 1.650 (1.95), 1.829 (0.94), 1.903 (0.70), 2.078 (1.25), 2.116 (1.17), 2.137 (1.01), 2.151 (1.17), 2.287 (0.78), 2.332 (3.43), 2.336 (1.64), 2.451 (2.58), 2.518 (14.91), 2.523 (10.69), 2.562 (16.00), 2.579 (2.50), 2.594 (2.81), 2.678 (1.48), 2.916 (1.09), 2.949 (1.72), 2.974 (1.64), 3.023 (2.34), 3.090 (2.34), 3.102 (2.50), 3.124 (2.34), 3.137 (2.19), 3.350 (5.70), 3.906 (1.64), 4.501 (1.25), 4.515 (1.25), 7.389 (2.34), 7.413 (2.97), 7.430 (2.73), 7.433 (2.65), 7.442 (2.89), 7.448 (5.07), 7.453 (2.97), 7.462 (2.50), 7.466 (2.58), 7.479 (1.01), 7.690 (4.60), 7.778 (6.56), 7.800 (5.78), 7.832 (2.65), 7.836 (2.65), 7.854 (2.50), 8.290 (0.86).  Example 1-B-D

(3S,10S,14R)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1- [(1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

₍

tri-tert-butyl (3S,10S,14R)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[(1 r,4S)-4-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate (30.0 mg, 21.8 µmol) was solubilised in DCM (280 µl), TFA (840 µl, 11 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (95 % purity, 25 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.820 (0.80), 0.851 (0.54), 1.091 (0.47), 1.119 (0.47), 1.232 (2.01), 1.310 (1.27), 1.325 (1.41), 1.468 (1.14), 1.489 (0.87), 1.584 (0.94), 1.618 (1.00), 1.665 (0.94), 1.707 (0.87), 1.729 (0.67), 1.745 (0.54), 1.857 (0.54), 1.907 (0.74), 2.022 (0.47), 2.053 (0.67), 2.074 (0.67), 2.190 (0.87), 2.206 (1.34), 2.227 (1.27), 2.244 (0.74), 2.318 (1.47), 2.322 (3.08), 2.326 (4.15), 2.331 (3.08), 2.336 (1.47), 2.518 (16.00), 2.522 (10.31), 2.636 (1.87), 2.659 (2.21), 2.664 (3.62), 2.668 (4.55), 2.673 (3.35), 2.678 (1.67), 2.899 (2.14), 2.957 (4.15), 2.996 (4.08), 3.081 (2.54), 3.115 (1.21), 3.429 (6.03), 3.991 (0.47), 4.011 (0.80), 4.023 (0.80), 4.043 (0.40), 4.066 (0.40), 4.086 (0.80), 4.101 (0.80), 4.465 (0.40), 4.488 (0.67), 4.501 (0.67), 6.350 (0.94), 6.364 (1.27), 6.383 (0.94), 7.384 (1.27), 7.388 (1.27), 7.408 (1.41), 7.418 (0.60), 7.431 (1.34), 7.435 (1.27), 7.452 (2.08), 7.466 (1.21), 7.469 (1.27), 7.483 (0.54), 7.689 (2.34), 7.785 (3.08), 7.805 (2.61), 7.838 (1.34), 7.856 (1.21), 7.916 (0.74), 8.068 (1.21).  Example 1-B-D- ²²⁷ Th [ ²²⁷ Th]Thorium-(3S,10S,14R)-3-[(naphthalen-2-yl)methyl]-1, 4,12-trioxo-1-{(1r,4S)-4-[(2- {4,7,10-tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclod odecan-1-yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]cyclohexyl}-2,5,11,13-tetraazahexade cane- 10,14,16-tricarboxylate 

 

(3S,10S,14R)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1- [(1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 7.1 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 96.7% by iTLC. Intermediate 6-Br

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1-bro monaphthalen- 2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,1 4,16-tricarboxylate )

N-Bromo-succinimide (7.60 mg, 42.7 µmol; CAS-RN:[128-08-5]) was added portionwise at r.t.

to a solution of tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen- 2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,1 4,16-tricarboxylate (32.0 mg, 38.8 µmol) in acetonitrile (1.9mL). After 3.5h further N-bromo-succinimide (15.2 mg, 85.4 µmol) was  

added and the mixture was stirred for further 3d in the dark at r.t.. Then the mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC (C18, acetonitrile/0.1% formic acid gradient) to give 13.0 mg of the final product (37% yield).

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.67), 1.353 (0.50), 1.382 (13.48), 1.389 (16.00), 1.907 (0.90), 2.327 (3.24), 2.331 (2.35), 2.336 (1.06), 2.518 (13.82), 2.523 (8.45), 2.562 (2.85), 2.669 (3.30), 2.673 (2.41), 2.678 (1.06), 7.868 (0.45).

Intermediate 7-Br

tri-tert-butyl (3S,10S,14S)-3-[(1-bromonaphthalen-2-yl)methyl]-1,4,12-triox o-1-[(1r,4S)-4- ({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraaza cyclododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate

N,N-Diisopropylethylamine (6.9 µl, 40 µmol; CAS-RN:[7087-68-5]) was added at r.t. to a solution of [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (22.8 mg, 39.9 µmol) and [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N-dimethylmetha niminium hexafluoridophosphate(1-) (14.9 mg, 39.4 µmol; CAS-RN:[94790-37-1]) in DMF (270 µl, 3.6 mmol; CAS-RN:[68-12-2]). After 15 min tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4- (aminomethyl)cyclohexyl]-3-[(1-bromonaphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (9.00 mg, 9.97 µmol) was added and the mixture stirred for 2h at r.t. After that the mixture was purified by preparative HPLC (C18, acetonitrile/0.1% formic acid gradient) to give 8.6 mg of the title compound (59% yield).

LC-MS (Method 1): R _t = 1.36 min; MS (ESIpos): m/z = 1459 [M+H] ⁺ Intermediate 7-3H

 

tri-tert-butyl (3S,10S,14S)-3-{[(1- ³ H)naphthalen-2-yl]methyl}-1,4,12-trioxo-1-[(1r,4S)-4-( {2- [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate

A mixture of 3.3 mg tri-tert-butyl (3S,10S,14S)-3-[(1-bromonaphthalen-2-yl)methyl]-1,4,12- trioxo-1-[(1r,4S)-4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethy l)-1,4,7,10-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate

(2.27 µmol) in 2.00 mL THF, 10 µL triethylamine and 11.8 mg 10% palladium on charcoal were stirred at a tritium pressure of 270 mbar for 2 h at room temperature. After freezing of the reaction mixture with liquid nitrogen the non-reacted tritium was adsorbed on a waste storage filled with charcoal and platinum oxide. The solvent was evaporated and the dry residue was dissolved in 2.5 mL THF/EtOH (1+1, v+v) and the catalyst was filtered off using a 0.45 µm PTFE syringe filter (Whatman). Labile tritium was removed by dissolving the residue in THF/EtOH (1+1, v+v) followed by evaporation (five times). The crude product solution was purified by RP- chromatography (method O) yielding 919.3 MBq (1.36 mg, 0.98 µmol) of the title compound with a specific activity of 933.7 GBq/mmol.

LC-MS (Method P): Rt = 1.33 ; m/z = 1378.9 (M+H)+, 1380.9 (M+H)+, 1382.9 (M+H)+

Radio-HPLC (Method Q): R _t = 20.9 min

Example 1-B-3H, 3H-PSMA-617

(3S,10S,14S)-3-{[(3- ³ H)naphthalen-2-yl]methyl}-1,4,12-trioxo-1-[(1r,4S)-4-( {2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid

 

750 MBq tri-tert-butyl (3S,10S,14S)-3-{[(1- ³ H)naphthalen-2-yl]methyl}-1,4,12-trioxo-1-[(1r,4S)- 4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraa zacyclododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate

(1.11 mg, 0.80 µmol) were dissolved in 2.00 mL 4 M HCl in dioxane/water (1.9+0.1, v+v) and stirred for 1.5 h at room temperature. The solvent war removed under reduced pressure and purified by RP-chromatography (method R) yielding 520.7 MBq (0.59 mg, 0.56 µmol) of the title compound with a specific activity of 926.6 GBq/mmol (70%, >99% radiochemical purity).

LC-MS (Method S): R _t = 1.11 min; m/z = 1042.1 (M+H) ⁺ , 1044.1 (M+H) ⁺ , 1046.1 (M+H) ⁺ Radio-HPLC (Method T): R _t = 10.0 min;

#2 Linker Intermediate 9

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(3-methylphenyl)methyl]- 3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-3-methyl-L-phenylalan ine (259 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 310 mg (75 % purity, 62 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.65 min; MS (ESIpos): m/z = 872 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (16.00), 1.383 (14.47), 2.235 (1.64), 2.297 (0.41), 2.518 (1.77), 2.522 (1.21), 4.131 (0.58), 4.152 (0.45), 7.081 (0.42), 7.403 (0.41), 7.657 (0.47), 7.868 (0.61), 7.887 (0.57).  Intermediate 10

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3-methylphenyl)propanoyl] amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

*

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(3-methylphenyl)methyl]- 3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (310 mg, 92 % purity, 327 µmol) was  

solubilised in DMF (8.6 ml), piperidine (650 µl, 6.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 130 mg (98 % purity, 60 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 650 [M+H] ⁺   Intermediate 11

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(3-methylphe nyl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3-methylphenyl)propanoyl] amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (130 mg, 200 µmol) and (1r,4r)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-car boxylic acid (114 mg, 301 µmol) were solubilised in DMF (1.5 ml), 4-methylmorpholine (66 µl, 600 µmol, CAS-RN: 109-02-4) and HATU (114 mg, 301 µmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 36.0 mg (18 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.64 min; MS (ESIpos): m/z = 1011 [M+H] ⁺   Intermediate 12

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3- methylphenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadec ane-10,14,16- tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(3-methylphe nyl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (36.0 mg, 35.6 µmol) was solubilised in DMF (940 µl), piperidine (70 µl, 710 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 12.0 mg (43 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.20 min; MS (ESIpos): m/z = 789 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.227 (0.49), 1.381 (14.82), 1.388 (16.00), 2.238 (2.41), 3.509 (1.21), 6.987 (0.47), 7.010 (0.51), 7.113 (0.45).  Example 2-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- methyl-L-phenylalanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3-met hylphenyl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (12.0 mg, 98 % purity, 14.9 µmol) was solubilised in DCM (1.0 ml), TFA (1.0 ml, 13 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.00 mg (95 % purity, 21 % yield) of the target compound.  

LC-MS (Method 1): R _t = 0.66 min; MS (ESIpos): m/z = 620 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.235 (2.71), 2.074 (2.44), 2.242 (11.93), 2.327 (16.00), 2.561 (2.71), 2.609 (2.44), 2.626 (2.44), 2.669 (15.46), 6.985 (2.44), 7.018 (2.71).  #3 Linker Intermediate 13

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-(3-phenylpr opyl)-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-5-phenyl-L-norvaline (268 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS- RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 300 mg (50 % purity, 39 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.67 min; MS (ESIpos): m/z = 886 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.379 (16.00), 2.298 (1.13), 2.518 (0.66), 2.523 (0.46), 7.161 (0.50), 7.163 (0.46), 7.165 (0.43), 7.173 (0.49), 7.179 (0.42), 7.181 (0.42), 7.247 (0.46), 7.264 (0.46), 7.283 (0.43), 7.403 (0.44), 7.874 (0.48), 7.893 (0.56).  Intermediate 14

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-5-phenylpentanoyl]amino}-1-t ert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate 

 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-(3-phenylpr opyl)-2-oxa-4,7,13,15- tetraazaoctadecane-12,16,18-tricarboxylate (300 mg, 80 % purity, 271 µmol) was solubilised in DMF (7.1 ml), piperidine (540 µl, 5.4 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 199 mg (90 % purity, 100 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.15 min; MS (ESIpos): m/z = 664 [M+H] ⁺   Intermediate 15

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -(3-phenylpropyl)- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-5-phenylpentanoyl]amino}-1-t ert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (199 mg, 300 µmol) and (1r,4r)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-car boxylic acid (171 mg, 450 µmol) were solubilised in DMF (2.3 ml), 4-methylmorpholine (99 µl, 900 µmol, CAS-RN: 109-02-4) and HATU (171 mg, 450 µmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 250 mg (81 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.66 min; MS (ESIpos): m/z = 1025 [M+H] ⁺  

 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.230 (0.87), 1.379 (16.00), 1.383 (11.49), 1.387 (12.23), 1.668 (0.40), 1.683 (0.40), 2.074 (0.83), 2.518 (1.21), 2.522 (0.86), 2.728 (0.96), 2.888 (1.21), 4.288 (0.61), 4.305 (0.42), 7.139 (0.53), 7.158 (0.74), 7.244 (0.47), 7.263 (0.56), 7.281 (0.43), 7.316 (0.75), 7.319 (0.68), 7.335 (0.50), 7.338 (0.45), 7.407 (0.61), 7.679 (0.55), 7.698 (0.49), 7.877 (0.72), 7.896 (0.62), 8.158 (0.82).  Intermediate 16

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-(3- phenylpropyl)-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -(3-phenylpropyl)-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (250 mg, 244 µmol) was solubilised in DMF (6.4 ml), piperidine (480 µl, 4.9 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 86.0 mg (44 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.18 min; MS (ESIpos): m/z = 803 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.383 (16.00), 1.385 (12.85), 1.390 (12.10), 2.518 (1.30), 2.523 (0.94), 2.540 (1.29), 2.559 (0.68), 7.137 (0.84), 7.157 (1.06), 7.244 (0.54), 7.261 (0.69), 8.408 (1.08).  Example 3-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-5- phenyl-L-norvalyl}-N ² -{[(1S)- 1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-(3- phenylpropyl)-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (13.4 mg, 98 % purity, 16.4 µmol) was solubilised in DCM (1.1 ml), TFA (1.1 ml, 15 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (95 % purity, 55 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.70 min; MS (ESIpos): m/z = 635 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.891 (1.19), 0.920 (1.72), 0.950 (1.32), 1.247 (3.11), 1.285 (2.45), 1.331 (3.37), 1.350 (2.91), 1.447 (1.85), 1.468 (2.71), 1.492 (3.44), 1.531 (1.92), 1.550 (2.45), 1.568 (2.78), 1.584 (2.51), 1.604 (2.38), 1.616 (2.25), 1.684 (1.45), 1.715 (3.44), 1.748 (2.12), 1.770 (1.59), 1.787 (1.65), 1.805 (1.98), 1.843 (0.79), 1.862 (0.46), 1.905 (0.79), 2.126 (1.45), 2.149 (2.38), 2.162 (2.25), 2.180 (1.39), 2.217 (0.99), 2.238 (1.52), 2.254 (0.86), 2.261 (0.99), 2.274 (0.93), 2.297 (0.46), 2.336 (1.19), 2.518 (16.00), 2.523 (12.36), 2.540 (12.43), 2.622 (2.45), 2.935 (0.86), 2.950 (1.19), 2.967 (1.52), 2.983 (1.32), 3.045 (1.45), 3.060 (1.72), 3.077 (1.52), 3.093 (1.12), 3.340 (4.96), 3.918 (2.18), 3.931 (2.78), 4.185 (1.39), 4.199 (1.59), 6.125 (1.06), 6.398 (1.65), 6.416 (1.65), 7.142 (5.36), 7.155 (5.36), 7.162 (7.47), 7.172 (3.24), 7.243 (6.21), 7.261 (6.28), 7.280 (2.91), 7.785 (1.26), 7.799 (2.31), 7.812 (1.26), 7.900 (2.12), 7.921 (2.05).  #4 Linker Intermediate 17

tri-tert-butyl (5S,12S,16S)-5-[3-(carbamoylamino)propyl]-1-(9H-fluoren-9-yl )-3,6,14-trioxo- 2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N5-carbamoyl-N2-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-ornit hine (257 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 310 mg (92 % purity, 76 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.43 min; MS (ESIpos): m/z = 868 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (16.00), 1.384 (9.41), 2.297 (0.94), 2.686 (0.68), 2.729 (0.43), 2.888 (0.55), 4.207 (0.47), 5.383 (0.47), 7.324 (0.54), 7.414 (0.60), 7.880 (0.74), 7.898 (0.62).  Intermediate 18

tri-tert-butyl (6S,13S,17S)-1,6-diamino-1,7,15-trioxo-2,8,14,16-tetraazanon adecane- 13,17,19-tricarboxylate 

 

tri-tert-butyl (5S,12S,16S)-5-[3-(carbamoylamino)propyl]-1-(9H-fluoren-9-yl )-3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (310 mg, 92 % purity, 329 µmol) was solubilised in DMF (8.6 ml), piperidine (650 µl, 6.6 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 212 mg (90 % purity, 90 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.385 (16.00), 1.392 (11.97), 1.397 (12.79), 2.297 (0.59), 2.518 (0.52), 5.397 (0.71), 8.286 (0.60).  Intermediate 19

tri-tert-butyl (6S,13S,17S)-1-amino-6-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,7,15-trioxo- 2,8,14,16-tetraazanonadecane-13,17,19-tricarboxylate 

 

tri-tert-butyl (6S,13S,17S)-1,6-diamino-1,7,15-trioxo-2,8,14,16-tetraazanon adecane-13,17,19- tricarboxylate (212 mg, 329 µmol) and (1r,4r)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carboxylic acid (187 mg, 493 µmol) were solubilised in DMF (2.5 ml), 4-methylmorpholine (110 µl, 990 µmol, CAS-RN: 109-02-4) and HATU (188 mg, 493 µmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 300 mg (90 % purity, 82 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.64), 1.255 (0.74), 1.275 (0.64), 1.315 (0.45), 1.380 (14.07), 1.384 (16.00), 1.389 (9.61), 1.392 (12.09), 1.668 (0.59), 1.694 (0.54), 2.297 (5.78), 2.322 (0.41), 2.327 (0.57), 2.331 (0.46), 2.518 (2.70), 2.522 (1.71), 2.664 (0.41), 2.669 (0.54), 2.673 (0.41), 2.818 (0.59), 4.200 (0.41), 4.286 (0.75), 4.294 (0.65), 4.303 (0.53), 4.310 (0.41), 5.367 (0.82), 6.297 (0.41), 7.143 (0.64), 7.163 (1.30), 7.181 (1.49), 7.231 (1.27), 7.235 (0.46), 7.249 (1.36), 7.267 (0.59), 7.303 (0.67), 7.320 (1.21), 7.322 (1.21), 7.338 (0.83), 7.341 (0.81), 7.392 (0.69), 7.410 (1.10), 7.429 (0.49), 7.680 (0.99), 7.698 (0.89), 7.879 (1.25), 7.897 (1.14).  Intermediate 20

formic acid-tri-tert-butyl (6S,13S,17S)-1-amino-6-{[(1r,4S)-4-(aminomethyl)cyclohexane- 1-carbonyl]amino}-1,7,15-trioxo-2,8,14,16-tetraazanonadecane -13,17,19-tricarboxylate

(1/1) 

tri-tert-butyl (6S,13S,17S)-1-amino-6-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,7,15-trioxo-2,8,14,16- tetraazanonadecane-13,17,19-tricarboxylate (300 mg, 90 % purity, 268 µmol) was solubilised in DMF (7.0 ml), piperidine (530 µl, 5.4 mmol) was added and the mixture was stirred under argon for 4h at rt. The mixture was evaporated and purified by preparative HPLC (C18,  

acetonitrile/water with 0.1% formic acid) to give 67.0 mg (98 % purity, 29 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.01 min; MS (ESIneg): m/z = 829 [M-H]- 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.273 (0.45), 1.359 (0.45), 1.385 (16.00), 1.388 (11.67), 1.395 (11.31), 2.518 (1.92), 2.523 (1.40), 2.534 (0.64), 2.551 (0.55), 5.382 (0.67), 8.412 (1.43).  Example 4-A

N ⁶ -{N ² -[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-N ⁵ -carbamoyl-L-ornithyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

formic acid—tri-tert-butyl (6S,13S,17S)-1-amino-6-{[(1r,4S)-4-(aminomethyl)cyclohexane- 1- carbonyl]amino}-1,7,15-trioxo-2,8,14,16-tetraazanonadecane-1 3,17,19-tricarboxylate (1/1) (20.0 mg, 98 % purity, 23.6 µmol) was solubilised in DCM (1.4 ml), TFA (1.4 ml, 19 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.2% ammonia) to give 7.50 mg (95 % purity, 49 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.931 (0.66), 1.266 (1.93), 1.289 (1.64), 1.326 (1.39), 1.344 (1.37), 1.360 (1.34), 1.440 (0.79), 1.461 (1.10), 1.475 (1.13), 1.494 (0.90), 1.577 (0.85), 1.595 (0.83), 1.717 (1.60), 1.746 (2.38), 2.158 (0.69), 2.185 (1.12), 2.201 (1.30), 2.210 (1.30), 2.230 (0.62), 2.327 (0.40), 2.522 (1.38), 2.539 (16.00), 2.627 (1.25), 2.641 (1.24), 2.665 (0.52), 2.669 (0.56), 2.922 (1.29), 3.018 (1.48), 3.032 (1.48), 3.558 (1.17), 3.777 (0.86), 3.896 (0.88), 3.915 (1.22), 3.929 (1.27), 3.955 (1.26), 3.972 (1.15), 4.116 (0.60), 4.137 (0.92), 4.151 (0.91), 4.171 (0.56), 5.586 (0.77), 6.243 (0.59), 6.260 (0.58), 6.327 (0.81), 6.346 (0.78), 6.446 (0.42), 7.829 (0.55), 7.842 (1.00), 7.855 (0.55), 7.887 (0.91), 7.907 (0.87), 8.247 (1.28).  #5 Linker  

Intermediate 21

tri-tert-butyl (5S,12S,16S)-5-[4-(carbamoylamino)butyl]-1-(9H-fluoren-9-yl) -3,6,14-trioxo- 2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N6-carbamoyl-N2-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-lysin e (266 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 310 mg (98 % purity, 80 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.44 min; MS (ESIpos): m/z = 882 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (16.00), 1.384 (8.62), 2.518 (0.45), 4.212 (0.49), 5.346 (0.40), 7.322 (0.47), 7.324 (0.46), 7.414 (0.62), 7.881 (0.65), 7.900 (0.55).  Intermediate 22

tri-tert-butyl (7S,14S,18S)-1,7-diamino-1,8,16-trioxo-2,9,15,17-tetraazaico sane-14,18,20- tricarboxylate 

 

tri-tert-butyl (5S,12S,16S)-5-[4-(carbamoylamino)butyl]-1-(9H-fluoren-9-yl) -3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (210 mg, 98 % purity, 234 µmol) was solubilised in DMF (2.5 ml), piperidine (460 µl, 4.7 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 131 mg (85 % purity, 72 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.272 (0.77), 1.289 (0.78), 1.310 (0.76), 1.327 (0.69), 1.385 (16.00), 1.391 (14.38), 1.396 (14.61), 2.907 (0.62), 2.922 (0.61), 3.051 (0.41), 3.242 (0.41), 3.951 (0.41), 5.372 (0.89), 6.295 (0.47), 6.316 (0.46), 6.339 (0.49), 6.360 (0.46), 8.289 (1.02).  Intermediate 23

tri-tert-butyl (7S,14S,18S)-1-amino-7-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,8,16-trioxo- 2,9,15,17-tetraazaicosane-14,18,20-tricarboxylate 

 

tri-tert-butyl (7S,14S,18S)-1,7-diamino-1,8,16-trioxo-2,9,15,17-tetraazaico sane-14,18,20- tricarboxylate (180 mg, 273 µmol) and (1r,4r)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carboxylic acid (156 mg, 410 µmol) were solubilised in DMF (2.1 ml), 4-methylmorpholine (90 µl, 820 µmol, CAS-RN: 109-02-4) and HATU

(156 mg, 410 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 119 mg (98 % purity, 42 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.231 (0.59), 1.252 (0.71), 1.269 (0.63), 1.289 (0.72), 1.315 (0.58), 1.343 (0.41), 1.380 (16.00), 1.384 (15.33), 1.391 (12.70), 1.667 (0.55), 1.702 (0.62), 2.297 (0.68), 2.821 (0.52), 2.893 (0.46), 2.908 (0.47), 4.286 (0.86), 4.304 (0.60), 5.342 (0.95), 6.245 (0.41), 6.290 (0.44), 6.310 (0.42), 7.303 (0.57), 7.321 (1.01), 7.340 (0.66), 7.392 (0.60), 7.410 (0.96), 7.429 (0.42), 7.681 (0.85), 7.699 (0.78), 7.879 (1.08), 7.898 (0.98).  Intermediate 24

tri-tert-butyl (7S,14S,18S)-1-amino-7-{[(1r,4S)-4-(aminomethyl)cyclohexane- 1- carbonyl]amino}-1,8,16-trioxo-2,9,15,17-tetraazaicosane-14,1 8,20-tricarboxylate 

 

)

tri-tert-butyl (7S,14S,18S)-1-amino-7-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,8,16-trioxo-2,9,15,17- tetraazaicosane-14,18,20-tricarboxylate (180 mg, 70 % purity, 123 µmol) was solubilised in DMF

(3.2 ml), piperidine (240 µl, 2.5 mmol) was added and the mixture was stirred under argon for 4h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 94.0 mg (85 % purity, 81 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.00 min; MS (ESIpos): m/z = 799 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.249 (0.41), 1.270 (0.42), 1.286 (0.43), 1.312 (0.41), 1.358 (0.41), 1.384 (16.00), 1.387 (11.85), 1.394 (11.13), 2.518 (1.20), 2.522 (0.82), 2.536 (0.52), 2.539 (0.42), 2.552 (0.50), 3.350 (0.48), 5.357 (0.64), 8.421 (0.62).  Example 5-A

N ⁶ -{N ² -[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-N ⁶ -carbamoyl-L-lysyl}-N ² -{[(1S)- 1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (7S,14S,18S)-1-amino-7-{[(1r,4S)-4-(aminomethyl)cyclohexane- 1-carbonyl]amino}- 1,8,16-trioxo-2,9,15,17-tetraazaicosane-14,18,20-tricarboxyl ate (30.0 mg, 98 % purity, 36.8 µmol) was solubilised in DCM (2.3 ml), TFA (2.3 ml, 29 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.00 mg (90 % purity, 19 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.929 (0.98), 1.263 (2.69), 1.290 (2.43), 1.333 (2.33), 1.473 (1.14), 1.556 (1.40), 1.710 (1.61), 1.738 (1.76), 1.796 (1.19), 2.074 (1.24), 2.136 (1.09), 2.155 (1.09), 2.219 (1.04), 2.518 (11.29), 2.523 (7.87), 2.540 (4.25), 2.620 (1.24), 2.876 (1.04), 2.945 (1.35), 2.961 (1.29), 2.993 (1.35), 3.072 (1.61), 3.354 (16.00), 3.882 (1.19), 3.927 (1.14), 4.108 (1.09), 4.122 (1.09), 5.537 (2.38), 6.162 (0.62), 6.370 (1.45), 7.781 (1.24), 7.930 (0.98), 7.948 (1.04).  #6 Linker Intermediate 25

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(3,4,5- trifluorophenyl)methyl]-2-oxa-4,7,13,15-tetraazaoctadecane-1 2,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-3,4,5-trifluoro-L- phenylalanine (285 mg, 646 µmol; CAS-RN:[205526-30-3]) were solubilised in DMF (3.3 ml), 4- methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 310 mg (95 % purity, 75 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.66 min; MS (ESIpos): m/z = 912 [M+H] ⁺   Intermediate 26

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3,4,5-trifluorophenyl)pro panoyl]amino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(3,4,5-tri fluorophenyl)methyl]-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (310 mg, 95 % purity, 323 µmol) was solubilised in DMF (3.5 ml), piperidine (640 µl, 6.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 243 mg of the target compound.  

LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 690 [M+H] ⁺   Intermediate 27

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(3,4,5- trifluorophenyl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3,4,5-trifluorophenyl)pro panoyl]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (243 mg, 92 % purity, 325 µmol) and (1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (185 mg, 487 µmol) were solubilised in DMF (2.5 ml), 4-methylmorpholine (110 µl, 970 µmol, CAS-RN: 109-02-4) and HATU (185 mg, 487 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 250 mg (98 % purity, 72 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.65 min; MS (ESIpos): m/z = 1051 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.379 (16.00), 1.384 (13.65), 1.387 (14.51), 2.518 (1.40), 2.523 (0.90), 2.728 (3.65), 2.888 (4.36), 4.289 (0.67), 4.305 (0.46), 7.312 (0.78), 7.315 (0.73), 7.331 (0.51), 7.387 (0.42), 7.406 (0.63), 7.675 (0.62), 7.694 (0.55), 7.877 (0.70), 7.896 (0.65), 7.938 (0.47), 7.951 (0.68), 8.162 (0.61).  Intermediate 28

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(3,4,5- trifluorophenyl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(3,4,5-trifluorophenyl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (250 mg, 98 % purity, 233 µmol) was solubilised in DMF (6.1 ml), piperidine (460 µl, 4.7 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 96.0 mg (98 % purity, 49 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.17 min; MS (ESIpos): m/z = 829 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.244 (0.43), 1.341 (0.42), 1.382 (14.92), 1.387 (16.00), 2.298 (2.72), 2.536 (0.82), 2.553 (0.65), 7.124 (0.44), 7.142 (0.70), 7.163 (0.97), 7.181 (0.76), 7.231 (0.59), 7.249 (0.66), 7.988 (0.45), 8.010 (0.44), 8.404 (1.15).  Example 6-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3, 4,5-trifluoro-L-phenylalanyl}- N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(3,4,5- trifluorophenyl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate (98.0 mg, 98 % purity, 116 µmol) was solubilised in DCM (7.1 ml), TFA (7.1 ml, 92 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative  

HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 21.0 mg (85 % purity, 23 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.70 min; MS (ESIpos): m/z = 661 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (1.11), 0.884 (2.74), 0.915 (3.04), 0.947 (1.26), 1.085 (0.74), 1.109 (1.63), 1.116 (1.63), 1.148 (1.63), 1.175 (1.19), 1.200 (1.78), 1.207 (1.78), 1.238 (3.85), 1.259 (3.78), 1.277 (3.56), 1.340 (3.33), 1.357 (3.63), 1.375 (2.37), 1.395 (1.41), 1.435 (2.67), 1.449 (2.96), 1.470 (2.30), 1.517 (1.93), 1.551 (1.70), 1.581 (2.22), 1.594 (3.26), 1.614 (3.33), 1.627 (2.15), 1.648 (2.37), 1.691 (3.11), 1.734 (2.52), 1.768 (2.15), 1.784 (1.93), 1.802 (1.56), 1.808 (1.63), 1.825 (1.56), 1.841 (1.11), 1.860 (0.59), 1.905 (1.11), 2.055 (1.26), 2.074 (3.11), 2.084 (2.30), 2.118 (1.56), 2.131 (1.33), 2.137 (1.26), 2.153 (2.07), 2.167 (2.07), 2.172 (2.00), 2.184 (1.33), 2.227 (1.56), 2.248 (2.22), 2.262 (1.33), 2.271 (1.63), 2.284 (1.33), 2.306 (0.81), 2.336 (1.41), 2.518 (16.00), 2.523 (11.11), 2.539 (0.89), 2.599 (7.26), 2.616 (7.19), 2.678 (1.41), 2.733 (1.70), 2.760 (2.22), 2.767 (2.59), 2.793 (2.30), 2.927 (1.33), 2.947 (3.33), 2.960 (4.37), 2.981 (2.89), 2.993 (2.30), 3.093 (2.00), 3.109 (2.59), 3.126 (2.30), 3.143 (1.85), 3.336 (4.22), 3.920 (2.00), 3.940 (4.07), 3.953 (4.15), 3.972 (1.85), 4.390 (1.26), 4.402 (1.41), 4.416 (2.00), 4.425 (2.07), 4.438 (1.41), 4.450 (1.26), 6.131 (1.48), 6.384 (2.67), 6.404 (2.59), 7.149 (4.52), 7.166 (5.19), 7.172 (5.19), 7.189 (4.74), 7.929 (1.85), 7.943 (3.41), 7.957 (1.78), 8.156 (3.04), 8.177 (2.89), 8.251 (0.74).  #7 Linker Intermediate 29

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(4-fluorophenyl)methyl]- 3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (287 mg, 70 % purity, 412 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-4-fluoro-L-phenylalan ine (251 mg, 618 µmol) were solubilised in DMF (3.2 ml), 4-methylmorpholine (140 µl, 1.2 mmol, CAS-RN: 109-02-4) and HATU (235 mg, 618 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 280 mg (95 % purity, 74 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.63 min; MS (ESIpos): m/z = 876 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.242 (0.63), 1.379 (16.00), 2.221 (0.48), 2.296 (0.73), 4.145 (1.16), 6.273 (0.61), 7.062 (0.63), 7.296 (1.17), 7.405 (0.73), 7.629 (0.70), 7.870 (0.72), 7.886 (0.74).  Intermediate 30

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(4-fluorophenyl)propanoyl] amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(4-fluorophenyl)methyl]- 3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (280 mg, 95 % purity, 304 µmol) was solubilised in DMF (8.0 ml), piperidine (600 µl, 6.1 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 160 mg (80 % purity, 65 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 653 [M+H] ⁺   Intermediate 31

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(4-fluorophe nyl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(4-fluorophenyl)propanoyl] amino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (160 mg, 245 µmol) and (1r,4r)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-car boxylic acid (140 mg, 368 µmol) were solubilised in DMF (1.9 ml), 4-methylmorpholine (81 µl, 740 µmol, CAS-RN: 109-02-4) and HATU (140 mg, 368 µmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 150 mg (60 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.62 min; MS (ESIpos): m/z = 1015 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.228 (0.43), 1.380 (16.00), 1.385 (13.83), 1.387 (15.17), 1.631 (0.44), 2.298 (1.35), 2.518 (1.75), 2.522 (1.15), 4.285 (0.72), 4.302 (0.49), 7.067 (0.65), 7.090 (0.41), 7.225 (0.47), 7.231 (0.69), 7.247 (0.62), 7.314 (0.79), 7.333 (0.52), 7.389 (0.44), 7.408 (0.68), 7.675 (0.65), 7.694 (0.58), 7.878 (0.82), 7.897 (0.71).  Intermediate 32

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(4- fluorophenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadec ane-10,14,16-tricarboxylate 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(4-fluorophe nyl)methyl]-1,4,12-trioxo-  

2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (150 mg, 80 % purity, 118 µmol) was solubilised in DMF (3.1 ml), piperidine (230 µl, 2.4 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 62.0 mg (66 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.17 min; MS (ESIpos): m/z = 793 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.347 (0.40), 1.383 (16.00), 1.387 (14.26), 1.389 (14.33), 2.518 (1.45), 2.523 (1.07), 2.527 (0.86), 7.039 (0.42), 7.062 (0.83), 7.084 (0.49), 7.208 (0.44), 7.222 (0.52), 7.229 (0.44), 8.421 (1.47).  Example 7-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-4- fluoro-L-phenylalanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(4-flu orophenyl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (13.8 mg, 98 % purity, 17.1 µmol) was solubilised in DCM (1.0 ml), TFA (1.0 ml, 14 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 3.00 mg (98 % purity, 28 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.64 min; MS (ESIpos): m/z = 625 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.868 (1.44), 0.898 (1.52), 0.924 (0.72), 1.096 (0.40), 1.128 (0.96), 1.161 (0.96), 1.204 (1.36), 1.239 (2.88), 1.261 (2.16), 1.320 (2.00), 1.337 (2.16), 1.352 (1.68), 1.437 (1.44), 1.452 (1.44), 1.470 (1.04), 1.504 (1.20), 1.541 (1.52), 1.569 (1.60), 1.686 (1.60), 1.751 (1.04), 1.777 (1.12), 1.808 (0.96), 1.834 (0.88), 1.898 (0.40), 2.049 (0.72), 2.074 (2.48), 2.117 (1.04), 2.139 (1.04), 2.153 (1.12), 2.223 (0.72), 2.248 (0.96), 2.268 (0.80), 2.302 (0.40), 2.332 (3.28), 2.336 (1.44), 2.518 (16.00), 2.523 (11.28), 2.539 (2.00), 2.572 (2.16), 2.586 (2.08), 2.678 (1.44), 2.729 (0.96), 2.755 (1.28), 2.763 (1.44), 2.789 (1.28), 2.917 (1.84), 2.928 (2.08), 2.951 (2.16), 2.962 (1.84), 3.062 (1.44), 3.076 (1.68), 3.093 (1.60), 3.110 (1.44),  

3.351 (4.48), 3.867 (1.36), 3.881 (1.44), 3.942 (1.36), 4.356 (0.72), 4.379 (1.28), 4.392 (1.28), 4.402 (0.80), 4.415 (0.72), 6.148 (0.64), 6.333 (1.12), 6.352 (1.04), 7.036 (3.12), 7.058 (6.24), 7.080 (3.68), 7.217 (3.20), 7.231 (3.84), 7.238 (3.28), 7.252 (2.48), 7.913 (1.52), 8.147 (1.20), 8.169 (1.20), 8.379 (1.04).  #8 Linker Intermediate 33

tri-tert-butyl (5S,12S,16S)-5-[(4-tert-butylphenyl)methyl]-1-(9H-fluoren-9- yl)-3,6,14-trioxo- 2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and 4-tert-butyl-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-phenyl alanine (287 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 340 mg (98 % purity, 85 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.75 min; MS (ESIpos): m/z = 914 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.180 (5.18), 1.235 (0.77), 1.381 (16.00), 2.297 (1.09), 4.073 (0.57), 6.275 (0.58), 7.168 (0.60), 7.189 (0.94), 7.218 (0.86), 7.231 (0.58), 7.249 (0.52), 7.274 (0.46), 7.293 (0.41), 7.395 (0.54), 7.411 (0.46), 7.865 (0.67), 7.883 (0.61).  Intermediate 34

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(4-tert-butylphenyl)propan oyl]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

 

tri-tert-butyl (5S,12S,16S)-5-[(4-tert-butylphenyl)methyl]-1-(9H-fluoren-9- yl)-3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (340 mg, 98 % purity, 365 µmol) was solubilised in DMF (9.6 ml), piperidine (720 µl, 7.3 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 232 mg (92 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.24 min; MS (ESIpos): m/z = 692 [M+H] ⁺   Intermediate 35

tri-tert-butyl (3S,10S,14S)-3-[(4-tert-butylphenyl)methyl]-1-{(1r,4S)-4-[({ [(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(4-tert-butylphenyl)propan oyl]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (232 mg, 336 µmol) and (1r,4r)-4- [({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexa ne-1-carboxylic acid (191 mg, 504 µmol) were solubilised in DMF (2.6 ml), 4-methylmorpholine (110 µl, 1.0 mmol, CAS-RN:  

109-02-4) and HATU (192 mg, 504 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 200 mg (57 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.72 min; MS (ESIpos): m/z = 1053 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.230 (9.12), 1.380 (16.00), 1.385 (14.22), 1.387 (15.49), 2.298 (0.69), 2.326 (0.41), 2.518 (1.55), 2.522 (1.02), 2.668 (0.46), 2.727 (1.20), 2.729 (1.29), 2.888 (1.44), 4.283 (0.69), 4.300 (0.46), 7.098 (0.51), 7.119 (0.71), 7.236 (0.82), 7.257 (0.77), 7.313 (0.80), 7.316 (0.77), 7.332 (0.53), 7.335 (0.49), 7.389 (0.42), 7.408 (0.65), 7.672 (0.62), 7.691 (0.55), 7.878 (0.80), 7.896 (0.65).  Intermediate 36

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(4-ter t- butylphenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadeca ne-10,14,16-tricarboxylate 

tri-tert-butyl (3S,10S,14S)-3-[(4-tert-butylphenyl)methyl]-1-{(1r,4S)-4-[({ [(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylate (200 mg, 190 µmol) was solubilised in DMF (5.0 ml), piperidine (380 µl, 3.8 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 116 mg (74 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.25 min; MS (ESIpos): m/z = 831 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.235 (12.06), 1.383 (16.00), 1.387 (14.27), 1.389 (14.85), 2.518 (0.83), 2.522 (0.58), 2.530 (0.61), 2.548 (0.56), 7.097 (0.66), 7.117 (0.87), 7.233 (1.03), 7.254 (0.74), 8.409 (1.16).  Example 8-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-4- tert-butyl-L-phenylalanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(4-ter t-butylphenyl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (21.0 mg, 98 % purity, 24.8 µmol) was solubilised in DCM (1.5 ml), TFA (1.5 ml, 20 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.00 mg (90 % purity, 27 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 663 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.236 (16.00), 2.331 (0.42), 2.518 (2.09), 2.522 (1.32), 2.539 (0.49), 2.673 (0.44), 3.350 (0.90), 7.105 (0.86), 7.125 (1.12), 7.236 (1.34), 7.257 (0.93).  #9 Linker Intermediate 37

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-{4-[(pyridi ne-3- carbonyl)amino]butyl}-2-oxa-4,7,13,15-tetraazaoctadecane-12, 16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N2-{[(9H-fluoren-9-yl)methoxy]carbonyl}-N6-(pyridine-3-carbo nyl)-L- lysine (306 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 280 mg (98 % purity, 68 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.47 min; MS (ESIpos): m/z = 944 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.377 (16.00), 2.297 (0.49), 2.522 (0.45), 4.204 (0.54), 7.310 (0.49), 7.409 (0.49), 7.877 (0.68), 7.896 (0.56).  Intermediate 38

tri-tert-butyl (7S,14S,18S)-7-amino-1,8,16-trioxo-1-(pyridin-3-yl)-2,9,15,1 7- tetraazaicosane-14,18,20-tricarboxylate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-{4-[(pyridi ne-3- carbonyl)amino]butyl}-2-oxa-4,7,13,15-tetraazaoctadecane-12, 16,18-tricarboxylate (280 mg, 297 µmol) was solubilised in DMF (7.8 ml), piperidine (590 µl, 5.9 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.2% ammonia) to give 111 mg (98 % purity, 51 % yield) of the target compound.

LC-MS (Method 2): R _t = 1.22 min; MS (ESIpos): m/z = 722 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.371 (1.15), 1.382 (16.00), 1.386 (13.46), 1.390 (12.62), 2.297 (0.53), 2.518 (1.35), 2.522 (0.84), 3.248 (0.68), 3.263 (0.64), 8.253 (1.43), 8.680 (0.48), 8.684 (0.49), 8.692 (0.49), 8.696 (0.44), 8.980 (0.53), 8.982 (0.60), 8.986 (0.58), 8.988 (0.55).  Intermediate 39

 

tri-tert-butyl (7S,14S,18S)-7-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,8,16-trioxo-1- (pyridin-3-yl)-2,9,15,17-tetraazaicosane-14,18,20-tricarboxy late 

tri-tert-butyl (7S,14S,18S)-7-amino-1,8,16-trioxo-1-(pyridin-3-yl)-2,9,15,1 7-tetraazaicosane- 14,18,20-tricarboxylate (111 mg, 154 µmol) and (1r,4r)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carboxylic acid (87.6 mg, 231 µmol) were solubilised in DMF (1.2 ml), 4-methylmorpholine (51 µl, 460 µmol, CAS-RN: 109-02-4) and HATU

(87.8 mg, 231 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 119 mg (98 % purity, 70 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.48 min; MS (ESIpos): m/z = 1083 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.239 (0.46), 1.378 (16.00), 1.386 (9.24), 2.297 (0.52), 2.518 (0.83), 2.522 (0.51), 4.293 (0.57), 7.320 (0.67), 7.322 (0.68), 7.338 (0.45), 7.341 (0.44), 7.410 (0.60), 7.684 (0.53), 7.702 (0.49), 7.879 (0.67), 7.898 (0.61), 8.978 (0.43), 8.982 (0.43).  Intermediate 40

formic acid—tri-tert-butyl (7S,14S,18S)-7-{[(1r,4S)-4-(aminomethyl)cyclohexane-1- carbonyl]amino}-1,8,16-trioxo-1-(pyridin-3-yl)-2,9,15,17-tet raazaicosane-14,18,20- tricarboxylate (1/1) 

 

z

O OH tri-tert-butyl (7S,14S,18S)-7-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,8,16-trioxo-1-(pyridin-3- yl)-2,9,15,17-tetraazaicosane-14,18,20-tricarboxylate (119 mg, 98 % purity, 108 µmol) was solubilised in DMF (2.8 ml), piperidine (210 µl, 2.2 mmol) was added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 44.0 mg (98 % purity, 44 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.05 min; MS (ESIpos): m/z = 861 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.240 (0.42), 1.382 (16.00), 1.389 (8.48), 2.518 (1.89), 2.523 (1.38), 3.231 (0.43), 3.250 (0.50), 8.418 (0.94), 8.979 (0.40), 8.983 (0.41).  Example 9-A

N ⁶ -{N ² -[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-N ⁶ -(pyridine-3-carbonyl)-L- lysyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

z

formic acid—tri-tert-butyl (7S,14S,18S)-7-{[(1r,4S)-4-(aminomethyl)cyclohexane-1- carbonyl]amino}-1,8,16-trioxo-1-(pyridin-3-yl)-2,9,15,17-tet raazaicosane-14,18,20- tricarboxylate (1/1) (13.4 mg, 98 % purity, 14.5 µmol) was solubilised in DCM (1.0 ml), TFA (1.0 ml, 13 mmol) was added and the mixture was stirred under argon at rt. The mixture was  

evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 8.00 mg (95 % purity, 76 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.49 min; MS (ESIpos): m/z = 693 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.858 (1.30), 0.888 (3.12), 0.916 (3.45), 0.944 (1.50), 1.143 (0.46), 1.253 (7.80), 1.314 (4.55), 1.330 (5.40), 1.349 (4.94), 1.427 (2.73), 1.446 (3.77), 1.463 (4.68), 1.478 (5.27), 1.496 (6.18), 1.512 (5.66), 1.533 (4.10), 1.548 (2.93), 1.571 (2.60), 1.602 (3.06), 1.623 (3.51), 1.636 (3.71), 1.657 (5.20), 1.670 (5.85), 1.702 (5.33), 1.750 (3.64), 1.767 (3.51), 1.787 (3.84), 1.820 (1.56), 1.839 (0.72), 2.073 (0.46), 2.107 (1.63), 2.137 (3.32), 2.164 (3.51), 2.177 (3.06), 2.197 (2.08), 2.204 (2.21), 2.226 (3.51), 2.247 (2.02), 2.262 (1.82), 2.284 (0.91), 2.322 (3.06), 2.326 (4.03), 2.331 (2.99), 2.517 (16.00), 2.522 (10.08), 2.539 (14.83), 2.627 (4.88), 2.664 (3.64), 2.668 (4.42), 2.673 (3.25), 2.949 (2.15), 2.965 (2.93), 2.983 (3.84), 2.997 (3.71), 3.022 (3.90), 3.038 (4.29), 3.055 (3.71), 3.072 (3.12), 3.219 (7.93), 3.236 (12.10), 3.252 (12.68), 3.268 (9.89), 3.385 (11.45), 3.944 (5.79), 4.108 (1.89), 4.129 (3.19), 4.143 (3.25), 4.164 (1.76), 6.151 (2.15), 6.166 (2.08), 6.390 (3.19), 6.410 (3.12), 7.471 (3.51), 7.483 (3.84), 7.490 (3.90), 7.502 (3.77), 7.766 (2.21), 7.780 (4.23), 7.792 (2.21), 7.874 (3.84), 7.894 (3.71), 8.182 (3.12), 8.187 (4.94), 8.191 (3.45), 8.202 (3.25), 8.207 (4.75), 8.211 (3.25), 8.232 (4.88), 8.677 (5.01), 8.686 (5.01), 8.764 (2.41), 8.777 (4.75), 8.791 (2.41), 8.995 (7.28), 8.998 (7.22).  #10 Linker Intermediate 41

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(3-fluorophenyl)methyl]- 3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (290 mg, 70 % purity, 416 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-3-fluoro-L-phenylalan ine (253 mg, 624 µmol) were solubilised in DMF (3.2 ml), 4-methylmorpholine (140 µl, 1.2 mmol, CAS-RN: 109-02-4) and HATU (237 mg, 624 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 310 mg (95 % purity, 81 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.63 min; MS (ESIpos): m/z = 876 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (16.00), 2.297 (1.42), 4.126 (0.41), 6.274 (0.44), 7.250 (0.42), 7.271 (0.42), 7.402 (0.41), 7.637 (0.56), 7.868 (0.61), 7.887 (0.55).  Intermediate 42

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3-fluorophenyl)propanoyl] amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(3-fluorophenyl)methyl]- 3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (310 mg, 92 % purity, 326 µmol) was solubilised in DMF (8.6 ml), piperidine (640 µl, 6.5 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 204 mg (96 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.14 min; MS (ESIpos): m/z = 654 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.384 (12.08), 1.392 (16.00), 2.327 (0.44), 2.518 (1.62), 2.523 (1.03), 2.665 (0.47), 2.668 (0.53), 7.010 (0.66), 7.013 (0.63), 7.032 (0.73), 8.194 (1.14).  Intermediate 43

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(3-fluorophe nyl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3-fluorophenyl)propanoyl] amino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (204 mg, 96 % purity, 300 µmol) and (1r,4r)-4- [({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexa ne-1-carboxylic acid (171 mg, 450 µmol) were solubilised in DMF (2.3 ml), 4-methylmorpholine (99 µl, 900 µmol, CAS-RN: 109- 02-4) and HATU (171 mg, 450 µmol) were added and the mixture was stirred under argon at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 80.0 mg (26 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.62 min; MS (ESIpos): m/z = 1015 [M+H] ⁺   Intermediate 44

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3- fluorophenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadec ane-10,14,16-tricarboxylate 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(3-fluorophe nyl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (80.0 mg, 80 % purity, 63.1 µmol) was solubilised in DMF (1.7 ml), piperidine (120 µl, 1.3 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 40.0 mg (80 % yield) of the target compound.

 

LC-MS (Method 1): Rt = 1.19 min; MS (ESIpos): m/z = 793 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.383 (15.49), 1.389 (16.00), 2.518 (1.98), 2.522 (1.57), 7.048 (0.44), 8.417 (1.51).  Example 10-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- fluoro-L-phenylalanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3-flu orophenyl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (23.5 mg, 98 % purity, 29.1 µmol) was solubilised in DCM (1.8 ml), TFA (1.8 ml, 23 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 11.0 mg (98 % purity, 59 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.63 min; MS (ESIpos): m/z = 625 [M+H] ⁺

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.48), 0.880 (1.18), 0.911 (1.25), 0.937 (0.62), 1.123 (0.69), 1.146 (0.83), 1.203 (0.90), 1.234 (1.73), 1.327 (1.45), 1.346 (1.59), 1.426 (1.11), 1.444 (1.18), 1.462 (0.90), 1.507 (0.83), 1.548 (0.90), 1.569 (1.18), 1.580 (1.32), 1.591 (1.18), 1.666 (1.25), 1.698 (1.11), 1.743 (0.76), 1.777 (0.90), 1.795 (0.62), 1.811 (0.62), 1.835 (0.69), 1.852 (0.48), 1.905 (1.11), 2.048 (0.55), 2.074 (4.23), 2.112 (0.69), 2.124 (0.62), 2.147 (0.90), 2.160 (0.90), 2.178 (0.55), 2.232 (0.62), 2.256 (0.90), 2.267 (0.62), 2.276 (0.69), 2.287 (0.55), 2.331 (2.98), 2.336 (1.39), 2.518 (15.38), 2.522 (9.77), 2.539 (1.39), 2.594 (2.42), 2.611 (2.49), 2.673 (2.98), 2.756 (0.83), 2.782 (0.97), 2.791 (1.18), 2.816 (1.04), 2.916 (0.62), 2.929 (0.83), 2.949 (1.87), 2.962 (1.94), 2.983 (1.11), 2.996 (0.97), 3.086 (1.04), 3.102 (1.32), 3.119 (1.32), 3.135 (1.11), 3.336 (16.00), 3.932 (1.59), 3.943 (1.73), 4.389 (0.55), 4.402 (0.62), 4.413 (0.90), 4.426 (0.97), 4.436 (0.62), 4.449 (0.48), 6.121 (0.62), 6.394 (1.11), 6.413 (1.11), 6.972 (0.76), 6.994 (1.52), 7.017 (0.97), 7.026 (1.45), 7.040 (2.01), 7.058 (3.05), 7.253 (0.97), 7.269 (1.18), 7.274 (1.45), 7.289 (1.59), 7.309 (0.69), 7.883 (0.83), 7.896 (1.39), 7.911 (0.69), 8.101 (1.39), 8.123 (1.32), 8.244 (0.97).  

#11 Linker Intermediate 45

tri-tert-butyl (5S,12S,16S)-5-(4-acetamidobutyl)-1-(9H-fluoren-9-yl)-3,6,14 -trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N6-acetyl-N2-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-lysine (265 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN:

109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 255 mg (80 % purity, 54 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.47 min; MS (ESIpos): m/z = 881 [M+H] ⁺   Intermediate 46

tri-tert-butyl (8S,15S,19S)-8-amino-2,9,17-trioxo-3,10,16,18-tetraazahenico sane-15,19,21- tricarboxylate 

 

tri-tert-butyl (5S,12S,16S)-5-(4-acetamidobutyl)-1-(9H-fluoren-9-yl)-3,6,14 -trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (255 mg, 80 % purity, 232 µmol) was solubilised in DMF (2.5 ml), piperidine (460 µl, 4.6 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 71.0 mg (98 % purity, 46 % yield)) of the target compound.

LC-MS (Method 1): R _t = 1.06 min; MS (ESIpos): m/z = 659 [M+H] ⁺   Intermediate 47

tri-tert-butyl (8S,15S,19S)-8-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-2,9,17-trioxo- 3,10,16,18-tetraazahenicosane-15,19,21-tricarboxylate 

 

tri-tert-butyl (8S,15S,19S)-8-amino-2,9,17-trioxo-3,10,16,18-tetraazahenico sane-15,19,21- tricarboxylate (71.0 mg, 98 % purity, 106 µmol) and (1r,4r)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carboxylic acid (60.2 mg, 159 µmol) were solubilised in DMF (810 µl), 4-methylmorpholine (35 µl, 320 µmol, CAS-RN: 109-02-4) and HATU

(60.3 mg, 159 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 61.0 mg (85 % purity, 48 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.48 min; MS (ESIpos): m/z = 1020 [M+H] ⁺   Intermediate 48

tri-tert-butyl (8S,15S,19S)-8-{[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbon yl]amino}- 2,9,17-trioxo-3,10,16,18-tetraazahenicosane-15,19,21-tricarb oxylate 

tri-tert-butyl (8S,15S,19S)-8-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-2,9,17-trioxo-3,10,16,18- tetraazahenicosane-15,19,21-tricarboxylate (61.0 mg, 85 % purity, 50.9 µmol) was solubilised in DMF (1.3 ml), piperidine (100 µl, 1.0 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 79.0 mg (73 % purity) of the target compound.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 798 [M+H] ⁺   Example 11-A

N ⁶ -{N ⁶ -acetyl-N ² -[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-L-lys yl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (8S,15S,19S)-8-{[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbon yl]amino}-2,9,17- trioxo-3,10,16,18-tetraazahenicosane-15,19,21-tricarboxylate (15.5 mg, 73 % purity, 14.2 µmol) was solubilised in DCM (870 µl), TFA (870 µl, 11 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 11.0 mg (89 % purity) of the target compound.

LC-MS (Method 1): R _t = 0.47 min; MS (ESIpos): m/z = 630 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.910 (0.76), 0.941 (0.86), 1.183 (0.55), 1.235 (1.01), 1.255 (1.47), 1.276 (1.49), 1.285 (1.37), 1.318 (1.53), 1.327 (1.39), 1.335 (1.43), 1.352 (1.88), 1.366 (1.40), 1.446 (0.63), 1.457 (0.72), 1.468 (1.03), 1.485 (1.02), 1.503 (0.95), 1.522 (0.67), 1.541 (0.53), 1.561 (0.46), 1.608 (0.53), 1.622 (0.57), 1.641 (0.45), 1.656 (0.40), 1.677 (0.43), 1.692 (0.50), 1.714 (0.93), 1.727 (1.31), 1.768 (16.00), 1.784 (1.15), 1.905 (0.50), 1.922 (0.57), 2.161 (0.68), 2.183 (0.43), 2.190 (0.43), 2.209 (0.75), 2.226 (1.15), 2.245 (1.27), 2.267 (0.66), 2.327 (0.41), 2.518 (1.93), 2.523 (1.20), 2.539 (2.24), 2.642 (1.01), 2.657 (1.48), 2.669 (1.24), 2.673 (1.25), 2.946 (0.78), 2.962 (1.88), 2.978 (2.09), 2.994 (1.29), 3.010 (0.78), 3.024 (0.69), 3.041 (0.44), 4.009 (0.49), 4.016 (0.76), 4.029 (0.77), 4.064 (0.43), 4.078 (0.53), 4.085 (0.87), 4.098 (0.92), 4.106 (0.81), 4.119 (0.81), 4.128 (0.68), 4.141 (0.69), 6.281 (1.27), 6.302 (1.26), 6.316 (1.40), 6.336 (1.29), 7.682 (1.36), 7.757 (1.29), 7.777 (1.60), 7.787 (1.27), 7.801 (0.62), 7.832 (0.60), 7.845 (1.18), 7.859 (0.58).  #11a Linker Intermediate 49

tri-tert-butyl (5S,12S,16S)-5-[([1,1'-biphenyl]-4-yl)methyl]-1-(9H-fluoren- 9-yl)-3,6,14- trioxo-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarbox ylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (280 mg, 70 % purity, 402 µmol) and (2S)-3-([1,1’-biphenyl]-4-yl)-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)propanoic acid (279 mg, 603 µmol) were solubilised in DMF (3.1 ml), 4-methylmorpholine (130 µl, 1.2 mmol, CAS-RN: 109-02-4) and HATU (229 mg, 603 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 380 mg (95 % purity, 96 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.71 min; MS (ESIpos): m/z = 934 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.376 (16.00), 2.223 (0.81), 2.997 (0.65), 4.137 (1.37), 6.277 (0.75), 7.377 (1.89), 7.394 (1.87), 7.582 (1.25), 7.651 (1.28), 7.880 (0.92).  Intermediate 50

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-([1,1'-biphenyl]-4-yl)prop anoyl]amino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

 

tri-tert-butyl (5S,12S,16S)-5-[([1,1’-biphenyl]-4-yl)methyl]-1-(9H-fluore n-9-yl)-3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (310 mg, 92 % purity, 306 µmol) was solubilised in DMF (8.0 ml), piperidine (600 µl, 6.1 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 254 mg (82 % purity, 96 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.26 min; MS (ESIpos): m/z = 712 [M+H] ⁺   Intermediate 51

tri-tert-butyl (3S,10S,14S)-3-[([1,1'-biphenyl]-4-yl)methyl]-1-{(1r,4S)-4-[ ({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-([1,1’-biphenyl]-4-yl)pr opanoyl]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (254 mg, 82 % purity, 293 µmol) and (1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (167 mg, 439 µmol) were solubilised in DMF (2.3 ml), 4-methylmorpholine (97 µl, 880 µmol, CAS-RN: 109-02-4) and HATU (167 mg, 439 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 78.0 mg (25 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.69 min; MS (ESIpos): m/z = 1073 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.233 (0.82), 1.378 (16.00), 1.384 (12.12), 2.298 (0.51), 2.323 (0.61), 2.327 (0.81), 2.332 (0.58), 2.523 (2.52), 2.665 (0.63), 2.669 (0.82), 2.673 (0.60), 2.729 (1.20), 2.794 (0.53), 2.888 (1.32), 4.282 (0.67), 4.298 (0.49), 7.289 (0.86), 7.308 (1.34), 7.325 (0.65), 7.378 (0.40), 7.397 (0.64), 7.417 (0.72), 7.437 (0.75), 7.546 (0.65), 7.565 (0.58), 7.620 (0.67), 7.639 (0.54), 7.668 (0.65), 7.687 (0.58), 7.866 (0.65), 7.885 (0.64).  Intermediate 52

formic acid—tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[([1,1' - biphenyl]-4-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexad ecane-10,14,16- tricarboxylate (1/1) 

 

tri-tert-butyl (3S,10S,14S)-3-[([1,1’-biphenyl]-4-yl)methyl]-1-{(1r,4S)-4 -[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylate (78.0 mg, 75 % purity, 54.6 µmol) was solubilised in DMF (1.4 ml), piperidine (110 µl, 1.1 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 27.0 mg (98 % purity, 54 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.26 min; MS (ESIpos): m/z = 851 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.381 (16.00), 1.386 (10.37), 2.518 (1.47), 2.522 (0.87), 7.285 (0.54), 7.306 (0.61), 7.441 (0.61), 7.540 (0.75), 7.561 (0.60), 7.617 (0.60), 7.635 (0.51), 8.424 (0.60).  Example 11a-A

(3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[([1 ,1'-biphenyl]-4-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid 

 

formic acid—tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[([1,1� ��- biphenyl]-4-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexad ecane-10,14,16-tricarboxylate

(1/1) (11.4 mg, 98 % purity, 12.5 µmol) was solubilised in DCM (770 µl), TFA (770 µl, 9.9 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.00 mg (98 % purity, 58 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.80 min; MS (ESIpos): m/z = 683 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.69), 0.883 (1.12), 0.913 (1.20), 0.936 (0.60), 1.160 (0.69), 1.184 (0.86), 1.215 (1.12), 1.237 (1.89), 1.258 (1.55), 1.339 (1.46), 1.352 (1.72), 1.437 (1.12), 1.570 (1.55), 1.693 (1.20), 1.752 (0.77), 1.783 (0.86), 1.816 (0.69), 1.841 (0.69), 1.905 (1.12), 2.068 (0.52), 2.097 (0.86), 2.128 (0.86), 2.151 (0.86), 2.167 (0.86), 2.234 (0.52), 2.254 (0.77), 2.278 (0.60), 2.289 (0.52), 2.332 (3.61), 2.336 (1.63), 2.518 (16.00), 2.523 (11.10), 2.586 (1.63), 2.604 (1.72), 2.673 (3.53), 2.678 (1.55), 2.790 (0.60), 2.813 (0.77), 2.824 (0.95), 2.848 (0.86), 2.925 (0.69), 2.938 (0.77), 2.957 (0.95), 2.972 (1.63), 2.986 (1.38), 3.007 (1.03), 3.020 (0.95), 3.106 (1.20), 3.122 (1.46), 3.139 (1.46), 3.154 (1.46), 3.940 (1.55), 4.412 (0.52), 4.434 (0.86), 4.447 (0.86), 4.470 (0.43), 6.123 (0.52), 6.400 (0.86), 6.420 (0.86), 7.293 (3.70), 7.314 (4.82), 7.333 (2.67), 7.352 (1.81), 7.421 (2.92), 7.441 (4.73), 7.459 (2.32), 7.541 (5.16), 7.561 (4.13), 7.621 (4.13), 7.639 (3.61), 7.642 (2.75), 7.888 (1.20), 8.114 (1.12), 8.135 (1.03), 8.262 (0.43).  #12 Linker Intermediate 53

  tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(2-methylphenyl)methyl]- 3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-2-methyl-L-phenylalan ine (259 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 168 mg (92 % purity, 41 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.66 min; MS (ESIpos): m/z = 872 [M+H] ⁺   Intermediate 54

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(2-methylphenyl)propanoyl] amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(2-methylphenyl)methyl]- 3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (168 mg, 92 % purity, 177 µmol) was solubilised in DMF (1.9 ml), piperidine (350 µl, 3.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 45.0 mg (90 % purity, 35 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.12 min; MS (ESIpos): m/z = 650 [M+H] ⁺   Intermediate 55

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(2-methylphe nyl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(2-methylphenyl)propanoyl] amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (45.0 mg, 90 % purity, 62.4 µmol) and (1r,4r)- 4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohe xane-1-carboxylic acid (35.5 mg, 93.6 µmol) were solubilised in DMF (480 µl), 4-methylmorpholine (21 µl, 190 µmol, CAS-RN:

109-02-4) and HATU (35.6 mg, 93.6 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 51.0 mg (93 % purity, 75 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 1011 [M+H] ⁺    

Intermediate 56

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(2- methylphenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadec ane-10,14,16- tricarboxylate 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(2-methylphe nyl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (53.0 mg, 80 % purity, 42.0 µmol) was solubilised in DMF (1.1 ml), piperidine (83 µl, 840 µmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 50.0 mg (80 % purity, 121 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 789 [M+H] ⁺   Example 12-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-2- methyl-L-phenylalanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(2-met hylphenyl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (12.0 mg, 98 % purity, 14.9 µmol) was solubilised in DCM (920 µl), TFA (920 µl, 12 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.20 mg (80 % purity, 54 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.66 min; MS (ESIpos): m/z = 621 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.884 (0.64), 0.916 (0.74), 1.156 (0.51), 1.210 (1.18), 1.233 (1.49), 1.300 (0.91), 1.314 (1.01), 1.437 (0.74), 1.566 (1.01), 1.606 (0.68), 1.675 (0.88), 1.702 (0.81), 1.751 (0.51), 1.790 (0.57), 1.807 (0.44), 1.829 (0.44), 2.074 (0.61), 2.096 (0.57), 2.130 (0.54), 2.155 (0.57), 2.172 (0.54), 2.248 (0.61), 2.269 (0.57), 2.288 (9.35), 2.332 (1.45), 2.518 (8.71), 2.522 (5.33), 2.539 (16.00), 2.601 (0.98), 2.673 (1.42), 2.678 (0.68), 2.728 (0.44), 2.751 (0.54), 2.762 (0.68), 2.786 (0.61), 2.921 (1.08), 2.936 (1.11), 2.956 (0.68), 2.971 (0.54), 3.053 (0.51), 3.068 (0.61), 3.086 (0.57), 3.101 (0.44), 3.330 (2.53), 3.918 (0.84), 3.930 (1.08), 4.421 (0.68), 4.435 (0.68), 6.117 (0.41), 6.392 (0.68), 6.411 (0.68), 7.039 (0.91), 7.050 (1.72), 7.057 (1.28), 7.061 (1.32), 7.066 (1.55), 7.079 (0.88), 7.087 (1.69), 7.103 (1.49), 7.122 (0.68), 7.825 (0.51), 7.839 (0.88), 8.083 (0.84), 8.104 (0.78).  #13 Linker Intermediate 57

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(4-methylphenyl)methyl]- 3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 70 % purity, 431 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-4-methyl-L-phenylalan ine (259 mg, 646 µmol) were solubilised in DMF (3.3 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (246 mg, 646 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 128 mg (98 % purity, 33 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.66 min; MS (ESIpos): m/z = 872 [M+H] ⁺   Intermediate 58

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(4-methylphenyl)propanoyl] amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-5-[(4-methylphenyl)methyl]- 3,6,14-trioxo-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (128 mg, 98 % purity, 144 µmol) was solubilised in DMF (1.5 ml), piperidine (280 µl, 2.9 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18,

 

acetonitrile/water with 0.1% formic acid) to give 73.0 mg (98 % purity, 77 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.14 min; MS (ESIpos): m/z = 650 [M+H] ⁺   Intermediate 59

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(4-methylphe nyl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(4-methylphenyl)propanoyl] amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (73.0 mg, 98 % purity, 110 µmol) and (1r,4r)- 4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohe xane-1-carboxylic acid (62.8 mg, 165 µmol) were solubilised in DMF (850 µl), 4-methylmorpholine (36 µl, 330 µmol, CAS-RN:

109-02-4) and HATU (62.9 mg, 165 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 73.0 mg (98 % purity, 64 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.62 min; MS (ESIpos): m/z = 1011 [M+H] ⁺   Intermediate 60

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(4- methylphenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadec ane-10,14,16- tricarboxylate 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(4-methylphe nyl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (73.0 mg, 98 % purity, 70.8 µmol) was solubilised in DMF (1.9 ml), piperidine (140 µl, 1.4 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 33.0 mg (80 % purity, 47 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.18 min; MS (ESIpos): m/z = 789 [M+H] ⁺   Example 13-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-4- methyl-L-phenylalanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(4-met hylphenyl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (10.8 mg, 98 % purity, 13.4 µmol) was solubilised in DCM (820 µl), TFA (820 µl, 11 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 3.30 mg (90 % purity, 36 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.67 min; MS (ESIpos): m/z = 621 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.882 (0.64), 0.913 (0.71), 1.153 (0.48), 1.210 (1.26), 1.232 (1.36), 1.296 (0.92), 1.313 (1.01), 1.439 (0.73), 1.543 (0.71), 1.565 (0.92), 1.603 (0.60), 1.624 (0.55), 1.672 (0.86), 1.701 (0.79), 1.751 (0.53), 1.783 (0.68), 1.799 (0.53), 1.821 (0.42), 2.074 (0.44), 2.098 (0.57), 2.128 (0.48), 2.159 (0.53), 2.173 (0.59), 2.241 (0.66), 2.263 (0.53), 2.288 (9.02), 2.331 (0.82), 2.518 (4.64), 2.522 (2.84), 2.539 (16.00), 2.601 (0.92), 2.673 (0.82), 2.727 (0.46), 2.749 (0.57), 2.761 (0.70), 2.784 (0.62), 2.896 (0.40), 2.921 (0.99), 2.934 (1.08), 2.955 (0.81), 2.969 (0.62), 3.042 (0.60), 3.058 (0.73), 3.074 (0.68), 3.091 (0.57), 3.349 (1.85), 3.915 (0.86), 3.929 (1.06), 4.422 (0.66), 4.438 (0.66), 6.135 (0.44), 6.388 (0.64), 6.407 (0.60), 7.038 (0.92), 7.042 (0.88), 7.049 (1.65), 7.056 (1.26), 7.061 (1.32), 7.065 (1.47), 7.079 (0.90), 7.086 (1.69), 7.102 (1.56), 7.122 (0.68), 7.854 (0.84), 8.094 (0.75), 8.115 (0.73).  #14 Linker Intermediate 61

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1-bro monaphthalen- 2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,1 4,16-tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (32.0 mg, 38.8 µmol) was solubilised in acetonitrile (1.1 ml), N-bromosuccinimide (7.60 mg, 42.7 µmol) was added and the mixture was stirred for 3.5h at rt. N-Bromosuccinimide (15.2 mg, 85.4 µmol) was added and the mixture was stirred in the dark over the weekend at rt. The mixture was filtered and evaporated.

The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 13.0 mg (37 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.24 min; MS (ESIpos): m/z = 903 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.67), 1.353 (0.50), 1.382 (13.48), 1.389 (16.00), 1.907 (0.90), 2.327 (3.24), 2.331 (2.35), 2.336 (1.06), 2.518 (13.82), 2.523 (8.45), 2.562 (2.85), 2.669 (3.30), 2.673 (2.41), 2.678 (1.06), 7.868 (0.45).  Example 14-A

(3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1- bromonaphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1-bro monaphthalen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (14.0 mg, 98 % purity, 15.2 µmol) was solubilised in DCM (930 µl), TFA (930 µl, 12 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 10.0 mg (85 % purity, 76 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.00 min; MS (ESIpos): m/z = 735 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.083 (3.33), 1.112 (3.88), 1.142 (4.07), 1.231 (14.61), 1.273 (6.57), 1.324 (7.86), 1.373 (6.20), 1.403 (6.84), 1.434 (6.10), 1.559 (7.68), 1.636 (9.90), 1.764 (6.01), 1.878 (4.07), 1.905 (4.81), 1.948 (3.98), 1.976 (3.70), 2.072 (2.87), 2.174 (6.01), 2.221 (7.95), 2.265 (5.92), 2.327 (8.51), 2.669 (6.75), 2.690 (3.14), 2.727 (11.84), 2.887 (13.32), 3.002 (16.00), 3.965 (4.53), 4.641 (3.14), 7.433 (4.53), 7.455 (4.81), 7.542 (3.24), 7.559 (5.46), 7.578 (4.07), 7.631 (3.98), 7.650 (5.27), 7.668 (3.14), 7.847 (6.20), 7.867 (5.92), 7.925 (7.21), 7.947 (7.58), 8.186 (7.58), 8.206 (7.40).  #15 Linker Intermediate 62

tri-tert-butyl (5S,12S,16S)-5-[(4-tert-butoxyphenyl)methyl]-3,6,14-trioxo-1 -phenyl-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (330 mg, 80 % purity, 541 µmol) and N-[(benzyloxy)carbonyl]-O-tert-butyl-L-tyrosine (201 mg, 541 µmol;

CAS-RN:[5545-54-0]) were solubilised in DMF (4.2 ml), 4-methylmorpholine (180 µl, 1.6 mmol, CAS-RN: 109-02-4) and HATU (309 mg, 812 µmol) were added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 310 mg (100 % purity, 68 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.59 min; MS (ESpos): m/z = 842 [M+H] ⁺  Intermediate 63

N ⁶ -{N-[(benzyloxy)carbonyl]-L-tyrosyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L- lysine 

 

tri-tert-butyl (5S,12S,16S)-5-[(4-tert-butoxyphenyl)methyl]-3,6,14-trioxo-1 -phenyl-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (310 mg, 369 µmol) was solubilised in DCM (3.6 ml), TFA (1.4 ml, 18 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 110 mg (90 % purity, 44 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.82 min; MS (ESIpos): m/z = 617 [M+H] ⁺

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.239 (2.26), 1.257 (3.03), 1.276 (2.75), 1.295 (1.41), 1.344 (2.68), 1.362 (2.75), 1.397 (0.85), 1.478 (0.99), 1.497 (1.20), 1.511 (1.55), 1.530 (1.13), 1.619 (1.13), 1.634 (1.41), 1.654 (1.34), 1.681 (1.27), 1.695 (1.48), 1.715 (1.76), 1.730 (1.34), 1.751 (0.70), 1.866 (0.56), 1.883 (1.06), 1.900 (1.48), 1.917 (1.48), 1.934 (0.78), 1.953 (0.56), 2.074 (3.81), 2.169 (0.42), 2.183 (0.63), 2.210 (2.33), 2.226 (4.02), 2.231 (3.03), 2.248 (3.95), 2.266 (2.04), 2.290 (0.56), 2.318 (1.34), 2.518 (16.00), 2.523 (10.85), 2.539 (1.97), 2.584 (1.34), 2.609 (1.62), 2.618 (1.90), 2.644 (1.69), 2.678 (1.34), 2.786 (1.83), 2.797 (1.97), 2.820 (1.41), 2.832 (1.27), 2.957 (0.63), 2.972 (1.34), 2.990 (2.11), 3.004 (2.26), 3.022 (1.97), 3.038 (1.69), 3.055 (1.13), 3.072 (0.56), 4.010 (1.06), 4.031 (2.11), 4.044 (2.33), 4.065 (2.75), 4.079 (3.17), 4.086 (3.88), 4.099 (3.45), 4.106 (2.47), 4.119 (1.69), 4.900 (1.83), 4.933 (6.84), 4.949 (7.68), 4.981 (1.83), 6.286 (4.09), 6.306 (7.40), 6.327 (4.09), 6.623 (8.39), 6.644 (9.23), 7.020 (7.75), 7.042 (7.47), 7.144 (0.56), 7.226 (5.00), 7.244 (6.84), 7.247 (5.99), 7.266 (1.13), 7.283 (4.09), 7.297 (3.10), 7.301 (3.95), 7.317 (7.05), 7.331 (4.16), 7.335 (6.34), 7.352 (2.19), 7.357 (1.55), 7.366 (3.67), 7.388 (3.45), 7.927 (1.55), 7.941 (3.10), 7.955 (1.69), 9.169 (1.69), 12.519 (0.70). Intermediate 64

N ⁶ -L-tyrosyl-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

N6-{N-[(benzyloxy)carbonyl]-L-tyrosyl}-N2-{[(1S)-1,3-dicarbo xypropyl]carbamoyl}-L-lysine (110 mg, 178 µmol) was solubilised in MeOH (5mL), Palladium on carbon (19.0 mg, 10 % purity, 17.8  

µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 3h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and concentrated under reduced pressure to give 69.0 mg (80 % yield) of the target compound. Intermediate 65

N ⁶ -(N-{(1r,4S)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}a mino)methyl]cyclohexane-1- carbonyl}-L-tyrosyl)-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

(1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (48.8 mg, 128 µmol) was solubilised in DMF (2.0 ml), 4-methylmorpholine (42 µl, 390 µmol, CAS- RN: 109-02-4) and HATU (48.9 mg, 128 µmol) were added and the mixture was stirred for 30min at rt. N6-L-tyrosyl-N2-{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysi ne (62.0 mg, 128 µmol) was added and it was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 72.0 mg (76 % purity, 50 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.09 min; MS (ESIpos): m/z = 845 [M+H] ⁺ Example 15-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-L- tyrosyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine 

 

N6-(N-{(1r,4S)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino )methyl]cyclohexane-1-carbonyl}- L-tyrosyl)-N2-{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine (70.0 mg, 82.9 µmol) was solubilised in DMF (640 µl), piperidine (160 µl, 1.7 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 38.0 mg (99 % purity, 73 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.57), 0.881 (0.92), 0.913 (0.92), 0.945 (0.42), 1.168 (0.71), 1.200 (1.27), 1.232 (2.34), 1.312 (1.13), 1.327 (1.20), 1.438 (0.92), 1.553 (1.06), 1.573 (1.35), 1.677 (0.99), 1.708 (0.92), 1.750 (0.57), 1.788 (0.78), 1.805 (0.57), 1.829 (0.50), 2.050 (0.42), 2.074 (1.77), 2.115 (0.50), 2.133 (0.42), 2.150 (0.64), 2.163 (0.71), 2.180 (0.42), 2.229 (0.50), 2.249 (0.71), 2.273 (0.57), 2.285 (0.42), 2.331 (3.12), 2.518 (16.00), 2.523 (10.41), 2.539 (7.65), 2.598 (1.70), 2.615 (2.12), 2.638 (0.78), 2.648 (0.99), 2.673 (3.75), 2.791 (0.78), 2.804 (0.85), 2.824 (0.64), 2.839 (0.57), 2.915 (0.57), 2.933 (0.64), 2.947 (0.50), 3.056 (0.57), 3.072 (0.71), 3.089 (0.64), 3.934 (1.27), 4.273 (0.42), 4.296 (0.78), 4.309 (0.78), 6.116 (0.50), 6.385 (0.92), 6.405 (0.85), 6.604 (3.75), 6.625 (3.89), 6.965 (3.54), 6.986 (3.12), 7.767 (0.64), 7.782 (1.06), 7.945 (1.13), 7.966 (1.06).  #16 Linker Intermediate 66

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-{[3-(pentaf luoro-lambda ⁶ - sulfanyl)phenyl]methyl}-2-oxa-4,7,13,15-tetraazaoctadecane-1 2,16,18-tricarboxylate 

 

*

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (470 mg, 964 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-3-(pentafluoro-lambda 6- sulfanyl)phenylalanine (495 mg, 964 µmol) were solubilised in DMF (7.4 ml), 4-methylmorpholine (320 µl, 2.9 mmol, CAS-RN: 109-02-4) and HATU (550 mg, 1.45 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 296 mg (87 % purity, 27 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.67 min; MS (ESIpos): m/z = 984 [M+H] ⁺   Intermediate 67

di-tert-butyl (2S)-2-({[(2S)-6-({(2S)-2-amino-3-[3-(pentafluoro-lambda ⁶ - sulfanyl)phenyl]propanoyl}amino)-1-tert-butoxy-1-oxohexan-2- yl]carbamoyl}amino)pentanedioate 

 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-{[3-(pentaf luoro-lambda6- sulfanyl)phenyl]methyl}-2-oxa-4,7,13,15-tetraazaoctadecane-1 2,16,18-tricarboxylate (295 mg, 300 µmol) was solubilised in DMF (2.3 ml), piperidine (590 µl, 6.0 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 210 mg (92 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.25 min; MS (ESIpos): m/z = 762 [M+H] ⁺   Intermediate 68

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -{[3-(pentafluoro-lambda ⁶ - sulfanyl)phenyl]methyl}-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-({(2S)-2-amino-3-[3-(pentafluoro-lambda6- sulfanyl)phenyl]propanoyl}amino)-1-tert-butoxy-1-oxohexan-2- yl]carbamoyl}amino)pentanedioate (240 mg, 315 µmol) and (1r,4r)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carboxylic acid (120 mg, 315 µmol) were solubilised in DMF (3.6 ml), 4-methylmorpholine (100 µl, 950 µmol, CAS-RN: 109-02-4) and HATU (126 mg, 331 µmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 53.0 mg (99 % purity, 15 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.65 min; MS (ESIpos): m/z = 1123 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.231 (0.49), 1.380 (16.00), 1.386 (14.23), 2.331 (0.96), 2.336 (0.43), 2.518 (5.06), 2.523 (3.34), 2.673 (0.96), 2.678 (0.43), 4.281 (0.67), 4.298 (0.45), 7.311 (0.74), 7.313 (0.69), 7.329 (0.47), 7.407 (0.63), 7.515 (0.65), 7.672 (0.61), 7.691 (0.58), 7.731 (0.52), 7.877 (0.67), 7.896 (0.63).   

Intermediate 69

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-{[3- (pentafluoro-lambda ⁶ -sulfanyl)phenyl]methyl}-2,5,11,13-tetraazahexadecane- 10,14,16- tricarboxylate 

°

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -{[3-(pentafluoro-lambda6- sulfanyl)phenyl]methyl}-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate (53.0 mg, 47.2 µmol) was solubilised in DMF (730 µl), piperidine (93 µl, 940 µmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 23.0 mg (95 % purity, 51 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.25 min; MS (ESIpos): m/z = 901 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.231 (0.43), 1.382 (13.02), 1.386 (16.00), 2.331 (0.77), 2.518 (4.42), 2.523 (2.75), 7.515 (0.68), 7.733 (0.43), 8.405 (0.63).  Example 16-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (pentafluoro-lambda ⁶ - sulfanyl)-L-phenylalanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-{[3- (pentafluoro-lambda6-sulfanyl)phenyl]methyl}-2,5,11,13-tetra azahexadecane-10,14,16- tricarboxylate (6.40 mg, 7.11 µmol) was solubilised in DCM (230 µl), TFA (270 µl, 3.6 mmol) was added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.50 mg (99 % purity, 48 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.77 min; MS (ESIpos): m/z = 733 [M+H] ⁺  

¹H-NMR (500 MHz, CHLOROFORM-d) d [ppm]: 7.263 (15.23), 7.278 (16.00), 7.288 (1.76), 7.292 (1.36), 7.499 (1.12), 7.658 (1.37).  #17 Linker Intermediate 70

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(quinolin- 2-yl)methyl]-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (567 mg, 1.16 mmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-3-quinolin-2-yl-L-ala nine (510 mg, 1.16 mmol) were solubilised in DMF (8.9 ml), 4-methylmorpholine (320 µl, 2.9 mmol, CAS-RN: 109- 02-4) and HATU (663 mg, 1.74 mmol) were added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated, diluted with water and extracted with DCM/isopropanol (4:1). The organic layer was dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 433 mg (95 % purity, 41 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.59 min; MS (ESIpos): m/z = 909 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.377 (16.00), 2.326 (0.51), 2.518 (2.42), 2.522 (1.48), 2.668 (0.51), 7.845 (0.41).  Intermediate 71

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-2-yl)propanoyl]a mino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(quinolin- 2-yl)methyl]-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (366 mg, 403 µmol) was solubilised in DMF (3.1 ml), piperidine (800 µl, 8.1 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 83.0 mg (100 % purity, 51 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.16 min; MS (ESIpos): m/z = 687 [M+H] ⁺

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.381 (16.00), 1.385 (14.41), 1.387 (12.59), 2.518 (1.14), 2.522 (0.70), 7.412 (0.69), 7.433 (0.70), 7.543 (0.40), 7.936 (0.77), 7.940 (0.48), 7.954 (0.63), 8.245 (0.69), 8.266 (0.48). Intermediate 72  

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-2-yl)propanoyl]a mino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (67.0 mg, 97.7 µmol) and (1r,4r)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-car boxylic acid (37.1 mg, 97.7 µmol) were solubilised in DMF (1.1 ml), 4-methylmorpholine (32 µl, 290 µmol, CAS-RN: 109-02- 4) and HATU (39.0 mg, 103 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 34.0 mg (95 % purity, 32 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.377 (13.17), 1.382 (16.00), 2.518 (1.44), 2.523 (0.95), 2.539 (1.28), 4.279 (0.52), 7.306 (0.50), 7.308 (0.48), 7.400 (0.49), 7.667 (0.47), 7.686 (0.44), 7.874 (0.51), 7.893 (0.49), 7.918 (0.45).  Intermediate 73

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3- [(quinolin-2-yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate 

 

₎

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(quinolin-2-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (63.0 mg, 60.2 µmol) was solubilised in DMF (1.9 ml), piperidine (120 µl, 1.2 mmol) was added and the mixture was stirred under argon for 1.5h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 28.0 mg (100 % purity, 56 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.14 min; MS (ESIpos): m/z = 826 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (12.72), 1.384 (16.00), 2.518 (1.92), 2.522 (1.20), 3.290 (0.52), 7.407 (0.42), 7.429 (0.42), 8.424 (0.71).  Intermediate 74

2,2'-{[(3-{[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11-bis (tert-butoxycarbonyl)-2,2-dimethyl- 4,9,17-trioxo-19-(quinolin-2-yl)-3-oxa-8,10,16-triazanonadec an-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl](2-{[1-(carboxymethyl )-3-hydroxy-6-methyl-2- oxopyridine-4(2H)-carbonyl]amino}ethyl)amino}propyl)azanediy l]bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl )]}diacetic acid 

 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (6.60 mg, 6.09 µmol)  in 660 µL NMP tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (5.10 mg, 6.19 µmol) in 510 µL NMP and PyAOP (4.45 mg, 8.53 µmol) in 380 µL NMP are mixed in a vial. DIPEA (8.0 µL, 46.0 µmol) is added. Reaction mix is diluted with 38% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm

C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 30-80% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 20 min) afforded 3.2 mg (28% yield) of the target product.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.16 min; MS (ESIpos): m/z = 1889.9  

[M+H] ⁺   Example 17-C monomer

N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)am ino]ethyl}carbamoyl)-3- hydroxy-6-methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)cycl ohexane-1-carbonyl]-3- (quinolin-2-yl)-L-alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

2,2'-{[(3-{[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11-bis (tert-butoxycarbonyl)-2,2-dimethyl- 4,9,17-trioxo-19-(quinolin-2-yl)-3-oxa-8,10,16-triazanonadec an-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl](2-{[1-(carboxymethyl )-3-hydroxy-6-methyl-2- oxopyridine-4(2H)-carbonyl]amino}ethyl)amino}propyl)azanediy l]bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl )]}diacetic acid (5.5 mg, 2.9 µmol)  is treated with 80% TFA in water (0.5 mL). Water (18 mL) is added and the reaction mixture is lyophilised affording 5.2 mg (100 % yield)

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.61 min; MS (ESIpos): m/z = 1721.8  

[M+H] ⁺    

Example 17-C monomer- ²³² Th

[N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-(hydroxy-kappaO)-6-methyl-2- (oxo-kappaO)-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]propyl}(2-{[1- (carboxymethyl)-3-(hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)-1 ,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2-(oxo- kappaO)pyridin-1(2H)-yl]acetamido}methyl)cyclohexane-1-carbo nyl]-3-(quinolin-2-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(4-)] ( ²³² Th)thorium 

N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6- methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)cyclohexane-1- carbonyl]-3-(quinolin-2-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine (1.00 mg, 0.581 µmol)is dissolved in 0.1 M TRIS buffer (420 µL). Th-232 solution (119 µL, 1µg/µl in HNO ₃ [2%])) is added. Reaction mixture is lyophilised affording1.10 mg (95 % purity, 92 % yield) 

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.38 min; MS (ESIpos): m/z = 1949.8  

[M+H] ⁺   Example 17-C monomer- ²²⁷ Th

[N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-(hydroxy-kappaO)-6-methyl-2- (oxo-kappaO)-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]propyl}(2-{[1- (carboxymethyl)-3-(hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)-1 ,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2-(oxo- kappaO)pyridin-1(2H)-yl]acetamido}methyl)cyclohexane-1-carbo nyl]-3-(quinolin-2-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(4-)] ( ²²⁷ Th)thorium 

 

N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6- methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)cyclohexane-1- carbonyl]-3-(quinolin-2-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine (5.3 µg) dissolved in 184 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.5 MBq/mL.1M carbonate buffer pH 9 (18 µL) was added and mixture incubated for 2 hrs. The labelling efficiency was determined to be 94% by iTLC.  Example 17-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (quinolin-2-yl)-L-alanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-2- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate (6.00 mg, 7.27 µmol) was solubilised in DCM (230 µl), TFA (280 µl, 3.6 mmol) was added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.80 mg (99 % purity, 58 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.55 min; MS (ESIpos): m/z = 658 [M+H] ⁺    

¹H-NMR (500 MHz, DMSO-d6) d [ppm]: 0.875 (4.33), 1.030 (1.31), 1.055 (3.10), 1.079 (3.02), 1.101 (1.31), 1.170 (1.47), 1.196 (4.16), 1.230 (9.31), 1.242 (7.76), 1.286 (1.80), 1.300 (3.76), 1.314 (6.94), 1.329 (8.00), 1.342 (5.22), 1.415 (4.49), 1.425 (4.90), 1.439 (4.73), 1.454 (3.35), 1.490 (3.92), 1.515 (3.43), 1.556 (2.78), 1.566 (3.59), 1.582 (3.02), 1.634 (3.43), 1.667 (5.80), 1.696 (5.88), 1.713 (5.14), 1.725 (5.80), 1.751 (6.04), 1.762 (4.65), 1.777 (4.00), 1.790 (2.94), 1.804 (2.04), 2.042 (2.37), 2.066 (4.08), 2.089 (2.12), 2.184 (3.76), 2.200 (4.73), 2.209 (4.73), 2.225 (5.14), 2.240 (3.18), 2.254 (2.04), 2.271 (1.14), 2.358 (3.59), 2.361 (4.90), 2.365 (3.67), 2.515 (16.00), 2.518 (13.80), 2.522 (10.69), 2.540 (2.04), 2.598 (7.10), 2.632 (4.33), 2.635 (5.55), 2.639 (4.16), 2.693 (0.73), 2.727 (3.92), 2.888 (4.98), 2.952 (3.02), 2.964 (4.16), 2.978 (4.90), 2.989 (4.33), 3.044 (4.57), 3.057 (5.39), 3.070 (4.73), 3.083 (3.59), 3.108 (5.06), 3.127 (5.80), 3.135 (6.53), 3.154 (6.12), 3.285 (7.10), 3.295 (7.92), 3.312 (6.94), 3.323 (6.37), 3.505 (3.43), 3.944 (6.04), 3.954 (7.10), 3.963 (7.10), 4.719 (2.86), 4.737 (5.14), 4.747 (5.14), 4.765 (2.61), 6.188 (3.43), 6.381 (4.16), 6.395 (3.92), 7.421 (11.27), 7.438 (11.10), 7.523 (5.22), 7.539 (9.71), 7.553 (6.04), 7.699 (5.14), 7.701 (5.14), 7.715 (8.73), 7.731 (4.98), 7.875 (3.84), 7.887 (6.69), 7.898 (4.33), 7.907 (11.10), 7.917 (13.31), 7.934 (10.04), 7.950 (0.65), 8.149 (5.88), 8.166 (5.55), 8.231 (15.51), 8.238 (11.18), 8.247 (13.39).  Intermediate 75

ethyl (1r,4r)-4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,1 0-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexane-1-carboxylate 

( ₎

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (1.00 g, 1.75 mmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N-dimethylmetha niminium hexafluoridophosphate(1-) (662 mg, 1.75 mmol; CAS-RN:94790-37-1) and N,N- diisopropylethylamine (820 µl, 4.8 mmol) were stirred in DMF (12 ml) for 30min at rt. ethyl (1r,4r)- 4-(aminomethyl)cyclohexane-1-carboxylate (294 mg, 1.59 mmol) was added and the mixture was stirred overnight at rt. The mixture was diluted with water and extracted with DCM. The organic phase was washed with brine, dried and evaporated to give 160.0 mg (14 % yield) of the target compound.

LC-MS (Method 3): Rt = 1.36 min; MS (ESIpos): m/z = 741 [M+H] ⁺

 

Intermediate 76

(1r,4r)-4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4, 7,10-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexane-1-carboxylic acid 

ethyl (1r,4r)-4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,1 0-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexane-1-carboxylate (270 mg, 365 µmol) was solubilised in MeOH

(2.2 ml), lithium hydroxide (3.6 ml, 1.0 M, 3.6 mmol) was added and the mixture was stirred overnight at rt. The mixture was neutralized with HCl (1.0 M) and extracted with DCM/MeOH

(4:1). The organic layer was dried and evaporated to give 150 mg (85 % purity, 80 % yield) of the target compound.

LC-MS (Method 3): Rt = 0.93 min; MS (ESIpos): m/z = 713 [M+H] ⁺ Intermediate 77

tri-tert-butyl (3S,10S,14S)-1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-1-[(1r, 4S)-4-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

 

(1r,4r)-4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,1 0-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexane-1-carboxylic acid (71.6 mg, 101 µmol) was solubilised in DMF (770 µl), 4-methylmorpholine (33 µl, 300 µmol, CAS-RN: 109-02-4) and HATU (45.9 mg, 121 µmol) were added and the mixture was stirred for 20min at rt. di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)- 2-amino-3-(quinolin-2-yl)propanoyl]amino}-1-tert-butoxy-1-ox ohexan-2- yl]carbamoyl}amino)pentanedioate (69.0 mg, 101 µmol) was added and it was stirred under argon for 3h at rt. The mixture was evaporated, diluted with water and extracted with DCM/isopropanol (4:1). The organic phase was dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 138 mg (99 % yield) of the target compound. Example 17-B

N ⁶ -{3-(quinolin-2-yl)-N-[(1r,4S)-4-({2-[4,7,10-tris(carb oxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexane-1-car bonyl]-L-alanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3S,10S,14S)-1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-1-[(1r, 4S)-4-({2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (100 mg, 72.5 µmol) was solubilised in DCM (2.8 ml), TFA (2.8 ml, 36 mmol) was added and the mixture was stirred under argon for 4.5h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 20.0 mg (95 % purity, 25 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.56 min; MS (ESIneg): m/z = 1042 [M-H]-  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.834 (0.51), 0.852 (0.45), 1.232 (1.73), 1.323 (1.16), 1.368 (0.51), 1.385 (1.29), 1.400 (0.77), 1.421 (0.39), 1.478 (0.71), 1.579 (0.71), 1.681 (0.90), 1.695 (0.84), 1.716 (0.64), 1.738 (1.09), 1.905 (0.51), 1.922 (0.58), 2.047 (0.45), 2.210 (0.77), 2.226 (1.16), 2.246 (1.22), 2.268 (0.71), 2.337 (1.35), 2.518 (15.87), 2.523 (10.41), 2.540 (2.44), 2.679 (1.41), 2.687 (0.96), 2.728 (1.73), 2.787 (0.71), 2.889 (2.57), 2.962 (3.28), 3.091 (0.58), 3.114 (0.77), 3.126 (0.77), 3.147 (0.84), 3.498 (2.06), 3.577 (0.51), 3.653 (0.45), 3.994 (0.64), 4.006 (0.64), 4.092 (0.71), 4.105 (0.64), 4.721 (0.45), 4.734 (0.51), 6.275 (0.90), 6.295 (0.84), 6.310 (1.03), 6.330 (0.96), 7.411 (1.61), 7.432 (1.54), 7.520 (0.58), 7.540 (1.16), 7.558 (0.77), 7.697 (0.58), 7.701 (0.64), 7.718 (1.09), 7.739 (0.71), 7.908 (1.80), 7.916 (1.67), 7.924 (1.29), 7.938 (1.54), 8.079 (0.64), 8.134 (16.00), 8.231 (1.35), 8.252 (1.67), 8.273 (0.39).  Example 17-B- ²³² Th

Thorium-{N ⁶ -[3-(quinolin-2-yl)-N-{4-[(2-{4,7,10-tris[(carboxy-kap paO)methyl]-1,4,7,10- tetraazacyclododecan-1-yl-kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]cyclohexane-1- carbonyl}-L-alanyl]-N ² -[(1,3-dicarboxypropyl)carbamoyl]-L-lysinate 

 

N ⁶ -{3-(quinolin-2-yl)-N-[(1r,4S)-4-({2-[4,7,10-tris(carb oxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexane-1-car bonyl]-L-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine (5.00 mg, 4.79 µmol) was solubilised in ammonium acetate (5.0 ml, 5.0 mmol, 1.0 M, prepared with ultrafiltered and autoclaved water), thorium solution (1.1 ml, 4.8 µmol, 1µg/µl in HNO ₃ [2%]) was added and the mixture was stirred for 3.5 hrs at 90°C.

The mixture was diluted with water and lyophilized. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.30 mg (95 % purity, 36 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.54 min; MS (ESIpos): m/z = 1272 [M+H] ⁺   Example 17-B- ²²⁷ Th

[ ²²⁷ Th]Thorium-{N ⁶ -[3-(quinolin-2-yl)-N-{(1r,4S)-4-[(2-{4,7,10-tris[(car boxy- kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1-yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]cyclohexane-1-carbonyl}-L-alanyl]-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysinate} 

 

N ⁶ -{3-(quinolin-2-yl)-N-[(1r,4S)-4-({2-[4,7,10-tris(carb oxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexane-1-car bonyl]-L-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine (2.6 µg) dissolved in 152 µL 400 mM sodium acetate buffer (pH 5.6) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5M HCl (2 µL) at 0.375 MBq/nmol specific activity and RAC of 6.0 MBq/mL at 90 °C for 40 min. The labelling efficiency was determined to be 90% by iTLC.  #18 Linker Intermediate 78

methyl N-(tert-butoxycarbonyl)-4-cyclobutyl-L-phenylalaninate 

Methyl 4-bromo-N-(tert-butoxycarbonyl)-L-phenylalaninate (853 mg, 2.38 mmol), and Ir(4',6'-dF- 5-CF3-ppy)2(4,4'-dtbbpy)PF6 (53.5 mg, 47.6 µmol) were dissolved in a reaction vial in (trifluoromethyl)benzene (51 ml). In a separate vial, 1,2-dimethoxyethane - dichloronickel (1:1) (2.62 mg, 11.9 µmol; CAS-RN:[29046-78-4]) and 4,4'-di-tert-butyl-2,2'-bipyridine (3.20 mg, 11.9 µmol; CAS-RN:[72914-19-3]) were stirred in N,N-dimethylacetamide (26 ml) for 5 min. The catalyst solution was added to the sealed reaction vial. The mixture was degassed by sparging for 20min. Then bromocyclobutane (1.0 ml, 11 mmol; CAS-RN:[4399-47-7]), 2,6-Lutidine (1.7 ml, 14 mmol; CAS-RN:[108-48-5]) and 1,1,1,3,3,3-hexamethyl-2-(trimethylsilyl)trisilane (730 µl, 2.4 mmol; CAS-RN:[1873-77-4]) were added. The vial was stirred in a water bath and irradiated by two 40W Kessil LED Aquarium lamps (A160WE tuna blue). The mixture was quenched with half sat. sodium hydrogen carbonate and extracted 3 times with EtOAc. The combined organic layers were dried and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, hexane/EtOAc gradient 0%-25%) to give 377 mg (47 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.145 (0.99), 1.163 (2.06), 1.180 (1.05), 1.235 (1.09), 1.306 (16.00), 1.921 (0.44), 1.944 (0.50), 1.970 (0.40), 1.978 (3.74), 2.010 (0.58), 2.017 (0.52), 2.032 (0.62), 2.038 (0.76), 2.062 (0.47), 2.225 (0.69), 2.232 (0.84), 2.238 (0.45), 2.245 (0.48), 2.252 (0.81), 2.259 (0.51), 2.508 (0.81), 2.513 (0.53), 2.790 (0.43), 2.815 (0.42), 2.908 (0.43), 2.921 (0.45), 3.452 (0.53), 3.596 (6.20), 4.007 (0.83), 4.025 (0.82), 7.127 (6.03), 7.256 (0.67), 7.277 (0.61). 

 

Intermediate 79

N-(tert-butoxycarbonyl)-4-cyclobutyl-L-phenylalanine 

methyl N-(tert-butoxycarbonyl)-4-cyclobutyl-L-phenylalaninate (733 mg, 2.20 mmol) was solubilised in THF (18 ml), lithium hydroxide (11 ml, 1.0 M, 11 mmol) was added and the mixture was stirred overnight at rt. The mixture was acidified with HCl (1.0 M) and extracted with DCM.

The organic layer was dried, evaporated and purified by flash chromatography (SiO2, DCM/Ethanol gradient 0%-20%) to give 530 mg (94 % purity, 71 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.31 min; MS (ESIpos): m/z = 320 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.247 (1.12), 1.314 (16.00), 1.931 (0.42), 1.953 (0.50), 2.023 (0.58), 2.029 (0.51), 2.045 (0.62), 2.050 (0.73), 2.074 (0.47), 2.235 (0.67), 2.241 (0.82), 2.247 (0.43), 2.254 (0.46), 2.261 (0.80), 2.268 (0.48), 2.518 (0.46), 2.948 (0.40), 3.459 (0.49), 5.758 (3.16), 7.057 (0.61), 7.078 (0.59), 7.114 (0.65), 7.135 (2.37), 7.147 (2.41), 7.167 (0.64).  Intermediate 80

tri-tert-butyl (6S,13S,17S)-6-[(4-cyclobutylphenyl)methyl]-2,2-dimethyl-4,7 ,15-trioxo-3- oxa-5,8,14,16-tetraazanonadecane-13,17,19-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (779 mg, 1.60 mmol) and N-(tert-butoxycarbonyl)-4-cyclobutyl-L-phenylalanine (510 mg, 1.60 mmol) were  

solubilised in DMF (12 ml), N,N-diisopropylethylamine (1.0 ml, 5.7 mmol) and T ₃ P (2.1 ml, 50 % purity in DMF, 3.5 mmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 370 mg (96 % purity, 28 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.65 min; MS (ESIpos): m/z = 790 [M+H] ⁺   Intermediate 81

N ⁶ -(4-cyclobutyl-L-phenylalanyl)-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (6S,13S,17S)-6-[(4-cyclobutylphenyl)methyl]-2,2-dimethyl-4,7 ,15-trioxo-3-oxa- 5,8,14,16-tetraazanonadecane-13,17,19-tricarboxylate (370 mg, 469 µmol) was solubilised in DCM (6.0 ml), TFA (5.4 ml, 70 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 115 mg (62 % purity, 29 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.72 min; MS (ESIpos): m/z = 521 [M+H] ⁺   Intermediate 82

N ⁶ -(4-cyclobutyl-N-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}-L-p henylalanyl)-N ² -{[(1S)- 1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

_{N '}

N6-(4-cyclobutyl-L-phenylalanyl)-N2-{[(1S)-1,3-dicarboxyprop yl]carbamoyl}-L-lysine (115 mg, 221 µmol) and (1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1- carboxylic acid (88.0 mg, 232 µmol) were solubilised in DMF (2.5 ml), 4-methylmorpholine (73 µl, 660 µmol, CAS-RN: 109-02-4) and HATU (88.2 mg, 232 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 68.0 mg (50 % purity, 17 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.29 min; MS (ESIpos): m/z = 882 [M+H] ⁺   Example 18-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-4- cyclobutyl-L-phenylalanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

N6-(4-cyclobutyl-N-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}-L-p henylalanyl)-N2-{[(1S)-1,3-  

dicarboxypropyl]carbamoyl}-L-lysine (68.0 mg, 77.1 µmol) was solubilised in DMF (1.2 ml), piperidine (150 µl, 1.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 7.00 mg (95 % purity, 13 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.80 min; MS (ESIpos): m/z = 661 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.867 (3.91), 0.898 (3.76), 1.137 (3.25), 1.168 (5.12), 1.232 (14.34), 1.322 (5.29), 1.450 (4.33), 1.504 (3.49), 1.540 (3.23), 1.572 (2.63), 1.674 (6.31), 1.767 (5.97), 1.785 (6.52), 1.898 (1.59), 1.919 (2.78), 1.943 (3.15), 1.967 (2.57), 1.983 (3.12), 2.005 (4.48), 2.028 (5.65), 2.051 (5.04), 2.078 (3.65), 2.171 (3.02), 2.208 (5.33), 2.228 (8.29), 2.249 (6.97), 2.328 (1.70), 2.598 (5.53), 2.669 (1.76), 2.695 (1.93), 2.727 (2.81), 2.753 (2.40), 2.891 (3.36), 2.914 (3.23), 2.927 (3.42), 2.961 (3.17), 3.043 (3.25), 3.420 (4.91), 3.442 (5.87), 3.463 (4.78), 3.556 (3.12), 3.596 (2.74), 3.937 (5.48), 4.373 (2.87), 4.386 (2.85), 6.187 (2.12), 6.385 (2.66), 6.401 (2.55), 7.076 (4.82), 7.096 (15.98), 7.107 (16.00), 7.127 (4.51), 7.874 (3.48), 8.034 (2.93), 8.054 (2.83), 8.288 (1.15). #19 Linker Intermediate 83

tri-tert-butyl (5S,12S,16S)-5-[(3,4-dimethoxyphenyl)methyl]-1-(9H-fluoren-9 -yl)-3,6,14- trioxo-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarbox ylate 

*

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (270 mg, 554 µmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-3-methoxy-O-methyl-L- tyrosine (248 mg, 554 µmol; CAS-RN:[184962-88-7]) were solubilised in DMF (4.3 ml), N,N-diisopropylethylamine (350 µl, 2.0 mmol) and T ₃ P (710 µl, 50 % purity in DMF, 1.2 mmol) were added and the mixture  

was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC

(C18, acetonitrile/water with 0.1% formic acid) to give 240 mg (91 % purity, 43 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.56 min; MS (ESIpos): m/z = 918 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.368 (1.44), 1.380 (16.00), 1.383 (14.03), 2.518 (2.11), 2.523 (1.43), 2.673 (0.47), 3.670 (2.29), 3.709 (2.41), 4.142 (1.57), 6.794 (0.56), 6.904 (0.43), 6.908 (0.42), 7.399 (0.41), 7.401 (0.42), 7.636 (0.52), 7.655 (0.47), 7.867 (0.63), 7.886 (0.58). Intermediate 84

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3,4-dimethoxyphenyl)propa noyl]amino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-5-[(3,4-dimethoxyphenyl)methyl]-1-(9H-fluoren-9 -yl)-3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (240 mg, 100 % purity, 262 µmol) was solubilised in DMF (2.8 ml), piperidine (520 µl, 5.2 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 100 mg (97 % purity, 53 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.48 min; MS (ESIpos): m/z = 1083 [M+H] ⁺

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.239 (0.46), 1.378 (16.00), 1.386 (9.24), 2.297 (0.52), 2.518 (0.83), 2.522 (0.51), 4.293 (0.57), 7.320 (0.67), 7.322 (0.68), 7.338 (0.45), 7.341 (0.44), 7.410 (0.60), 7.684 (0.53), 7.702 (0.49), 7.879 (0.67), 7.898 (0.61), 8.978 (0.43), 8.982 (0.43). Intermediate 85

tri-tert-butyl (3S,10S,14S)-3-[(3,4-dimethoxyphenyl)methyl]-1-{(1r,4S)-4-[( {[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(3,4-dimethoxyphenyl)propa noyl]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (115 mg, 165 µmol) and (1r,4r)-4- [({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexa ne-1-carboxylic acid (94.2 mg, 248 µmol) were solubilised in DMF (1.3 ml), 4-methylmorpholine (55 µl, 500 µmol, CAS-RN: 109- 02-4) and HATU (94.4 mg, 248 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 48.0 mg (77 % purity, 21 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.56 min; MS (ESIpos): m/z = 1057 [M+H] ⁺   Intermediate 86

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3,4- dimethoxyphenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexa decane-10,14,16- tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-3-[(3,4-dimethoxyphenyl)methyl]-1-{(1r,4S)-4-[( {[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylate (47.0 mg, 44.5 µmol) was solubilised in DMF (680 µl), piperidine (88 µl, 890 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 14.0 mg (90 % purity, 34 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.15 min; MS (ESIpos): m/z = 835 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.230 (0.58), 1.250 (0.45), 1.383 (16.00), 1.388 (12.74), 1.391 (13.44), 2.451 (0.48), 2.468 (0.74), 2.518 (4.00), 2.523 (2.70), 3.689 (3.86), 3.710 (3.69), 6.796 (0.71), 6.817 (0.65), 6.822 (0.60), 6.827 (0.51).  Example 19-A

 

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- methoxy-O-methyl-L-tyrosyl}- N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3,4- dimethoxyphenyl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexa decane-10,14,16-tricarboxylate

(14.0 mg, 16.8 µmol) was solubilised in DCM (650 µl), TFA (520 µl, 6.7 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 3.00 mg (95 % purity, 26 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.59 min; MS (ESIpos): m/z = 667 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.877 (1.06), 0.905 (1.15), 1.139 (0.73), 1.172 (0.90), 1.243 (2.08), 1.341 (1.63), 1.456 (1.29), 1.519 (1.01), 1.552 (1.07), 1.602 (0.78), 1.678 (1.82), 1.748 (1.06), 1.769 (1.23), 2.072 (1.04), 2.094 (0.95), 2.127 (0.67), 2.145 (0.67), 2.184 (0.98),  

2.210 (1.09), 2.229 (0.96), 2.326 (1.12), 2.331 (0.92), 2.350 (0.98), 2.539 (0.81), 2.603 (1.69), 2.668 (1.65), 2.691 (1.18), 2.700 (1.12), 2.727 (2.60), 2.851 (1.24), 2.861 (1.38), 2.887 (3.00), 2.964 (1.41), 3.064 (1.83), 3.082 (1.80), 3.098 (1.71), 3.164 (2.16), 3.418 (8.19), 3.686 (16.00), 3.706 (14.18), 3.863 (0.87), 3.943 (2.08), 4.336 (0.62), 4.357 (1.03), 4.370 (1.03), 6.185 (0.62), 6.403 (0.72), 6.695 (1.23), 6.716 (1.69), 6.795 (2.57), 6.815 (1.88), 6.836 (2.88), 7.885 (1.12), 8.035 (0.95), 8.055 (0.93).  #20 Linker Intermediate 87

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-{3-[(pyrazi ne-2- carbonyl)amino]propyl}-2-oxa-4,7,13,15-tetraazaoctadecane-12 ,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (287 mg, 97 % purity, 571 µmol) and N2-{[(9H-fluoren-9-yl)methoxy]carbonyl}-N5-(pyrazine-2-carbo nyl)- L-ornithine (394 mg, 856 µmol) were solubilised in DMF (4.4 ml), 4-methylmorpholine (190 µl, 1.7 mmol, CAS-RN: 109-02-4) and HATU (326 mg, 856 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 411 mg (95 % purity, 74 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.41 min; MS (ESIpos): m/z = 931 [M+H] ⁺  

¹H-NMR (500 MHz, DMSO-d6) d [ppm]: 1.368 (5.96), 1.377 (16.00), 2.298 (0.53), 2.515 (0.71), 2.518 (0.62), 2.522 (0.47), 4.206 (0.49), 6.282 (0.44), 7.310 (0.56), 7.388 (0.52), 7.402 (0.50), 7.873 (0.79), 7.888 (0.68), 8.721 (0.43), 8.723 (0.46), 8.726 (0.41), 8.860 (0.73), 8.865 (0.68), 9.177 (0.53), 9.179 (0.55).   

Intermediate 88

tri-tert-butyl (6S,13S,17S)-6-amino-1,7,15-trioxo-1-(pyrazin-2-yl)-2,8,14,1 6- tetraazanonadecane-13,17,19-tricarboxylate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-{3-[(pyrazi ne-2- carbonyl)amino]propyl}-2-oxa-4,7,13,15-tetraazaoctadecane-12 ,16,18-tricarboxylate (405 mg, 94 % purity, 409 µmol) was solubilised in DMF (4.4 ml), piperidine (810 µl, 8.2 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 285 mg (94 % purity, 92 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 709 [M+H] ⁺   Intermediate 89

tri-tert-butyl (6S,13S,17S)-6-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,7,15-trioxo-1- (pyrazin-2-yl)-2,8,14,16-tetraazanonadecane-13,17,19-tricarb oxylate 

 

tri-tert-butyl (6S,13S,17S)-6-amino-1,7,15-trioxo-1-(pyrazin-2-yl)-2,8,14,1 6- tetraazanonadecane-13,17,19-tricarboxylate (285 mg, 93 % purity, 374 µmol) and (1r,4r)-4- [({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexa ne-1-carboxylic acid (213 mg, 562 µmol) were solubilised in DMF (2.9 ml), 4-methylmorpholine (120 µl, 1.1 mmol, CAS-RN:

109-02-4) and HATU (214 mg, 562 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 72.0 mg (46 % purity, 8 % yield) and 63.0 mg (66 % purity, 10 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.48 min; MS (ESIpos): m/z = 1070 [M+H] ⁺   Intermediate 90

tri-tert-butyl (6S,13S,17S)-6-{[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbon yl]amino}- 1,7,15-trioxo-1-(pyrazin-2-yl)-2,8,14,16-tetraazanonadecane- 13,17,19-tricarboxylate 

 

tri-tert-butyl (6S,13S,17S)-6-({(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-carbonyl}amin o)-1,7,15-trioxo-1-(pyrazin-2- yl)-2,8,14,16-tetraazanonadecane-13,17,19-tricarboxylate (63.0 mg, 66 % purity, 38.9 µmol) was solubilised in DMF (1.0 ml), piperidine (77 µl, 780 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 19.0 mg (37 % purity, 21 % yield) and 23.0 mg (82 % purity, 57 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.02 min; MS (ESIpos): m/z = 848 [M+H] ⁺   Example 20-A

N ⁶ -{N ² -[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-N ⁵ -(pyrazine-2-carbonyl)-L- ornithyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (6S,13S,17S)-6-{[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbon yl]amino}-1,7,15- trioxo-1-(pyrazin-2-yl)-2,8,14,16-tetraazanonadecane-13,17,1 9-tricarboxylate (23.0 mg, 82 % purity, 22.3 µmol) was solubilised in DCM (1.4 ml), TFA (1.4 ml, 18 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 9.00 mg (93 % purity, 55 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.49 min; MS (ESIpos): m/z = 680 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.901 (3.70), 0.931 (4.06), 1.230 (8.78), 1.245 (8.24), 1.263 (9.01), 1.318 (5.37), 1.337 (6.93), 1.354 (6.93), 1.486 (9.91), 1.545 (4.96), 1.562 (4.66), 1.583 (4.18), 1.621 (4.36), 1.751 (14.51), 1.904 (0.96), 2.072 (0.66), 2.083 (0.72), 2.122 (2.15), 2.151 (3.70), 2.194 (6.27), 2.213 (8.78), 2.322 (2.87), 2.326 (3.64), 2.331 (2.75), 2.522 (9.79), 2.539 (13.73), 2.632 (6.69), 2.664 (4.54), 2.668 (4.90), 2.673 (3.82), 2.991 (6.99), 3.004 (8.54), 3.020 (7.16), 3.050 (4.72), 3.164 (4.78), 3.277 (13.85), 3.290 (14.27), 3.384 (9.73), 3.502 (10.27), 3.965 (6.69), 3.979 (7.82), 3.997 (7.82), 4.014 (6.33), 4.164 (5.31), 4.176 (5.07), 4.275 (1.61), 4.515 (1.01), 6.223 (3.34), 6.241 (3.22), 6.345 (4.42), 6.365 (4.18), 7.340 (0.54), 7.815 (5.55), 7.848 (5.31), 7.868 (4.96), 7.984 (0.48), 8.202 (5.01), 8.720 (10.63), 8.855 (11.94), 8.860 (10.93), 8.949 (3.52), 8.964 (6.99), 8.979 (3.52), 9.166 (15.88), 9.169 (16.00), 9.310 (0.60).  #21 Linker Intermediate 91

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-4,12-dioxo-1-(6-oxo -1,6- dihydropyridin-2-yl)-2,5,11,13-tetraazahexadecane-10,14,16-t ricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (403 mg, 25 % purity, 147 µmol) and 6-oxo- 1,6-dihydropyridine-2-carbaldehyde (19.8 mg, 96 % purity, 154 µmol; CAS-RN:[358751-77-6]) were solubilised in MeOH (1.2 ml), acetic acid (8.4 µl, 150 µmol) was added and sodium cyanoborohydride (27.7 mg, 441 µmol) was added carefully. The mixture was stirred for 45min at rt. The mixture was evaporated, diluted with DMF and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 72.0 mg (90 % purity, 56 % yield) of the target compound.

LC-MS (Method 1): R _t = 135.00 min; MS (ESIpos): m/z = 793 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.382 (16.00), 2.518 (0.99), 2.522 (0.61), 7.795 (0.43), 7.816 (0.44).  Intermediate 92

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12- trioxo-2-[(6-oxo-1,6-dihydropyridin-2-yl)methyl]-2,5,11,13-t etraazahexadecane-10,14,16- tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-4,12-dioxo-1-(6-oxo -1,6-dihydropyridin- 2-yl)-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (65.0 mg, 82.1 µmol) and (1r,4r)-4- [({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexa ne-1-carboxylic acid (46.7 mg, 123 µmol) were solubilised in DMF (630 µl), 4-methylmorpholine (27 µl, 250 µmol, CAS-RN:

109-02-4) and HATU (46.8 mg, 123 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 10.0 mg (11 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.62 min; MS (ESIpos): m/z = 1155 [M+H] ⁺   Intermediate 93

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2- yl)methyl]-1,4,12-trioxo-2-[(6-oxo-1,6-dihydropyridin-2-yl)m ethyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

 

d

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12-trioxo-2-[(6- oxo-1,6-dihydropyridin-2-yl)methyl]-2,5,11,13-tetraazahexade cane-10,14,16-tricarboxylate

(41.0 mg, 90 % purity, 32.0 µmol) was solubilised in DMF (840 µl), piperidine (63 µl, 640 µmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.00 mg (85 % purity, 14 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.22 min; MS (ESIneg): m/z = 930 [M-H]-  Example 21-A

(3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(na phthalen-2-yl)methyl]-1,4,12- trioxo-2-[(6-oxo-1,6-dihydropyridin-2-yl)methyl]-2,5,11,13-t etraazahexadecane-10,14,16- tricarboxylic acid 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(napht halen-2-yl)methyl]- 1,4,12-trioxo-2-[(6-oxo-1,6-dihydropyridin-2-yl)methyl]-2,5, 11,13-tetraazahexadecane-  

10,14,16-tricarboxylate (5.00 mg, 5.37 µmol) was solubilised in DCM (330 µl), TFA (330 µl, 4.3 mmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated to give 1.50 mg (90 % purity, 33 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.75 min; MS (ESIpos): m/z = 764 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.234 (13.49), 1.313 (8.46), 1.538 (6.40), 1.624 (8.69), 2.179 (5.26), 2.197 (5.71), 2.227 (5.94), 2.327 (14.86), 2.636 (5.26), 2.668 (16.00), 3.873 (4.80), 3.954 (6.63), 4.522 (5.03), 6.068 (5.49), 6.090 (5.71), 7.207 (4.80), 7.431 (6.63), 7.464 (11.66), 7.476 (11.89), 7.667 (9.37), 7.784 (8.46), 7.836 (9.83), 7.856 (12.80).  #22 Linker Intermediate 94

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(quinolin- 3-yl)methyl]-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (1.33 g, 2.74 mmol) and N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-3-quinolin-3-yl-L-ala nine (1.20 g, 2.74 mmol; CAS-RN:[281655-61-6]) were solubilised in DMF (21 ml), 4-methylmorpholine (900 µl, 8.2 mmol, CAS-RN: 109-02-4) and HATU (1.25 g, 3.28 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was diluted with water and extracted with DCM.

The organic phase was washed with brine, dried and evaporated. The residue was by by flash chromatography (SiO2, hexane/EtOAc gradient 0%-100%) and preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 680 mg (100 % purity, 27 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.56 min; MS (ESIpos): m/z = 909 [M+H] ⁺  

 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.357 (16.00), 2.517 (0.67), 2.522 (0.45), 4.083 (1.34), 7.349 (0.56), 7.368 (0.45), 7.527 (0.45), 7.546 (0.56), 7.821 (0.90), 7.840 (0.78), 8.821 (0.67), 8.826 (0.56).  Intermediate 95

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-3-yl)propanoyl]a mino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(quinolin- 3-yl)methyl]-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (680 mg, 749 µmol) was solubilised in DMF (5.8 ml), piperidine (1.5 ml, 15 mmol) was added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 254 mg (100 % purity, 49 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.11 min; MS (ESIpos): m/z = 687 [M+H] ⁺ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.363 (15.61), 1.366 (16.00), 1.895 (0.39), 2.517 (0.59), 2.522 (0.39), 8.094 (0.39), 8.098 (0.39), 8.237 (0.88), 8.728 (0.69), 8.734 (0.59).  Intermediate 96

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-3-yl)propanoyl]a mino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (90.0 mg, 131 µmol) and (1r,4r)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexane-1-car boxylic acid (54.8 mg, 144 µmol) were solubilised in DMF (2.0 ml), 4-methylmorpholine (43 µl, 390 µmol, CAS-RN: 109-02- 4) and HATU (54.9 mg, 144 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 42.0 mg (100 % purity, 31 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.54 min; MS (ESIpos): m/z = 1048 [M+H] ⁺   Intermediate 97

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3- [(quinolin-3-yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(quinolin-3-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (35.0 mg, 33.4 µmol) was solubilised in DMF (510 µl), piperidine (66 µl, 670 µmol) was added and the mixture was stirred under argon for 2h at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 22.0 mg (86 % purity, 69 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.220 (0.40), 1.231 (0.44), 1.380 (16.00), 1.383 (13.04), 1.387 (10.91), 2.331 (0.42), 2.518 (2.30), 2.523 (1.49), 2.673 (0.43), 8.042 (0.42), 8.084 (0.47), 8.089 (0.44), 8.415 (0.70), 8.762 (0.64), 8.767 (0.61).  Example 22-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (quinolin-3-yl)-L-alanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-3- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate (10.0 mg, 12.1 µmol) was solubilised in DCM (390 µl), TFA (190 µl, 2.4 mmol) was added and the mixture was stirred under argon for 43h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (95 % purity, 72 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.52 min; MS (ESIpos): m/z = 658 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.830 (0.53), 0.861 (0.83), 0.892 (0.65), 0.970 (0.47), 1.002 (0.41), 1.188 (0.65), 1.232 (1.83), 1.252 (1.18), 1.316 (0.89), 1.334 (1.00), 1.353 (0.89), 1.384 (1.42), 1.392 (1.42), 1.461 (0.89), 1.476 (0.77), 1.496 (0.77), 1.582 (0.41), 1.596 (0.47), 1.636 (1.12), 1.672 (0.94), 1.692 (0.89), 1.714 (0.83), 1.728 (0.83), 1.750 (0.41), 1.907 (0.53), 1.924 (0.47), 2.085 (0.59), 2.209 (0.65), 2.227 (1.00), 2.246 (1.12), 2.268 (0.59), 2.337 (1.18),  

2.518 (16.00), 2.523 (10.57), 2.534 (3.37), 2.548 (5.49), 2.562 (2.66), 2.598 (0.89), 2.614 (1.36), 2.629 (0.89), 2.674 (2.54), 2.679 (1.18), 2.927 (0.47), 2.950 (0.65), 2.961 (0.89), 2.985 (0.83), 2.997 (0.71), 3.011 (0.59), 3.044 (0.47), 3.060 (0.59), 3.077 (0.47), 3.139 (0.65), 3.150 (0.71), 3.173 (0.59), 3.185 (0.53), 3.532 (4.37), 4.016 (0.71), 4.029 (0.71), 4.069 (0.41), 4.082 (0.53), 4.090 (0.77), 4.102 (0.77), 4.565 (0.41), 4.574 (0.59), 4.587 (0.59), 6.276 (1.12), 6.297 (1.06), 6.311 (1.18), 6.332 (1.06), 7.569 (1.00), 7.586 (2.18), 7.604 (1.77), 7.694 (0.71), 7.698 (0.77), 7.715 (1.18), 7.732 (0.59), 7.736 (0.59), 7.876 (1.12), 7.894 (1.00), 7.972 (1.42), 7.993 (1.18), 8.033 (1.30), 8.053 (1.83), 8.123 (1.48), 8.786 (2.01), 8.791 (1.83).  Intermediate 98 monomer

2,2'-{[(3-{[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11-bis (tert-butoxycarbonyl)-2,2-dimethyl- 4,9,17-trioxo-19-(quinolin-3-yl)-3-oxa-8,10,16-triazanonadec an-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl](2-{[1-(carboxymethyl )-3-hydroxy-6-methyl-2- oxopyridine-4(2H)-carbonyl]amino}ethyl)amino}propyl)azanediy l]bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl )]}diacetic acid 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.38 mg, 4.97 µmol) in 540 µL NMP, tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (5.00 mg, 6.06 µmol) in 500 µL NMP and PyAOP (4.74 mg, 9.09 µmol) in 474 µL NMP are mixed in a vial. DIPEA (8.7 µL, 50 µmol) is added. Reaction mix is quenched with water (2 mL), diluted with 38% ACN/water/0.1% TFA (6 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA;

ACN Gradient: 30-80% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm tR product:

27 min) affording 2.9 mg of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 1.77 min; MS (ESIpos): m/z = 1891.0  

[M+H] ⁺  

 

Intermediate 99 dimer

2,2'-{propane-1,3-diylbis[{[2-({1-[2-({[(1S,4r)-4-{[(7S,1 1S,18S)-7,11-bis(tert- butoxycarbonyl)-2,2-dimethyl-4,9,17-trioxo-19-(quinolin-3-yl )-3-oxa-8,10,16- triazanonadecan-18-yl]carbamoyl}cyclohexyl]methyl}amino)-2-o xoethyl]-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl}amino)ethyl]azan ediyl}ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) ]}diacetic acid 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.38 mg, 4.97 µmol) in 540 µL NMP, tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (5.00 mg, 6.06 µmol) in 500 µL NMP and PyAOP (4.74 mg, 9.09 µmol) in 474 µL NMP are mixed in a vial. DIPEA (8.7 µL, 50 µmol) is added. Reaction mix is quenched with water (2 mL), diluted with 38% ACN/water/0.1% TFA (6 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA;

ACN Gradient: 30-80% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm tR product:

33 min) affording 2.7 mg of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.33 min; MS (ESIpos): m/z = 1348.8  

[M+2H] ²⁺   Intermediate 100 trimer

{4-[(2-{(3-{bis[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11 -bis(tert-butoxycarbonyl)-2,2- dimethyl-4,9,17-trioxo-19-(quinolin-3-yl)-3-oxa-8,10,16-tria zanonadecan-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl]amino}propyl)[2-({1-[ 2-({[(1S,4r)-4- {[(7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-4, 9,17-trioxo-19-(quinolin-3-yl)- 3-oxa-8,10,16-triazanonadecan-18-yl]carbamoyl}cyclohexyl]met hyl}amino)-2-oxoethyl]- 3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl}amino)ethyl]amino}ethyl)carbamoyl]-3-hydroxy-6-meth yl-2-oxopyridin-1(2H)-

 

yl}acetic acid

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.38 mg, 4.97 µmol) in 540 µL NMP, tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (5.00 mg, 6.06 µmol) in 500 µL NMP and PyAOP (4.74 mg, 9.09 µmol) in 474 µL NMP are mixed in a vial. DIPEA (8.7 µL, 50 µmol) is added. Reaction mix is quenched with water (2 mL), diluted with 38% ACN/water/0.1% TFA (6 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA;

ACN Gradient: 30-80% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm tR product:

37 min) affording 1.4 mg of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.66 min; MS (ESIpos): m/z = 1752.8  

[M+2H] ²⁺   Example 22-C monomer

N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)am ino]ethyl}carbamoyl)-3- hydroxy-6-methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)cycl ohexane-1-carbonyl]-3- (quinolin-3-yl)-L-alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

2,2'-{[(3-{[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11-bis (tert-butoxycarbonyl)-2,2-dimethyl- 4,9,17-trioxo-19-(quinolin-3-yl)-3-oxa-8,10,16-triazanonadec an-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl](2-{[1-(carboxymethyl )-3-hydroxy-6-methyl-2- oxopyridine-4(2H)-carbonyl]amino}ethyl)amino}propyl)azanediy l]bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl )]}diacetic acid (2.90 mg, 1.53 µmol) is treated with 80% TFA in water (0.5 mL). Water (18 mL) is and the reaction mixture is lyophilised affording 2.90 mg (95 % purity, 104 % yield) of the target compound  

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 0.83 min; MS (ESIpos): m/z = 1221.8  

[M+H] ⁺   Example 22-C monomer- ²²⁷ Th

[N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-(hydroxy-kappaO)-6-methyl-2- (oxo-kappaO)-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]propyl}(2-{[1- (carboxymethyl)-3-(hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)-1 ,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2-(oxo- kappaO)pyridin-1(2H)-yl]acetamido}methyl)cyclohexane-1-carbo nyl]-3-(quinolin-3-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(4-)] ( ²²⁷ Th)thorium 

N ⁶ -{N-[(1r,4S)-4-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6-  

methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)cyclohexane -1-carbonyl]-3-(quinolin-3-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine (4 µg) dissolved in 147 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.6 MBq/mL.1M carbonate buffer pH 9 (15 µL) was added and mixture incubated for 60 min. The labelling efficiency was determined to be 97% by iTLC.  Example 22-C dimer

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)az anediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis{1,4, 12-trioxo-3-[(quinolin-3- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ic acid} 

2,2'-{propane-1,3-diylbis[{[2-({1-[2-({[(1S,4r)-4-{[(7S,1 1S,18S)-7,11-bis(tert-butoxycarbonyl)- 2,2-dimethyl-4,9,17-trioxo-19-(quinolin-3-yl)-3-oxa-8,10,16- triazanonadecan-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl]azanediyl}ethane-2,1- diylcarbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid (2.70 mg, 1.00 µmol) is treated with 80% TFA in water (0.5 mL). Water (18 mL) is and the reaction mixture is lyophilised affording 2.8 mg of the target compound

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 0.93 min; MS (ESIpos): m/z = 1180.6  

[M+2H] ²⁺   Example 22-C dimer- ²²⁷ Th

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-(hydroxy- kappaO)-6-methyl-2-(oxo-kappaO)-1,2-dihydropyridine-4- carbonyl]amino}ethyl)azanediyl]ethane-2,1-diylcarbamoyl[3-(h ydroxy-kappaO)-6-methyl- 2-(oxo-kappaO)pyridine-4,1(2H)-diyl](1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis{1,4, 12-trioxo-3-[(quinolin-3- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ato}(4-)( ²²⁷ Th)thorium 

 

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)azan ediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis{1,4, 12-trioxo-3-[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid} (8 µg) dissolved in 193 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.5 MBq/mL.1M carbonate buffer pH 9 (19 µL) was added and mixture incubated for 60 min. The labelling efficiency was determined to be 87% by iTLC. Example 22-C trimer

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{[(3-{[2-({1-[2-({[(1S,4r )-4-{[(2S)-1-{[(5S)-5-carboxy-5- ({[(1S)-1,3-dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1- oxo-3-(quinolin-3- yl)propan-2-yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl ]-3-hydroxy-6-methyl-2- oxopyridine-4(2H)-carbonyl}amino)ethyl](2-{[1-(carboxymethyl )-3-hydroxy-6-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino}propyl) azanediyl]bis[(ethane- 2,1-diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)- diyl)(1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl]}bis{1,4, 12-trioxo-3-[(quinolin-3- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ic acid} 

 

{4-[(2-{(3-{bis[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18S)-7,11 -bis(tert-butoxycarbonyl)-2,2-dimethyl- 4,9,17-trioxo-19-(quinolin-3-yl)-3-oxa-8,10,16-triazanonadec an-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl]amino}propyl)[2-({1-[ 2-({[(1S,4r)-4-{[(7S,11S,18S)- 7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-4,9,17-trioxo-19- (quinolin-3-yl)-3-oxa-8,10,16- triazanonadecan-18-yl]carbamoyl}cyclohexyl]methyl}amino)-2-o xoethyl]-3-hydroxy-6-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl}amino)ethyl]amino}ethyl)c arbamoyl]-3-hydroxy-6-methyl-2- oxopyridin-1(2H)-yl}acetic acid (1.40 mg, 0.400 µmol) is treated with 80% TFA in water (0.5 mL) 01:00. Water (18 mL) is and the reaction mixture is lyophilised affording 2.8 mg of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 1.02 min; MS (ESIpos): m/z = 1500.0  

[M+2H] ²⁺   Example 22-C trimer- ²²⁷ Th

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{[(3-{[2-({1-[2-({[(1S,4r )-4-{[(2S)-1-{[(5S)-5-carboxy-5- ({[(1S)-1,3-dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1- oxo-3-(quinolin-3- yl)propan-2-yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl ]-3-(hydroxy-kappaO)-6- methyl-2-(oxo-kappaO)pyridine-4(2H)-carbonyl}amino)ethyl](2- {[1-(carboxymethyl)-3- (hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)-1,2-dihydropyridine -4- carbonyl]amino}ethyl)amino}propyl)azanediyl]bis[(ethane-2,1- diyl)carbamoyl[3- (hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)pyridine-4,1(2H)-diy l](1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl]}bis{1,4, 12-trioxo-3-[(quinolin-3- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ato}(4-)( ²²⁷ Th)thorium 

 

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{[(3-{[2-({1-[2-({[(1S,4r )-4-{[(2S)-1-{[(5S)-5-carboxy-5-({[(1S)- 1,3-dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(q uinolin-3-yl)propan-2- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxopyridine-4(2H)- carbonyl}amino)ethyl](2-{[1-(carboxymethyl)-3-hydroxy-6-meth yl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino}propyl)azanediyl]bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)(1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl]}bis{1,4, 12-trioxo-3-[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid} (9 µg) dissolved in 172 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.5 MBq/mL.1M carbonate buffer pH 9 (17 µL) was added and mixture incubated for 4 hrs. The labelling efficiency was determined to be 90% by iTLC.  #23 Linker Intermediate 101

tri-tert-butyl (4S,11S,15S)-1-[4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino )phenyl]-4- [(naphthalen-2-yl)methyl]-2,5,13-trioxo-3,6,12,14-tetraazahe ptadecane-11,15,17- tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (319 mg, 90 % purity, 419 µmol) and [4-({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)phenyl]acetic acid (130 mg, 349 µmol) were solubilised in DMF (2.7 ml), 4-methylmorpholine (260 µl, 1.0 mmol, CAS-RN: 109-02-4) and COMU (150 mg, 349 µmol) were added and the mixture was stirred under argon for 2h at rt. The mixture was diluted with brine and extracted 3 times with DCM. The combined organic phases were dried and evaporated and purified by by flash chromatography (SiO2, DCM/Ethanol gradient 0%-10%) to give 219 mg (80 % purity, 40 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.64 min; MS (ESIpos): m/z = 1041 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.250 (0.58), 1.268 (1.11), 1.286 (0.64), 1.377 (12.53), 1.381 (16.00), 2.518 (3.47), 2.522 (2.21), 2.673 (0.62), 2.737 (9.57), 3.053 (1.07), 3.065 (1.32), 3.077 (1.21), 3.301 (0.42), 3.547 (1.08), 3.559 (1.10), 3.570 (1.01), 7.348 (0.59), 7.366 (0.40), 7.408 (0.40), 7.428 (0.74), 7.435 (0.51), 7.639 (0.42), 7.744 (0.81), 7.760 (0.59), 7.765 (0.64), 7.902 (0.65), 7.921 (0.56).  Intermediate 102

tri-tert-butyl (4S,11S,15S)-1-(4-aminophenyl)-4-[(naphthalen-2-yl)methyl]-2 ,5,13-trioxo- 3,6,12,14-tetraazaheptadecane-11,15,17-tricarboxylate 

 

tri-tert-butyl (4S,11S,15S)-1-[4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino )phenyl]-4- [(naphthalen-2-yl)methyl]-2,5,13-trioxo-3,6,12,14-tetraazahe ptadecane-11,15,17-tricarboxylate

(219 mg, 85 % purity, 179 µmol) was solubilised in DMF (1.4 ml), piperidine (350 µl, 3.6 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 126 mg (90 % purity, 77 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.40 min; MS (ESIpos): m/z = 819 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.382 (13.17), 1.386 (16.00), 2.518 (1.35), 2.522 (0.83), 3.166 (0.42), 3.200 (0.41), 4.833 (0.55), 6.281 (0.53), 6.322 (0.74), 6.343 (0.78), 6.703 (0.67), 6.724 (0.60), 7.464 (0.44), 7.648 (0.42).  Example 23-A

(4S,11S,15S)-1-(4-aminophenyl)-4-[(naphthalen-2-yl)methyl ]-2,5,13-trioxo-3,6,12,14- tetraazaheptadecane-11,15,17-tricarboxylic acid 

2

tri-tert-butyl (4S,11S,15S)-1-(4-aminophenyl)-4-[(naphthalen-2-yl)methyl]-2 ,5,13-trioxo- 3,6,12,14-tetraazaheptadecane-11,15,17-tricarboxylate (17.0 mg, 90 % purity, 18.7 µmol) was solubilised in DCM (1 ml), TFA (29 µl, 370 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.30 mg (95 % purity, 41 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.75 min; MS (ESIpos): m/z = 650 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.206 (3.53), 1.212 (3.38), 1.225 (3.83), 1.262 (1.93), 1.279 (3.66), 1.297 (3.83), 1.316 (2.35), 1.404 (0.53), 1.423 (1.05), 1.440 (1.53), 1.456 (1.98), 1.475 (1.63), 1.494 (0.58), 1.543 (0.75), 1.561 (1.50), 1.575 (1.93), 1.594 (1.65), 1.608 (1.05), 1.628 (0.55), 1.669 (0.53), 1.689 (1.33), 1.707 (1.63), 1.724 (2.13), 1.741 (1.68), 1.752 (0.75), 1.761 (0.80), 1.839 (0.63), 1.856 (1.45), 1.872 (1.93), 1.891 (1.95), 1.905 (1.10), 1.925 (0.58), 2.190 (0.53), 2.214 (3.18), 2.221 (3.18), 2.235 (4.61), 2.240 (4.68), 2.252 (2.65), 2.261 (2.60), 2.283 (0.50), 2.332 (1.05), 2.518 (6.49), 2.523 (4.01), 2.539 (0.48), 2.673 (1.05), 2.727 (6.54),  

2.736 (1.55), 2.887 (8.14), 2.902 (2.45), 2.924 (3.03), 2.935 (4.78), 2.957 (4.13), 2.966 (2.38), 2.985 (1.13), 2.996 (1.25), 3.013 (2.28), 3.028 (2.70), 3.045 (2.25), 3.062 (4.03), 3.075 (3.73), 3.095 (2.70), 3.109 (2.50), 3.135 (3.81), 3.171 (9.19), 3.200 (9.29), 3.235 (5.33), 3.546 (1.08), 3.982 (1.45), 3.995 (1.93), 4.002 (2.93), 4.015 (3.00), 4.022 (1.83), 4.035 (1.45), 4.057 (1.48), 4.078 (3.03), 4.091 (3.08), 4.112 (1.38), 4.492 (1.40), 4.506 (1.78), 4.514 (2.80), 4.527 (2.88), 4.535 (1.73), 4.548 (1.43), 6.282 (4.86), 6.302 (6.44), 6.323 (16.00), 6.328 (5.33), 6.340 (5.11), 6.344 (15.10), 6.351 (1.93), 6.703 (13.32), 6.724 (11.77), 6.972 (0.48), 6.993 (0.50), 7.316 (0.65), 7.339 (4.76), 7.343 (4.51), 7.360 (4.76), 7.364 (4.88), 7.430 (1.53), 7.433 (1.88), 7.447 (4.76), 7.450 (4.33), 7.460 (5.03), 7.466 (7.71), 7.470 (5.33), 7.480 (3.91), 7.484 (4.46), 7.497 (1.80), 7.501 (1.35), 7.655 (8.41), 7.763 (7.16), 7.773 (5.26), 7.784 (6.76), 7.791 (4.33), 7.795 (3.51), 7.838 (4.08), 7.842 (4.38), 7.860 (3.96), 7.950 (1.13), 7.977 (2.18), 7.991 (4.28), 8.005 (2.08), 8.060 (4.96), 8.081 (4.63), 8.147 (1.60), 8.213 (0.43), 8.217 (0.43).  Intermediate 103

tri-tert-butyl (4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1-(4- {2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}phenyl)-3,6,12,14- tetraazaheptadecane-11,15,17-tricarboxylate 

N-[9,12-bis(2-tert-butoxy-2-oxoethyl)-2,2,6-trimethyl-4-oxo- 3-oxa-6,9,12-triazatetradecan-14- yl]-N-ethylglycine (132 mg, 225 µmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (84.2 mg, 222 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (36 µl, 220 µmol) were stirred in DMF (2 ml) for 10min at rt. tri- tert-butyl (4S,11S,15S)-1-(4-aminophenyl)-4-[(naphthalen-2-yl)methyl]-2 ,5,13-trioxo-3,6,12,14-  

tetraazaheptadecane-11,15,17-tricarboxylate (57.5 mg, 80 % purity, 56.2 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 45.0 mg (70 % purity, 41 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.26 min; MS (ESIpos): m/z = 1373 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.47), 1.342 (6.22), 1.380 (16.00), 1.384 (15.09), 1.408 (3.07), 1.429 (2.35), 1.467 (3.51), 2.084 (1.13), 2.331 (0.45), 2.518 (2.53), 2.522 (1.58), 2.539 (0.43), 2.673 (0.46), 2.888 (0.67), 2.896 (0.46), 3.065 (0.43), 7.367 (0.51), 7.462 (0.40), 7.754 (0.42), 7.775 (0.51).  Example 23-B

(4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1- (4-{2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}phenyl)-3,6,12,14- tetraazaheptadecane-11,15,17-tricarboxylic acid 

N Z

tri-tert-butyl (4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1-(4- {2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}phenyl)-3,6,12,14- tetraazaheptadecane-11,15,17-tricarboxylate (40.0 mg, 29.1 µmol) was solubilised in DCM (560 µl), TFA (166 mg, 1.46 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 3.80 mg (90 % purity, 11 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.70 min; MS (ESIpos): m/z = 1037 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.833 (0.45), 0.852 (0.83), 1.232 (5.86), 1.247 (2.29), 1.256 (2.29), 1.295 (1.49), 1.333 (0.83), 1.348 (1.60), 1.445 (0.61), 1.563 (0.59), 1.692 (0.51),  

1.712 (0.64), 1.726 (0.51), 1.897 (0.59), 1.914 (0.59), 2.178 (0.61), 2.210 (0.91), 2.230 (1.36), 2.247 (1.46), 2.268 (0.88), 2.518 (5.35), 2.523 (3.86), 2.540 (7.48), 2.674 (1.41), 2.727 (1.97), 2.806 (1.01), 2.888 (2.42), 2.993 (2.98), 3.094 (1.54), 3.114 (1.44), 3.129 (1.46), 3.356 (16.00), 3.506 (3.83), 3.992 (0.85), 4.005 (0.83), 4.025 (0.51), 4.064 (0.53), 4.085 (0.93), 4.098 (0.96), 4.529 (0.72), 4.544 (0.69), 6.293 (0.67), 6.314 (0.69), 6.344 (0.56), 6.998 (1.73), 7.019 (1.70), 7.358 (1.14), 7.376 (1.33), 7.460 (1.97), 7.670 (1.60), 7.687 (0.61), 7.773 (2.32), 7.795 (2.26), 7.816 (0.53), 7.843 (0.91), 7.861 (1.06), 7.951 (0.45), 8.289 (0.43).  Example 23-B- ²²⁷ Th

[ ²²⁷ Th]Thorium-(4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2, 5,13-trioxo-1-[4-(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)phenyl]-3,6,12,14-tetraazaheptadecane-11,15 ,17- tricarboxylate

(4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1-(4- {2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetamido}phenyl)-3,6,12, 14-tetraazaheptadecane- 11,15,17-tricarboxylic acid (1.00 mg, 0.965 µmol) was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 3.1 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 75.3% by iTLC.  #24 Linker Intermediate 104

 

tri-tert-butyl (3S,10S,14S)-1-{4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]phenyl}-3-[(naphthalen-2-yl )methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (290 mg, 95 % purity, 402 µmol) and 4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]benzoic acid (150 mg, 402 µmol) were solubilised in DMF (3.1 ml), 4-methylmorpholine (180 µl, 1.6 mmol, CAS-RN: 109-02-4) and HATU (229 mg, 603 µmol) were added and the mixture was stirred under argon for 1h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 109 mg (95 % purity, 25 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.62 min; MS (ESIpos): m/z = 1041 [M+H] ⁺   Intermediate 105

tri-tert-butyl (3S,10S,14S)-1-[4-(aminomethyl)phenyl]-3-[(naphthalen-2-yl)m ethyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

tri-tert-butyl (3S,10S,14S)-1-{4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amin o)methyl]phenyl}-3- [(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahe xadecane-10,14,16-tricarboxylate

 

(267 mg, 80 % purity, 205 µmol) was solubilised in DMF (2.1 ml), piperidine (390 µl, 2.1 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 56.0 mg (70 % purity, 23 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.23 min; MS (ESIpos): m/z = 819 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.49), 1.367 (2.07), 1.380 (15.66), 1.527 (0.45), 1.538 (0.56), 1.586 (0.81), 1.592 (0.81), 2.326 (0.76), 2.331 (0.56), 2.518 (3.05), 2.522 (1.92), 2.669 (0.80), 2.673 (0.59), 2.922 (0.90), 3.178 (0.45), 3.205 (0.52), 3.216 (0.59), 3.274 (0.92), 3.288 (1.05), 3.302 (1.19), 3.314 (1.26), 3.327 (1.40), 3.342 (1.48), 5.758 (16.00), 7.399 (0.46), 7.434 (0.46), 7.750 (0.49), 7.771 (0.74), 7.781 (0.60), 7.795 (0.77).  Example 24-A

(3S,10S,14S)-1-[4-(aminomethyl)phenyl]-3-[(naphthalen-2-y l)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3S,10S,14S)-1-[4-(aminomethyl)phenyl]-3-[(naphthalen-2-yl)m ethyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (25.0 mg, 80 % purity, 24.4 µmol) was solubilised in DCM (310 µl), TFA (1.0 ml, 490 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.00 mg (90 % purity, 11 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 650 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.850 (0.59), 1.170 (0.59), 1.231 (4.32), 1.246 (1.94), 1.255 (2.85), 1.295 (2.06), 1.332 (1.43), 1.347 (2.57), 1.380 (1.66), 1.461 (0.83), 1.608 (1.90), 1.807 (0.59), 2.142 (1.03), 2.246 (0.79), 2.331 (1.74), 2.518 (11.21), 2.522 (7.01), 2.539 (1.07), 2.669 (2.46), 2.673 (1.90), 2.728 (1.62), 2.736 (16.00), 2.887 (1.82), 2.954 (1.54), 3.052 (3.41), 3.064 (3.72), 3.076 (3.72), 3.171 (3.05), 3.197 (3.25), 3.248 (4.40), 3.458 (9.07), 3.504 (8.67), 3.546 (8.79), 3.558 (8.36), 3.570 (7.80), 3.930 (3.33), 4.746 (0.91), 6.159 (0.48), 6.414 (0.59),  

7.417 (3.09), 7.432 (4.32), 7.447 (2.73), 7.494 (1.35), 7.514 (1.43), 7.777 (5.70), 7.799 (6.14), 7.830 (1.82), 8.141 (0.79), 8.770 (0.51).  Intermediate 106

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[4- ({2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)phenyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (89.6 mg, 156 µmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N-dimethylmetha niminium hexafluoridophosphate(1-) (58.6 mg, 155 µmol; CAS-RN:94790-37-1) and N,N- diisopropylethylamine (28 µl, 160 µmol) were stirred in DMF (1 ml) for 10min at rt. tri-tert-butyl (3S,10S,14S)-1-[4-(aminomethyl)phenyl]-3-[(naphthalen-2-yl)m ethyl]-1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (40.0 mg, 80 % purity, 39.1 µmol) was added and the mixture was stirred for 5d at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 35.0 mg (85 % purity, 55 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 1373 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.337 (2.40), 1.379 (16.00), 1.382 (15.72), 1.399 (4.09), 1.434 (0.72), 1.452 (1.62), 2.518 (1.50), 2.523 (0.95), 2.727 (1.28), 2.888 (1.59), 7.434 (0.44), 7.753 (0.48), 7.762 (0.42), 7.773 (0.62), 7.791 (0.70). 

 

Example 24-B

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1- [4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)phenyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[4- ({2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}methyl)phenyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (36.0 mg, 26.2 µmol) was solubilised in DCM (840 µl), TFA (2.0 ml, 26 mmol) was added and the mixture was stirred under argon over the weekend at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (95 % purity, 21 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 1037 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.233 (7.44), 1.365 (5.45), 1.485 (2.67), 1.605 (2.50), 1.719 (2.45), 1.885 (2.78), 2.233 (6.28), 2.328 (3.22), 2.668 (6.22), 2.743 (3.50), 2.991 (11.78), 3.047 (11.89), 3.166 (9.98), 3.229 (10.77), 3.418 (16.00), 4.019 (4.01), 4.076 (3.87), 4.295 (4.82), 4.735 (2.56), 6.325 (5.45), 7.331 (5.52), 7.434 (6.59), 7.493 (3.66), 7.513 (3.79), 7.722 (3.98), 7.801 (14.38), 8.107 (3.06), 8.662 (2.74).  Example 24-B- ²²⁷ Th

[ ²²⁷ Th]Thorium (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-{4- [(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]phenyl}-2,5,11,13-tetraazahexadecane -10,14,16- tricarboxylate   

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[4- ({2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl]- 2,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylic acid (1.00 mg, 0.965 µmol) was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 3.1 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 83.2% by iTLC.  #25 Linker Intermediate 107

5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]pyri dine-2-carboxylic acid 

 

5-(aminomethyl)pyridine-2-carboxylic acid (410 mg, 97 % purity, 2.61 mmol) was solubilised in 1,4-dioxane (5.0 ml), sodium carbonate (5.8 ml, 2.0 M, 12 mmol) and (9H-fluoren-9-yl)methyl carbonochloridate (751 mg, 2.90 mmol) were added and the mixture was stirred for 2d at rt. HCl (20 ml, 2.0 M) was added dropwise and extracted with DCM. The organic phase was washed with brine, dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 160 mg (95 % purity, 16 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.99 min; MS (ESIpos): m/z = 375 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.68), 2.336 (1.28), 2.518 (16.00), 2.523 (10.94), 2.678 (1.22), 3.159 (11.07), 3.172 (11.21), 4.082 (1.08), 4.095 (2.16), 4.108 (2.03), 4.121 (0.74), 4.216 (1.22), 4.233 (2.63), 4.249 (1.69), 4.275 (4.79), 4.290 (4.73), 4.368 (7.36), 4.385 (6.21), 7.304 (2.43), 7.320 (5.74), 7.338 (3.71), 7.397 (3.78), 7.416 (6.14), 7.434 (2.84), 7.674 (5.81), 7.693 (5.20), 7.741 (1.96), 7.747 (1.96), 7.761 (2.23), 7.766 (2.23), 7.884 (6.21), 7.903 (5.74), 7.957 (1.42), 7.972 (2.77), 7.993 (4.19), 8.014 (3.24), 8.134 (1.76), 8.573 (3.71), 8.577 (3.71).  Intermediate 108

 

tri-tert-butyl (10S,14S)-1-{5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)m ethyl]pyridin- 2-yl}-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-te traazahexadecane-10,14,16- tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (129 mg, 85 % purity, 160 µmol) and 5-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]pyridine-2-carbox ylic acid (70.5 mg, 85 % purity, 160 µmol) were solubilised in DMF (1.2 ml), 4-methylmorpholine (79 µl, 640 µmol, CAS-RN: 109- 02-4) and HATU (102 mg, 240 µmol) were added and the mixture was stirred under argon at rt.

The mixture was diluted with brine and extracted 3 times with DCM. The combined organic layers

 

were dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 144 mg (90 % purity, 78 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.63 min; MS (ESIpos): m/z = 1042 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.368 (6.92), 1.376 (16.00), 2.518 (1.11), 2.522 (0.71), 2.888 (0.41), 4.362 (0.44), 7.306 (0.41), 7.401 (0.45), 7.420 (0.50), 7.660 (0.41), 7.681 (0.59), 7.764 (0.41), 7.871 (0.42), 7.890 (0.41).  Intermediate 109

tri-tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(naphthalen- 2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate 

tri-tert-butyl (3S,10S,14S)-1-{5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amin o)methyl]pyridin-2- yl}-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetr aazahexadecane-10,14,16- tricarboxylate (142 mg, 90 % purity, 123 µmol) was solubilised in DMF (1.9 ml), piperidine (105 mg, 1.23 mmol) was added and the mixture was stirred under argon for 1h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 66.0 mg (95 % purity, 62 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.21 min; MS (ESIpos): m/z = 819 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.42), 1.376 (10.35), 1.380 (16.00), 1.383 (11.88), 1.558 (0.42), 1.626 (0.57), 1.639 (0.69), 2.518 (1.88), 2.523 (1.18), 2.996 (0.83), 3.011 (1.02), 3.024 (0.72), 4.149 (0.88), 7.430 (0.41), 7.441 (0.49), 7.451 (0.43), 7.680 (0.51), 7.758 (0.61), 8.016 (1.00), 8.679 (0.75).  Example 25-A

(3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(naphthal en-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(naphthalen- 2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (20.0 mg, 90 % purity, 22.0 µmol) was solubilised in DCM (420 µl), TFA (251 mg, 2.20 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 11.9 mg (95 % purity, 79 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.72 min; MS (ESIpos): m/z = 651 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.848 (0.61), 1.229 (7.23), 1.244 (7.51), 1.263 (6.62), 1.341 (6.67), 1.360 (7.29), 1.377 (5.11), 1.412 (1.28), 1.432 (2.12), 1.448 (2.85), 1.465 (3.55), 1.484 (2.85), 1.551 (1.98), 1.567 (2.85), 1.582 (3.57), 1.602 (3.16), 1.616 (2.51), 1.682 (0.87), 1.702 (2.09), 1.722 (2.85), 1.737 (3.97), 1.755 (3.83), 1.776 (2.15), 1.797 (3.27), 1.814 (4.13), 1.832 (3.38), 1.847 (2.01), 1.866 (0.92), 2.070 (0.89), 2.199 (7.73), 2.218 (13.26), 2.237 (6.45), 2.322 (1.42), 2.326 (1.82), 2.331 (1.40), 2.522 (4.80), 2.539 (10.86), 2.665 (1.62), 2.669 (1.98), 2.673 (1.62), 2.684 (0.92), 2.725 (9.05), 2.885 (11.06), 3.003 (3.10), 3.019 (4.22), 3.036 (7.04), 3.050 (8.43), 3.062 (7.12), 3.078 (3.99), 3.165 (7.23), 3.180 (3.74), 3.200 (4.33), 3.214 (6.90), 3.235 (7.20), 3.247 (7.20), 3.260 (7.60), 3.281 (4.58), 3.294 (4.10), 3.504 (4.27), 3.950 (9.16), 3.970 (11.59), 3.983 (12.73), 4.003 (13.29), 4.021 (11.42), 4.038 (8.35), 4.107 (14.21), 4.793 (3.60), 4.814 (5.86), 4.828 (6.06), 4.848 (3.38), 6.245 (4.83), 6.264 (4.66), 6.341 (6.20), 6.361 (5.92), 7.358 (7.65), 7.362 (7.57), 7.379 (8.18), 7.382 (8.15), 7.409 (2.60), 7.422 (8.40), 7.426 (13.35), 7.436 (15.22), 7.445 (13.88), 7.450 (8.38), 7.462 (2.60), 7.691 (14.94), 7.749 (7.68), 7.760 (16.00), 7.771 (6.95), 7.781 (11.39), 7.815 (6.87), 7.822 (5.78), 7.838 (6.39), 7.947 (2.01), 7.961 (4.24), 7.981 (11.62), 7.995 (6.09), 8.220 (3.97), 8.235 (7.12), 8.248 (3.63), 8.678 (8.29), 8.691 (9.83), 8.712 (7.96).  Intermediate 110

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[5- ({2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)pyridin-2- yl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate   

₎

N-[9,12-bis(2-tert-butoxy-2-oxoethyl)-2,2,6-trimethyl-4-oxo- 3-oxa-6,9,12-triazatetradecan-14- yl]-N-ethylglycine (119 mg, 202 µmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (84.1 mg, 200 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (29 µl, 150 µmol) were stirred in DMF (970 µl) for 10min at rt. tri- tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(naphthalen- 2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (46.0 mg, 90 % purity, 50.5 µmol) was added and the mixture was stirred for 5h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 52.0 mg (80 % purity, 60 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m/z = 1374 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.40), 1.313 (2.15), 1.322 (2.42), 1.371 (6.50), 1.380 (16.00), 1.395 (2.85), 1.400 (3.76), 1.435 (3.47), 1.463 (1.08), 1.477 (1.59), 2.518 (1.81), 2.522 (1.16), 2.669 (0.49), 2.888 (0.45).  Example 25-B

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1- [5-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)pyridin-2-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[5- ({2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}methyl)pyridin-2-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (50.0 mg, 80 % purity, 29.1 µmol) was solubilised in DCM (740 µl), TFA (670 µl, 8.7 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 7.10 mg (95 % purity, 22 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.72 min; MS (ESIpos): m/z = 1038 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (1.76), 1.344 (0.81), 1.462 (0.41), 1.575 (0.41), 1.731 (0.47), 1.751 (0.47), 1.907 (0.61), 2.074 (1.02), 2.084 (0.68), 2.206 (0.95), 2.225 (1.69), 2.244 (0.81), 2.331 (2.92), 2.518 (16.00), 2.522 (10.24), 2.539 (1.02), 2.544 (0.95), 2.576 (0.95), 2.673 (3.05), 2.678 (1.42), 2.910 (1.29), 3.024 (2.58), 3.089 (1.29), 3.440 (1.90), 3.985 (0.54), 4.068 (0.54), 4.382 (0.95), 4.779 (0.54), 4.793 (0.54), 6.298 (0.47), 6.330 (0.54), 6.352 (0.54), 7.370 (0.81), 7.390 (0.88), 7.428 (1.36), 7.437 (1.90), 7.446 (1.36), 7.702 (1.56), 7.773 (1.90), 7.794 (1.69), 7.816 (0.88), 7.839 (1.02), 7.865 (0.61), 7.936 (0.47), 8.142 (1.22), 8.184 (0.68), 8.615 (0.68), 8.689 (0.68), 8.710 (0.68), 8.793 (0.47).  Example 25-B- ²²⁷ Th

[ ²²⁷ Th]Thorium (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-{5- [(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]pyridin-2-yl}-2,5,11,13-tetraazahexa decane- 10,14,16-tricarboxylate 

 

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[5- ({2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)pyridin- 2-yl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid (1.00 mg, 0.964 µmol) was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 3.1 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 92.7% by iTLC.

  Intermediate 114D

tri-tert-butyl (3S,10S,14R)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-3-[(naphthale n-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate

 

A mixture of di-tert-butyl (2R)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}- 1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (584 mg, 94 % purity, 801 µmol), 5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]pyridin e-2-carboxylic acid (450 mg, 1.20 mmol), 4-methylmorpholine (260 µl, 2.4 mmol), HATU (457 mg, 1.20 mmol), and DMF

(6.2mL) was stirred at r.t. for 3h. The mixture was concentrated under reduced pressure and purified by preparative HPLC (C18, acetonitrile/1%TFA) to give the title compound (100mg, 95% purity, 32% yield).

LC-MS (OA01a01): R _t = 1.61 min; MS (ESIpos): m/z = 1042 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) delta [ppm]: 1.206 (0.21), 1.231 (0.26), 1.361 (3.25), 1.365 (16.00), 1.389 (9.58), 2.158 (0.17), 2.175 (0.20), 2.179 (0.26), 2.200 (0.24), 2.322 (0.17), 2.326 (0.23), 2.331 (0.17), 2.518 (0.85), 2.522 (0.56), 2.668 (0.23), 2.673 (0.17), 3.202 (0.17), 3.224 (0.25), 3.239 (0.19), 3.955 (0.17), 3.970 (0.19), 4.009 (0.16), 4.016 (0.23), 4.030 (0.22), 4.220 (0.24), 4.237 (0.19), 4.249 (0.37), 4.264 (0.35), 4.363 (0.58), 4.379 (0.48), 5.759 (0.45), 6.293 (0.30), 6.314 (0.55), 6.335 (0.31), 7.287 (0.23), 7.306 (0.52), 7.325 (0.35), 7.357 (0.25), 7.382 (0.40), 7.402 (0.58), 7.421 (0.66), 7.433 (0.39), 7.438 (0.29), 7.444 (0.40), 7.660 (0.51), 7.681 (0.73), 7.727 (0.18), 7.732 (0.19), 7.747 (0.40), 7.752 (0.37), 7.764 (0.56), 7.785 (0.39), 7.814 (0.23), 7.825 (0.22), 7.838 (0.19), 7.871 (0.56), 7.890 (0.53), 7.898 (0.46), 7.918 (0.30), 7.941 (0.25), 8.214 (0.21), 8.481 (0.33), 8.485 (0.33), 8.630 (0.26), 8.652 (0.24).

Intermediate 115D

 

tri-tert-butyl (3S,10S,14R)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(naphthalen- 2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate

A mixture of tri-tert-butyl (3S,10S,14R)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-3-[(naphthale n-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (100 mg, 96.0 µmol), piperidine (190 µl, 1.9 mmol), and DMF (2.5 mL) was stirred at r.t. for 3h. After that the mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (C18, acetonitrile/1%

TFA) to give the title compound (75.0 mg, 95 % purity, 91 % yield).

LC-MS (Method 1): R _t = 1.30 min; MS (ESIpos): m/z = 820 [M+H] ⁺ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.353 (0.44), 1.371 (16.00), 1.392 (10.35), 2.518 (1.91), 2.523 (1.37), 3.221 (0.40), 3.236 (0.40), 3.339 (1.51), 3.857 (1.52), 6.318 (0.46), 7.429 (0.42), 7.438 (0.56), 7.448 (0.47), 7.681 (0.50), 7.764 (0.62), 7.915 (1.43), 7.919 (1.22), 8.235 (1.71), 8.581 (0.58).

Example 25-A-D

(3S,10S,14R)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(naphthal en-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid

 

A mixture of tri-tert-butyl (3S,10S,14R)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(naphthalen- 2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (10.0 mg, 12.2 µmol), TFA (300µL), and DCM (1.0 mL) was stirred at r.t. for 2h. After that the mixture was concentrated under reduced pressure and purified by preparative HPLC (C18, acetonitrile/1% formic acid) to give the title compound (6.20 mg, 95 % purity, 74 % yield).

LC-MS (Method 1): R _t = 0.74 min; MS (ESIpos): m/z = 652 [M+H] ⁺ 1H-NMR (400 MHz, DMSO-d6) delta [ppm]: 1.233 (2.32), 1.253 (1.93), 1.271 (0.90), 1.337 (1.93), 1.356 (2.17), 1.374 (1.40), 1.439 (0.63), 1.458 (0.85), 1.473 (1.09), 1.492 (0.85), 1.548 (0.77), 1.563 (0.98), 1.583 (0.92), 1.597 (0.53), 1.617 (0.44), 1.636 (0.57), 1.650 (0.94), 1.669 (1.16), 1.684 (0.88), 1.703 (0.44), 1.732 (0.39), 1.753 (1.05), 1.770 (1.23), 1.787 (0.92), 1.803 (0.68), 1.824 (0.31), 2.073 (16.00), 2.108 (0.39), 2.121 (0.53), 2.145 (1.38), 2.164 (1.55), 2.173 (1.53), 2.193 (2.21), 2.214 (1.14), 2.231 (0.88), 2.252 (0.35), 2.518 (4.36), 2.522 (2.91), 2.539 (1.90), 2.995 (0.85), 3.010 (1.31), 3.027 (1.90), 3.052 (2.06), 3.066 (1.99), 3.083 (1.44), 3.099 (1.05), 3.180 (1.49), 3.200 (1.66), 3.213 (2.56), 3.234 (2.69), 3.247 (2.65), 3.260 (2.82), 3.280 (1.93), 3.294 (1.82), 3.637 (3.02), 3.933 (2.30), 3.953 (3.00), 3.965 (3.44), 3.984 (3.48), 4.000 (2.95), 4.029 (8.05), 4.788 (1.01), 4.803 (1.27), 4.809 (1.73), 4.823 (1.75), 4.843 (0.94), 6.246 (1.12), 6.262 (1.09), 6.452 (1.82), 6.472 (1.75), 7.357 (2.43), 7.361 (2.45), 7.379 (2.56), 7.382 (2.65), 7.404 (0.46), 7.409 (0.81), 7.421 (2.65), 7.426 (4.55), 7.435 (5.10), 7.444 (4.73), 7.449 (2.78), 7.461 (0.85), 7.691 (4.64), 7.751 (2.39), 7.760 (5.17), 7.775 (2.21), 7.781 (3.68), 7.814 (2.17), 7.822 (1.73), 7.837 (1.99), 7.941 (1.01), 7.961 (6.54), 7.989 (0.63), 8.197 (1.14), 8.211 (2.85), 8.225 (1.12), 8.653 (3.94), 8.708 (2.74), 8.729 (2.63).

Example 25-C-D

(3S,10S,14R)-1-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)am ino]ethyl}carbamoyl)-3-  

hydroxy-6-methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)p yridin-2-yl]-3-[(naphthalen- 2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,1 4,16-tricarboxylic acid

2,2'-{({3-[(2-{[1-(2-{[(6-{[(7R,11S,18S)-7,11-bis(tert-butox ycarbonyl)-2,2-dimethyl-19- (naphthalen-2-yl)-4,9,17-trioxo-3-oxa-8,10,16-triazanonadeca n-18-yl]carbamoyl}pyridin-3- yl)methyl]amino}-2-oxoethyl)-3-hydroxy-6-methyl-2-oxo-1,2-di hydropyridine-4- carbonyl]amino}ethyl)(2-{[1-(carboxymethyl)-3-hydroxy-6-meth yl-2-oxopyridine-4(2H)- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid (2.40 mg, 1.27 µmol) is treated with 90% TFA in water (1 mL). Water (15 mL) is added and solution lyophilised affording 2.3 mg of the target compound. LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 2.13 min; MS (ESIpos): m/z = 1715.8  

[M+H] ⁺   Example 25-C-D- ²²⁷ Th (3S,10S,14R)-1-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)- 3-(hydroxy-kappaO)-6-methyl- 2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]propy l}(2-{[1-(carboxymethyl)- 3-(hydroxy-kappaO)-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2- oxopyridin-1(2H)-yl]acetamido}methyl)pyridin-2-yl]-3-[(napht halen-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylato( 4-)( ²²⁷ Th)thorium

 

(3S,10S,14R)-1-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethy l)-3-hydroxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6- methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)pyridin-2-yl]- 3-[(naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid (4 µg dissolved in 147 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in

0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.6 MBq/mL.1M carbonate

buffer pH 9 (15 µL) was added and mixture incubated for 60 min. The labelling efficiency was determined to be 97% by iTLC. #26 Linker Intermediate 111

 

6-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]pyri dine-3-carboxylic acid 

6-(aminomethyl)pyridine-3-carboxylic acid (140.0 mg, 920 µmol) was solubilised in 1,4-dioxane (1.1 ml, 13 mmol), Na2CO3 (4.60 ml, 2.0 M, 9.2 mmol) and (9H-fluoren-9-yl)methyl carbonochloridate (261.8 mg, 1.01 mmol) were added. The mixture was stirred over the weekend at rt under nitrogen atmosphere. The mixture was diluted with brine and extracted with DCM.

The organic phase was dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 139 mg (85 % purity, 40 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.10 min; MS (ESIpos): m/z = 375 [M+H] ⁺

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.84), 2.331 (1.80), 2.518 (8.29), 2.523 (5.45), 2.728 (13.57), 2.737 (5.28), 2.888 (16.00), 3.053 (0.46), 3.065 (0.59), 3.077 (0.59), 3.546 (0.54), 3.558 (0.59), 3.571 (0.50), 3.719 (3.64), 3.732 (4.31), 3.737 (4.65), 3.749 (4.06), 3.981 (1.93), 3.999 (3.48), 4.016 (1.51), 4.234 (1.21), 4.251 (2.64), 4.267 (1.68), 4.330 (4.73), 4.345 (4.69),  

4.374 (6.12), 4.391 (4.65), 5.069 (1.63), 5.082 (3.06), 5.095 (1.47), 7.205 (0.50), 7.280 (3.64), 7.282 (4.06), 7.298 (9.97), 7.301 (9.21), 7.317 (9.26), 7.320 (8.46), 7.337 (5.91), 7.359 (7.25), 7.379 (7.75), 7.398 (3.64), 7.408 (3.43), 7.427 (5.36), 7.445 (2.30), 7.533 (0.42), 7.552 (0.42), 7.656 (7.58), 7.674 (6.91), 7.711 (5.40), 7.730 (4.77), 7.799 (0.71), 7.818 (0.71), 7.849 (8.29), 7.868 (7.62), 7.896 (5.70), 7.914 (5.15), 7.950 (2.14), 7.974 (1.26), 7.990 (2.55), 8.006 (1.17), 8.211 (2.30), 8.217 (2.35), 8.232 (2.14), 8.237 (2.09), 8.975 (4.61), 8.979 (4.65), 13.364 (0.50). Intermediate 112

tri-tert-butyl (3S,10S,14S)-1-{6-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-3-yl}-3-[(naphthale n-2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (122 mg, 178 µmol) and 6-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridine-3-carboxylic acid (66.7 mg, 178 µmol) were solubilised in DMF (4.1 ml), 4-methylmorpholine (78 µl, 710 µmol, CAS-RN: 109-02-4) and HATU

(102 mg, 267 µmol) were added and the mixture was stirred under argon at rt. The mixture was diluted with brine and extracted with DCM. The organic phase was dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 94.0 mg (85 % purity, 43 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.60 min; MS (ESIpos): m/z = 1042 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.46), 1.377 (13.29), 1.381 (16.00), 2.518 (2.09), 2.523 (1.36), 2.728 (0.46), 2.888 (0.57), 4.356 (0.49), 7.418 (0.59), 7.435 (0.49), 7.791 (0.42), 7.801 (0.52), 7.813 (0.49), 7.888 (0.47), 7.907 (0.44). 

 

Intermediate 113

 

tri-tert-butyl (3S,10S,14S)-1-[6-(aminomethyl)pyridin-3-yl]-3-[(naphthalen- 2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate 

tri-tert-butyl (3S,10S,14S)-1-{6-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amin o)methyl]pyridin-3- yl}-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetr aazahexadecane-10,14,16- tricarboxylate (93.0 mg, 85 % purity, 75.9 µmol) was solubilised in DMF (1.8 ml), piperidine (32.3 mg, 380 µmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 47.0 mg (85 % purity, 64 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.24 min; MS (ESIpos): m/z = 820 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (13.40), 1.385 (16.00), 2.518 (1.27), 2.523 (0.79), 2.888 (0.48), 3.984 (0.69), 7.441 (0.42), 7.790 (0.45), 7.801 (0.62), 7.810 (0.45), 8.867 (0.56).  Example 26-A

(3S,10S,14S)-1-[6-(aminomethyl)pyridin-3-yl]-3-[(naphthal en-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3S,10S,14S)-1-[6-(aminomethyl)pyridin-3-yl]-3-[(naphthalen- 2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (15.0 mg, 18.3 µmol) was solubilised in DCM (350 µl), TFA (20.9 mg, 183 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 10.0 mg (95 % purity, 80 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.71 min; MS (ESIpos): m/z = 651 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (1.16), 1.272 (1.23), 1.290 (1.16), 1.360 (1.10), 1.378 (1.16), 1.459 (0.65), 1.481 (0.52), 1.594 (0.84), 1.613 (0.97), 1.631 (0.84), 1.789 (0.45), 1.812 (0.58), 1.829 (0.52), 1.846 (0.39), 1.906 (0.52), 2.084 (3.74), 2.159 (0.65), 2.178 (0.71), 2.236 (0.52), 2.259 (0.77), 2.279 (0.52), 2.295 (0.45), 2.332 (2.71), 2.336 (1.29), 2.518 (16.00), 2.522 (10.26), 2.673 (2.71), 2.678 (1.23), 3.032 (0.71), 3.048 (1.03), 3.063 (1.16), 3.079 (1.16), 3.095 (1.16), 3.112 (1.03), 3.128 (1.29), 3.160 (1.61), 3.186 (1.81), 3.269 (4.39), 3.973 (1.42), 3.986 (1.42), 4.112 (5.48), 4.742 (0.45), 4.754 (0.52), 4.767 (0.65), 4.775 (0.65), 6.167 (0.52), 6.399 (0.97), 6.420 (0.90), 7.408 (0.45), 7.421 (1.29), 7.425 (1.23), 7.439 (2.13), 7.458 (1.23), 7.471 (0.52), 7.491 (1.74), 7.511 (2.19), 7.529 (1.42), 7.785 (3.16), 7.806 (2.71), 7.816 (3.81), 7.839 (1.23), 8.171 (1.42), 8.176 (1.48), 8.183 (1.03), 8.191 (1.81), 8.196 (1.68), 8.206 (1.16), 8.903 (2.19), 8.907 (2.13), 9.063 (0.90), 9.084 (0.90).  Intermediate 114

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[6- ({2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)pyridin-3- yl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

N-[9,12-bis(2-tert-butoxy-2-oxoethyl)-2,2,6-trimethyl-4-oxo- 3-oxa-6,9,12-triazatetradecan-14- yl]-N-ethylglycine (73.3 mg, 125 µmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (46.6 mg, 123 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (16 µl, 93 µmol) were stirred in DMF (720 µl) for 10min at rt. tri- tert-butyl (3S,10S,14S)-1-[6-(aminomethyl)pyridin-3-yl]-3-[(naphthalen- 2-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (30.0 mg, 85 % purity, 31.1 µmol) was added and the mixture was stirred for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 30.0 mg (70 % purity, 49 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.36 min; MS (ESIpos): m/z = 1374 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.256 (0.42), 1.277 (2.27), 1.295 (2.44), 1.380 (12.53), 1.385 (16.00), 1.414 (0.66), 1.433 (3.11), 1.462 (0.47), 2.518 (1.54), 2.523 (0.98), 2.686 (1.88), 2.727 (5.20), 2.888 (6.62), 7.438 (0.43), 7.799 (0.56), 7.950 (0.84).  Example 26-B

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1- [6-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)pyridin-3-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[6- ({2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}methyl)pyridin-3-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (28.0 mg, 70 % purity, 14.3 µmol) was solubilised in DCM (460 µl), TFA (16.3 mg, 143 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 14.0 mg (95 % purity, 90 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.68 min; MS (ESIpos): m/z = 1038 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.137 (1.27), 1.233 (1.54), 1.255 (1.54), 1.274 (1.34), 1.352 (1.41), 1.371 (1.41), 1.473 (0.60), 1.489 (0.67), 1.509 (0.60), 1.604 (0.67), 1.625 (0.67), 1.670 (0.47), 1.684 (0.60), 1.706 (0.74), 1.720 (0.54), 1.897 (0.67), 1.907 (0.94), 1.914 (0.74), 2.115 (0.60), 2.200 (0.94), 2.217 (1.54), 2.237 (1.61), 2.258 (0.87), 2.331 (2.81), 2.336 (1.34), 2.518 (16.00), 2.522 (10.64), 2.539 (0.94), 2.673 (2.95), 2.678 (1.41), 2.729 (0.80), 2.796 (1.34), 2.888 (1.00), 2.983 (6.56), 3.058 (1.81), 3.102 (1.14), 3.135 (1.47), 3.162 (1.41), 3.491 (2.95), 3.517 (3.55), 3.995 (0.54), 4.015 (1.00), 4.028 (1.00), 4.048 (0.47), 4.066 (0.60), 4.086 (1.14), 4.099 (1.21), 4.120 (0.60), 4.409 (1.74), 4.421 (1.67), 4.744 (0.67), 6.292 (1.61), 6.304 (1.87), 6.313 (1.74), 6.325 (1.61), 7.374 (1.34), 7.395 (1.41), 7.405 (0.74), 7.409 (0.74), 7.422 (1.61), 7.426 (1.47), 7.442 (2.41), 7.457 (1.41), 7.461 (1.54), 7.474 (0.67), 7.497 (1.47), 7.521 (1.67), 7.793 (3.75), 7.812 (4.75), 7.840 (1.47), 8.039 (0.87), 8.060 (0.80), 8.133 (1.61), 8.193 (1.21), 8.763 (0.94), 8.815 (1.87), 8.933 (0.67), 8.951 (0.60).  Example 26-B- ²²⁷ Th

 

[ ²²⁷ Th]Thorium (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-{6- [(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]pyridin-3-yl}-2,5,11,13-tetraazahexa decane- 10,14,16-tricarboxylate 

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[6- ({2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)pyridin- 3-yl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid (1.00 mg, 0.964 µmol) was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 3.1 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 87% by iTLC.  #27 Linker Intermediate 115

tri-tert-butyl (3S,10S,14S)-1-{3-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]phenyl}-3-[(naphthalen-2-yl )methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (136 mg, 90 % purity, 179 µmol) and 3-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]benzoic acid (66.9 mg, 179 µmol) were solubilised in DMF (2.8 ml), 4-methylmorpholine (79 µl, 720 µmol, CAS-RN: 109-02-4) and COMU (153 mg, 358 µmol) were added and the mixture was stirred under argon for 2h at rt. The mixture was diluted with brine and extracted with DCM. The organic phase was dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 50.0 mg (95 % purity, 25 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.65 min; MS (ESIpos): m/z = 1041 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.49), 1.377 (16.00), 2.518 (1.84), 2.523 (1.15), 2.728 (0.80), 2.888 (1.02), 7.674 (0.44), 7.691 (0.54).  Intermediate 116

tri-tert-butyl (3S,10S,14S)-1-[3-(aminomethyl)phenyl]-3-[(naphthalen-2-yl)m ethyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

 

tri-tert-butyl (3S,10S,14S)-1-{3-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amin o)methyl]phenyl}-3- [(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahe xadecane-10,14,16-tricarboxylate

(49.0 mg, 47.1 µmol) was solubilised in DMF (1.1 ml), piperidine (47 µl, 470 µmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 30.0 mg (85 % purity, 66 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.23 min; MS (ESIpos): m/z = 819 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.233 (0.47), 1.380 (15.69), 1.383 (16.00), 2.332 (0.60), 2.518 (3.16), 2.523 (2.13), 2.728 (0.42), 2.888 (0.54), 7.438 (0.45), 7.457 (0.45), 7.790 (0.54), 7.797 (0.67), 7.810 (0.42).  Example 27-A

(3S,10S,14S)-1-[3-(aminomethyl)phenyl]-3-[(naphthalen-2-y l)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3S,10S,14S)-1-[3-(aminomethyl)phenyl]-3-[(naphthalen-2-yl)m ethyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (10.0 mg, 85 % purity, 10.4 µmol) was  

solubilised in DCM (330 µl), TFA (118 mg, 1.04 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (95 % purity, 84 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.75 min; MS (ESIpos): m/z = 650 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.137 (0.94), 1.232 (1.30), 1.359 (0.94), 1.376 (0.86), 1.787 (1.01), 1.806 (1.08), 1.824 (0.50), 2.084 (1.37), 2.247 (0.94), 2.257 (1.08), 2.276 (0.50), 2.332 (3.03), 2.336 (1.30), 2.518 (16.00), 2.523 (10.45), 2.678 (1.30), 2.960 (0.43), 3.941 (0.58), 3.968 (0.65), 3.979 (0.58), 4.035 (3.24), 4.775 (0.65), 4.792 (0.65), 6.339 (0.65), 6.358 (0.65), 7.388 (0.72), 7.407 (1.95), 7.419 (1.01), 7.426 (1.59), 7.436 (2.09), 7.442 (1.08), 7.454 (1.01), 7.489 (1.23), 7.506 (1.51), 7.523 (1.08), 7.682 (1.01), 7.701 (0.86), 7.786 (1.80), 7.807 (2.31), 7.815 (1.73), 7.821 (2.74), 7.837 (1.01), 8.008 (0.72), 8.230 (1.37), 9.102 (0.65), 9.124 (0.58).  Intermediate 117

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[3- ({2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)phenyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate  *

N-[9,12-bis(2-tert-butoxy-2-oxoethyl)-2,2,6-trimethyl-4-oxo- 3-oxa-6,9,12-triazatetradecan-14- yl]-N-ethylglycine (46.5 mg, 79.0 µmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (29.6 mg, 78.0 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (10 µl, 59 µmol) were stirred in DMF (760 µl) for 10min at rt. tri- tert-butyl (3S,10S,14S)-1-[3-(aminomethyl)phenyl]-3-[(naphthalen-2-yl)m ethyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (19.0 mg, 85 % purity, 19.7 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by

 

preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 21.0 mg (80 % purity, 62 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.40 min; MS (ESIpos): m/z = 1373 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.233 (0.50), 1.347 (3.02), 1.380 (14.40), 1.384 (16.00), 1.396 (4.35), 1.403 (2.48), 1.434 (1.48), 1.442 (1.18), 1.451 (1.04), 1.461 (3.39), 2.332 (0.60), 2.518 (3.38), 2.523 (2.16), 3.117 (0.44), 3.544 (0.43), 7.403 (0.48), 7.438 (0.52), 7.793 (0.62).  Example 27-B

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1- [3-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)phenyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[3- ({2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}methyl)phenyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (18.0 mg, 80 % purity, 10.5 µmol) was solubilised in DCM (330 µl), TFA (12.0 mg, 105 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 7.50 mg (95 % purity, 66 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.74 min; MS (ESIpos): m/z = 1037 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.137 (0.63), 1.232 (1.20), 1.279 (0.85), 1.297 (0.78), 1.353 (0.49), 1.373 (0.70), 1.392 (0.85), 1.621 (0.42), 1.713 (0.42), 1.907 (1.13), 2.084 (1.06), 2.207 (0.70), 2.229 (1.06), 2.331 (3.10), 2.336 (1.41), 2.518 (16.00), 2.523 (10.22), 2.620 (0.92), 2.673 (3.17), 2.678 (1.48), 3.018 (3.03), 3.110 (1.41), 3.258 (2.54), 3.438 (2.33), 4.009 (0.56), 4.023 (0.56), 4.042 (0.49), 4.061 (0.49), 4.077 (0.56), 4.279 (0.42), 4.698 (0.49), 4.718 (0.49), 6.366 (0.78), 6.385 (0.78), 7.285 (0.56), 7.398 (1.27), 7.415 (1.48), 7.430 (1.76), 7.435 (1.76), 7.453 (0.92), 7.500 (0.92), 7.521 (0.99), 7.562 (0.49), 7.579 (0.42), 7.785 (1.55), 7.806 (2.19), 7.824 (1.62), 7.851 (1.20), 7.990 (0.63), 8.137 (1.13), 8.228 (0.42), 8.751 (0.42).   

Example 27-B- ²²⁷ Th

[ ²²⁷ Th]Thorium (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-{3- [(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]phenyl}-2,5,11,13-tetraazahexadecane -10,14,16- tricarboxylate 

(3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1,4,12-trioxo-1-[3- ({2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl]- 2,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylic acid (1.00 mg, 0.965 µmol) was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 3.1 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 95.7% by iTLC.  #28 Linker Intermediate 118

tri-tert-butyl (4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-1-[3-(4-nitrophenyl )-1H-pyrazol-1- yl]-2,5,13-trioxo-3,6,12,14-tetraazaheptadecane-11,15,17-tri carboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (100 mg, 146 µmol) and [3-(4-nitrophenyl)- 1H-pyrazol-1-yl]acetic acid (32.8 mg, 133 µmol) were solubilised in DMF (3.1 ml), 4- methylmorpholine (59 µl, 530 µmol, CAS-RN: 109-02-4) and HATU (75.7 mg, 199 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 57.0 mg (90 % purity, 38 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.53 min; MS (ESIpos): m/z = 915 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.379 (16.00), 2.518 (1.05), 2.522 (0.66), 2.728 (0.40), 2.888 (0.52), 6.867 (0.63), 6.873 (0.55), 7.708 (0.60), 7.714 (0.55), 7.795 (0.46), 7.818 (0.44), 7.971 (0.70), 7.993 (0.84), 8.217 (0.92), 8.240 (0.72).  Intermediate 119

tri-tert-butyl (4S,11S,15S)-1-[3-(4-aminophenyl)-1H-pyrazol-1-yl]-4-[(napht halen-2- yl)methyl]-2,5,13-trioxo-3,6,12,14-tetraazaheptadecane-11,15 ,17-tricarboxylate 

 

tri-tert-butyl (4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-1-[3-(4-nitrophenyl )-1H-pyrazol-1-yl]- 2,5,13-trioxo-3,6,12,14-tetraazaheptadecane-11,15,17-tricarb oxylate (57.0 mg, 90 % purity, 56.1 µmol) was solubilised in MeOH (1.1 ml), palladium on carbon (600 µg, 10 % purity, 5.6 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 48.0 mg (85 % purity, 82 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.41 min; MS (ESIpos): m/z = 885 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.353 (0.46), 1.377 (8.35), 1.380 (16.00), 1.383 (14.91), 2.331 (0.52), 2.518 (2.91), 2.523 (1.83), 2.673 (0.52), 5.142 (0.46), 6.404 (0.52), 6.410 (0.50), 6.536 (0.57), 6.558 (0.59), 7.381 (0.76), 7.402 (0.54), 7.455 (0.43), 7.508 (0.52), 7.514 (0.50).  Example 28-A

(4S,11S,15S)-1-[3-(4-aminophenyl)-1H-pyrazol-1-yl]-4-[(na phthalen-2-yl)methyl]-2,5,13- trioxo-3,6,12,14-tetraazaheptadecane-11,15,17-tricarboxylic acid 

 

tri-tert-butyl (4S,11S,15S)-1-[3-(4-aminophenyl)-1H-pyrazol-1-yl]-4-[(napht halen-2-yl)methyl]- 2,5,13-trioxo-3,6,12,14-tetraazaheptadecane-11,15,17-tricarb oxylate (15.0 mg, 17.0 µmol) was solubilised in DCM (540 µl), TFA (65 µl, 850 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (80 % purity, 40 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 716 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.192 (1.29), 1.211 (1.36), 1.231 (1.49), 1.270 (1.42), 1.289 (1.49), 1.413 (0.47), 1.431 (0.88), 1.446 (0.81), 1.466 (0.68), 1.550 (0.54), 1.563 (0.75), 1.583 (0.68), 1.676 (0.54), 1.690 (0.68), 1.711 (0.88), 1.725 (0.61), 1.886 (0.54), 1.907 (1.42), 1.921 (0.81), 2.208 (1.08), 2.226 (1.63), 2.245 (1.90), 2.266 (0.95), 2.336 (1.29), 2.518 (16.00), 2.522 (10.64), 2.539 (0.88), 2.673 (2.92), 2.678 (1.29), 2.924 (0.68), 2.938 (1.22), 2.958 (1.22), 2.971 (1.22), 2.992 (0.95), 3.022 (0.68), 3.038 (0.81), 3.055 (0.68), 3.071 (0.41), 3.096 (0.95), 3.109 (1.02), 3.129 (0.75), 3.143 (0.68), 3.982 (0.54), 4.003 (1.15), 4.015 (1.15), 4.036 (0.54), 4.066 (0.68), 4.079 (0.81), 4.087 (1.29), 4.100 (1.36), 4.107 (0.75), 4.121 (0.61), 4.549 (0.47), 4.569 (1.08), 4.583 (1.08), 4.603 (0.47), 4.703 (1.02), 4.743 (3.05), 4.766 (3.05), 4.806 (1.15), 6.259 (1.97), 6.280 (1.83), 6.304 (2.10), 6.324 (2.03), 6.408 (4.20), 6.414 (4.14), 6.547 (3.39), 6.569 (3.46), 6.585 (0.47), 6.591 (0.47), 7.361 (1.56), 7.365 (1.63), 7.386 (6.44), 7.407 (4.47), 7.431 (0.54), 7.444 (2.17), 7.446 (2.58), 7.457 (3.39), 7.467 (2.92), 7.470 (2.24), 7.482 (0.54), 7.511 (4.47), 7.517 (4.34), 7.591 (0.54), 7.596 (0.68), 7.601 (0.61), 7.658 (0.68), 7.672 (2.98), 7.777 (2.51), 7.790 (1.63), 7.799 (2.78), 7.814 (1.36), 7.829 (1.49), 7.840 (1.29), 7.853 (1.36), 8.090 (0.75), 8.105 (1.56), 8.119 (0.95), 8.349 (1.69), 8.369 (1.63).  Intermediate 120

 

tri-tert-butyl (4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1-[3- (4-{2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1-yl]acetamido}phenyl)-1H- pyrazol-1-yl]-3,6,12,14-tetraazaheptadecane-11,15,17-tricarb oxylate 

N-[9,12-bis(2-tert-butoxy-2-oxoethyl)-2,2,6-trimethyl-4-oxo- 3-oxa-6,9,12-triazatetradecan-14- yl]-N-ethylglycine (82.6 mg, 140 µmol), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (52.5 mg, 139 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (18 µl, 110 µmol) were stirred in DMF (1.4 ml) for 10min at rt. tri- tert-butyl (4S,11S,15S)-1-[3-(4-aminophenyl)-1H-pyrazol-1-yl]-4-[(napht halen-2-yl)methyl]- 2,5,13-trioxo-3,6,12,14-tetraazaheptadecane-11,15,17-tricarb oxylate (31.0 mg, 35.1 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 40.0 mg (80 % purity, 63 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.35 min; MS (ESIpos): m/z = 1439 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.354 (2.79), 1.380 (16.00), 1.411 (1.40), 1.432 (1.31), 1.439 (1.17), 1.451 (0.62), 1.467 (2.57), 2.331 (0.73), 2.518 (4.01), 2.523 (2.57), 2.673 (0.73), 7.606 (0.55).  Example 28-B

 

(4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1- [3-(4-{2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}phenyl)-1H-pyrazol-1- yl]-3,6,12,14-tetraazaheptadecane-11,15,17-tricarboxylic acid 

tri-tert-butyl (4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1-[3- (4-{2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}phenyl)-1H-pyrazol-1-yl]- 3,6,12,14-tetraazaheptadecane-11,15,17-tricarboxylate (39.0 mg, 90 % purity, 24.4 µmol) was solubilised in DCM (1.6 ml), TFA (190 µl, 2.4 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 10.0 mg (90 % purity, 33 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 1102 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.137 (0.81), 1.190 (1.22), 1.210 (1.22), 1.232 (1.69), 1.268 (1.42), 1.287 (1.42), 1.427 (0.54), 1.441 (0.68), 1.462 (0.54), 1.547 (0.54), 1.560 (0.68), 1.580 (0.54), 1.677 (0.47), 1.691 (0.68), 1.712 (0.74), 1.725 (0.61), 1.907 (1.28), 1.919 (0.68), 2.207 (0.95), 2.225 (1.42), 2.244 (1.62), 2.266 (0.81), 2.336 (1.35), 2.518 (16.00), 2.522 (10.53), 2.539 (0.68), 2.673 (2.97), 2.678 (1.35), 2.727 (0.61), 2.888 (1.69), 2.978 (4.66), 2.999 (5.00), 3.016 (4.73), 3.107 (1.22), 3.121 (1.28), 3.141 (1.08), 3.155 (1.01), 3.559 (2.90), 3.583 (2.77), 3.979 (0.47), 3.999 (0.95), 4.012 (1.01), 4.032 (0.47), 4.065 (0.61), 4.078 (0.68), 4.086 (1.15), 4.098 (1.15), 4.106 (0.61), 4.119 (0.54), 4.552 (0.47), 4.572 (0.95), 4.586 (1.08), 4.606 (0.47), 4.751 (0.88), 4.792 (1.76), 4.829 (1.82), 4.870 (0.95), 6.271 (1.35), 6.291 (1.28), 6.316 (1.42),  

6.337 (1.35), 6.584 (3.11), 6.590 (2.97), 7.376 (1.49), 7.397 (1.62), 7.437 (0.47), 7.449 (1.69), 7.452 (2.57), 7.463 (2.97), 7.471 (2.70), 7.476 (1.96), 7.488 (0.54), 7.597 (3.04), 7.602 (2.90), 7.642 (1.22), 7.663 (4.93), 7.684 (3.85), 7.791 (2.63), 7.811 (3.17), 7.821 (1.35), 7.837 (1.42), 7.845 (1.15), 7.860 (1.28), 8.097 (1.08), 8.133 (1.62), 8.443 (1.22), 8.463 (1.22), 10.209 (1.42).  Example 28-B- ²²⁷ Th

[ ²²⁷ Th]Thorium (4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1-{3- [4-(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)phenyl]-1H-pyrazol-1-yl}-3,6,12,14-tetraaza heptadecane- 11,15,17-tricarboxylate 

(4S,11S,15S)-4-[(naphthalen-2-yl)methyl]-2,5,13-trioxo-1-[3- (4-{2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetamido}phenyl)-1H-pyra zol-1-yl]-3,6,12,14- tetraazaheptadecane-11,15,17-tricarboxylic acid was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 3.1 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 79.2% by iTLC.  #29 Linker Intermediate 121

tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1-(4-nitrophenoxy)- 1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(naphthalen-2-yl)propanoyl ]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (40.0 mg, 80 % purity, 46.7 µmol) was solubilised in THF (1.9 ml), 4-nitrophenyl carbonochloridate (11.3 mg, 56.1 µmol) was added and the mixture was stirred for 3h at 60°C. The mixture was evaporated to give 39.7 mg (99 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.55 min; MS (ESIpos): m/z = 851 [M+H] ⁺   Intermediate 122

tri-tert-butyl (5S,12S,16S)-1-[(1r,4S)-4-{[(tert-butoxycarbonyl)amino]methy l}cyclohexyl]- 5-[(naphthalen-2-yl)methyl]-3,6,14-trioxo-2,4,7,13,15-pentaa zaoctadecane-12,16,18- tricarboxylate 

tert-butyl {[(1r,4r)-4-(aminomethyl)cyclohexyl]methyl}carbamate (13.6 mg, 56.1 µmol) was solubilised in DCM (1.5 ml), N,N-diisopropylethylamine (41 µl, 230 µmol) and tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1-(4-nitrophenoxy)- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (39.7 mg, 46.7 µmol) were added and the mixture was stirred for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 30.0 mg (85 % purity, 57 % yield) of the target compound.  

LC-MS (Method 1): R _t = 1.57 min; MS (ESIpos): m/z = 954 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.743 (0.41), 1.352 (3.00), 1.366 (7.77), 1.380 (16.00), 2.181 (0.43), 2.518 (2.02), 2.523 (1.22), 2.728 (0.43), 5.758 (10.86), 7.622 (0.41), 7.775 (0.55), 7.796 (0.51).  Example 29-A

(5S,12S,16S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-5-[(na phthalen-2-yl)methyl]-3,6,14- trioxo-2,4,7,13,15-pentaazaoctadecane-12,16,18-tricarboxylic acid 

tri-tert-butyl (5S,12S,16S)-1-[(1r,4S)-4-{[(tert-butoxycarbonyl)amino]methy l}cyclohexyl]-5- [(naphthalen-2-yl)methyl]-3,6,14-trioxo-2,4,7,13,15-pentaaza octadecane-12,16,18- tricarboxylate (29.0 mg, 30.4 µmol) was solubilised in DCM (970 µl), TFA (230 µl, 3.0 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.20 mg (90 % purity, 27 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 685 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.763 (1.11), 0.794 (3.34), 0.820 (3.41), 0.851 (1.39), 1.137 (2.78), 1.175 (0.70), 1.232 (1.32), 1.286 (1.88), 1.300 (2.02), 1.313 (2.09), 1.415 (1.32), 1.430 (1.46), 1.449 (1.11), 1.616 (1.95), 1.663 (1.11), 1.699 (2.43), 1.726 (2.02), 1.762 (1.04), 1.779 (1.11), 1.795 (0.77), 1.814 (0.56), 1.906 (0.77), 2.084 (12.52), 2.115 (1.18), 2.162 (0.42), 2.181 (0.56), 2.199 (1.18), 2.217 (1.11), 2.232 (0.83), 2.240 (0.97), 2.261 (1.74), 2.282 (0.90), 2.298 (0.90), 2.318 (1.60), 2.322 (3.13), 2.327 (4.03), 2.331 (2.99), 2.336 (1.39), 2.518 (16.00), 2.523 (10.43), 2.590 (3.69), 2.607 (3.55), 2.636 (0.77), 2.659 (1.95), 2.664 (3.48), 2.669 (5.01), 2.673 (3.62), 2.678 (1.74), 2.854 (1.25), 2.870 (2.50), 2.888 (1.95), 2.904 (2.16), 2.926 (1.32), 3.034 (1.25), 3.047 (1.46), 3.067 (1.18), 3.081 (1.04), 3.131 (0.97), 3.147 (1.32), 3.164 (1.25), 3.181 (1.11), 3.932 (1.11), 3.947 (1.53), 3.966 (1.53), 3.978 (1.18), 4.334 (0.63), 4.356 (1.25), 4.370 (1.32), 4.393 (0.63), 6.176 (0.77), 6.306 (1.18), 6.327 (1.11), 6.539 (0.97), 6.729 (0.70), 7.341 (2.02), 7.344 (2.09), 7.362 (2.23), 7.365 (2.16), 7.418 (0.70), 7.422 (0.83), 7.435 (2.16),  

7.439 (1.95), 7.448 (2.16), 7.453 (3.69), 7.459 (2.37), 7.467 (1.81), 7.471 (2.09), 7.485 (0.83), 7.489 (0.63), 7.653 (3.83), 7.781 (6.54), 7.801 (5.63), 7.838 (1.95), 7.843 (2.02), 7.860 (1.81), 8.195 (1.60).  #30 Linker Intermediate 123

tri-tert-butyl (5S,12S,16S)-1-[1-(tert-butoxycarbonyl)piperidin-4-yl]-5-[(n aphthalen-2- yl)methyl]-3,6,14-trioxo-2,4,7,13,15-pentaazaoctadecane-12,1 6,18-tricarboxylate 

tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (12 µl, 56 µmol) was solubilised in DCM (1.5 ml), N,N-diisopropylethylamine (41 µl, 230 µmol) and tri-tert-butyl (3S,10S,14S)-3-[(naphthalen- 2-yl)methyl]-1-(4-nitrophenoxy)-1,4,12-trioxo-2,5,11,13-tetr aazahexadecane-10,14,16- tricarboxylate (39.7 mg, 46.7 µmol) were added and the mixture was stirred for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 40.0 mg (80 % purity, 74 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.56 min; MS (ESIpos): m/z = 926 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.350 (1.71), 1.375 (10.29), 1.379 (16.00), 2.327 (0.69), 2.331 (0.46), 2.518 (2.63), 2.523 (1.71), 2.669 (0.69), 2.673 (0.46), 3.321 (0.46), 7.450 (0.46), 7.780 (0.46), 7.800 (0.46).  Example 30-A

(5S,12S,16S)-5-[(naphthalen-2-yl)methyl]-3,6,14-trioxo-1- (piperidin-4-yl)-2,4,7,13,15- pentaazaoctadecane-12,16,18-tricarboxylic acid 

 

tri-tert-butyl (5S,12S,16S)-1-[1-(tert-butoxycarbonyl)piperidin-4-yl]-5-[(n aphthalen-2-yl)methyl]- 3,6,14-trioxo-2,4,7,13,15-pentaazaoctadecane-12,16,18-tricar boxylate (39.0 mg, 80 % purity, 33.7 µmol) was solubilised in DCM (1.1 ml), TFA (260 µl, 3.4 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 3.20 mg (90 % purity, 13 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.69 min; MS (ESIpos): m/z = 657 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.137 (4.47), 1.204 (1.05), 1.232 (2.45), 1.270 (2.72), 1.302 (4.26), 1.319 (4.33), 1.433 (1.19), 1.449 (1.40), 1.467 (1.47), 1.488 (1.26), 1.531 (2.03), 1.573 (2.31), 1.611 (1.33), 1.657 (1.12), 1.676 (1.12), 1.741 (1.40), 1.760 (3.63), 1.779 (3.84), 1.797 (1.61), 1.905 (0.98), 2.084 (0.70), 2.115 (1.96), 2.188 (0.49), 2.205 (1.05), 2.226 (1.82), 2.243 (3.07), 2.252 (1.82), 2.261 (1.47), 2.273 (2.86), 2.293 (1.47), 2.311 (1.12), 2.318 (1.54), 2.322 (3.07), 2.326 (4.33), 2.332 (3.35), 2.336 (1.47), 2.518 (16.00), 2.522 (10.62), 2.539 (0.98), 2.647 (0.91), 2.660 (2.31), 2.664 (4.26), 2.668 (6.01), 2.673 (4.96), 2.678 (3.21), 2.700 (2.10), 2.730 (1.96), 2.751 (1.26), 2.765 (1.61), 2.821 (0.98), 2.834 (1.19), 2.853 (1.33), 2.869 (2.17), 2.892 (1.82), 2.903 (2.59), 2.925 (2.86), 2.938 (1.54), 2.955 (1.19), 3.048 (1.96), 3.061 (2.31), 3.081 (1.82), 3.095 (1.68), 3.160 (3.14), 3.178 (3.35), 3.193 (3.00), 3.211 (2.52), 3.227 (2.38), 3.915 (1.89), 3.932 (1.89), 3.973 (0.91), 3.994 (1.82), 4.004 (1.82), 4.025 (0.91), 4.338 (1.05), 4.352 (1.26), 4.360 (2.03), 4.374 (2.03), 4.396 (0.98), 6.139 (1.33), 6.157 (1.33), 6.301 (2.03), 6.321 (1.96), 6.919 (1.33), 7.055 (1.05), 7.352 (3.14), 7.355 (3.21), 7.373 (3.35), 7.376 (3.42), 7.417 (0.91), 7.421 (1.19), 7.434 (3.21), 7.438 (3.07), 7.445 (3.28), 7.451 (6.64), 7.457 (3.56), 7.464 (3.07), 7.468 (3.21), 7.482 (1.19), 7.485 (0.91), 7.665 (5.94), 7.782 (9.08), 7.804 (9.71), 7.816 (1.75), 7.836 (2.93), 7.841 (3.00), 7.859 (2.86), 8.198 (2.24).  #31 Linker Intermediate 124

 

tri-tert-butyl (5S,12S,16S)-5-[(5-bromopyridin-2-yl)methyl]-1-(9H-fluoren-9 -yl)-3,6,14- trioxo-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarbox ylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (300 mg, 95 % purity, 584 µmol) and 3-(5-bromopyridin-2-yl)-N-{[(9H-fluoren-9-yl)methoxy]carbony l}-L- alanine (410 mg, 877 µmol; CAS-RN:[282734-37-6]) were solubilised in DMF (4.5 ml), 4- methylmorpholine (190 µl, 1.8 mmol, CAS-RN: 109-02-4) and HATU (333 mg, 877 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 254 mg (80 % purity, 37 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.62 min; MS (ESIpos): m/z = 938 [M+H] ⁺   Intermediate 125

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(5-bromopyridin-2-yl)propa noyl]amino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-5-[(5-bromopyridin-2-yl)methyl]-1-(9H-fluoren-9 -yl)-3,6,14-trioxo-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (254 mg, 80 % purity, 217 µmol) was  

solubilised in DMF (5.7 ml), piperidine (430 µl, 4.3 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 38.0 mg (94 % purity, 23 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.09 min; MS (ESIpos): m/z = 717 [M+H] ⁺   Intermediate 126

tri-tert-butyl (3S,10S,14S)-3-[(5-bromopyridin-2-yl)methyl]-1-{(1r,4S)-4-[( {[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

N N

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(5-bromopyridin-2-yl)propa noyl]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (38.0 mg, 94 % purity, 50.0 µmol) and (1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (28.4 mg, 75.0 µmol) were solubilised in DMF (380 µl), 4-methylmorpholine (16 µl, 150 µmol, CAS-RN: 109-02-4) and HATU (28.5 mg, 75.0 µmol) were added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 27.0 mg (95 % purity, 48 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.60 min; MS (ESIpos): m/z = 1077 [M+H] ⁺   Intermediate 127  

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(5-bro mopyridin-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

tri-tert-butyl (3S,10S,14S)-3-[(5-bromopyridin-2-yl)methyl]-1-{(1r,4S)-4-[( {[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylate (27.0 mg, 90 % purity, 22.6 µmol) was solubilised in DMF (590 µl), piperidine (45 µl, 450 µmol) was added and the mixture was stirred under argon at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 23.0 mg (27 % purity, 32 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.14 min; MS (ESIpos): m/z = 856 [M+H] ⁺   Example 31-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (5-bromopyridin-2-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(5-bro mopyridin-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (23.0 mg, 27 % purity, 7.27 µmol) was solubilised in DCM (500 µl), TFA (500 µl, 6.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.80 mg (85 % purity, 48 % yield) of the target compound.

LC-MS (Method 2): R _t = 0.60 min; MS (ESIpos): m/z = 687 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.850 (0.75), 0.913 (0.54), 1.107 (11.59), 1.165 (0.47), 1.232 (3.05), 1.333 (0.75), 1.352 (0.68), 1.437 (0.61), 1.574 (0.54), 1.670 (0.54), 2.056 (0.41), 2.331 (2.92), 2.336 (1.29), 2.518 (16.00), 2.523 (9.97), 2.539 (1.02), 2.593 (0.88), 2.612 (0.88), 2.669 (4.07), 2.673 (2.92), 2.678 (1.36), 2.889 (0.68), 2.915 (0.61), 2.926 (0.68), 2.948 (0.61), 3.073 (0.68), 3.093 (0.75), 3.108 (0.61), 3.804 (0.54), 3.934 (0.68), 6.400 (0.47), 6.421 (0.41), 7.222 (1.08), 7.242 (1.08), 7.798 (0.61), 7.918 (0.88), 7.924 (0.88), 7.938 (0.81), 7.945 (0.81), 8.089 (0.54), 8.112 (0.54), 8.571 (1.29), 8.576 (1.29).  #32 Linker Intermediate 128

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-3-yl)prop-2- enoate 

0

1,8-Diazabicyclo(5.4.0)undec-7-ene (5.7 ml, 38 mmol) was added dropwise to a solution of methyl (benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (12.6 g, 38.2 mmol) in DCM

(120 ml). After stirring at rt for 10 min, a solution of quinoline-3-carbaldehyde (5.00 g, 31.8 mmol) in DCM (30 ml) was added. The reaction was stirred at rt for 2 h. The reaction solvent was removed under reduced pressure. The residue was diluted with EtOAc. The solution was washed with 1M HCl and brine. The organic phase was dried and filtered. The filtrate was concentrated under reduced pressure and purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-50%) to give 5.62 g (96 % purity, 47 % yield) of the target compound.

1H NMR (400 MHz, CDCl ₃ ): d [ppm] = 9.03 (d, 1H), 8.21 (s, 1H), 8.08 (d, 1H), 7.78-7.66 (m, 2H), 7.59-7.51 (m, 1H), 7.50 (s, 1H), 7.39-7.23 (m, 5H), 6.75 ( s, 1H), 5.10 (s, 2H), 3.89 (s, 3H).  

  Intermediate 129

methyl N-[(benzyloxy)carbonyl]-3-quinolin-3-yl-D-alaninate 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-3-yl)prop-2- enoate (4.12 g, 96 % purity, 10.9 mmol) in MeOH (200 ml) was added (R)-[Rh(COD)(MaxPHOS)]BF4 (388 mg, 655 µmol). The reaction mixture was stirred at rt for 40 h under hydrogen atmosphere (2.5 MPa). The reaction mixture was evaporated under reduced pressure. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 20%-50%) to give 3.78 g (92 % purity, 87 % yield) of the target compound. Intermediate 130

N-[(benzyloxy)carbonyl]-3-quinolin-3-yl-D-alanine 

To a solution of methyl N-[(benzyloxy)carbonyl]-3-quinolin-3-yl-D-alaninate (3.54 g, 9.71 mmol) in pyridine (70 ml) was added lithium iodide (15 ml, 29 mmol; CAS-RN:[10377-51-2]) at rt. The reaction mixture was refluxed for 16 h. The reaction solution was concentrated under reduced pressure and dissolved in water. The pH of the mixture was adjusted to 5 with HCl (1 M). The mixture was extracted with EtOAc. The organic phase was washed with brine, dried, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (C18,

 

acetonitrile/water with 0.1% formic acid) to give 1.54 g (98 % purity, 44 % yield) of the target compound.

LC-MS (Method D): R _t = 0.599 min; MS (ESIpos): m/z = 351.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 12.87 (s, 1H), 8.83 (d, 1H), 8.20 (d, 1H), 8.01 (d, 1H), 7.90 (d, 1H), 7.79 (d, 1H), 7.81-7.75 (m, 1H), 7.64-7.56 (m, 1H), 7.31-7.16 (m, 5H), 4.95 (s, 2H), 4.44-4.27 (m, 1H), 3.33-3.28 (m, 1H), 3.07 (dd, 1H).

Intermediate 131

tri-tert-butyl (5R,12S,16S)-3,6,14-trioxo-1-phenyl-5-[(quinolin-3-yl)methyl ]-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (600 mg, 1.23 mmol) and N-[(benzyloxy)carbonyl]-3-quinolin-3-yl-D-alanine (431 mg, 1.23 mmol) were solubilised in DMF (9.5 ml), 4-methylmorpholine (340 µl, 3.1 mmol, CAS-RN: 109-02-4) and HATU (655 mg, 1.72 mmol) were added and the mixture was stirred under argon overnight at rt.

The residue was diluted with DCM/propan-2-ol, washed with water and brine. The organic layer was dried and evaporated. The residue was purified by flash chromatography (SiO2, DCM/Ethanol gradient 0%-5%) to give 470 mg (99 % purity, 46 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.43 min; MS (ESIpos): m/z = 821 [M+H] ⁺   Intermediate 132

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(quinolin-3-yl)propanoyl]a mino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

 

tri-tert-butyl (5R,12S,16S)-3,6,14-trioxo-1-phenyl-5-[(quinolin-3-yl)methyl ]-2-oxa-4,7,13,15- tetraazaoctadecane-12,16,18-tricarboxylate (470 mg, 573 µmol) was solubilised in MeOH (4.6 ml), palladium on carbon (61.0 mg, 10 % purity, 57.3 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 2h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 380 mg (90 % purity, 87 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.13 min; MS (ESIpos): m/z = 687 [M+H] ⁺   Intermediate 133

tri-tert-butyl (3R,10S,14S)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-1,4,12-trioxo -3-[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(quinolin-3-yl)propanoyl]a mino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (330 mg, 481 µmol) and 5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridine-2-carboxylic acid (164 mg, 437 µmol) were solubilised in DMF (2 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU

(233 mg, 612 µmol) were added and the mixture was stirred under argon for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 340 mg (88 % purity, 60 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.53 min; MS (ESIpos): m/z = 1043 [M+H] ⁺   Intermediate 134

tri-tert-butyl (3R,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 -[(quinolin-3- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate 

tri-tert-butyl (3R,10S,14S)-1-{5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amin o)methyl]pyridin-2- yl}-1,4,12-trioxo-3-[(quinolin-3-yl)methyl]-2,5,11,13-tetraa zahexadecane-10,14,16- tricarboxylate (340 mg, 326 µmol) was solubilised in DMF (5.0 ml), piperidine (650 µl, 6.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 115 mg (99 % purity, 43 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.08 min; MS (ESIpos): m/z = 821 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.362 (0.88), 1.373 (7.59), 1.379 (16.00), 2.518 (0.98), 2.522 (0.63), 3.873 (0.83), 7.907 (0.47), 8.728 (0.48), 8.734 (0.53).  Example 32-A

 

N ⁶ -{N-[5-(aminomethyl)pyridine-2-carbonyl]-3-(quinolin-3 -yl)-L-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine   

tri-tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 -[(quinolin-3- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate (50.0 mg, 85 % purity, 51.8 µmol) was solubilised in DCM (1.7 ml), TFA (800 µl, 10 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.50 mg (99 % purity, 16 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.53 min; MS (ESIpos): m/z = 653 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (1.13), 1.288 (1.57), 1.326 (0.94), 1.342 (0.88), 1.392 (1.26), 1.410 (1.13), 1.422 (1.13), 1.443 (0.94), 1.459 (0.76), 1.605 (0.88), 1.658 (0.57), 1.671 (0.76), 1.691 (0.94), 1.706 (0.76), 1.727 (0.63), 1.747 (0.94), 1.764 (1.13), 1.781 (0.76), 1.798 (0.57), 1.906 (0.44), 2.074 (4.98), 2.168 (1.20), 2.186 (1.95), 2.206 (2.27), 2.227 (1.01), 2.244 (0.63), 2.331 (2.77), 2.336 (1.26), 2.518 (16.00), 2.522 (9.95), 2.673 (2.83), 2.678 (1.32), 2.686 (1.64), 3.070 (2.46), 3.084 (2.46), 3.226 (1.89), 3.247 (2.27), 3.259 (3.21), 3.281 (3.65), 3.301 (3.91), 3.312 (4.35), 3.333 (3.72), 3.345 (3.46), 3.944 (1.32), 3.959 (1.76), 3.978 (1.76), 3.990 (1.45), 4.011 (0.94), 4.032 (4.60), 4.799 (0.69), 4.813 (0.82), 4.821 (1.26), 4.835 (1.32), 4.856 (0.63), 6.124 (0.94), 6.140 (0.94), 6.411 (1.76), 6.431 (1.70), 7.523 (1.13), 7.526 (1.13), 7.543 (2.46), 7.560 (1.57), 7.563 (1.57), 7.656 (1.39), 7.659 (1.45), 7.673 (1.26), 7.677 (2.46), 7.681 (1.76), 7.694 (1.32), 7.698 (1.32), 7.835 (2.27), 7.854 (1.95), 7.909 (1.89), 7.927 (4.91), 7.945 (2.65), 7.952 (2.90), 7.957 (2.39), 7.973 (1.07), 7.978 (1.07), 8.111 (3.02), 8.115 (3.09), 8.172 (1.95), 8.205 (0.88), 8.219 (1.76), 8.232 (0.88), 8.656 (3.09), 8.731 (4.28), 8.736 (4.16), 8.903 (2.14), 8.925 (2.02).  Intermediate 135

 

tri-tert-butyl (3R,10S,14S)-1,4,12-trioxo-3-[(quinolin-3-yl)methyl]-1-[5-({ 2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)pyridin-2- yl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (249 mg, 435 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (163 mg, 430 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (53 µl, 330 µmol) were stirred in DMF (4.2 ml) for 10min at rt. tri- tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 -[(quinolin-3-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (105 mg, 85 % purity, 109 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 45.0 mg (30 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.33 min; MS (ESIpos): m/z = 1375 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.41), 1.322 (1.44), 1.374 (5.64), 1.379 (16.00), 1.392 (2.09), 1.435 (1.52), 2.518 (4.14), 2.523 (2.69), 2.673 (0.78), 2.687 (0.46), 2.888 (0.41). Example 32-B

N ⁶ -{3-(quinolin-3-yl)-N-[5-({2-[4,7,10-tris(carboxymethy l)-1,4,7,10-tetraazacyclododecan- 1-yl]acetamido}methyl)pyridine-2-carbonyl]-D-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3R,10S,14S)-1,4,12-trioxo-3-[(quinolin-3-yl)methyl]-1-[5-({ 2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}methyl)pyridin-2-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (20.0 mg, 14.5 µmol) was solubilised in DCM (470 µl), TFA (220 µl, 2.9 mmol) was added and the mixture was stirred under argon over the weekend at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 1.50 mg (97 % purity, 10 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (1.82), 1.316 (0.63), 1.334 (0.77), 1.352 (0.84), 1.455 (0.49), 1.470 (0.56), 1.590 (0.49), 1.694 (0.42), 1.715 (0.56), 1.731 (0.49), 1.871 (0.49), 2.084 (1.12), 2.205 (0.91), 2.226 (1.19), 2.233 (1.19), 2.254 (0.70), 2.327 (3.91), 2.331 (2.93), 2.336 (1.40), 2.518 (16.00), 2.523 (9.99), 2.589 (1.26), 2.669 (4.19), 2.673 (3.07), 2.678 (1.54), 2.915 (1.68), 3.037 (3.21), 3.103 (1.89), 3.995 (0.77), 4.008 (0.84), 4.028 (0.49), 4.050 (0.49), 4.070 (0.77), 4.084 (0.77), 4.104 (0.42), 4.389 (1.19), 4.805 (0.63), 4.821 (0.63), 6.300 (1.33), 6.320 (1.33), 7.529 (0.70), 7.547 (1.40), 7.564 (0.84), 7.661 (0.84), 7.664 (0.84), 7.682 (1.40), 7.699 (0.70), 7.845 (2.03), 7.863 (1.47), 7.938 (2.03), 7.959 (1.68), 8.111 (1.82), 8.243 (0.56), 8.629 (0.77), 8.743 (2.31), 8.748 (2.24), 8.771 (1.12), 8.791 (1.26).

Example 32-B 227Th

 

{N ⁶ -[3-(quinolin-3-yl)-N-{5-[(2-{4,7,10-tris[(carboxy-kap paO)methyl]-1,4,7,10- tetraazacyclododecan-1-yl-kappa ² N ¹ ,N ⁴ }acetamido)methyl]pyridine-2-carbonyl}-D- alanyl]-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(3-)} ( ²²⁷ Th)thorium

N ⁶ -{3-(quinolin-3-yl)-N-[5-({2-[4,7,10-tris(carboxymethy l)-1,4,7,10-tetraazacyclododecan-1- yl]acetamido}methyl)pyridine-2-carbonyl]-D-alanyl}-N2-{[(1S) -1,3-dicarboxypropyl]carbamoyl}- L-lysine was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 98,2% by iTLC.

 

  #33 Linker Intermediate 136

methyl 1-methoxynaphthalene-2-carboxylate 

 

To a solution of 1-hydroxynaphthalene-2-carboxylic acid (10.0 g, 53.1 mmol) and potassium carbonate (22.0 g, 159 mmol) in acetone (200 ml) was added iodomethane (13 ml, 210 mmol) at room temperature. The reaction mixture was refluxed for 16 hours. The mixture was filtered through a pad of celite and the filtrate was concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 100: 1 to 50:1) to give methyl 1-methoxy-2-naphthoate (5.00 g, 44% yield) as yellow oil. 

LC-MS (Method C): R _t = 0.930 min; MS (ESIpos): m/z = 217.1 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 8.19 (d, 1H), 7.99 (d, 1H), 7.78-7.50 (m, 2H), 7.69- 7.62 (m, 2H), 3.97 (s, 3H), 3.90 (s, 3H). Intermediate 137

(1-methoxynaphthalen-2-yl)methanol 

To a solution of methyl 1-methoxynaphthalene-2-carboxylate (14.0 g, 64.7 mmol) in toluene (200 ml) was added diisobutylaluminum hydride (97 ml, 1.0 M in toluene, 97 mmol; CAS-RN:[1191- 15-7]) at -40 °C. The mixture was stirred at 0 °C for 1 h. The mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The organic phase was washed with brine, dried and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-50%) to give 12.0 g (98 % yield) of the target compound. Intermediate 138

1-methoxynaphthalene-2-carbaldehyde 

 

To a solution of morpholine (1.7 ml, 19 mmol) in tetrahydrofuran (40 ml) was added diisobutylaluminum hydride (1M in toluene) at 0 °C. The mixture was stirred at 0 °C for 3 hours.

A solution of methyl 1-methoxynaphthalene-2-carboxylate (2.00 g, 9.25 mmol) in tetrahydrofuran (5 ml) was added into the above mixture. The reaction mixture was stirred for 10 minutes.

Diisobutylaluminum hydride (1M in toluene) was added. After stirring for 0.5 hour, the mixture was quenched with hydrochloric acid (1M) and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether:

ethyl acetate = 50: 1 to 10: 1) to give 1-methoxy-2-naphthaldehyde (600 mg, 35% yield) as yellow oil.

LC-MS (Method D): R _t = 0.769 min; MS (ESIpos): m/z = 187.1 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 10.5 (s, 1H), 8.26 (d, 1H), 8.03 (d, 1H), 7.81-7.68 (m, 4H), 4.12 (s, 3H).

Intermediate 139

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(1-methoxynaphthalen-2 -yl)prop-2-enoate 

To a solution of methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (1.28 g, 3.87 mmol) in dichloromethane (10 ml) was added 1,8-diazabicyclo(5.4.0)undec-7-ene (0.580 ml, 3.9 mmol) at room temperature. After stirring for 0.5 hour, a solution of 1-methoxynaphthalene-2- carbaldehyde (600 mg, 3.22 mmol) in dichloromethane (5 ml) was added into above mixture.

The reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether:

ethyl acetate = 10: 1 to 3: 1) to give methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(1-methoxy- 2-naphthyl)acrylate (1.10 g, 87% yield) as brown oil.  

LC-MS (Method D): R _t = 0.899 min; MS (ESIpos): m/z = 392.1 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.19 (s, 1H), 8.11-8.09 (m, 1H), 7.96-7.93 (m, 1H), 7.75-7.67 (m, 1H), 7.61-7.60 (m, 1H), 7.60-7.58 (m, 3H), 7.34 (br.s, 5H), 5.08 (s, 2H), 3.87 (s, 3H), 3.76 (s, 3H). Intermediate 140

methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(1-methoxynaphthalen-2 -yl)propanoate 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(1-methoxynaphthalen-2 -yl)prop- 2-enoate (8.9 g, 22.7 mmol) in methanol (200 ml) were added (R)-[Rh(COD)(MaxPhos)]BF ₄ (270 mg, 0.455 mmol). After stirring at room temperature for 16 hours under hydrogen atmosphere (15 psi), the mixture was concentrated to give a residue. The residue was purified by column chromatography (200 - 300 mesh, ethyl acetate) to give methyl (2S)-2- {[(benzyloxy)carbonyl]amino}-3-(1-methoxy-2-naphthyl) propanoate (8.5 g, 95% yield) as a yellow oil.

LC-MS (Method D): R _t = 0.886 min; MS (ESIpos): m/z = 350.1 [M+H] ⁺ . Intermediate 141

(2R)-2-{[(benzyloxy)carbonyl]amino}-3-(1-methoxynaphthale n-2-yl)propanoic acid 

 

To a solution of methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(1-methoxynaphthalen-2 - yl)propanoate (8.50 g, 21.6 mmol) in tetrahydrofuran (100 ml) was added lithium hydroxide (16 ml, 2M in water) at room temperature. The reaction mixture was stirred at 25 °C for 1 hour. The pH of the mixture was adjusted to 5 with hydrochloric acid (1M). The mixture was extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue. The residue was purified by preparative HPLC

(Instrument: Shimadzu LC-20AP; Column: Phenomenex luna C18250*50mm*10 µm; eluent A:

0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-28 min 40-70% B; flow 100 ml/min;

temperature: room temperature; Detector: UV 220/254 nm) to give (2S)-2- {[(benzyloxy)carbonyl]amino}-3-(1-methoxy-2-naphthyl)propano ic acid (4.98 g, 99% purity, 60% yield) as a white solid.

LC-MS (Method D): R _t = 0.914 min; MS (ESIpos): m/z = 402.1 [M+23] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 8.02 (d, 1H), 7.92 (d, 1H), 7.70 (d, 1H), 7.63 (d, 2H), 7.62-7.61 (m, 2H), 7.43 (d, 1H), 7.35-7.07 (m, 5H), 4.98 (s, 2H), 4.40-4.34 (m, 2H), 3.89 (s, 3H), 3.39-3.34 (m, 1H), 3.02-2.96 (m, 1H),. Intermediate 142

tri-tert-butyl (5R,12S,16S)-5-[(1-methoxynaphthalen-2-yl)methyl]-3,6,14-tri oxo-1-phenyl- 2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (918 mg, 1.88 mmol) and (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(1-methoxynaphthalen-2 -yl)propanoic acid (750 mg, 1.98 mmol) were solubilised in DMF (14 ml), 4-methylmorpholine (620 µl, 5.6 mmol, CAS-RN: 109-02-4) and HATU (859 mg, 2.26 mmol) were added and the mixture was stirred under argon for 2h at rt. The residue was diluted with DCM/propan-2-ol, washed with water and brine. The organic layer was dried and evaporated. The residue was purified by

 

preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 380 mg (95 % purity, 23 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.57 min; MS (ESIpos): m/z = 850 [M+H] ⁺   Intermediate 143

di-tert-butyl (2S)-2-({[(2R)-6-{[(2S)-2-amino-3-(1-methoxynaphthalen-2- yl)propanoyl]amino}-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl} amino)pentanedioate 

N _'

tri-tert-butyl (5R,12S,16S)-5-[(1-methoxynaphthalen-2-yl)methyl]-3,6,14-tri oxo-1-phenyl-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (380 mg, 448 µmol) was solubilised in MeOH (2.7 ml), Palladium on carbon (47.6 mg, 10 % purity, 44.8 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred at rt under hydrogen atmosphere.

The mixture was filtered over Celite, washed with MeOH and DCM and evaporated to give 228 mg (97 % purity, 69 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.25 min; MS (ESIpos): m/z = 716 [M+H] ⁺   Intermediate 144

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]- 3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13 -tetraazahexadecane- 10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(1-methoxynaphthalen-2-yl) propanoyl]amino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (228 mg, 319 µmol) and (1r,4r)-4- ({[(benzyloxy)carbonyl]amino}methyl)cyclohexane-1-carboxylic acid (92.9 mg, 319 µmol) were solubilised in DMF (4.9 ml), 4-methylmorpholine (88 µl, 800 µmol, CAS-RN: 109-02-4) and HATU

(146 mg, 383 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 95.0 mg (100 % purity, 30 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.57 min; MS (ESIpos): m/z = 989 [M+H] ⁺   Intermediate 145

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1- methoxynaphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetra azahexadecane-10,14,16- tricarboxylate 

 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]-3-[(1- methoxynaphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetra azahexadecane-10,14,16- tricarboxylate (100 mg, 101 µmol) was solubilised in MeOH (200 µl) and THF (410 µl), Palladium on carbon (10.8 mg, 10 % purity, 10.1 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 2h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 110 mg (70 % purity, 89 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.22 min; MS (ESIpos): m/z = 855 [M+H] ⁺   Example 33-A

(3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1- methoxynaphthalen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylic acid 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1-met hoxynaphthalen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (50.0 mg, 58.5 µmol) was stirred in TFA (900 µl, 12 mmol) under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 14.0 mg (99 % purity, 35 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.74 min; MS (ESIpos): m/z = 687 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.847 (2.04), 0.878 (2.25), 0.910 (0.98), 1.081 (0.56), 1.113 (1.26), 1.146 (1.40), 1.183 (1.54), 1.230 (2.81), 1.258 (2.95), 1.302 (1.89), 1.317 (1.82), 1.337 (1.75), 1.353 (1.89), 1.369 (1.61), 1.401 (2.04), 1.422 (2.11), 1.433 (2.04), 1.511 (1.61), 1.541 (1.40), 1.608 (2.18), 1.630 (2.53), 1.642 (2.81), 1.680 (2.60), 1.714 (1.33), 1.774 (2.25), 1.795 (2.25), 2.034 (0.91), 2.063 (1.68), 2.074 (3.23), 2.094 (0.91), 2.125 (0.63), 2.145 (0.84), 2.161 (1.68), 2.180 (1.61), 2.193 (1.12), 2.214 (1.19), 2.236 (2.04), 2.258 (1.19), 2.272 (1.12),  

2.294 (0.56), 2.323 (3.09), 2.327 (4.28), 2.332 (3.23), 2.522 (16.00), 2.556 (1.75), 2.569 (2.88), 2.587 (4.35), 2.603 (2.67), 2.618 (1.12), 2.635 (0.77), 2.665 (3.23), 2.669 (4.35), 2.673 (3.30), 2.910 (1.47), 2.935 (2.39), 2.945 (2.60), 2.969 (3.02), 3.077 (1.47), 3.093 (1.82), 3.110 (1.61), 3.174 (2.53), 3.187 (2.88), 3.208 (2.88), 3.221 (2.95), 3.335 (7.23), 3.934 (3.30), 3.946 (2.53), 4.509 (0.91), 4.532 (1.75), 4.545 (1.75), 4.568 (0.91), 6.119 (1.26), 6.444 (1.05), 7.369 (4.14), 7.391 (4.84), 7.454 (1.47), 7.471 (3.30), 7.487 (2.67), 7.491 (2.67), 7.502 (2.53), 7.506 (2.60), 7.523 (3.16), 7.540 (1.47), 7.575 (4.70), 7.596 (3.72), 7.822 (1.12), 7.867 (3.86), 7.886 (3.44), 7.988 (3.72), 8.008 (3.37), 8.189 (0.77), 8.222 (1.47).  Intermediate 146

tri-tert-butyl (3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12-tri oxo-1-[(1r,4S)- 4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraa zacyclododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

j

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (207 mg, 361 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (135 mg, 356 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (46 µl, 270 µmol) were stirred in DMF (1.4 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1-met hoxynaphthalen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (110 mg, 70 % purity, 90.2 µmol) was added and the mixture was stirred at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 155.0 mg (80 % purity, 98 % yield) of the target compound.

 

LC-MS (Method 1): Rt = 1.28 min; MS (ESIpos): m/z = 1410 [M+H] ⁺   Example 33-B

(3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12- trioxo-1-[(1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12-tri oxo-1-[(1r,4S)-4-({2- [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate (195 mg, 138 µmol) was solubilised in DCM (450 µl), TFA (210 µl, 2.8 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 37.0 mg (100 % purity, 25 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.73 min; MS (ESIneg): m/z = 1071 [M-H]- 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.802 (0.86), 0.833 (1.04), 0.852 (0.52), 1.116 (0.52), 1.145 (0.52), 1.237 (2.11), 1.301 (0.91), 1.317 (1.29), 1.333 (1.25), 1.352 (1.08), 1.413 (0.39), 1.432 (0.52), 1.449 (0.56), 1.468 (0.47), 1.510 (0.60), 1.541 (0.65), 1.600 (0.91), 1.634 (1.16), 1.663 (1.12), 1.699 (0.99), 1.718 (0.78), 1.735 (0.52), 1.885 (0.52), 1.902 (0.56), 2.049 (0.65), 2.075 (0.78), 2.085 (0.69), 2.208 (0.86), 2.223 (1.34), 2.245 (1.29), 2.262 (0.69), 2.318 (1.04), 2.323 (1.98), 2.327 (2.72), 2.332 (1.90), 2.336 (0.91), 2.456 (0.52), 2.518 (9.75), 2.523 (6.68), 2.649 (2.11), 2.660 (2.54), 2.665 (3.28), 2.669 (3.71), 2.673 (2.63), 2.678 (1.42), 2.907 (2.03), 2.979 (5.05), 2.997 (4.40), 3.097 (2.20), 3.182 (1.25), 3.203 (1.29), 3.216 (1.34), 3.339 (3.58), 3.880 (16.00), 3.957 (0.43), 3.976 (0.82), 3.989 (0.86), 4.008 (0.43), 4.073 (0.78), 4.087 (0.78), 4.107 (0.39), 4.517 (0.73), 4.531 (0.73), 6.295 (0.95), 6.314 (1.55), 6.335 (0.91), 7.359 (1.94), 7.380 (2.24), 7.451 (0.65), 7.468 (1.51), 7.488 (1.21), 7.504 (1.21), 7.522 (1.42), 7.539 (0.69),  

7.578 (1.94), 7.600 (1.60), 7.867 (2.37), 7.886 (1.94), 7.989 (1.77), 8.010 (1.73), 8.075 (0.95), 8.138 (0.47).

Example 33-B 227Th

(3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12- trioxo-1-{5-[(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ² N ¹ ,N ⁴ }acetamido)methyl]pyridin-2-yl}-2,5,11,13-tetraazahexa decane-10,14,16- tricarboxylato(3-)( ²²⁷ Th)thorium

 (3S,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12-t rioxo-1-[(1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 96,3% by iTLC. 

  Intermediate 147

tri-tert-butyl (3R,10S,14S)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-3-[(1-methoxy naphthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate   

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(1-methoxynaphthalen-2-yl) propanoyl]amino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (295 mg, 413 µmol) and 5-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]pyridine-2-carbox ylic acid (154 mg, 413 µmol) were solubilised in DMF (6.3 ml), 4-methylmorpholine (110 µl, 1.0 mmol, CAS-RN: 109-02-4) and HATU (188 mg, 495 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 160.0 mg (80 % purity, 29 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.63 min; MS (ESIpos): m/z = 1072 [M+H] ⁺   Intermediate 148

tri-tert-butyl (3R,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(1-methoxyna phthalen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

 

tri-tert-butyl (3R,10S,14S)-1-{5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amin o)methyl]pyridin-2- yl}-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,1 1,13-tetraazahexadecane- 10,14,16-tricarboxylate (160 mg, 149 µmol) was solubilised in DMF (2.3 ml), piperidine (300 µl, 3.0 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 110 mg (100 % purity, 87 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.25 min; MS (ESIpos): m/z = 850 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.358 (0.78), 1.370 (7.12), 1.379 (16.00), 2.518 (0.58), 3.224 (0.41), 3.242 (0.42), 3.866 (1.06), 3.890 (2.63), 7.383 (0.40), 7.906 (0.81), 8.263 (0.60), 8.591 (0.41).  Intermediate 149

 

tri-tert-butyl (3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12-tri oxo-1-[5-({2- [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1- yl]acetamido}methyl)pyridin-2-yl]-2,5,11,13-tetraazahexadeca ne-10,14,16-tricarboxylate 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (108 mg, 188 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (70.6 mg, 186 µmol; CAS-RN:94790-37-1)

 

and N,N-diisopropylethylamine (23 µl, 140 µmol) were stirred in DMF (1.8 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-3-[(1-methoxyna phthalen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (40.0 mg, 47.1 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 41.0 mg (100 % purity, 62 % yield) of the target compound.

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.315 (2.41), 1.324 (2.68), 1.370 (6.86), 1.380 (16.00), 1.396 (2.77), 1.437 (2.21), 2.518 (1.96), 2.522 (1.23), 2.669 (0.49), 3.873 (1.66), 3.881 (0.61). Example 33a-B

(3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12- trioxo-1-[5-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)pyridin-2-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12-tri oxo-1-[5-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1-yl]acetamido}methyl)pyridin-2- yl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (40.0 mg, 28.5 µmol) was solubilised in DCM (920 µl), TFA (440 µl, 5.7 mmol) was added and the mixture was stirred under argon over the weekend at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 6.00 mg (98 % purity, 19 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.72 min; MS (ESIpos): m/z = 1068 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.206 (0.77), 1.225 (1.40), 1.241 (1.35), 1.259 (0.68), 1.293 (0.44), 1.309 (0.77), 1.328 (0.98), 1.347 (0.89), 1.364 (0.58), 1.442 (0.51), 1.458 (0.63),  

1.477 (0.49), 1.560 (0.44), 1.577 (0.56), 1.595 (0.54), 1.673 (0.42), 1.688 (0.56), 1.708 (0.68), 1.723 (0.51), 1.866 (0.44), 1.884 (0.61), 1.901 (0.65), 2.202 (0.95), 2.217 (1.63), 2.224 (1.21), 2.239 (1.58), 2.257 (0.84), 2.336 (0.51), 2.460 (0.58), 2.463 (0.68), 2.518 (5.22), 2.522 (3.35), 2.601 (1.84), 2.660 (0.65), 2.918 (2.68), 3.023 (5.40), 3.114 (2.82), 3.191 (1.40), 3.225 (2.47), 3.245 (3.42), 3.259 (3.21), 3.473 (4.19), 3.682 (0.42), 3.900 (16.00), 3.962 (0.54), 3.983 (0.98), 3.996 (1.00), 4.015 (0.51), 4.055 (0.56), 4.069 (0.72), 4.075 (1.09), 4.088 (1.09), 4.096 (0.68), 4.109 (0.54), 4.383 (1.82), 4.397 (1.79), 4.762 (0.47), 4.784 (0.86), 4.799 (0.91), 4.820 (0.44), 6.284 (1.44), 6.304 (2.49), 6.325 (1.35), 7.362 (2.31), 7.383 (2.63), 7.439 (0.68), 7.443 (0.70), 7.459 (1.58), 7.476 (1.28), 7.480 (1.28), 7.492 (1.21), 7.496 (1.26), 7.513 (1.54), 7.516 (1.30), 7.530 (0.75), 7.533 (0.72), 7.562 (2.35), 7.584 (1.89), 7.835 (1.68), 7.843 (1.96), 7.855 (2.28), 7.862 (1.84), 7.931 (1.23), 7.951 (0.84), 7.985 (1.68), 8.006 (1.54), 8.112 (0.58), 8.126 (1.14), 8.139 (0.79), 8.616 (2.07), 8.728 (1.26), 8.751 (1.40), 8.772 (1.09), 8.786 (0.56).

Example 33a-B-227Th

(3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,12- trioxo-1-{5-[(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ² N ¹ ,N ⁴ }acetamido)methyl]pyridin-2-yl}-2,5,11,13-tetraazahexa decane-10,14,16- tricarboxylato(3-)( ²²⁷ Th)thorium

 (3R,10S,14S)-3-[(1-methoxynaphthalen-2-yl)methyl]-1,4,1 2-trioxo-1-[5-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)pyridin-2-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375  

MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 94,4% by iTLC. 

  #34 Linker Intermediate 150

tri-tert-butyl (3S,10S,14S)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-1,4,12-trioxo -3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-2-yl)propanoyl]a mino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (83.0 mg, 121 µmol) and (2S)-5-[({[(9H-fluoren- 9-yl)methoxy]carbonyl}amino)methyl]pyridine-2-carboxylic acid (64.0 mg, 85 % purity, 145 µmol) were solubilised in DMF (940 µl), 4-methylmorpholine (79 µl, 480 µmol, CAS-RN: 109-02-4) and HATU (102 mg, 182 µmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 82.0 mg (65 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.57 min; MS (ESIpos): m/z = 1043 [M+H] ⁺ NMR:   Intermediate 151

tri-tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 -[(quinolin-2- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate 

 

tri-tert-butyl (3S,10S,14S)-1-{5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amin o)methyl]pyridin-2- yl}-1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-2,5,11,13-tetraa zahexadecane-10,14,16- tricarboxylate (80.0 mg, 76.8 µmol) was solubilised in DMF (1.2 ml), piperidine (150 µl, 1.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 37.0 mg (97 % purity, 57 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.14 min; MS (ESIpos): m/z = 821 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.368 (9.95), 1.376 (14.05), 1.379 (16.00), 2.323 (0.51), 2.327 (0.67), 2.665 (0.51), 2.669 (0.69), 3.389 (0.78), 3.405 (0.62), 3.859 (1.63), 7.399 (0.59), 7.421 (0.60), 7.898 (0.45), 7.912 (1.51), 7.914 (1.48), 7.951 (0.45), 8.227 (0.55), 8.232 (0.54), 8.248 (0.48), 8.626 (0.64).  Example 34-A

N ⁶ -{N-[5-(aminomethyl)pyridine-2-carbonyl]-3-(quinolin-2 -yl)-L-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 -[(quinolin-2- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate (10.0 mg, 12.2 µmol) was solubilised in DCM (390 µl), TFA (190 µl, 2.4 mmol) was added and the mixture was stirred  

under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 3.50 mg (98 % purity, 43 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.55 min; MS (ESIpos): m/z = 653 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.208 (1.47), 1.230 (1.88), 1.274 (0.71), 1.290 (1.18), 1.307 (1.59), 1.326 (1.41), 1.343 (0.88), 1.379 (0.65), 1.392 (0.76), 1.412 (0.76), 1.428 (0.65), 1.531 (0.88), 1.549 (0.71), 1.657 (0.41), 1.670 (0.76), 1.690 (1.00), 1.705 (0.76), 1.724 (0.71), 1.744 (1.00), 1.763 (1.12), 1.779 (0.76), 1.796 (0.59), 2.151 (0.47), 2.174 (1.24), 2.194 (1.41), 2.201 (1.29), 2.222 (2.00), 2.242 (1.00), 2.259 (0.76), 2.332 (2.53), 2.336 (1.24), 2.518 (12.18), 2.522 (7.94), 2.678 (1.29), 2.686 (0.65), 2.966 (1.12), 2.982 (1.53), 2.999 (2.06), 3.013 (2.24), 3.025 (2.29), 3.039 (2.29), 3.057 (1.94), 3.074 (1.71), 3.407 (16.00), 3.423 (15.24), 3.917 (1.24), 3.938 (2.18), 3.951 (3.00), 3.970 (2.18), 4.033 (6.47), 4.070 (0.59), 4.904 (0.76), 4.920 (1.76), 4.941 (1.71), 4.957 (0.71), 6.123 (1.06), 6.140 (1.00), 6.334 (1.88), 6.354 (1.82), 7.421 (4.24), 7.442 (4.12), 7.525 (1.29), 7.527 (1.24), 7.545 (2.53), 7.562 (1.59), 7.565 (1.53), 7.710 (1.35), 7.714 (1.35), 7.731 (2.41), 7.735 (1.65), 7.749 (1.29), 7.752 (1.29), 7.901 (2.41), 7.920 (2.24), 7.951 (8.29), 7.955 (6.06), 7.978 (2.47), 8.080 (1.00), 8.094 (1.94), 8.108 (0.94), 8.233 (3.35), 8.254 (3.12), 8.704 (3.59), 9.210 (2.47), 9.231 (2.35).  Intermediate 152 monomer

2,2'-{({3-[(2-{[1-(2-{[(6-{[(7S,11S,18S)-7,11-bis(tert-bu toxycarbonyl)-2,2-dimethyl-4,9,17- trioxo-19-(quinolin-2-yl)-3-oxa-8,10,16-triazanonadecan-18-y l]carbamoyl}pyridin-3- yl)methyl]amino}-2-oxoethyl)-3-hydroxy-6-methyl-2-oxo-1,2-di hydropyridine-4- carbonyl]amino}ethyl)(2-{[1-(carboxymethyl)-3-hydroxy-6-meth yl-2-oxopyridine-4(2H)- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy- 6-methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid 

 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (3.50 mg, 3.23 µmol) and tri-tert-butyl (3S,10S,14S)- 1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[(quinolin-2 -yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (2.65 mg, 3.23 µmol) are dissolved in NMP (610 µL). DIPEA (3.8 µL, 22 µmol) is added. PyAOP (2.1 mg, 4.04 µmol) in NMP 170 µL is added.

Reaction mix is diluted with 20% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-70% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 28 min) afforded 1.6 mg (26% yield) of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.14 min; MS (ESIpos): m/z = 1885.0  

[M+H] ⁺   Intermediate 153 dimer

2-[4-[2-[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butoxy-5-[[( 1S)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-2-oxo-1-(2- quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2-oxo -ethyl]-3-hydroxy-6- methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl-[3-[2-[[1-[2-[[ 6-[[(1S)-2-[[(5S)-6-tert-butoxy- 5-[[(1S)-4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-butyl]car bamoylamino]-6-oxo- hexyl]amino]-2-oxo-1-(2-quinolylmethyl)ethyl]carbamoyl]-3-py ridyl]methylamino]-2-oxo- ethyl]-3-hydroxy-6-methyl-2-oxo-pyridine-4-carbonyl]amino]et hyl-[2-[[1-(carboxymethyl)- 3-hydroxy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]propyl]amino]ethylcarbamoyl]-3-hy droxy-6-methyl-2-oxo-1- pyridyl]acetic acid

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (3.50 mg, 3.23 µmol) and tri-tert-butyl (3S,10S,14S)- 1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[(quinolin-2 -yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (2.65 mg, 3.23 µmol) are dissolved in NMP (610  

µL). DIPEA (3.8 µL, 22 µmol) is added. PyAOP (2.1 mg, 4.04 µmol) in NMP 170 µL is added.

Reaction mix is diluted with 20% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-70% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 35 min) afforded 1.5 mg (17% yield) of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.35 min; MS (ESIpos): m/z = 1344.0  

[M+2H] ²⁺   Intermediate 154 trimer

2-[4-[2-[3-[bis[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butox y-5-[[(1S)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-2-oxo-1-(2- quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2-oxo -ethyl]-3-hydroxy-6- methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2 -[[1-[2-[[6-[[(1S)-2-[[(5S)-6- tert-butoxy-5-[[(1S)-4-tert-butoxy-1-tert-butoxycarbonyl-4-o xo-butyl]carbamoylamino]-6- oxo-hexyl]amino]-2-oxo-1-(2-quinolylmethyl)ethyl]carbamoyl]- 3-pyridyl]methylamino]-2- oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydroxy-6-methy l-2-oxo-1-pyridyl]acetic acid

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (3.50 mg, 3.23 µmol) and tri-tert-butyl (3S,10S,14S)-  

1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[(quinoli n-2-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (2.65 mg, 3.23 µmol) are dissolved in NMP (610 µL). DIPEA (3.8 µL, 22 µmol) is added. PyAOP (2.1 mg, 4.04 µmol) in NMP 170 µL is added.

Reaction mix is diluted with 20% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-70% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 40 min) afforded 1.0 mg (9% yield) of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.90 min; MS (ESIpos): m/z = 1744.4  

[M+2H] ²⁺   Intermediate 155 tetramer

ditert-butyl (2S)-2-[[(1S)-5-[[(2S)-2-[[5-[[[2-[4-[2-[3-[bis[2-[[1-[2-[[6 -[[(1S)-2-[[(5S)-6-tert- butoxy-5-[[(1S)-4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-bu tyl]carbamoylamino]-6-oxo- hexyl]amino]-2-oxo-1-(2-quinolylmethyl)ethyl]carbamoyl]-3-py ridyl]methylamino]-2-oxo- ethyl]-3-hydroxy-6-methyl-2-oxo-pyridine-4-carbonyl]amino]et hyl]amino]propyl-[2-[[1-[2- [[6-[[(1S)-2-[[(5S)-6-tert-butoxy-5-[[(1S)-4-tert-butoxy-1-t ert-butoxycarbonyl-4-oxo- butyl]carbamoylamino]-6-oxo-hexyl]amino]-2-oxo-1-(2-quinolyl methyl)ethyl]carbamoyl]- 3-pyridyl]methylamino]-2-oxo-ethyl]-3-hydroxy-6-methyl-2-oxo -pyridine-4- carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydroxy-6-methy l-2-oxo-1- pyridyl]acetyl]amino]methyl]pyridine-2-carbonyl]amino]-3-(2- quinolyl)propanoyl]amino]- 1-tert-butoxycarbonyl-pentyl]carbamoylamino]pentanedioate

 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (3.50 mg, 3.23 µmol) and tri-tert-butyl (3S,10S,14S)- 1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[(quinolin-2 -yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (2.65 mg, 3.23 µmol) are dissolved in NMP (610 µL). DIPEA (3.8 µL, 22 µmol) is added. PyAOP (2.1 mg, 4.04 µmol) in NMP 170 µL is added.

Reaction mix is diluted with 20% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-70% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 43 min) afforded 1.1 mg (8% yield) of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 3.15 min; MS (ESIpos): m/z = 1430.4  

[M+3H] ³⁺  

  Example 34-C monomer

N ⁶ -{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-hyd roxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)am ino]ethyl}carbamoyl)-3- hydroxy-6-methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)pyri dine-2-carbonyl]-3- (quinolin-2-yl)-L-alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine   

2,2'-{({3-[(2-{[1-(2-{[(6-{[(7S,11S,18S)-7,11-bis(tert-bu toxycarbonyl)-2,2-dimethyl-4,9,17-trioxo- 19-(quinolin-2-yl)-3-oxa-8,10,16-triazanonadecan-18-yl]carba moyl}pyridin-3-yl)methyl]amino}- 2-oxoethyl)-3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4-c arbonyl]amino}ethyl)(2-{[1- (carboxymethyl)-3-hydroxy-6-methyl-2-oxopyridine-4(2H)- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid (1.60 mg, 0.849 µmol) is treated with 90% TFA in water (0.5 mL). Water (18 mL) is added and solution lyophilised.affording 1.40 mg (95 % purity, 91 % yield) of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.47 min; MS (ESIpos): m/z = 1716.7  

[M+H] ⁺   Example 34-C dimer

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)az anediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl})bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid} 

 

2-[4-[2-[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butoxy-5-[[( 1S)-4-tert-butoxy-1-tert-butoxycarbonyl-4- oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino]-2-oxo-1-(2-quin olylmethyl)ethyl]carbamoyl]-3- pyridyl]methylamino]-2-oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-p yridine-4-carbonyl]amino]ethyl- [3-[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butoxy-5-[[(1S)-4-te rt-butoxy-1-tert-butoxycarbonyl-4-oxo- butyl]carbamoylamino]-6-oxo-hexyl]amino]-2-oxo-1-(2-quinolyl methyl)ethyl]carbamoyl]-3- pyridyl]methylamino]-2-oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-p yridine-4-carbonyl]amino]ethyl- [2-[[1-(carboxymethyl)-3-hydroxy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]propyl]amino]ethylcarbamoyl]-3-hy droxy-6-methyl-2-oxo-1- pyridyl]acetic acid (1.50 mg, 0.558 µmol) is treated with 90% TFA in water (0.5 mL). Water (8 mL) is added and solution subjected to preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA;

ACN, Gradient: 10-50% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product:

24 min) affording 1.00 mg (95 % purity, 72 % yield) of the target compound. 

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.65 min; MS (ESIpos): m/z = 1175.5  

[M+2H] ²⁺   Example 34-C trimer

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2 S)-1-{[(5S)-5-carboxy-5-({[(1S)-1,3- dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(quino lin-2-yl)propan-2- yl]carbamoyl}pyridin-3-yl)methyl]amino}-2-oxoethyl)-3-hydrox y-6-methyl-2-oxopyridine- 4(2H)-carbonyl]amino}ethyl)(2-{[1-(carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}azanediy l)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl )(1-oxoethane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl]}bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid} 

 

2-[4-[2-[3-[bis[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butox y-5-[[(1S)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-2-oxo-1-(2- quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2-oxo -ethyl]-3-hydroxy-6-methyl-2-oxo- pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[[6-[ [(1S)-2-[[(5S)-6-tert-butoxy-5-[[(1S)- 4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-butyl]carbamoylami no]-6-oxo-hexyl]amino]-2-oxo-1- (2-quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2- oxo-ethyl]-3-hydroxy-6-methyl-2- oxo-pyridine-4-carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3- hydroxy-6-methyl-2-oxo-1- pyridyl]acetic acid (1.00 mg, 0.287 µmol) is treated with 90% TFA in water (0.5 mL). Water (8 mL) is added and solution subjected to preparative HPLC (Äkta pure system, Column:

Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN

Gradient: 10-50% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR: 27 min affording 500 µg (95 % purity, 56 % yield) of the target compound. 

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.78 min; MS (ESIpos): m/z = 1492.4  

[M+2H] ²⁺   Example 34-C tetramer

(2S)-2-[[(1S)-5-[[(2S)-2-[[5-[[[2-[4-[2-[3-[bis[2-[[1-[2- [[6-[[(1S)-2-[[(5S)-5-carboxy-5-[[(1S)- 1,3-dicarboxypropyl]carbamoylamino]pentyl]amino]-2-oxo-1-(2- quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2-oxo -ethyl]-3-hydroxy-6- methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2 -[[1-[2-[[6-[[(1S)-2-[[(5S)-5- carboxy-5-[[(1S)-1,3-dicarboxypropyl]carbamoylamino]pentyl]a mino]-2-oxo-1-(2-  

quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2- oxo-ethyl]-3-hydroxy-6- methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl]amino]ethylcarb amoyl]-3-hydroxy-6- methyl-2-oxo-1-pyridyl]acetyl]amino]methyl]pyridine-2-carbon yl]amino]-3-(2- quinolyl)propanoyl]amino]-1-carboxy-pentyl]carbamoylamino]pe ntanedioic acid

ditert-butyl (2S)-2-[[(1S)-5-[[(2S)-2-[[5-[[[2-[4-[2-[3-[bis[2-[[1-[2-[[6 -[[(1S)-2-[[(5S)-6-tert-butoxy- 5-[[(1S)-4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-butyl]car bamoylamino]-6-oxo-hexyl]amino]- 2-oxo-1-(2-quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methyla mino]-2-oxo-ethyl]-3-hydroxy-6- methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2 -[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert- butoxy-5-[[(1S)-4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-bu tyl]carbamoylamino]-6-oxo- hexyl]amino]-2-oxo-1-(2-quinolylmethyl)ethyl]carbamoyl]-3-py ridyl]methylamino]-2-oxo-ethyl]-3- hydroxy-6-methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl]amino ]ethylcarbamoyl]-3-hydroxy-6- methyl-2-oxo-1-pyridyl]acetyl]amino]methyl]pyridine-2-carbon yl]amino]-3-(2- quinolyl)propanoyl]amino]-1-tert-butoxycarbonyl-pentyl]carba moylamino]pentanedioate (1.10 mg, 0.256 µmol) is treated with 90% TFA in water (0.5 mL). Water (18 mL) is added and solution affording 900 µg (95 % purity, 92 % yield) of the target compound  

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.83 min; MS (ESIpos): m/z = 1206.2  

[M+3H] ³⁺   Intermediate 156

 

tri-tert-butyl (3S,10S,14S)-1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-1-[5-({ 2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)pyridin-2- yl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (64.3 mg, 112 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (42.0 mg, 111 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (14 µl, 84 µmol) were stirred in DMF (1.1 ml) for 10min at rt. tri- tert-butyl (10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[( quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (23.0 mg, 28.0 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 20.0 mg (80 % purity, 41 % yield) of the target compound.

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.81), 1.324 (3.29), 1.366 (9.69), 1.375 (12.53), 1.379 (16.00), 1.395 (7.35), 1.401 (4.60), 1.439 (2.25), 2.336 (0.86), 2.518 (9.92), 2.522 (6.40), 2.539 (0.68), 2.660 (0.86), 2.687 (0.50), 3.558 (0.59), 7.905 (0.59), 7.925 (0.63), 8.588 (0.45). Example 34-B

N ⁶ -{3-(quinolin-2-yl)-N-[5-({2-[4,7,10-tris(carboxymethy l)-1,4,7,10-tetraazacyclododecan- 1-yl]acetamido}methyl)pyridine-2-carbonyl]-L-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine   

tri-tert-butyl (3S,10S,14S)-1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-1-[5-({ 2-[4,7,10-tris(2-tert- butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetam ido}methyl)pyridin-2-yl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (18.0 mg, 13.1 µmol) was solubilised in DCM (420 µl), TFA (200 µl, 2.6 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.50 mg (95 % purity, 38 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.196 (0.54), 1.231 (1.42), 1.290 (0.61), 1.382 (3.44), 1.386 (2.97), 1.907 (0.47), 2.228 (0.74), 2.327 (3.98), 2.331 (2.97), 2.336 (1.35), 2.518 (16.00), 2.523 (10.26), 2.539 (1.62), 2.586 (0.68), 2.669 (4.12), 2.673 (2.97), 2.678 (1.35), 2.917 (0.88), 3.012 (1.89), 3.096 (0.81), 3.397 (2.03), 3.456 (1.08), 3.875 (0.61), 4.394 (0.68), 6.293 (0.61), 7.407 (1.01), 7.427 (0.95), 7.540 (0.74), 7.560 (0.47), 7.714 (0.41), 7.718 (0.41), 7.735 (0.68), 7.871 (0.47), 7.894 (0.74), 7.913 (0.68), 7.966 (0.95), 7.988 (0.74), 8.110 (0.41), 8.229 (0.95), 8.249 (0.88), 8.656 (0.47), 9.164 (0.47), 9.185 (0.47).

Example 34-C monomer ²²⁷ Th

[N6-{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-(hy droxy-kappaO)-6-methyl-2-oxo- 1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{ [1-(carboxymethyl)-3- (hydroxy-kappaO)-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2- oxopyridin-1(2H)-yl]acetamido}methyl)pyridine-2-carbonyl]-3- (quinolin-2-yl)-L-alanyl}- N2-{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(4-)](227 Th)thorium

 

N6-{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-hydr oxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6- methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)pyridine-2-car bonyl]-3-(quinolin-2-yl)-L-alanyl}- N2-{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine (2.0 µg) dissolved in 84 µL 30 mM citrate

buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at

0.3 MBq/nmol specific activity and RAC of 4.6 MBq/mL.1M carbonate buffer pH 9 (8 µL) was added and mixture incubated for 90 min. The labelling efficiency was determined to be 97% by iTLC. Example 34-C dimer ²²⁷ Th

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-(hydroxy- kappaO)-6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino} ethyl)azanediyl]ethane- 2,1-diylcarbamoyl[3-(hydroxy-kappaO)-6-methyl-2-oxopyridine- 4,1(2H)-diyl](1- oxoethane-2,1-diyl)azanediylmethylenepyridine-5,2-diyl})bis{ 1,4,12-trioxo-3-[(quinolin-2- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ato}(4-)( ²²⁷ Th)thorium

 

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)azan ediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl})bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid} (2.0 µg) dissolved in 63 µL 30 mM citrate

buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at

0.3 MBq/nmol specific activity and RAC of 4.5 MBq/mL.1M carbonate buffer pH 9 (6 µL) was added and mixture incubated for 90 min. The labelling efficiency was determined to be 94% by iTLC. Example 34-C trimer ²²⁷ Th

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2 S)-1-{[(5S)-5-carboxy-5-({[(1S)-1,3- dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(quino lin-2-yl)propan-2- yl]carbamoyl}pyridin-3-yl)methyl]amino}-2-oxoethyl)-3-(hydro xy-kappaO)-6-methyl-2- oxopyridine-4(2H)-carbonyl]amino}ethyl)(2-{[1-(carboxymethyl )-3-(hydroxy-kappaO)-6- methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl[3- (hydroxy-kappaO)-6-methyl-2-oxopyridine-4,1(2H)-diyl](1-oxoe thane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl]}bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylato}(4-)( ²²⁷ Th)thorium

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2 S)-1-{[(5S)-5-carboxy-5-({[(1S)-1,3- dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(quino lin-2-yl)propan-2-  

yl]carbamoyl}pyridin-3-yl)methyl]amino}-2-oxoethyl)-3-hyd roxy-6-methyl-2-oxopyridine-4(2H)- carbonyl]amino}ethyl)(2-{[1-(carboxymethyl)-3-hydroxy-6-meth yl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)(1-oxoethane-2,1-diyl)azan ediylmethylenepyridine-5,2- diyl]}bis{1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16- tricarboxylic acid} (4.0 µg) dissolved in 84 µL 30 mM citrate buffer (pH 5.5) containing 0.5

mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.5 MBq/mL.1M carbonate buffer pH 9 (8 µL) was added and mixture incubated for 90 min. The labelling efficiency was determined to be 99% by iTLC.

Example 34-C tetramer ²²⁷ Th

(2S)-2-[[(1S)-5-[[(2S)-2-[[5-[[[2-[4-[2-[3-[bis[2-[[1-[2- [[6-[[(1S)-2-[[(5S)-5-carboxy-5-[[(1S)-1,3- dicarboxypropyl]carbamoylamino]pentyl]amino]-2-oxo-1-(2-quin olylmethyl)ethyl]carbamoyl]-3- pyridyl]methylamino]-2-oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-p yridine-4- carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[[6-[[(1S)-2-[[( 5S)-5-carboxy-5-[[(1S)-1,3- dicarboxypropyl]carbamoylamino]pentyl]amino]-2-oxo-1-(2-quin olylmethyl)ethyl]carbamoyl]-3- pyridyl]methylamino]-2-oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-p yridine-4- carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydroxy-6-methy l-2-oxo-1- pyridyl]acetyl]amino]methyl]pyridine-2-carbonyl]amino]-3-(2- quinolyl)propanoyl]amino]-1- carboxy-pentyl]carbamoylamino]pentanedioic acid (4.0 µg) dissolved in 69 µL 30 mM citrate  

buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.5 MBq/mL. 1M carbonate buffer pH 9 (7 µL) was added and mixture incubated for 4 hrs and 30 min. The labelling efficiency was determined to be 95% by iTLC.  Example 34-B ²²⁷ Th

{N ⁶ -[3-(quinolin-2-yl)-N-{5-[(2-{4,7,10-tris[(carboxy-kap paO)methyl]-1,4,7,10- tetraazacyclododecan-1-yl-kappa ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido)methyl]pyridine-2-carbonyl}-L- alanyl]-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(4-)} ( ²²⁷ Th)thorate(1-)

N ⁶ -{3-(quinolin-2-yl)-N-[5-({2-[4,7,10-tris(carboxymethy l)-1,4,7,10-tetraazacyclododecan-1- yl]acetamido}methyl)pyridine-2-carbonyl]-L-alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L- lysine (1.00 mg, 0.963 µmol) was dissolved in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.9 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 79.6% by iTLC.  Intermediate 78 D

tri-tert-butyl (5S,12S,16R)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(quinolin- 2-yl)methyl]-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate

 

di-tert-Butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-2-yl)propanoyl]a mino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (183 mg, 70 % purity, 262 µmol) and N-{[(9H- fluoren-9-yl)methoxy]carbonyl}-3-quinolin-2-yl-L-alanine (115 mg, 262 µmol) were solubilised in DMF (2.0 ml) at 0°C, then N-methylmorpholine (87 µl, 790 µmol) and HATU (150 mg, 393 µmol) were added and the mixture was stirred under argon at 0°C for 45 min. The mixture was diluted with DCM and water, the organic phase was filtered through a phase separator and concentrated under reduced pressure. The crude material was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 83.0 mg (34 % yield) of the target compound. LC-MS (Method 1): R _t = 1.58 min; MS (ESIpos): m/z = 909 [M+H}+. Intermediate 79 D

di-tert-butyl (2R)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-2-yl)propanoyl]a mino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate

A mixture of tri-tert-butyl (5S,12S,16R)-1-(9H-fluoren-9-yl)-3,6,14-trioxo-5-[(quinolin- 2- yl)methyl]-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-trica rboxylate (100 mg, 110 µmol), piperidine (110 µl, 1.1 mmol; CAS-RN:[110-89-4]), and DMF (1.7mL) was stirred at r.t. for 2h.

 

After that the mixture was concentrated under reduced pressure and the crude product was

purified by preparative HPLC (C18, acetonitrile/0.1% ammonium hydroxide gradient) to give

the title compound (48 mg, 64% yield).

LC-MS (Method 1): R _t = 1.34 min; MS (ESIpos): m/z = 689 [M+H}+. Intermediate 156 D

tri-tert-butyl (3S,10S,14R)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-1,4,12-trioxo -3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate

To a solution of di-tert-butyl (2R)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-2-yl)propanoyl]a mino}- 1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (48.0 mg, 70.0 µmol) in DMF

(1.1mL) was added at r.t.5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]pyr idine-2- carboxylic acid (39.3 mg, 105 µmol), HATU (47.9 mg, 126 µmol; CAS-RN:[148893-10-1]), and

4-methylmorpholine (23 µl, 210 µmol; CAS-RN:[109-02-4]). After stirring for 1h the mixture was diluted with DCM and water, the organic phase was washed with brine and filtered through a phase separator. The filtrate was concentrated under reduced pressure and purified by

preparative HPLC (C18, acetonitrile/0.1% formic acid gradient) to give the title compound (30.0 mg, 41% yield).

LC-MS (Method 1): R _t = 1.56 min; MS (ESIpos): m/z = 1043 [M+H}+.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.360 (16.00), 1.387 (9.10), 2.323 (0.75), 2.327 (1.04), 2.331 (0.75), 2.518 (3.26), 2.523 (2.41), 2.665 (0.77), 2.669 (1.06), 2.673 (0.73), 4.363 (0.53),

4.380 (0.44), 7.308 (0.50), 7.403 (0.80), 7.424 (0.48), 7.664 (0.50), 7.685 (0.53), 7.874 (0.53),

7.894 (0.84), 7.916 (0.41), 7.943 (0.41), 8.225 (0.43). Intermediate 157 D

tri-tert-butyl (3S,10S,14R)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 -[(quinolin-2- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate

 

A mixture of tri-tert-butyl (3S,10S,14R)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-1,4,12-trioxo -3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (30.0 mg, 28.8 µmol), piperidine (57 µl, 580 µmol; CAS-RN:[110-89-4]) and DMF was stirred at r.t. for 2h. After that the mixture was

concentrated under reduced pressure and purified by preparative HPLC (C18, acetonitrile,

0.1% ammonium hydroxide gradient) to give 20.0mg of the title compound (85% yield).

LC-MS (Method 2): R _t = 1.33 min; MS (ESIpos): m/z = 821 [M+H]+

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.026 (5.62), 0.852 (1.07), 1.232 (10.46), 1.366

(16.00), 1.391 (8.80), 2.323 (1.43), 2.327 (2.01), 2.331 (1.43), 2.336 (0.64), 2.518 (6.40), 2.523

(4.72), 2.665 (1.46), 2.669 (2.02), 2.673 (1.41), 2.678 (0.61), 3.387 (0.47), 3.804 (1.19), 5.229

(0.96), 7.401 (0.56), 7.422 (0.54), 7.892 (1.39), 7.896 (1.44), 8.227 (0.42), 8.605 (0.53).

  Intermediate 157 D monomer

2,2'-{({3-[(2-{[1-(2-{[(6-{[(7R,11S,18S)-7,11-bis(tert-bu toxycarbonyl)-2,2-dimethyl-4,9,17- trioxo-19-(quinolin-2-yl)-3-oxa-8,10,16-triazanonadecan-18-y l]carbamoyl}pyridin-3- yl)methyl]amino}-2-oxoethyl)-3-hydroxy-6-methyl-2-oxo-1,2-di hydropyridine-4- carbonyl]amino}ethyl)(2-{[1-(carboxymethyl)-3-hydroxy-6-meth yl-2-oxopyridine-4(2H)- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy- 6-methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid

 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (7.9 mg, 7.32 µmol) and tri-tert-butyl (3S,10S,14R)- 1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[(quinolin-2 -yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (3.00 mg, 3.66 µmol) are dissolved in NMP (610 µL). DIPEA (8.9 µl, 51 µmol) is added. PyAOP (2.86 mg, 5.49 µmol) in NMP 170 µL is added.

Reaction mix is diluted with 20% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-70% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 27 min) afforded 2.5 mg (36% yield) of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.25 min; MS (ESIpos): m/z = 1884.9  

[M+H] ⁺   Intermediate 158 D dimer

2-[4-[2-[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butoxy-5-[[( 1R)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-2-oxo-1-(2- quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2-oxo -ethyl]-3-hydroxy-6- methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl-[3-[2-[[1-[2-[[ 6-[[(1S)-2-[[(5S)-6-tert-butoxy- 5-[[(1R)-4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-butyl]car bamoylamino]-6-oxo- hexyl]amino]-2-oxo-1-(2-quinolylmethyl)ethyl]carbamoyl]-3-py ridyl]methylamino]-2-oxo- ethyl]-3-hydroxy-6-methyl-2-oxo-pyridine-4-carbonyl]amino]et hyl-[2-[[1-(carboxymethyl)- 3-hydroxy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]propyl]amino]ethylcarbamoyl]-3-hy droxy-6-methyl-2-oxo-1- pyridyl]acetic acid

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1-di yl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (7.9 mg, 7.32 µmol) and tri-tert-butyl (3S,10S,14R)- 1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[(quinolin-2 -yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (3.00 mg, 3.66 µmol) are dissolved in NMP (610 µL). DIPEA (8.9 µl, 51 µmol) is added. PyAOP (2.86 mg, 5.49 µmol) in NMP 170 µL is added.

Reaction mix is diluted with 20% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2)  

100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-70% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 28 min) afforded 1.6 mg (16% yield) of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.69 min; MS (ESIpos): m/z = 1343.7  

[M+2H] ²⁺

  Intermediate 159 D trimer

2-[4-[2-[3-[bis[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butox y-5-[[(1R)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-2-oxo-1-(2- quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2-oxo -ethyl]-3-hydroxy-6- methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2 -[[1-[2-[[6-[[(1S)-2-[[(5S)-6- tert-butoxy-5-[[(1R)-4-tert-butoxy-1-tert-butoxycarbonyl-4-o xo-butyl]carbamoylamino]-6- oxo-hexyl]amino]-2-oxo-1-(2-quinolylmethyl)ethyl]carbamoyl]- 3-pyridyl]methylamino]-2- oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydroxy-6-methy l-2-oxo-1-pyridyl]acetic acid

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (7.9 mg, 7.32 µmol) and tri-tert-butyl (3S,10S,14R)- 1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3-[(quinolin-2 -yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (3.00 mg, 3.66 µmol) are dissolved in NMP (610 µL). DIPEA (8.9 µl, 51 µmol) is added. PyAOP (2.86 mg, 5.49 µmol) in NMP 170 µL is added.

Reaction mix is diluted with 20% ACN/water/0.1% TFA (8 mL) and the products purified by preparative HPLC (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm, Mobile phase: Water/0.1% TFA; ACN Gradient: 20-70% B over 40 min

 

Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 28 min) afforded 0.9 mg (7% yield) of the target compound.

LC-MS (Method K, gradient: 10-70% B over 3 min): R _t = 2.98 min; MS (ESIpos): m/z = 1744.4  

[M+2H] ²⁺   Example 34-C-D monomer

N ⁶ -{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-hyd roxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)am ino]ethyl}carbamoyl)-3- hydroxy-6-methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)pyri dine-2-carbonyl]-3- (quinolin-2-yl)-L-alanyl}-N ² -{[(1R)-1,3-dicarboxypropyl]carbamoyl}-L-lysine

2,2'-{({3-[(2-{[1-(2-{[(6-{[(7R,11S,18S)-7,11-bis(tert-bu toxycarbonyl)-2,2-dimethyl-4,9,17-trioxo- 19-(quinolin-2-yl)-3-oxa-8,10,16-triazanonadecan-18-yl]carba moyl}pyridin-3-yl)methyl]amino}- 2-oxoethyl)-3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4-c arbonyl]amino}ethyl)(2-{[1- (carboxymethyl)-3-hydroxy-6-methyl-2-oxopyridine-4(2H)- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid (2.50 mg, 1.33 µmol) is treated with 90% TFA in water (1 mL). Water (15 mL) is added and solution lyophilised.affording 2.30 mg (95% purity, 96% yield) of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.78 min; MS (ESIpos): m/z = 1716.6  

[M+H] ⁺   Example 34-C-D dimer

(3S,10S,14R,3'S,10'S,14'R)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)az anediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl})bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid}

 

2-[4-[2-[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butoxy-5-[[( 1R)-4-tert-butoxy-1-tert-butoxycarbonyl-4- oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino]-2-oxo-1-(2-quin olylmethyl)ethyl]carbamoyl]-3- pyridyl]methylamino]-2-oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-p yridine-4-carbonyl]amino]ethyl- [3-[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butoxy-5-[[(1R)-4-te rt-butoxy-1-tert-butoxycarbonyl-4-oxo- butyl]carbamoylamino]-6-oxo-hexyl]amino]-2-oxo-1-(2-quinolyl methyl)ethyl]carbamoyl]-3- pyridyl]methylamino]-2-oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-p yridine-4-carbonyl]amino]ethyl- [2-[[1-(carboxymethyl)-3-hydroxy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]propyl]amino]ethylcarbamoyl]-3-hy droxy-6-methyl-2-oxo-1- pyridyl]acetic acid (1.60 mg, 0.595 µmol) is treated with 90% TFA in water (1 mL). Water (15 mL) is added and solution lyophilised.affording 1.40 mg (95% purity, 95% yield) of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 2.06 min; MS (ESIpos): m/z = 1175.5  

[M+2H] ²⁺   Example 34-C-D trimer

(3S,10S,14R,3'S,10'S,14'R)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2 S)-1-{[(5S)-5-carboxy-5-({[(1R)-1,3- dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(quino lin-2-yl)propan-2- yl]carbamoyl}pyridin-3-yl)methyl]amino}-2-oxoethyl)-3-hydrox y-6-methyl-2-oxopyridine- 4(2H)-carbonyl]amino}ethyl)(2-{[1-(carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}azanediy l)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl )(1-oxoethane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl]}bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid}

 

2-[4-[2-[3-[bis[2-[[1-[2-[[6-[[(1S)-2-[[(5S)-6-tert-butox y-5-[[(1R)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-2-oxo-1-(2- quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2-oxo -ethyl]-3-hydroxy-6-methyl-2-oxo- pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[[6-[ [(1S)-2-[[(5S)-6-tert-butoxy-5-[[(1R)- 4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-butyl]carbamoylami no]-6-oxo-hexyl]amino]-2-oxo-1- (2-quinolylmethyl)ethyl]carbamoyl]-3-pyridyl]methylamino]-2- oxo-ethyl]-3-hydroxy-6-methyl-2- oxo-pyridine-4-carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3- hydroxy-6-methyl-2-oxo-1- pyridyl]acetic acid (900 µg, 0.26 µmol) is treated with 90% TFA in water (1 mL). Water (15 mL) is added and solution lyophilised.affording 0.8 mg (95% purity, 99% yield) of the target

compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 2.14 min; MS (ESIpos): m/z = 1491.8  

[M+2H] ²⁺   Example 34-C-D monomer 227Th

[N ⁶ -{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-(hy droxy-kappaO)-6-methyl-2-(oxo- kappaO)-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]pro pyl}(2-{[1- (carboxymethyl)-3-(hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)-1 ,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2-(oxo- kappaO)pyridin-1(2H)-yl]acetamido}methyl)pyridine-2-carbonyl ]-3-(quinolin-2-yl)-L- alanyl}-N ² -{[(1R)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(4-)] ( ²²⁷ Th)thorium

 

N ⁶ -{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-hyd roxy-6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{[1-( carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]ethyl}carbamoyl)-3-hydroxy-6- methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)pyridine-2-car bonyl]-3-(quinolin-2-yl)-L-alanyl}- N ² -{[(1R)-1,3-dicarboxypropyl]carbamoyl}-L-lysine (5.0 µg) dissolved in 158 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at

0.3 MBq/nmol specific activity and RAC of 4.6 MBq/mL.1M carbonate buffer pH 9 (16 µL) was added and mixture incubated for 90 min. The labelling efficiency was determined to be 94% by iTLC. Example 34-C-D dimer 227Th

(3S,10S,14R,3'S,10'S,14'R)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-(hydroxy- kappaO)-6-methyl-2-(oxo-kappaO)-1,2-dihydropyridine-4- carbonyl]amino}ethyl)azanediyl]ethane-2,1-diylcarbamoyl[3-(h ydroxy-kappaO)-6-methyl- 2-(oxo-kappaO)pyridine-4,1(2H)-diyl](1-oxoethane-2,1-diyl)az anediylmethylenepyridine- 5,2-diyl})bis{1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-2,5,11 ,13-tetraazahexadecane- 10,14,16-tricarboxylato}(4-)( ²²⁷ Th)thorium

)

(3S,10S,14R,3'S,10'S,14'R)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)azan ediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1-  

diyl)azanediylmethylenepyridine-5,2-diyl})bis{1,4,12-trio xo-3-[(quinolin-2-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid} (6.0 µg) dissolved in 166 µL 30 mM citrate

buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at

0.3 MBq/nmol specific activity and RAC of 4.6 MBq/mL.1M carbonate buffer pH 9 (17 µL) was added and mixture incubated for 90 min. The labelling efficiency was determined to be 95% by iTLC. Example 34-C-D trimer 227Th (3S,10S,14R,3'S,10'S,14'R)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2S)- 1-{[(5S)-5-carboxy-5-({[(1R)-1,3- dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(quino lin-2-yl)propan-2- yl]carbamoyl}pyridin-3-yl)methyl]amino}-2-oxoethyl)-3-(hydro xy-kappaO)-6-methyl-2- (oxo-kappaO)pyridine-4(2H)-carbonyl]amino}ethyl)(2-{[1-(carb oxymethyl)-3-(hydroxy- kappaO)-6-methyl-2-(oxo-kappaO)-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl[3- (hydroxy-kappaO)-6-methyl-2-(oxo-kappaO)pyridine-4,1(2H)-diy l](1-oxoethane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl]}bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylato}(4-)( ²²⁷ Th)thorium

(3S,10S,14R,3'S,10'S,14'R)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2 S)-1-{[(5S)-5-carboxy-5-({[(1R)-1,3- dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(quino lin-2-yl)propan-2- yl]carbamoyl}pyridin-3-yl)methyl]amino}-2-oxoethyl)-3-hydrox y-6-methyl-2-oxopyridine-4(2H)-  

carbonyl]amino}ethyl)(2-{[1-(carboxymethyl)-3-hydroxy-6-m ethyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6- methyl-2-oxopyridine-4,1(2H)-diyl)(1-oxoethane-2,1-diyl)azan ediylmethylenepyridine-5,2- diyl]}bis{1,4,12-trioxo-3-[(quinolin-2-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16- tricarboxylic acid} (8.0 µg) dissolved in 162 µL 30 mM citrate buffer (pH 5.5) containing 0.5

mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.6 MBq/mL.1M carbonate buffer pH 9 (16 µL) was added and mixture incubated for 90 min. The labelling efficiency was determined to be 68% by iTLC.

Example 34-C monomer ⁸⁹ Zr

[N6-{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-(hy droxy-kappaO)-6-methyl-2-oxo- 1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl}(2-{ [1-(carboxymethyl)-3- (hydroxy-kappaO)-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]ethyl}carbamoyl)-3-(hydroxy-kappa O)-6-methyl-2- oxopyridin-1(2H)-yl]acetamido}methyl)pyridine-2-carbonyl]-3- (quinolin-2-yl)-L-alanyl}- N2-{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(4-)](89Z r)zirconium

25 µl Zr(Ox) ₂ (~43 MBq) were transferred into a 2 ml Eppendorf tube, then 200 µl 1M HEPES pH8 / 0.02% TWEEN 20 were added and vortexed gently on circular tube shaker (@~550rpm) for 3 minutes @ RT. Then 50 µl N6-{N-[5-({2-[4-({2-[{3-[bis(2-{[1-(carboxymethyl)-3-hydroxy -6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]propyl}(2-{[1-(carboxymethyl)- 3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amin o}ethyl)amino]ethyl}carbamoyl)- 3-hydroxy-6-methyl-2-oxopyridin-1(2H)-yl]acetamido}methyl)py ridine-2-carbonyl]-3-(quinolin-2- yl)-L-alanyl}-N2-{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lys ine dissolved in 30mM Citrate pH

5.5, 70mM NaCl, 0.5mg/ml pABA, 2mM EDTA was added (2.9µg; 1.69nmol). Then 725µl 0.1M

HEPES pH7.5 was added to give 1000µl total volume. Incubation was performed by vortex  

gently on circular tube shaker (@~550rpm) for 90 minutes @ room temperature. Purification

was performed on C18 cartridge and fractions analyzed by HPLC. The product containing

fraction (90µL) was diluted with 810 µl saline and 130µL of the resulting solution again

checked via HPLC. to determine 15.56 MBq of the final product in the remaining 770µl saline solution. Example 34-C dimer ⁸⁹ Zr

(3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-(hydroxy- kappaO)-6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino} ethyl)azanediyl]ethane- 2,1-diylcarbamoyl[3-(hydroxy-kappaO)-6-methyl-2-oxopyridine- 4,1(2H)-diyl](1- oxoethane-2,1-diyl)azanediylmethylenepyridine-5,2-diyl})bis{ 1,4,12-trioxo-3-[(quinolin-2- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ato}(4-)( ⁸⁹ Zr)zirconium

30 µl Zr(Ox) ₂ (~51 MBq) were transferred into a 2 ml Eppendorf tube, then 240 µl 1M HEPES pH8 / 0.02% TWEEN 20 were added and vortexed gently on circular tube shaker (@~550rpm) for 3 minutes @ RT. Then 68 µl (3S,10S,14S,3'S,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2-{[1 - (carboxymethyl)-3-hydroxy-6-methyl-2-oxo-1,2-dihydropyridine -4- carbonyl]amino}ethyl)azanediyl]ethane-2,1-diylcarbamoyl(3-hy droxy-6-methyl-2-oxopyridine- 4,1(2H)-diyl)(1-oxoethane-2,1-diyl)azanediylmethylenepyridin e-5,2-diyl})bis{1,4,12-trioxo-3- [(quinolin-2-yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylic acid} dissolved in

30mM Citrate pH 5.5, 70mM NaCl, 0.5mg/ml pABA, 2mM EDTA was added (3.97µg ;

1.69nmol). Then 662 µl 0.1M HEPES pH7.5 was added to give 1000µl total volume. Incubation was performed by vortex gently on circular tube shaker (@~550rpm) for 90 minutes @ room temperature. Purification was performed on C18 cartridge and fractions analyzed by HPLC.

The product containing fraction (115µL) was diluted with 1035 µl saline and 165µL of the

resulting solution again checked via HPLC to determine 21.18 MBq of the final product in the remaining 985µl saline solution.

 

Example 34-C trimer ⁸⁹ Zr

(3S,10S,14S,3'S,10'S,14'S)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2 S)-1-{[(5S)-5-carboxy-5-({[(1S)-1,3- dicarboxypropyl]carbamoyl}amino)pentyl]amino}-1-oxo-3-(quino lin-2-yl)propan-2- yl]carbamoyl}pyridin-3-yl)methyl]amino}-2-oxoethyl)-3-(hydro xy-kappaO)-6-methyl-2- oxopyridine-4(2H)-carbonyl]amino}ethyl)(2-{[1-(carboxymethyl )-3-(hydroxy-kappaO)-6- methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]propyl}azanediyl)bis[(ethane-2,1- diyl)carbamoyl[3- (hydroxy-kappaO)-6-methyl-2-oxopyridine-4,1(2H)-diyl](1-oxoe thane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl]}bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylato}(4-)( ⁸⁹ Zr)zirconium

30 µl Zr(Ox) ₂ (~46 MBq) were transferred into a 2 ml Eppendorf tube, then 240 µl 1M HEPES pH8 / 0.02% TWEEN 20 were added and vortexed gently on circular tube shaker (@~550rpm) for 3 minutes @ RT. Then 87 µl (3S,10S,14S,3'S,10'S,14'S)-1,1'-{({3-[(2-{[1-(2-{[(6-{[(2S)- 1- {[(5S)-5-carboxy-5-({[(1S)-1,3-dicarboxypropyl]carbamoyl}ami no)pentyl]amino}-1-oxo-3- (quinolin-2-yl)propan-2-yl]carbamoyl}pyridin-3-yl)methyl]ami no}-2-oxoethyl)-3-hydroxy-6- methyl-2-oxopyridine-4(2H)-carbonyl]amino}ethyl)(2-{[1-(carb oxymethyl)-3-hydroxy-6-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amino]propyl} azanediyl)bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl )(1-oxoethane-2,1- diyl)azanediylmethylenepyridine-5,2-diyl]}bis{1,4,12-trioxo- 3-[(quinolin-2-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylic acid} dissolved in 30mM Citrate pH 5.5, 70mM

NaCl, 0.5mg/ml pABA, 2mM EDTA was added (5.05µg ; 1.69nmol). Then 643 µl 0.1M HEPES  

pH7.5 was added to give 1000µl total volume. Incubation was performed by vortexing gently on circular tube shaker (@~550rpm) for 90 minutes @ room temperature.Purification was

performed on C18 cartridge and fractions analyzed by HPLC. The product containing fraction

(120µL) was diluted with 1080 µl saline and 120µL of the resulting solution again checked with

HPLC to determine 21 MBq of the final product in the remaining 985µl saline solution.

  #35 Linker Intermediate 160

methyl 4-methoxynaphthalene-2-carboxylate 

To a solution of 4-hydroxynaphthalene-2-carboxylic acid (5.00 g, 26.6 mmol) in N,N- dimethylformamide (100 ml) were added potassium carbonate (11.0 g, 79.7 mmol) and iodomethane (8.3 ml, 130 mmol) at room temperature. After stirring at room temperature for 12 hours. Water was added to the reaction mixture. The mixture was extracted with ethyl acetate.

The organic layer was evporated under reduced pressure to give a residue. The residue was purified by chromatography (1000 mesh, petroleum ether: ethyl acetate = 1: 0, then 100: 1) to give methyl 4-methoxy-2-naphthoate (5.70 g, 97 % purity, 96 % yield) as a yellow oil.  

LC-MS (Method D): R _t = 0.841 min; MS (ESIpos): m/z = 217.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d = 8.22 (s, 1H), 8.20-8.16 (m, 1H), 8.11-8.05 (m, 1H), 7.68-7.59 (m, 2H), 7.34 (d, 1H), 4.02 (s, 3H), 3.91 (s, 3H). Intermediate 161

(4-methoxynaphthalen-2-yl)MeOH 

 

To a solution of methyl 4-methoxynaphthalene-2-carboxylate (5.70 g, 97% purity, 25.6 mmol) in toluene (70 ml) was added diisobutylaluminum hydride (38 ml, 1M in toluene, 38 mmol) at -40 °C. After stirring at 0 °C for 1 hours, the mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 20: 1, then 10: 1) to give (4-methoxy-2-naphthyl)methanol (5.00 g, 97% purity) as a yellow soild.

LC-MS (Method D): R _t = 0.728 min; MS (ESIpos): m/z = 189.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d = 8.12 (d, 1H), 7.83 (d, 1H), 7.54-7.37 (m, 3H), 6.95 (s, 1H), 5.33 (t, 1H), 4.67 (d, 2H), 3.97 (s, 3H). Intermediate 162

4-methoxynaphthalene-2-carbaldehyde 

To a solution of (4-methoxynaphthalen-2-yl)methanol (5.00 g, 97% purity, 25.8 mmol) in dichloromethane (50 ml) was added 3,3,3-triacetoxy-3-iodophthalide (13.1 g, 30.9 mmol) at room temperature. After stirring at 25 °C for 1 hour, the mixture was filtered through a pad of celite and the filtrate was concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 100: 1 then 50: 1) to give 4- methoxy-2-naphthaldehyde (4.30 g, 98% purity, 88% yield) as a light yellow solid.

LC-MS (Method D): R _t = 0.797 min; MS (ESIpos): m/z = 187.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d = 10.09 (s, 1H), 8.23-8.15 (m, 2H), 8.14-8.07 (m, 1H), 7.75- 7.64 (m, 2H), 7.24 (s, 1H), 4.03 (s, 3H). Intermediate 163

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxynaphthalen-2 -yl)prop-2-enoate 

 

1,8-Diazabicyclo(5.4.0)undec-7-ene (0.470 ml, 3.2 mmol) was added dropwise to a solution of methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (1.05 g, 3.16 mmol) in dichloromethane (10 ml). After stirring at room temperature for 10 minutes, a solution of 4- methoxynaphthalene-2-carbaldehyde (500 mg, 98% purity, 2.63 mmol) in dichloromethane (5 ml) was added. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and diluted with ethyl acetate (40 ml). The solution was washed with hydrochloric acid (1M) and brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 10: 1, then 5: 1, then 3:1) to give a residue. The residue was triturated with methyl tertiary butyl ether (10 ml). The suspension was filtered and the filter cake was collected to give methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxy-2- naphthyl)acrylate (700 mg, 95% purity, 65% yield) as a white solid.

LC-MS (Method D): R _t = 0.899 min; MS (ESIpos): m/z = 392.1 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d = 9.33 (br.s, 1H), 8.17-8.04 (m, 1H), 7.91-7.68 (m, 2H), 7.64- 7.49 (m, 2H), 7.48-6.94 (m, 7H), 5.13 (s, 2H), 3.88 (s, 3H), 3.75 (s, 3H). Intermediate 164

methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxynaphthalen-2 -yl)propanoate 

 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxynaphthalen-2 -yl)prop- 2-enoate (3.80 g, 98% purity, 9.51 mmol) in methanol (200 ml) were added (R)- [Rh(COD)(MaxPhos)]BF ₄ (0.06 eq) (338 mg, 571 µmol). The reaction mixture was stirred at room temperature for 16 hours under hydrogen atmosphere (2.5 MPa). The reaction mixture was evaporated under reduced pressure to give a residue. The residue was dissolved in dichloromethane, the solution was filtered through silica gel (200-300 mesh). The filtrate was evaporated under reduced pressure to give methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-(4- methoxy-2-naphthyl)propanoate (4.00 g, 94% purity, 100% yield) as a yellow oil.

LC-MS (Method D): R _t = 0.874 min; MS (ESIpos): m/z = 394.1 [M+H] ⁺ . Intermediate 165

(2R)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxynaphthale n-2-yl)propanoic acid 

To a solution of methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxynaphthalen-2 - yl)propanoate (4.00 g, 94% purity, 9.56 mmol) in pyridine (70 ml, 870 mmol) was added lithium iodide anhydrous (3.84 g, 28.7 mmol) at room temperature. The reaction mixture was heated to 110 °C and stirred at 110 °C for 12 hours. The reaction mxture was cooled to room tempetature, concentrated and dissolved in methanol. The solution was adjusted to pH 5-6 through  

hydrochloric acid (1M). The solution was evaporated under reduced pressure and purified by preparative HPLC (Instrument: Shimadzu LC-20AP; Column: Phenomenex luna C18 250*100mm*10 µm; eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-48 min 40-70% B; flow 400 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give (2S)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxy-2-naphthyl) propanoic acid (2.60 g, 99% purity, 71% yield) as a yellow solid.

LC-MS (Method D): R _t = 0.853 min; MS (ESIpos): m/z = 380.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d = 12.76 (s, 1H), 8.09 (d, 1H), 7.74 (dd, 2H), 7.55-7.42 (m, 2H), 7.32 (s, 1H), 7.29-7.09 (m, 5H), 6.93 (s, 1H), 4.96 (s, 2H), 4.41-4.30 (m, 1H), 3.93 (s, 3H), 3.23 (dd, 1H), 2.99 (dd, 1H). Intermediate 166

tri-tert-butyl (5R,12S,16S)-5-[(4-methoxynaphthalen-2-yl)methyl]-3,6,14-tri oxo-1-phenyl- 2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2R)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (626 mg, 98 % purity, 1.26 mmol) and (2S)-2-{[(benzyloxy)carbonyl]amino}-3-(4-methoxynaphthalen-2 - yl)propanoic acid (477 mg, 1.26 mmol) were solubilised in DMF (9.7 ml), 4-methylmorpholine (550 µl, 5.0 mmol, CAS-RN: 109-02-4) and HATU (718 mg, 1.89 mmol) were added and the mixture was stirred under argon for 2h at rt. The mixture was diluted with brine and extracted with DCM. The organic phase was dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 541 mg (95 % purity, 48 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.59 min; MS (ESIpos): m/z = 850 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.382 (12.58), 1.387 (16.00), 2.518 (1.13), 2.522 (0.74), 3.930 (1.97), 4.905 (0.44), 4.929 (0.44), 6.929 (0.44), 7.223 (0.49), 7.227 (0.43), 7.235 (0.64), 7.240 (0.48), 7.297 (0.40). 

 

Intermediate 167

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(4-methoxynaphthalen-2- yl)propanoyl]amino}-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl} amino)pentanedioate 

tri-tert-butyl (5R,12S,16S)-5-[(4-methoxynaphthalen-2-yl)methyl]-3,6,14-tri oxo-1-phenyl-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (540 mg, 90 % purity, 572 µmol) was solubilised in MeOH (4.6 ml), Palladium on carbon (6.09 mg, 10 % purity, 57.2 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 3h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 380 mg (95 % purity, 88 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.21 min; MS (ESIpos): m/z = 716 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.381 (16.00), 1.385 (13.49), 1.388 (11.58), 2.518 (0.79), 2.522 (0.53), 2.727 (0.67), 2.729 (0.67), 2.888 (0.81), 3.022 (0.42), 3.036 (0.42), 3.951 (3.67), 6.831 (0.64), 6.834 (0.65), 7.249 (0.62).  Intermediate 168

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]- 3-[(4-methoxynaphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13 -tetraazahexadecane- 10,14,16-tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(4-methoxynaphthalen-2-yl) propanoyl]amino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (379 mg, 95 % purity, 504 µmol) and (1r,4r)-4-({[(benzyloxy)carbonyl]amino}methyl)cyclohexane-1- carboxylic acid (147 mg, 504 µmol) were solubilised in DMF (3.9 ml), 4-methylmorpholine (220 µl, 2.0 mmol, CAS-RN: 109- 02-4) and HATU (287 mg, 755 µmol) were added and the mixture was stirred under argon 1h at rt. The mixture was diluted with brine and extracted with DCM. The organic phase was dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 338 mg (90 % purity, 61 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.57 min; MS (ESIpos): m/z = 989 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.376 (15.76), 1.384 (16.00), 2.518 (1.26), 2.522 (0.87), 2.539 (14.58), 2.801 (0.39), 3.420 (0.71), 3.426 (0.55), 3.433 (0.47), 3.941 (3.15), 4.985 (1.50), 6.867 (0.63), 7.237 (0.79), 7.315 (0.47), 7.318 (0.47), 7.329 (1.10), 7.336 (1.18), 7.351 (0.47), 7.355 (0.47).  Intermediate 169

tri-tert-butyl (3R,10S,14S)-3-[(4-methoxy-2-naphthyl)methyl]-1-{trans-4-[2- (methylamino)ethyl]cyclohexyl}-1,4,12-trioxo-2,5,11,13-tetra azahexadecane-10,14,16- tricarboxylate

 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]-3-[(4- methoxynaphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetra azahexadecane-10,14,16- tricarboxylate (335 mg, 90 % purity, 305 µmol) was solubilised in MeOH (2.5 ml), Palladium on carbon (33.0 mg, 10 % purity, 31 µmol) was added and the mixture was purged with hydrogen.

The mixture was stirred at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 29.0 mg (11 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.22 min; MS (ESIpos): m/z = 883 [M+H]+

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (16.00), 1.389 (15.25), 1.905 (0.61), 1.940 (0.62), 1.958 (0.59), 2.059 (5.71), 2.331 (0.41), 2.518 (2.26), 2.523 (1.41), 2.673 (0.41), 3.946 (3.37), 6.874 (0.66), 7.241 (0.65), 7.928 (0.52). Example 35-A

(3R,10S,14S)-3-[(4-methoxynaphthalen-2-yl)methyl]-1-{(1r, 4S)-4-[2- (methylamino)ethyl]cyclohexyl}-1,4,12-trioxo-2,5,11,13-tetra azahexadecane-10,14,16- tricarboxylic acid 

tri-tert-butyl (3R,10S,14S)-3-[(4-methoxynaphthalen-2-yl)methyl]-1-{(1r,4S) -4-[2- (methylamino)ethyl]cyclohexyl}-1,4,12-trioxo-2,5,11,13-tetra azahexadecane-10,14,16- tricarboxylate (11.1 mg, 12.6 µmol) was solubilised in DCM (400 µl), TFA (190 µl, 2.5 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 4.50 mg (90 % purity, 45 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.77 min; MS (ESIpos): m/z = 715 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.795 (0.39), 1.232 (1.63), 1.287 (0.46), 1.352 (0.52), 1.433 (0.52), 1.629 (0.46), 1.761 (0.65), 1.907 (1.11), 2.202 (0.59), 2.224 (1.04), 2.245 (0.85), 2.275 (2.42), 2.332 (2.81), 2.336 (1.24), 2.518 (16.00), 2.522 (10.06), 2.673 (2.81), 2.678 (1.24), 2.903 (0.39), 3.022 (0.65), 3.037 (0.52), 3.063 (0.52), 3.076 (0.59), 3.098 (0.46), 3.110 (0.46), 3.946 (6.92), 3.973 (0.46), 3.985 (0.52), 4.002 (0.52), 4.020 (0.46), 6.336 (0.46), 6.356 (0.46),  

6.886 (1.31), 7.254 (1.24), 7.404 (0.59), 7.407 (0.72), 7.424 (0.59), 7.428 (0.59), 7.447 (0.52), 7.450 (0.52), 7.468 (0.72), 7.724 (0.78), 7.744 (0.65), 7.876 (0.39), 8.033 (0.46), 8.045 (0.85), 8.066 (0.72).  #36 Linker Intermediate 170

isoquinoline-3-carbaldehyde 

To a mixture of methyl isoquinoline-3-carboxylate (7.80 g, 41.7 mmol) in toluene (160 ml) was added diisobutylaluminum hydride (1M in toluene, 63 ml, 63 mmol) dropwise at -60 °C under nitrogen atmosphere. The reaction mixture was stirred at -60 °C for 2 hours. The mixture was quenched with saturated ammonium chloride solution and saturated potassium tartrate. After stirring at room temperature for 16 hours, the mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (200-300 mesh, petroleum ether: ethyl acetate = 50: 1 to 1: 1) to give isoquinoline-3-carbaldehyde (2.60 g, 90% purity, 36% yield) as a yellow solid.

1H-NMR (400 MHz, CDCl ₃ ): d [ppm] = 10.30 (s, 1H), 9.42 (s, 1H), 9.43 (s, 1H), 8.11 (dd, 1H), 8.06 (dd, 1H), 7.86-7.82 (m, 2H). Intermediate 171

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-3-yl)prop -2-enoate 

o

To a solution of methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (10.9 g, 33.0 mmol) in dichloromethane (100 ml) was added 1,8-diazabicyclo(5.4.0)undec-7-ene (4.9 ml, 33 mmol) dropwise at 0-5 °C. After stirring for 30 minutes, a solution of isoquinoline-3-carbaldehyde (4.80 g, 90% purity, 27.5 mmol) in dichloromethane (100 ml) was added dropwise at 0-5 °C.

After stirring at room temperature for 2 hours, the reaction mixture (combined with another batch) was diluted with water and extracted with ethyl acetate. The organic phase was washed with  

brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (200-300 mesh, petroleum ether: ethyl acetate = 20: 1 to 5: 1) to give methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-3-yl)acry late (7.20 g, 99% purity, 72% yield) as a yellow solid.

1H-NMR (400 MHz, CDCl ₃ ): d [ppm] = 11.05 (s, 1H), 9.13 (s, 1H), 7.89 (d, 1H), 7.73 (d, 1H), 7.64 (td, 1H), 7.56-7.52 (m, 2H), 7.34-7.28 (m, 5H), 6.45 (s, 1H), 5.13 (s, 2H), 3.78 (s, 3H). Intermediate 172

(rac)-methyl N-[(benzyloxy)carbonyl]-3-isoquinolin-3-yl-alaninate 

To a solution of (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-3-yl)prop -2-enoate (4.70 g, 99% purity, 12.8 mmol) and nickel(II) chloride hexahydrate (305 mg, 1.28 mmol) in methanol (120 ml)/tetrahydrofuran (40 ml) was added sodium borohydride (1.46 g, 38.5 mmol) in three portions at 0 °C. After stirring at room temperature for 16 hours, the reaction mixture (combined with another batch) was diluted with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulfate, filtered and the filtrate was concentrated under reduce pressure to afford methyl N-[(benzyloxy)carbonyl]-3-isoquinolin-3-ylalaninate 4.45 g (90% purity, 86% yield) as a yellow oil.

LC-MS (Method C): R _t = 0.782 min; MS (ESIpos): m/z = 365.2 [M+H] ⁺ . Intermediate 173

(rac)-N-[(benzyloxy)carbonyl]-3-isoquinolin-3-yl-alanine� �

 

To a solution of methyl N-[(benzyloxy)carbonyl]-3-isoquinolin-3-yl-alaninate (3.85 g, 90 % purity, 9.51 mmol) in THF (100 ml) and water (100 ml) was added lithium hydroxide (1.20 g, 28.5 mmol).

The reaction mixture was stirred at rt for 2 h. The reaction mixture was acidified to pH = 3 by HCl (6 M), diluted with water and extracted with EtOAc. The organic phase was washed with brine, dried and concentrated in vacuo to give 2.20 g (94 % purity, 62 % yield) of the target compound. Intermediate 174

N-[(benzyloxy)carbonyl]-3-isoquinolin-3-yl-L-alanine 

(Rac)N-[(benzyloxy)carbonyl]-3-isoquinolin-3-yl-L-alanine (2.20 g, 94% purity, 5.90 mmol) was separated by preparative-SFC (Instrument: Waters 80Q SFC; Column: Chiralcel OD column, 250×25 mm×10 mm particle size; Mobile Phase: Phase A for Supercritical carbon dioxide, Phase B for ethanol (0.1% ammonia water); Isocratic elution: 40% Phase B (60% Phase A); flow:

70g/min; cycle time: 4.6 mintues; Back Pressure: 100 bar to keep the carbon dioxide in Supercritical flow; temperature: room temperature; Detector: UV 220 nm) to give (Ent)N- [(benzyloxy)carbonyl]-3-isoquinolin-3-ylalanine (stereoisomer 1, first eluting, SFC retention time:

1.595, 865 mg, 97% purity) as an off-white solid and (Ent)N-[(benzyloxy)carbonyl]-3-isoquinolin- 3-ylalanine (stereoisomer 2, second eluting, SFC retention time: 1.969, specific rotation (calc.):

-9.458°, concentration: 0.1882 g/100ml in methanol, 25 °C, 698 mg, 97% purity) as a white solid.

 

SFC (Method:Cellucoat-Isopropanol (diethylamine)-20-3 ml-35T) Instrument: SHIMADZU-2020;

Column: Cellucoat 50×4.6mm I.D., 3 mm Mobile phase: 20% iso-propanol (0.05% diethylamine) in carbon dioxide from 5% to 40%; Flow rate: 3mL/min; temperature: 35 °C; Detector: 220 nm.

LC-MS (Method C): R _t = 0.637 min; MS (ESIpos): m/z = 351.0 [M+H] ⁺ .

1H-NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.27 (s, 1H), 8.10 (d, 1H), 7.88 (d, 1H), 7.76 (td, 1H), 7.65-7.62 (m, 3H), 7.26-7.21 (m, 5H), 4.98-4.91 (m, 2H), 4.60-4.56 (m, 1H), 3.37-3.36 (m, 1H), 3.19-3.13 (m, 1H).

LC-MS (Method C): R _t = 0.637 min; MS (ESIpos): m/z = 351.1 [M+H] ⁺ .

1H-NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.27 (s, 1H), 8.10 (d, 1H), 7.88 (d, 1H), 7.76 (td, 1H), 7.67-7.63 (m, 3H), 7.26-7.21 (m, 5H), 4.98-4.91 (m, 2H), 4.60-4.56 (m, 1H), 3.37-3.36 (m, 1H), 3.19-3.13 (m, 1H). Intermediate 175

tri-tert-butyl (5S,12S,16S)-5-[(isoquinolin-3-yl)methyl]-3,6,14-trioxo-1-ph enyl-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (615 mg, 1.26 mmol) and N-[(benzyloxy)carbonyl]-3-isoquinolin-3-yl-L-alanine (442 mg, 1.26 mmol) were solubilised in DMF (9.7 ml), 4-methylmorpholine (350 µl, 3.2 mmol, CAS-RN: 109-02-4) and HATU (671 mg, 1.77 mmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was diluted with DCM/propan-2-ol and washed with water and brine. The organic layer was dried, evaporated and purified by flash chromatography (SiO2, DCM/Ethanol gradient 0%-5%) to give 400 mg (100 % purity, 39 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.45 min; MS (ESIpos): m/z = 821 [M+H] ⁺  

 

Intermediate 176

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(isoquinolin-3-yl)propanoy l]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5S,12S,16S)-5-[(isoquinolin-3-yl)methyl]-3,6,14-trioxo-1-ph enyl-2-oxa-4,7,13,15- tetraazaoctadecane-12,16,18-tricarboxylate (400 mg, 488 µmol) was solubilised in MeOH (4.0 ml), Palladium on carbon (51.9 mg, 10 % purity, 48.8 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 6h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 300 mg (94 % purity, 84 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.12 min; MS (ESIpos): m/z = 687 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (16.00), 1.384 (14.04), 1.387 (12.11), 1.396 (2.81), 2.518 (0.65), 2.522 (0.41), 7.620 (0.67), 7.883 (0.46), 9.249 (0.71).  Intermediate 177

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]- 3-[(isoquinolin-3-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraaz ahexadecane-10,14,16- tricarboxylate 

 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(isoquinolin-3-yl)propanoy l]amino}-1-tert-butoxy- 1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (300 mg, 437 µmol) and (1r,4r)-4- ({[(benzyloxy)carbonyl]amino}methyl)cyclohexane-1-carboxylic acid (127 mg, 437 µmol) were solubilised in DMF (5 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU

(200 mg, 525 µmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 165 mg (95 % purity, 37 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.46 min; MS (ESIpos): m/z = 960 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.231 (0.50), 1.379 (15.54), 1.381 (16.00), 2.332 (0.88), 2.518 (4.87), 2.522 (3.17), 2.673 (0.90), 4.984 (1.28), 7.319 (0.42), 7.331 (0.99), 7.337 (1.05), 7.356 (0.40), 7.577 (0.61), 7.823 (0.44), 9.226 (0.71).  Intermediate 178

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-3- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]-3- [(isoquinolin-3-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazah exadecane-10,14,16-tricarboxylate

(170 mg, 177 µmol) was solubilised in MeOH (1.4 ml), Palladium on carbon (18.9 mg, 10 % purity, 17.7 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 8h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 120 mg (95 % purity, 78 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.09 min; MS (ESIpos): m/z = 826 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.231 (0.59), 1.380 (16.00), 2.203 (0.47), 2.327 (1.25), 2.331 (0.92), 2.518 (5.46), 2.523 (3.39), 2.669 (1.23), 2.673 (0.88), 3.109 (0.51), 7.580 (0.69), 9.226 (0.59).  Example 36-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (isoquinolin-3-yl)-L-alanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-3-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (25.0 mg, 30.3 µmol) was solubilised in DCM (970 µl), TFA (580 µl, 7.6 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 4.50 mg (99 % purity, 22 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.54 min; MS (ESIpos): m/z = 657 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.858 (2.88), 1.046 (1.85), 1.073 (1.85), 1.153 (4.74), 1.172 (9.55), 1.189 (6.66), 1.214 (5.70), 1.230 (5.70), 1.275 (4.19), 1.291 (4.12), 1.415 (3.30), 1.448 (3.23), 1.558 (1.99), 1.642 (4.67), 1.684 (3.78), 1.741 (4.26), 1.751 (2.88), 1.779 (2.40), 1.986 (15.66), 2.056 (2.13), 2.073 (14.90), 2.083 (4.19), 2.156 (1.51), 2.171 (2.82), 2.186 (2.88), 2.204 (2.13), 2.239 (2.95), 2.255 (1.99), 2.518 (16.00), 2.522 (9.96), 2.539 (1.72), 2.574 (5.49), 2.589 (5.01), 2.934 (2.88), 2.952 (3.50), 2.967 (3.50), 3.044 (5.08), 3.067 (5.36), 3.079 (5.22),  

3.102 (4.67), 3.236 (8.93), 3.249 (9.75), 3.269 (10.23), 3.282 (10.64), 3.387 (13.32), 3.923 (4.46), 3.934 (5.29), 3.999 (2.13), 4.016 (4.19), 4.034 (4.05), 4.052 (1.79), 4.677 (2.20), 4.686 (2.61), 4.700 (2.54), 4.722 (1.10), 6.156 (2.06), 6.390 (2.13), 6.409 (2.33), 6.892 (0.96), 7.589 (3.16), 7.607 (10.03), 7.624 (3.85), 7.704 (3.09), 7.724 (5.29), 7.744 (3.30), 7.806 (3.30), 7.830 (6.66), 7.850 (4.46), 8.059 (5.97), 8.080 (5.42), 8.120 (2.61), 8.141 (2.40), 8.213 (0.76), 8.243 (2.06), 9.226 (10.64). Intermediate 179

tri-tert-butyl (3S,10S,14S)-3-[(isoquinolin-3-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2- [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (69.4 mg, 121 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (45.4 mg, 120 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (17 µl, 110 µmol) were stirred in DMF (1.2 ml) for 10min at rt. tri- tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-3-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (25.0 mg, 30.3 µmol) was  

added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 25.0 mg (97 % purity, 58 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.38 min; MS (ESIpos): m/z = 1380 [M+H] ⁺   Example 36-B

N ⁶ -{3-(isoquinolin-3-yl)-N-[(1r,4S)-4-({2-[4,7,10-tris(c arboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexane-1-car bonyl]-L-alanyl}-N ² - {[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-3-[(isoquinolin-3-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate (25.0 mg, 18.1 µmol) was solubilised in DCM (580 µl), TFA (280 µl, 3.6 mmol) was added and the mixture was stirred under argon over the weekend at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 5.00 mg (95 % purity, 25 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.54 min; MS (ESIpos): m/z = 1044 [M+H] ⁺  

 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (1.37), 1.352 (0.44), 2.332 (2.68), 2.336 (1.18), 2.518 (16.00), 2.523 (10.89), 2.539 (1.74), 2.678 (1.25), 2.983 (1.12), 3.072 (0.62), 3.427 (1.37), 7.599 (0.87), 7.726 (0.44), 7.837 (0.56), 8.061 (0.87), 8.081 (0.62), 9.229 (1.12).

Example 36-B 227Th

{N ⁶ -[3-(isoquinolin-3-yl)-N-{(1r,4S)-4-[(2-{4,7,10-tris[( carboxy-kappaO)methyl]-1,4,7,10- tetraazacyclododecan-1-yl-kappa ² N ¹ ,N ⁴ }acetamido)methyl]cyclohexane-1- carbonyl}alanyl]-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysinato(3-)} ( ²²⁷ Th)thorium

N ⁶ -{3-(isoquinolin-3-yl)-N-[(1r,4S)-4-({2-[4,7,10-tris(c arboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexane-1-car bonyl]-L-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 98,5% by iTLC. 

 

 

#37 Linker Intermediate 180

2-bromo-4-nitronaphthalen-1-amine 

To a solution of 4-nitronaphthalen-1-amine (6.00 g, 31.9 mmol) in acetonitrile (100 ml) were added ammonium acetate (246 mg, 3.19 mmol) and N-bromosuccinimide (5.96 g, 33.5 mmol) at room temperature. The reaction mixture was stirred at room temperature for 0.5 hour. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water and extracted with ethyl acetate. The combined organic layers were washed brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give 2-bromo-4-nitronaphthalen-1-amine (7.00 g 82% yield) as a yellow solid. 

LC-MS (Method C): R _t = 0.895 min; MS (M+H): m/z = 267.1 

1H-NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 8.78 (d, 1H), 8.54 (s, 1H), 8.45 (d, 1H), 7.70-7.85 (m, 1H), 7.54-7.64 (m, 1H), 7.50 (s, 2H). Intermediate 181

3-bromo-1-nitronaphthalene 

To a solution of 2-bromo-4-nitronaphthalen-1-amine (5.00 g, 18.7 mmol) in dichloromethane (200 ml) was added boron trifluoride diethyl etherate (3.6 ml) in one portion at -15 °C. After stirring for 15 minutes, a solution of tert-butyl nitrite (2.7 ml) in dichloromethane (50 ml) was added dropwise at -15 °C. After stirring for 1 hour, hypophosphorous (24.7 g, 50% purity, 187 mmol) acid and copper(I) oxide (133.94 mg) were added. The reaction mixture was stirred at 0 °C for 1 hour. The mixture was neutralized to pH=7 with saturated sodium bicarbonate and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum to give a residue.  

The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 100: 1 to 1: 1) to give 3-bromo-1-nitronaphthalene (3.50 g, 74% yield) as a brown solid.

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 8.67 (d, 1H), 8.44 (d, 1H), 8.26 (dd, 1H), 8.23 (dd, 1H), 7.82 (td, 1H), 7.78 (td, 1H). Intermediate 182

3-bromonaphthalen-1-amine 

To a solution of 3-bromo-1-nitronaphthalene (3.50 g, 13.9 mmol) in a mixed solvent of ethanol (60 ml), tetrahydrofuran (60 ml) and water (60 ml) were added iron (3.10 g, 55.5 mmol) and ammonium chloride (4.46 g, 83.3 mmol) in one portion. After stirring at 75 °C for 2 hours, the reaction mixture was filtered and the filtrate was washed with saturated sodium bicarbonate. The aqueous phase was extracted with ethyl acetate. The combined oragnic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give a residue. The residue was purified by column chromatography on silica gel (200-300 mesh, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give 3-bromonaphthalen-1-amine (2.60 g, 84% yield) as a yellow solid.

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 8.06 (d, 1H), 7.68 (d, 1H), 7.44 (td, 1H), 7.42 (td, 1H), 7.26 (d, 1H), 6.78 (d, 1H), 6.08 (s, 2H). Intermediate 183

di-tert-butyl (3-bromonaphthalen-1-yl)-2-imidodicarbonate 

To a solution of 3-bromonaphthalen-1-amine (2.60 g, 11.7 mmol) in toluene (100 ml) were added 4-dimethylaminopyridine (5.72 g, 46.8 mmol) and di-tert-butyl dicarbonate (8.1 ml, 35 mmol).

After stirring at 100 °C for 16 hours, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum to give di-tert-butyl (3-bromo- 1-naphthyl)-2-imidodicarbonate (4.00 g, 81% yield) as a yellow solid.  

¹ H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 8.26 (d, 1H), 8.03-7.93 (m, 1H), 7.70-7.50 (m, 4H), 1.32 (s, 18H). Intermediate 184

tert-butyl (3-bromonaphthalen-1-yl)carbamate 

To a solution of di-tert-butyl (3-bromonaphthalen-1-yl)-2-imidodicarbonate (4.00 g, 9.47 mmol)  in methanol (50 ml) was added potassium carbonate (3.27 g, 23.7 mmol) in one portion. After stirring at 25 °C for 16 hours, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum to give tert-butyl (3-bromo-1- naphthyl)carbamate (3.00 g, 98% yield) as a yellow solid.

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.51 (s, 1H), 8.14-8.06 (m, 1H), 7.99-7.78 (m, 3H), 7.61-7.48 (m, 2H), 1.50 (s, 9H). Intermediate 185

tert-butyl (3-bromonaphthalen-1-yl)methylcarbamate 

To a solution of tert-butyl (3-bromonaphthalen-1-yl)carbamate (3.04 g, 9.44 mmol)  in N,N- dimethylformamide (30 ml) was added sodium hydride (415 mg, 60% purity, 10.4 mmol) at 0 °C.

After stirring for 10 minutes, iodomethane (1.8 ml, 28 mmol) was added in a portion. After stirring at 20 °C for 16 hours, the reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum to give a residue. The residue was purified by column chromatography on silica gel (200-300 mesh, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give tert-butyl (3-bromo-1-naphthyl)methylcarbamate (3.17 g, 99% yield) as yellow oil.

 

¹ H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 8.18 (s, 1H), 7.97 (d, 1H), 7.74-7.68 (m, 1H), 7.66-7.57 (m, 3H), 3.24 (s, 3H), 1.43-0.95 (m, 9H). Intermediate 186

tert-butyl (3-formylnaphthalen-1-yl)methylcarbamate 

To a solution of tert-butyl (3-bromonaphthalen-1-yl)methylcarbamate (3.04 g, 9.04 mmol) in tetrahydrofuran (50 ml) was added n-butyllithium (5.4 ml, 2.5 M, 14 mmol) dropwise at -50 °C.

After stirring at -50 °C for 10 minutes, a solution of N,N-dimethylformamide (1 ml) in tetrahydrofuran (10 ml) was added dropwise. After stirring at 25 °C for 3 hours, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum to give a residue. The residue was purified by column chromatography on silica gel (200-300 mesh, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give tert-butyl (3- formyl-1-naphthyl)methylcarbamate (1.50 g, 58% yield) as yellow oil.

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 10.15 (s, 1H), 8.57 (s, 1 H), 8.23 (d, 1H), 7.85-7.67 (m, 4H), 3.27 (s, 3H), 1.49-1.10 (m, 9H). Intermediate 187

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}prop-2-enoate 

 

To a solution of methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (2.09 g, 6.31 mmol) in dichloromethane (60 ml) was added 1,8-diazabicyclo(5.4.0)undec-7-ene (0.940 ml, 6.3 mmol) in a portion. After stirring at 0 °C for 10 minutes, a solution of tert-butyl (3- formylnaphthalen-1-yl)methylcarbamate (1.50 g, 5.26 mmol) in dichloromethane (90 ml) was added. After stirring at 0 °C for 2 hours, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with ethyl acetate (40 ml) and washed with hydrochloric acid (1M) and brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuum and purified by column chromatography on silica gel (1000 mesh, petroleum ether: ethyl acetate = 50: 1 to 3: 1) to give methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (1.65 g, 64% yield) as yellow oil.

LC-MS (Method C): R _t = 1.029 min; MS (M-100+1): m/z = 391.2 

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.36 (br.s, 1H), 8.16 (s, 1H), 7.92 (d, 1H), 7.51-7.83 (m, 4 H), 7.04-7.51 (m, 6H), 4.76-5.31 (m, 2H), 3.51-3.87 (m, 3H), 3.21 (s, 3H), 1.16-1.69 (m, 9H). Intermediate 188

methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoate 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-{4-[(tert-butoxycarbon yl)(methyl) amino]naphthalen-2-yl}prop-2-enoate (1.00 g, 2.04 mmol) in methanol (60 ml) were added (R)- [Rh(COD)(MaxPhos)]BF ₄ (72.5 mg, 0.122 mmol). After stirring at room temperature for 16 hours under hydrogen atmosphere (50 psi), the reaction mixture was concentrated and purified by column chromatography on silica gel (200-300 mesh, petroleum ether: ethyl acetate = 1: 0 to 1:

1) to give methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-{4-[(tert-butoxycarbon yl)(methyl)amino] naphthalen-2-yl}propanoate (850 mg, 95% purity, 80% yield) as a yellow solid. 

 

¹ H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 7.88 (d, 2H), 7.75-7.64 (m, 2H), 7.58-7.47 (m, 2H), 7.40-7.14 (m, 6H), 4.95 (s, 2 H), 4.47-4.35 (m, 1 H), 3.72-3.56 (m, 3H), 3.25-3.20 (m, 1H), 3.17 (s, 3 H), 3.11-2.96 (m, 1H), 1.50-1.14 (m, 9H). Intermediate 189

(2R)-2-{[(benzyloxy)carbonyl]amino}-3-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoic acid 

To a solution of methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoate (1.10 g, 95% purity, 2.12 mmol) in tetrahydrofuran (5.7 ml) was added lithium hydroxide (1.3 ml, 2M in water, 2.5 mmol) at room temperature. After stirring at 25 °C for 16 hours, the reaction mixture was concentrated and purified by preparative HPLC (Instrument:ACSWH-GX-Q; Column: Shim-pack C18 150*25*10 µm; eluent A: water (0.225% formic acid), eluent B: acetonitrile; gradient: 0-8 min 55-75% B;

flow 25 ml/min; temperature: RT; Detector: UV 220/254 nm.) to give (2S)-2- {[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)propanoic acid as a yellow solid. The product was dissloved in a mixed solvent of acetonitrile and water, lyophilized to give (2S)-2- {[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)propanoic acid (830 mg, 99% purity, 81% yield) as a yellow solid.

LC-MS (Method D): R _t = 0.880 min; MS (M-55): m/z = 423.1

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 12.8 (s, 1 H), 7.95-7.80 (m, 1 H), 7.80-7.58 (m, 3 H), 7.58-7.45 (m, 2 H), 7.36 (s, 1H), 7.32-7.04 (m, 5H), 4.94 (s, 2H), 4.42-4.22 (m, 1H), 3.25-3.22 (m, 1H), 3.17 (s, 3H), 3.07-2.92 (m, 1H), 1.60-1.00 (m, 9H).

Intermediate 190

   

tri-tert-butyl (5R,12S,16S)-5-({4-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2- yl}methyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13,15-tetraazaoc tadecane-12,16,18- tricarboxylate 

di-tert-butyl N-{[(2R)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (1.11 g, 2.28 mmol) and (2S)-2-{[(benzyloxy)carbonyl]amino}-3-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoic acid (1.09 g, 2.28 mmol) were solubilised in DMF (14 ml), 4-methylmorpholine (630 µl, 5.7 mmol, CAS-RN: 109-02-4) and HATU (1.21 g, 3.19 mmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was diluted with DCM/propan-2-ol, washed with water and brine, dried, evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 380 mg (99 % purity, 17 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.62 min; MS (ESIpos): m/z = 949 [M+H] ⁺   Intermediate 191

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoyl]amino }-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate 

 

tri-tert-butyl (5R,12S,16S)-5-({4-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2-yl}methyl)- 3,6,14-trioxo-1-phenyl-2-oxa-4,7,13,15-tetraazaoctadecane-12 ,16,18-tricarboxylate (200 mg, 211 µmol) was solubilised in MeOH (5 ml), Palladium on carbon (2.24 mg, 10 % purity, 21.1 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 2h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 165 mg (99 % purity, 95 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.25 min; MS (ESIpos): m/z = 815 [M+H] ⁺   Intermediate 192

 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]- 3-({4-[(tert-butoxycarbonyl)(methyl)amino]naphthalen-2-yl}me thyl)-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoyl]amino }-1-tert-butoxy-1-oxohexan-2-  

yl]carbamoyl}amino)pentanedioate (530 mg, 651 µmol) and (1r,4r)-4- ({[(benzyloxy)carbonyl]amino}methyl)cyclohexane-1-carboxylic acid (209 mg, 716 µmol) were solubilised in DMF (10 ml), 4-methylmorpholine (180 µl, 1.6 mmol, CAS-RN: 109-02-4) and HATU (297 mg, 781 µmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 560 mg (95 % purity, 75 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.60 min; MS (ESIpos): m/z = 1088 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.139 (0.89), 1.380 (16.00), 1.389 (11.45), 2.518 (1.32), 2.523 (0.82), 2.729 (0.41), 2.888 (0.50), 3.203 (0.80), 4.983 (0.79), 4.986 (0.80), 7.316 (0.49), 7.318 (0.49), 7.329 (1.18), 7.337 (1.01), 7.355 (0.41), 8.165 (0.44). Intermediate 193

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({4-[(t ert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}methyl)-1,4,12- trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]-3-({4- [(tert-butoxycarbonyl)(methyl)amino]naphthalen-2-yl}methyl)- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (560 mg, 515 µmol) was solubilised in MeOH (5 ml), Palladium on carbon (54.8 mg, 10 % purity, 51.5 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 3h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 55.0 mg (100 % purity, 11 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.29 min; MS (ESIpos): m/z = 954 [M+H] ⁺   Example 37-A

 

(3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-{[4- (methylamino)naphthalen-2- yl]methyl}-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylic acid 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({4-[(t ert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}methyl)-1,4,12- trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (55.0 mg, 57.7 µmol) was solubilised in DCM (1.9 ml), TFA (890 µl, 12 mmol) was added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 24.0 mg (98 % purity, 60 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.65 min; MS (ESIpos): m/z = 686 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.821 (0.61), 0.852 (1.98), 0.882 (1.83), 0.907 (0.84), 1.092 (0.46), 1.123 (0.99), 1.154 (0.99), 1.176 (0.76), 1.208 (1.22), 1.232 (2.82), 1.271 (2.59), 1.307 (1.37), 1.323 (1.22), 1.343 (1.30), 1.359 (1.30), 1.376 (1.30), 1.394 (1.37), 1.408 (1.68), 1.430 (1.68), 1.444 (1.45), 1.525 (1.22), 1.563 (1.14), 1.605 (1.68), 1.619 (1.98), 1.640 (2.06), 1.681 (1.52), 1.720 (0.99), 1.768 (1.37), 1.791 (1.60), 1.803 (1.68), 1.825 (1.07), 1.840 (0.69), 2.052 (0.76), 2.075 (9.30), 2.081 (1.45), 2.113 (0.69), 2.125 (0.69), 2.139 (0.76), 2.144 (0.76), 2.161 (1.37), 2.174 (1.30), 2.180 (1.30), 2.193 (0.84), 2.224 (0.99), 2.246 (1.52), 2.260 (0.84), 2.268 (0.99), 2.281 (0.91), 2.303 (0.46), 2.336 (1.45), 2.518 (16.00), 2.523 (11.35), 2.574 (2.13), 2.590 (2.82), 2.602 (2.29), 2.619 (0.61), 2.635 (0.46), 2.678 (1.45), 2.805 (1.14), 2.837 (12.11), 2.862 (1.52), 2.888 (0.53), 2.960 (0.76), 2.975 (1.07), 2.994 (2.29), 3.007 (2.59), 3.029 (1.52), 3.042 (1.37), 3.065 (1.22), 3.079 (1.52), 3.096 (1.22), 3.112 (0.99), 3.334 (8.91), 3.913 (1.30), 3.934 (2.59), 3.946 (2.59), 3.965 (1.30), 4.444 (0.84), 4.458 (0.91), 4.467 (1.45), 4.480 (1.45), 4.489 (0.84), 4.502 (0.76), 6.145 (1.07), 6.217 (0.91), 6.335 (5.10), 6.337 (5.18), 6.434 (0.99), 6.450 (0.99), 6.910 (5.18), 7.283 (1.30), 7.286 (1.37), 7.299 (2.13), 7.303 (2.82), 7.320 (2.13), 7.323 (2.06), 7.347 (2.06), 7.350 (2.13), 7.367 (3.05), 7.385 (1.45), 7.610 (3.05), 7.629 (2.74), 7.837 (1.14), 7.993 (2.90), 8.014 (2.67), 8.114 (0.76), 8.221 (1.45).  Intermediate 194 monomer

2,2'-{[(3-{[2-({1-[2-({[(1S,4r)-4-{[(7S,11S,18R)-7,11-bis (tert-butoxycarbonyl)-19-{4-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}-2,2-dimethyl-4 ,9,17-trioxo-3-oxa-  

8,10,16-triazanonadecan-18-yl]carbamoyl}cyclohexyl]methyl }amino)-2-oxoethyl]-3- hydroxy-6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl}amino) ethyl](2-{[1- (carboxymethyl)-3-hydroxy-6-methyl-2-oxopyridine-4(2H)- carbonyl]amino}ethyl)amino}propyl)azanediyl]bis[(ethane-2,1- diyl)carbamoyl(3-hydroxy- 6-methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.00 mg, 4.62 µmol) and tri-tert-butyl (3R,10S,14S)- 1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({4-[(tert-butoxycar bonyl)(methyl)amino]naphthalen-2- yl}methyl)-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (6.38 mg, 6.69 µmol) are dissolved in NMP (1.14 mL). DIPEA (4.0 µL, 23 µmol) is added. PyAOP (2.4, 4.6 µmol) in NMP (960 µL) is added. Reaction mix is diluted with 30% ACN/water/0.1% TFA (6 mL) and the products purified by preparative (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 30- 80% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 27 min) affording 1.0 mg of the target compound.

LC-MS (Method K, gradient: 50-100% B over 3 min): R _t = 0.48 min; MS (ESIpos): m/z = 875.6  

[M+2H] ²⁺ Intermediate 195 dimer

2,2'-{propane-1,3-diylbis[{[2-({1-[2-({[(1S,4r)-4-{[(7S,1 1S,18R)-7,11-bis(tert- butoxycarbonyl)-19-{4-[(tert-butoxycarbonyl)(methyl)amino]na phthalen-2-yl}-2,2- dimethyl-4,9,17-trioxo-3-oxa-8,10,16-triazanonadecan-18- yl]carbamoyl}cyclohexyl]methyl}amino)-2-oxoethyl]-3-hydroxy- 6-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl}amino)ethyl]azanediyl}ethane-2,1- diylcarbamoyl(3-hydroxy- 6-methyl-2-oxopyridine-4,1(2H)-diyl)]}diacetic acid

 

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.00 mg, 4.62 µmol) and tri-tert-butyl (3R,10S,14S)- 1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({4-[(tert-butoxycar bonyl)(methyl)amino]naphthalen-2- yl}methyl)-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (6.38 mg, 6.69 µmol) are dissolved in NMP (1.14 mL). DIPEA (4.0 µL, 23 µmol) is added. PyAOP (2.4, 4.6 µmol) in NMP (960 µL) is added. Reaction mix is diluted with 30% ACN/water/0.1% TFA (6 mL) and the products purified by preparative (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 30- 80% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 37 min) affording 1.6 mg of the target compound.

LC-MS (Method K, gradient: 50-100% B over 3 min): R _t = 1.42 min; MS (ESIpos): m/z = 1477.4  

[M+2H] ²⁺ Intermediate 196 trimer

2-[4-[2-[3-[bis[2-[[1-[2-[[4-[[(1R)-2-[[(5S)-6-tert-butox y-5-[[(1R)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-1-[[4-[tert- butoxycarbonyl(methyl)amino]-2-naphthyl]methyl]-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo- pyridine-4-carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[[4-[ [(1R)-2-[[(5S)-6-tert-butoxy-5- [[(1S)-4-tert-butoxy-1-tert-butoxycarbonyl-4-oxo-butyl]carba moylamino]-6-oxo- hexyl]amino]-1-[[4-[tert-butoxycarbonyl(methyl)amino]-2-naph thyl]methyl]-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo- pyridine-4-carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydr oxy-6-methyl-2-oxo-1-

 

pyridyl]acetic acid

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (5.00 mg, 4.62 µmol) and tri-tert-butyl (3R,10S,14S)- 1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({4-[(tert-butoxycar bonyl)(methyl)amino]naphthalen-2- yl}methyl)-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (6.38 mg, 6.69 µmol) are dissolved in NMP (1.14 mL). DIPEA (4.0 µL, 23 µmol) is added. PyAOP (2.4, 4.6 µmol) in NMP (960 µL) is added. Reaction mix is diluted with 30% ACN/water/0.1% TFA (6 mL) and the products purified by preparative (RP-HPLC using Äkta pure system, Column: Phenomenex Luna 5 µm C18(2) 100Å, 250 x 21.2 mm Mobile phase: Water/0.1% TFA; ACN Gradient: 30- 80% B over 40 min Flow: 10 mL/min Detection: UV 280/335 nm, tR product: 47 min) affording 1.4 mg of the target compound.

LC-MS (Method K, gradient: 50-100% B over 3 min): R _t = 2.23 min; MS (ESIpos): m/z = 1944.0  

[M+2H] ²⁺ Example 37-C dimer

(3R,10S,14S,3'R,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy- 6-methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)az anediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis(3-{[ 4-(methylamino)naphthalen- 2-yl]methyl}-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,1 4,16-tricarboxylic acid) 

 

2,2'-{propane-1,3-diylbis[{[2-({1-[2-({[(1S,4r)-4-{[(7S,1 1S,18R)-7,11-bis(tert-butoxycarbonyl)-19- {4-[(tert-butoxycarbonyl)(methyl)amino]naphthalen-2-yl}-2,2- dimethyl-4,9,17-trioxo-3-oxa- 8,10,16-triazanonadecan-18-yl]carbamoyl}cyclohexyl]methyl}am ino)-2-oxoethyl]-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl}amino)ethyl]azan ediyl}ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) ]}diacetic acid (1.60 mg, 0.542 µmol) is treated with 90% TFA in water (0.5 mL). Water (18 mL) is added and the reaction mixture is lyophilised affording 1.60 mg (95 % purity, 122 % yield) of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.56 min; MS (ESIpos): m/z = 1208.7  

[M+2H] ²⁺ Example 37-C dimer- ²²⁷ Th

(3^R,10S,14S,3'R,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2 -{[1-(carboxymethyl)-3- (hydroxy-kappaO)-6-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)azanediyl]ethane-2,1-diylcarbamoyl[3-(h ydroxy-kappaO)-6-methyl- 2-oxopyridine-4,1(2H)-diyl](1-oxoethane-2,1-diyl)azanediylme thylene(1r,4S)cyclohexane- 4,1-diyl})bis(3-{[4-(methylamino)naphthalen-2-yl]methyl}-1,4 ,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylato)(4-)( ²²⁷ Th)thorium 

(3R,10S,14S,3'R,10'S,14'S)-1,1'-(propane-1,3-diylbis{[(2- {[1-(carboxymethyl)-3-hydroxy-6- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)azan ediyl]ethane-2,1- diylcarbamoyl(3-hydroxy-6-methyl-2-oxopyridine-4,1(2H)-diyl) (1-oxoethane-2,1- diyl)azanediylmethylene(1r,4S)cyclohexane-4,1-diyl})bis(3-{[ 4-(methylamino)naphthalen-2- yl]methyl}-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylic acid) (6.0 µg) dissolved in 153 µL 30 mM citrate buffer (pH 5.5) containing 0.5 mg/mL pABA was mixed with thorium-227 in 0.5 M HCl (2 µL) at 0.3 MBq/nmol specific activity and RAC of 4.5 MBq/mL.1M

 

carbonate buffer pH 9 (15 µL) was added and mixture incubated for 80 min. The labelling efficiency was determined to be 99% by iTLC. Example 37-C trimer

(2S)-2-[[(1S)-1-carboxy-5-[[(2R)-2-[[4-[[[2-[4-[2-[2-[[1- [2-[[4-[[(1R)-2-[[(5S)-5-carboxy-5- [[(1S)-1,3-dicarboxypropyl]carbamoylamino]pentyl]amino]-1-[[ 4-(methylamino)-2- naphthyl]methyl]-2-oxo-ethyl]carbamoyl]cyclohexyl]methylamin o]-2-oxo-ethyl]-3- hydroxy-6-methyl-2-oxo-pyridine-4-carbonyl]amino]ethyl-[3-[2 -[[1-[2-[[4-[[(1S)-2-[[(5S)-5- carboxy-5-[[(1S)-1,3-dicarboxypropyl]carbamoylamino]pentyl]a mino]-1-[[4- (methylamino)-2-naphthyl]methyl]-2-oxo-ethyl]carbamoyl]cyclo hexyl]methylamino]-2- oxo-ethyl]-3-hydroxy-6-methyl-2-oxo-pyridine-4-carbonyl]amin o]ethyl-[2-[[1- (carboxymethyl)-3-hydroxy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]propyl]amino]ethylcarbamoyl]-3-hy droxy-6-methyl-2-oxo-1- pyridyl]acetyl]amino]methyl]cyclohexanecarbonyl]amino]-3-[4- (methylamino)-2- naphthyl]propanoyl]amino]pentyl]carbamoylamino]pentanedioic acid) 

2-[4-[2-[3-[bis[2-[[1-[2-[[4-[[(1R)-2-[[(5S)-6-tert-butox y-5-[[(1R)-4-tert-butoxy-1-tert- butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-oxo-hexyl]amino ]-1-[[4-[tert- butoxycarbonyl(methyl)amino]-2-naphthyl]methyl]-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]propyl-[2-[[1-[2-[[4-[[(1S)-2-[[( 5S)-6-tert-butoxy-5-[[(1S)-4-tert- butoxy-1-tert-butoxycarbonyl-4-oxo-butyl]carbamoylamino]-6-o xo-hexyl]amino]-1-[[4-[tert- butoxycarbonyl(methyl)amino]-2-naphthyl]methyl]-2-oxo- ethyl]carbamoyl]cyclohexyl]methylamino]-2-oxo-ethyl]-3-hydro xy-6-methyl-2-oxo-pyridine-4- carbonyl]amino]ethyl]amino]ethylcarbamoyl]-3-hydroxy-6-methy l-2-oxo-1-pyridyl]acetic acid

 

(1.40 mg, 0.36 µmol) is treated with 90% TFA in water (0.5 mL). Water (18 mL) is added and the reaction mixture is lyophilised affording 1.5 mg (135 % yield) of the target compound.

LC-MS (Method K, gradient: 10-50% B over 3 min): R _t = 1.74 min; MS (ESIpos): m/z = 1541.6  

[M+2H] ²⁺   Intermediate 197

tri-tert-butyl (3R,10S,14S)-3-({4-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2- yl}methyl)-1,4,12-trioxo-1-[(1r,4S)-4-({2-[4,7,10-tris(2-ter t-butoxy-2-oxoethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexyl]-2,5,1 1,13-tetraazahexadecane- 10,14,16-tricarboxylate 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (228 mg, 399 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (149 mg, 394 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (51 µl, 300 µmol) were stirred in DMF (1.5 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({4-[(t ert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}methyl)-1,4,12- trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (95.0 mg, 99.7 µmol) was added and the mixture was stirred at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 100 mg (95 % purity, 63 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.56 min; MS (ESIpos): m/z = 1508 [M+H] ⁺    

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.144 (0.73), 1.232 (0.40), 1.239 (0.60), 1.255 (0.50), 1.269 (0.43), 1.359 (1.57), 1.382 (16.00), 1.390 (10.54), 1.406 (3.66), 1.412 (2.25), 1.480 (2.74), 2.332 (0.55), 2.518 (3.25), 2.523 (2.09), 2.539 (0.62), 2.673 (0.58), 2.909 (0.47), 3.201 (0.55).  Example 37-B

 

(3R,10S,14S)-3-{[4-(methylamino)naphthalen-2-yl]methyl}-1 ,4,12-trioxo-1-[(1r,4S)-4-({2- [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylic

acid 

tri-tert-butyl (3R,10S,14S)-3-({4-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2-yl}methyl)- 1,4,12-trioxo-1-[(1r,4S)-4-({2-[4,7,10-tris(2-tert-butoxy-2- oxoethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexyl]-2,5,1 1,13-tetraazahexadecane- 10,14,16-tricarboxylate (100 mg, 66.3 µmol) was solubilised in DCM (2.1 ml), TFA (1.0 ml, 13 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 35.0 mg (99 % purity, 49 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.68 min; MS (ESIpos): m/z = 1071 [M+H] ⁺  

¹H-NMR (600 MHz, DMSO-d6) d [ppm]: 0.000 (6.39), 0.816 (0.95), 0.835 (0.82), 1.116 (0.59), 1.137 (0.59), 1.224 (1.55), 1.236 (2.17), 1.248 (1.94), 1.260 (1.07), 1.288 (0.43), 1.299 (0.64), 1.311 (0.92), 1.323 (1.10), 1.336 (1.17), 1.349 (1.25), 1.361 (1.02), 1.372 (0.67), 1.441 (0.58), 1.454 (0.81), 1.464 (0.95), 1.476 (0.82), 1.489 (0.49), 1.516 (0.92), 1.533 (0.86), 1.595 (1.32), 1.607 (1.35), 1.616 (1.00), 1.642 (0.63), 1.669 (0.94), 1.684 (0.92), 1.699 (1.05), 1.708 (0.97), 1.718 (0.67), 1.723 (0.95), 1.733 (0.66), 1.881 (0.56), 1.893 (0.77), 1.904 (0.77), 1.909 (0.74), 1.917 (0.48), 2.068 (0.71), 2.217 (1.07), 2.227 (1.02), 2.233 (1.33), 2.236 (1.45), 2.242 (1.19),  

2.247 (1.35), 2.251 (1.32), 2.262 (1.10), 2.279 (0.43), 2.385 (0.79), 2.388 (1.05), 2.391 (0.77), 2.519 (3.29), 2.522 (3.34), 2.525 (2.75), 2.610 (0.53), 2.613 (0.95), 2.616 (1.22), 2.619 (0.99), 2.622 (0.59), 2.663 (1.66), 2.747 (1.65), 2.825 (1.07), 2.840 (16.00), 2.865 (3.70), 2.905 (2.32), 2.973 (5.19), 2.993 (4.13), 3.006 (4.69), 3.016 (5.60), 3.117 (2.11), 3.173 (0.79), 3.195 (0.79), 3.480 (2.04), 3.500 (2.83), 3.633 (0.64), 3.696 (0.59), 3.975 (0.81), 3.989 (1.37), 3.997 (1.22), 4.002 (0.86), 4.011 (0.64), 4.067 (0.69), 4.076 (1.10), 4.081 (1.27), 4.090 (1.33), 4.094 (0.79), 4.103 (0.58), 4.449 (0.43), 4.463 (0.82), 4.473 (1.04), 4.487 (0.66), 6.316 (1.27), 6.335 (4.95), 6.349 (1.66), 6.905 (3.54), 6.914 (0.59), 7.288 (1.02), 7.290 (1.12), 7.302 (2.07), 7.315 (1.53), 7.355 (1.63), 7.368 (2.16), 7.380 (1.22), 7.615 (2.19), 7.628 (1.93), 7.897 (0.94), 7.970 (0.66), 7.983 (0.64), 7.997 (2.49), 8.011 (2.26), 8.079 (0.76), 8.156 (0.79), 8.318 (0.76). Example 37-B 227Th

(3R,10S,14S)-3-{[4-(methylamino)naphthalen-2-yl]methyl}-1 ,4,12-trioxo-1-{(1r,4S)-4-[(2- {4,7,10-tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclod odecan-1-yl- kappa ² N ¹ ,N ⁴ }acetamido)methyl]cyclohexyl}-2,5,11,13-tetraazahexade cane-10,14,16- tricarboxylato(3-)( ²²⁷ Th)thorium

(3R,10S,14S)-3-{[4-(methylamino)naphthalen-2-yl]methyl}-1,4, 12-trioxo-1-[(1r,4S)-4-({2- [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 95.5% by iTLC. #38 Linker

 

Intermediate 198

3,4-dihydro-2H-1-benzopyran 

To a solution 2,3-dihydro-4H-1-benzopyran-4-one (30.0 g, 202 mmol) in acetic acid (300 ml) was added palladium on carbon (2.15 g, 20.2 mmol) at room temperature. After stirring at room temperature for 12 hours under hydrogen (50 psi) atmosphere, the reaction mixture was filtered through celite. The filtrate was evaporated under reduced pressure to give chromane (27.0 g, 99% yield) as yellow oil.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 7.07-6.99 (m, 2H), 6.79 (td, 1H), 6.74-6.67 (m, 1H), 4.11 (t, 2H), 2.72 (t, 2H), 1.96-1.83 (m, 2H). Intermediate 199

3,4-dihydro-2H-1-benzopyran-6-carbaldehyde 

To a solution of 3,4-dihydro-2H-1-benzopyran (27.0 g, 201 mmol) in 1,2-dichloroethane (270 ml) were added N,N-dimethylformamide (31 ml, 400 mmol) and phosphorus oxychloride (38 ml, 400 mmol) at room temperature. The reaction mixture was heated to 85 °C and stirred at 85 °C for 12 hours. The reaction mixture was quenched with water. The mixture was extracted with ethyl acetate. The organic layer was evaporated under reduced pressure to give a residue. The residue was purified by chromatography (1000 mesh, petroluem ether: ethyl acetate = 100: 1, then 50: 1, then 20: 1) to give chromane-6-carbaldehyde (11.5 g, 35 % yield) as a colorless oil.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 9.80 (s, 1H), 7.67-7.55 (m, 2H), 6.90 (d, 1H), 4.23 (t, 2H), 2.81 (t, 2H), 1.98-1.91 (m, 2H). Intermediate 200

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(3,4-dihydro-2H-1-benz opyran-6-yl)prop-2- enoate 

 

1,8-Diazabicyclo(5.4.0)undec-7-ene (13 ml, 85 mmol) was added dropwise to a solution of methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (28.2 g, 85.1 mmol) in dichloromethane (250 ml). After stirring at room temperature for 10 minutes, a solution of 3,4- dihydro-2H-1-benzopyran-6-carbaldehyde (11.5 g, 70.9 mmol) in dichloromethane (100 ml) was added. The reaction was stirred at room temperature for 2 hours. The reaction solvent was removed under reduced pressure to give a residue. The residue was diluted with ethyl acetate (40 ml). The solution was washed with hydrochloric acid (1M) and brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 1: 0, then 20: 1, then 12:1) to give methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(3,4-dihydro-2H-chrome n-6- yl)acrylate (16.9 g, 95% purity, 62% yield) as colorless oil.

LC-MS (Method D): R _t = 0.848 min; MS (ESIpos): m/z = 368.1 [M+H] ⁺ . Intermediate 201

methyl N-[(benzyloxy)carbonyl]-3-(3,4-dihydro-2H-1-benzopyran-6-yl) -D-alaninate 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(3,4-dihydro-2H-1-benz opyran-6- yl)prop-2-enoate (5.00 g, 13.6 mmol) in methanol (200 ml) were added (R)- [Rh(COD)(MaxPhos)]BF ₄ (484 mg, 0.817 mmol). After stirring at room temperature for 16 hours under hydrogen atmosphere (50 psi), the reaction mixture was filtered through silica gel (200- 300 mesh). The filtrate was evaporated under reduced pressure to give methyl N-  

[(benzyloxy)carbonyl]-3-(3,4-dihydro-2H-chromen-6-yl)-L-a laninate (5.10 g, 94% purity, 95% yield) as a yellow oil.

LC-MS (Method D): R _t = 0.857 min; MS (ESIpos): m/z = 370.1 [M+H] ⁺ . Intermediate 202

N-[(benzyloxy)carbonyl]-3-(3,4-dihydro-2H-1-benzopyran-6- yl)-D-alanine 

To a solution of methyl N-[(benzyloxy)carbonyl]-3-(3,4-dihydro-2H-chromen-6-yl)-D-al aninate (5.10 g, 13.8 mmol) in pyridine (80 ml) was added lithium iodide anhydrous (5.5 g, 41.4 mmol) at room temperature. The reaction mixture was heated to 110 °C and stirred at 110 °C for 24 hours. The reaction mxture was cooled to room tempetature and evaporated under reduced pressure to give a residue. The residue was dissolved in methanol. The pH was adjusted to 5~6 through hydrochloric acid (1M). The solution was evaporated under reduced pressure and purified by preparative HPLC (Instrument: Shimadzu LC-20AP; Column: Phenomenex luna C18 250*100mm*10 µm; eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-30 min 30-60% B; flow 100 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give N-[(benzyloxy)carbonyl]-3-(3,4-dihydro-2H-chromen-6-yl)-L-al anine (2.42 g, 98% purity, 48% yield) as a yellow solid.

LC-MS (Method D): R _t = 0.803 min; MS (ESIpos): m/z = 356.1 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 7.58 (d, 1H), 7.38-7.17 (m, 5H), 6.96-6.88 (m, 2H), 6.61 (d, 1H), 4.98 (s, 2H), 4.15-4.02 (m, 3H), 2.93 (dd, 1H), 2.76-2.70 (m, 1H), 2.68-2.61 (m, 2H), 1.94-1.78 (m, 2H). Intermediate 203

tri-tert-butyl (5R,12S,16S)-5-[(3,4-dihydro-2H-1-benzopyran-6-yl)methyl]-3, 6,14-trioxo-1- phenyl-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarbox ylate 

 

di-tert-butyl N-{[(2R)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (1.11 g, 2.28 mmol) and N-[(benzyloxy)carbonyl]-3-(3,4-dihydro-2H-1-benzopyran-6-yl) -L-alanine (810 mg, 2.28 mmol) were solubilised in DMF (14 ml), 4-methylmorpholine (630 µl, 5.7 mmol, CAS- RN: 109-02-4) and HATU (1.21 g, 3.19 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was diluted with DCM/propan-2-ol, washed with water and brine, dried, evaporated and purified by flash chromatography (SiO2, DCM/Ethanol gradient 0%- 5%) and preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 583 mg (95 % purity, 29 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.51 min; MS (ESIpos): m/z = 826 [M+H] ⁺   Intermediate 204

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(3,4-dihydro-2H-1-benzopyr an-6- yl)propanoyl]amino}-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl} amino)pentanedioate 

 

tri-tert-butyl (5R,12S,16S)-5-[(3,4-dihydro-2H-1-benzopyran-6-yl)methyl]-3, 6,14-trioxo-1- phenyl-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarbox ylate (580 mg, 703 µmol) was solubilised in MeOH (5.7 ml), Palladium on carbon (74.8 mg, 10 % purity, 70.3 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 3h at 40°C under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 488 mg (97 % purity, 97 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.09 min; MS (ESIpos): m/z = 692 [M+H] ⁺   Intermediate 205

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]- 3-[(3,4-dihydro-2H-1-benzopyran-6-yl)methyl]-1,4,12-trioxo-2 ,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

(1r,4r)-4-({[(benzyloxy)carbonyl]amino}methyl)cyclohexane-1- carboxylic acid (226 mg, 777 µmol) was solubilised in DMF (11 ml), 4-methylmorpholine (190 µl, 1.8 mmol, CAS-RN: 109-02- 4) and HATU (322 mg, 848 µmol) were added, stirred for 20min at rt, di-tert-butyl (2S)-2-({[(2S)- 6-{[(2R)-2-amino-3-(3,4-dihydro-2H-1-benzopyran-6-yl)propano yl]amino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (488 mg, 706 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 360 mg (75 % purity, 40 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.52 min; MS (ESIpos): m/z = 965 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.385 (16.00), 1.392 (9.79), 1.396 (8.35), 2.522 (0.67), 4.046 (0.43), 4.059 (0.50), 4.996 (1.14), 6.275 (0.52), 6.296 (0.48), 6.849 (0.79), 7.323 (0.41), 7.339 (1.03), 7.344 (1.24).  Intermediate 206

 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3,4-d ihydro-2H-1- benzopyran-6-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexa decane-10,14,16- tricarboxylate 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-({[(benzyloxy)carbonyl]amino}methy l)cyclohexyl]-3-[(3,4- dihydro-2H-1-benzopyran-6-yl)methyl]-1,4,12-trioxo-2,5,11,13 -tetraazahexadecane-10,14,16- tricarboxylate (360 mg, 373 µmol) was solubilised in MeOH (3.0 ml), Palladium on carbon (39.7 mg, 10 % purity, 37.3 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 2h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 324 mg (93 % purity, 97 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.18 min; MS (ESIpos): m/z = 831 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.385 (16.00), 1.394 (8.51), 1.396 (9.15), 2.224 (0.91), 2.326 (0.44), 2.518 (1.02), 2.522 (0.63), 6.276 (0.46), 6.298 (0.43), 6.852 (0.56). Example 38-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (3,4-dihydro-2H-1-benzopyran- 6-yl)-D-alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3,4-d ihydro-2H-1- benzopyran-6-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexa decane-10,14,16-tricarboxylate  

(20.0 mg, 24.1 µmol) was solubilised in DCM (780 µl), TFA (930 µl, 12 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.00 mg (95 % purity, 12 % yield) of the target compound.

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.89), 0.889 (1.24), 0.918 (1.03), 1.231 (2.61), 1.248 (2.82), 1.312 (1.51), 1.327 (1.30), 1.342 (1.17), 1.352 (1.17), 1.444 (1.30), 1.561 (1.44), 1.595 (1.99), 1.694 (1.58), 1.799 (1.65), 1.854 (2.20), 1.863 (2.47), 1.879 (2.13), 2.050 (0.62), 2.080 (1.03), 2.109 (1.03), 2.121 (0.82), 2.146 (1.10), 2.164 (1.17), 2.230 (0.82), 2.246 (1.03), 2.270 (0.82), 2.282 (0.69), 2.305 (0.41), 2.318 (1.44), 2.322 (3.02), 2.326 (4.05), 2.331 (3.02), 2.336 (1.37), 2.461 (5.36), 2.518 (16.00), 2.523 (9.82), 2.581 (1.30), 2.598 (2.82), 2.637 (3.02), 2.659 (4.19), 2.664 (4.94), 2.669 (5.91), 2.673 (4.39), 2.791 (1.17), 2.803 (1.37), 2.824 (1.10), 2.837 (0.96), 2.982 (1.10), 3.041 (1.17), 3.336 (4.05), 3.913 (2.06), 4.048 (2.88), 4.061 (3.85), 4.073 (2.75), 4.264 (0.62), 4.286 (1.10), 4.299 (1.17), 4.322 (0.62), 6.111 (0.62), 6.425 (0.55), 6.559 (3.09), 6.581 (3.36), 6.866 (5.22), 6.882 (1.65).  Intermediate 207

tri-tert-butyl (3R,10S,14S)-3-[(3,4-dihydro-2H-1-benzopyran-6-yl)methyl]-1, 4,12-trioxo-1- [(1r,4S)-4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7, 10-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

 

)

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (190 mg, 332 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (125 mg, 328 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (47 µl, 290 µmol) were stirred in DMF (3.2 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(3,4-d ihydro-2H-1- benzopyran-6-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexa decane-10,14,16-tricarboxylate

(69.0 mg, 83.1 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 55.0 mg (90 % purity, 43 % yield) of the target compound.

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.385 (16.00), 1.394 (9.98), 1.396 (10.45), 1.405 (3.63), 1.414 (1.34), 1.420 (2.50), 1.484 (2.08), 2.518 (3.44), 2.522 (2.39), 6.274 (0.43), 6.295 (0.40). Example 38-B

N ⁶ -{3-(3,4-dihydro-2H-1-benzopyran-6-yl)-N-[(1r,4S)-4-({ 2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)cyclohex ane-1-carbonyl]-D- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

 

tri-tert-butyl (3R,10S,14S)-3-[(3,4-dihydro-2H-1-benzopyran-6-yl)methyl]-1, 4,12-trioxo-1- [(1r,4S)-4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7, 10-tetraazacyclododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate (53.0 mg, 38.3 µmol) was solubilised in DCM (1.2 ml), TFA (590 µl, 7.7 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 11.0 mg (95 % purity, 26 % yield) of the target compound.

LC-MS (Method 1): Rt = 0.46 min; MS (ESIneg): m/z = 1043 [M-H]-  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.784 (0.76), 0.814 (0.83), 1.228 (1.45), 1.319 (1.18), 1.352 (1.18), 1.463 (0.90), 1.594 (1.25), 1.722 (0.62), 1.738 (0.48), 1.907 (2.08), 2.032 (0.69), 2.209 (0.76), 2.224 (1.25), 2.246 (1.25), 2.263 (0.69), 2.318 (1.45), 2.322 (3.05), 2.326 (4.02), 2.331 (3.05), 2.336 (1.52), 2.518 (16.00), 2.522 (9.77), 2.659 (2.49), 2.664 (3.88), 2.669 (4.71), 2.673 (3.53), 2.678 (1.87), 2.729 (1.59), 2.889 (2.84), 2.979 (4.09), 3.095 (1.66), 3.152 (1.66), 3.185 (1.80), 3.436 (6.03), 3.987 (0.69), 4.000 (0.69), 4.073 (0.76), 4.086 (0.69), 4.534 (0.62), 6.298 (0.97), 6.317 (1.25), 6.333 (0.83), 7.555 (1.18), 7.576 (1.25), 7.699 (1.66), 7.714 (1.73), 7.731 (2.08), 7.993 (2.08), 8.015 (1.66), 8.075 (1.11), 8.136 (0.90), 8.437 (3.19), 8.451 (2.70), 9.233 (3.74). Example 38-B 227Th  

{N ⁶ -[3-(3,4-dihydro-2H-1-benzopyran-6-yl)-N-{(1r,4S)-4-[( 2-{4,7,10-tris[(carboxy- kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1-yl- kappa ² N ¹ ,N ⁴ }acetamido)methyl]cyclohexane-1-carbonyl}-D-alanyl]-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysinato(3-)}( ²²⁷ Th)thorium

Xxx

N ⁶ -{3-(3,4-dihydro-2H-1-benzopyran-6-yl)-N-[(1r,4S)-4-({ 2-[4,7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexane-1-car bonyl]-D-alanyl}-N ² -{[(1S)-1,3- dicarboxypropyl]carbamoyl}-L-lysine was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 79.9% by iTLC.

#39 Linker Intermediate 208

methyl 3-aminonaphthalene-2-carboxylate 

 

To a solution of 3-amino-2-naphthoic acid (10.0 g, 53.4 mmol) in methanol (100 ml) was added thionyl chloride (11.69 ml, 160.3 mmol) dropwise at 0 °C. After refluxing for 16 hours, the reaction mixture was concentrated and dissolved in ethyl acetate. The solution was washed with saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (1000 mesh, petroleum ether : ethyl acetate = 10 : 1, then 5: 1, then 3 : 1) to give methyl 3-amino-2-naphthoate (5 g, 46% yield) as a yellow solid.

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 8.51 (s, 1H), 7.72 (d, 1H), 7.54 (d, 1H), 7.43 (t, 1H), 7.21 (t, 1H), 7.00 (s, 1H), 3.98 (s, 3H). Intermediate 209

methyl 3-[(tert-butoxycarbonyl)amino]naphthalene-2-carboxylate 

A mixture of methyl 3-aminonaphthalene-2-carboxylate (5.00 g, 99% purity, 24.6 mmol) in di- tert-butyl dicarbonate (10 ml, 44 mmol) was stirred at 60 °C for 16 hours. The reaction mixture was triturated with petroleum ether. The suspension was filtered. The solid cake was washed with petroleum ether and dried under reduced pressure to give methyl 3-[(tert- butoxycarbonyl)amino]-2-naphthoate (5.60 g, 90% purity, 68% yield) as a white solid.

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 10.23 (s, 1H), 8.83 (s, 1H), 8.64 (s, 1H), 7.82 (d, 2H), 7.55 (t, 1H), 7.41 (t, 1H), 4.02 (s, 3H), 1.59 (s, 9H). Intermediate 210

methyl 3-[(tert-butoxycarbonyl)(methyl)amino]naphthalene-2-carboxyl ate 

 

To a solution of methyl 3-[(tert-butoxycarbonyl)amino]naphthalene-2-carboxylate (5.60 g, 90% purity, 16.7 mmol) in N,N-dimethylformamide (45 ml) was added sodium hydride (2.01 g, 60% purity, 50.2 mmol) at 0 °C under nitrogen atmosphere. After stirring at 25 °C for 1 hour, iodomethane (4.2 ml, 67 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 2 hours. The mixture was quenched with saturated ammonium chloride, and extracted with ethyl acetate (combined with two other batches). The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chormatography on silica gel (1000 mesh, petroleum ether: ethyl acetate = 20: 1 to 5: 1) to give methyl 3-[(tert- butoxycarbonyl)(methyl)amino]-2-naphthoate (8.30 g, 95% purity) as a yellow solid.

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 8.47 (s, 1H), 7.98-7.88 (m, 1H), 7.87-7.79 (m, 1H), 7.74- 7.66 (m, 1H), 7.64-7.50 (m, 2H), 3.96 (s, 3H), 3.35 (s, 3H), 1.32 (s, 9H). Intermediate 211

[3-(methylamino)naphthalen-2-yl]MeOH 

To a solution of methyl 3-[(tert-butoxycarbonyl)(methyl)amino]naphthalene-2-carboxyl ate (4.95 g, 95% purity, 14.91 mmol) in tetrahydrofuran (50 ml) was added lithium borohydride (974.5 mg, 44.73 mmol) at 0 °C. After stirring at 50 °C for 16 hours, the mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 5: 1) to give [3-(methylamino) -2-naphthyl]methanol (3.34 g, 80% purity, 96% yield) as a white solid.

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 7.68 (d, 2H), 7.52 (s, 1H), 7.38 (t, 1H), 7.22 (t, 1H), 6.86 (s, 1H), 4.80 (s, 2H), 2.97 (s, 3H). Intermediate 212

3-(methylamino)naphthalene-2-carbaldehyde   

To a solution of [3-(methylamino)naphthalen-2-yl]methanol (3.92 g, 80% purity, 16.8 mmol) in acetonitrile (50 ml) was added manganese(IV) oxide (7.29 g, 83.8 mmol) at room temperature.

After stirring at 80 °C for 16 hours, the suspension was filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (1000 mesh, petroleum ether) to give 3-(methylamino)-2-naphthaldehyde (1.94, 62% yield) as a yellow solid.

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 10.06 (s, 1H), 8.04 (s, 1H), 7.74 (d, 1H), 7.61 (d, 2H), 7.46 (t, 1H), 7.19 (t, 1H), 6.80 (s, 1H), 2.99 (s, 3H). Intermediate 213

tert-butyl (3-formylnaphthalen-2-yl)methylcarbamate 

A solution of 3-(methylamino)naphthalene-2-carbaldehyde (2.01 g, 10.9 mmol) in di-tert-butyl dicarbonate (5.0 ml, 22 mmol) was stirred at 100 °C for 16 hours. The mixture was purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 30: 1 to 5: 1) to give tert- butyl (3-formyl-2-naphthyl)methylcarbamate (1.58 g, 51% yield) as a white solid.

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 10.20 (s, 1H), 8.43 (s, 1H), 8.01 (d, 1H), 7.87 (d, 1H), 7.70 (s, 1H), 7.67-7.52 (m, 2H), 3.41 (s, 3H), 1.34 (s, 9H). Intermediate 214

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-{3-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}prop-2-enoate 

 

1,8-Diazabicyclo(5.4.0)undec-7-ene (0.990 ml, 6.6 mmol) was added dropwise to a solution of methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (2.20 g, 6.64 mmol) in dichloromethane (20 ml). After stirring at room temperature for 10 minutes, a solution of tert- butyl (3-formylnaphthalen-2-yl)methylcarbamate (1.58 g, 5.54 mmol) in dichloromethane (10 ml) was added. After stirring at room temperature for 2 hours, the reaction mixture was concentrated and diluted with ethyl acetate. The solution was washed with hydrochloric acid (1M) and brine.

The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 10: 1 then 2: 1) to give methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-3- yl)acrylate (1.78 g, 66% yield) as a yellow solid.

LC-MS (Method D): R _t = 1.095 min; MS (ESIpos): m/z = 513.1 [M+Na] ⁺

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 7.93-7.87 (m, 1H), 7.70 (d, 1H), 7.61-7.57 (m, 2H), 7.40- 7.39 (m, 2H), 7.22-7.18 (m, 6H), 6.38-6.37 (m, 1H), 5.01 (d, 2H), 3.75 (s, 3H), 3.15 (s, 3H), 1.40- 1.26 (m, 9H). Intermediate 215

methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-{3-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoate 

 

To a solution of methyl methyl (2E)-2-{[(benzyloxy)carbonyl]amino}-3-{3-[(tert-butoxycarbon yl) (methyl)amino]naphthalen-2-yl}prop-2-enoate (1.48 g, 3.02 mmol) in methanol (50 ml) was added (R)-[Rh(COD)(MaxPhos)]BF ₄ (179 mg, 0.302 mmol). After stirring at room temperature for 24 hours under hydrogen atmosphere (50 psi), the reaction mixture was evaporated under reduced pressure to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 2: 1) to give methyl (2S)-2-{[(benzyloxy)carbonyl]amino}- 3-{3-[(tert-butoxycarbonyl)(methyl)amino]-2-naphthyl}propano ate (1.48 g, 100% yield) as yellow oil. Intermediate 216

(2R)-2-{[(benzyloxy)carbonyl]amino}-3-{3-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoic acid 

To a solution of methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-{3-[(tert-butoxycarbon yl) (methyl)amino]naphthalen-2-yl}propanoate (1.45 g, 2.94 mmol) in tetrahydrofuran (15 ml) was added lithium hydroxide aqueous solution (1.8 ml, 2.0 M, 3.5 mmol). After stirring at room temperature for 1 hour, the pH of the solution was adjusted to 5 with hydrochloric acid (1 M).

The reaction solution was concentrated under reduced pressure and dissolved in ethyl acetate.

The solution was washed with water and brine. The organic phase was dried over anhydrous  

sodium sulfate, filtered and concentrated under reduced pressure. The residue (combined with another batch) was purified by preparative HPLC (Instrument: ACSWH-PREP-HPLC-D; Column:

Phenomenex Synergi Max-RP 250*50mm*10 µm; eluent A: 0.01% ammonium bicarbonate in water, eluent B: acetonitrile; gradient: 5-22 min 30-70% B; flow 100 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give (2S)-2-{[(benzyloxy)carbonyl]amino}-3-{3-[(tert- butoxycarbonyl)(methyl)amino]-2-naphthyl} propanoic acid (1.16 g, 99% purity, 82% yield) as a yellow solid.

LC-MS (Method C): R _t = 0.965 min; MS (ESIpos): m/z = 501.2 [M+Na] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 7.86-7.72 (m, 4H), 7.49-7.47 (m, 2H), 7.31-7.06 (m, 6H), 4.97-4.88 (m, 2H), 4.33-4.13 (m, 1H), 3.35-3.31 (m, 1H), 3.20-3.15 (m, 3H), 2.95-2.84 (m, 1H), 1.49-1.26 (m, 9H). Intermediate 217

tri-tert-butyl (5R,12S,16S)-5-({3-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2- yl}methyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13,15-tetraazaoc tadecane-12,16,18- tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (713 mg, 1.46 mmol) and (2R)-2-{[(benzyloxy)carbonyl]amino}-3-{3-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoic acid (700 mg, 1.46 mmol) were solubilised in DMF (11 ml), 4-methylmorpholine (400 µl, 3.7 mmol, CAS-RN: 109-02-4) and HATU (779 mg, 2.05 mmol) were added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 320 mg (95 % purity, 22 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.66 min; MS (ESIpos): m/z = 949 [M+H] ⁺  

 

Intermediate 218

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-{3-[(tert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}propanoyl]amino }-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5R,12S,16S)-5-({3-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2-yl}methyl)- 3,6,14-trioxo-1-phenyl-2-oxa-4,7,13,15-tetraazaoctadecane-12 ,16,18-tricarboxylate (456 mg, 481 µmol) was solubilised in MeOH (3.9 ml), Palladium on carbon (51.2 mg, 10 % purity, 48.1 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 3h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 227 mg (95 % purity, 55 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.27 min; MS (ESIpos): m/z = 815 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.235 (1.14), 1.257 (1.03), 1.383 (16.00), 1.387 (11.18), 1.488 (0.48), 2.522 (1.09), 3.189 (0.56), 7.817 (0.56), 7.843 (0.45).  Intermediate 219

tri-tert-butyl (3R,10S,14S)-3-({3-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2- yl}methyl)-1-{(1r,4S)-4-[({[(9H-fluoren-9-yl)methoxy]carbony l}amino)methyl]cyclohexyl}- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate 

 

(1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (111 mg, 291 µmol) was solubilised in DMF (4.1 ml), 4-methylmorpholine (87 µl, 790 µmol, CAS- RN: 109-02-4) and HATU (111 mg, 291 µmol) were added, stirred for 20min at rt, di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-{3-[(tert-butoxycarbonyl)( methyl)amino]naphthalen-2- yl}propanoyl]amino}-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl} amino)pentanedioate (227 mg, 95 % purity, 265 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 180 mg (98 % purity, 57 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.71 min; MS (ESIpos): m/z = 1176 [M+H] ⁺   Intermediate 220

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({3-[(t ert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}methyl)-1,4,12- trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

 

tri-tert-butyl (3R,10S,14S)-3-({3-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2-yl}methyl)-1- {(1r,4S)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methy l]cyclohexyl}-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (180 mg, 153 µmol) was solubilised in DMF (2.4 ml), piperidine (300 µl, 3.1 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 151 mg (92 % purity, 95 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.29 min; MS (ESIpos): m/z = 954 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.242 (1.48), 1.383 (16.00), 1.390 (10.35), 1.450 (0.42), 1.489 (0.59), 1.715 (1.27), 1.870 (1.17), 2.085 (3.32), 2.518 (1.32), 2.523 (0.87), 3.194 (0.60).  Example 39-A

(3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-{[3- (methylamino)naphthalen-2- yl]methyl}-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylic acid 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({3-[(t ert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}methyl)-1,4,12- trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (11.3 mg, 11.9 µmol) was solubilised in DCM (380 µl), TFA (180 µl, 2.4 mmol) was added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC to give 2.00 mg (90 % purity, 22 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.74 min; MS (ESIpos): m/z = 686 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (1.30), 0.886 (1.19), 0.913 (0.54), 1.148 (0.70), 1.175 (0.76), 1.233 (2.16), 1.263 (2.27), 1.291 (1.84), 1.328 (0.86), 1.352 (0.81), 1.383 (1.03), 1.430 (1.35), 1.444 (1.41), 1.544 (0.92), 1.594 (1.19), 1.607 (1.35), 1.627 (1.51), 1.643 (1.57), 1.679 (1.46), 1.715 (0.81), 1.763 (0.92), 1.786 (1.19), 2.052 (0.54), 2.081 (0.92), 2.111 (0.49), 2.145 (0.54), 2.163 (0.86), 2.181 (0.92), 2.194 (0.59), 2.227 (0.54), 2.249 (0.92), 2.269 (0.65), 2.284 (0.59), 2.327 (3.14), 2.332 (2.27), 2.336 (1.08), 2.518 (16.00), 2.523 (9.78), 2.539 (2.32),  

2.596 (1.78), 2.669 (3.19), 2.673 (2.32), 2.823 (9.95), 2.848 (1.19), 2.872 (0.97), 2.978 (1.73), 2.989 (1.78), 3.012 (1.24), 3.026 (1.08), 3.099 (1.35), 3.117 (1.30), 3.933 (1.78), 3.944 (1.78), 4.444 (0.54), 4.466 (0.97), 4.478 (1.03), 4.500 (0.49), 6.139 (0.59), 6.424 (0.49), 6.668 (4.92), 7.059 (1.08), 7.079 (2.11), 7.097 (1.30), 7.237 (1.30), 7.255 (2.11), 7.272 (1.14), 7.432 (4.22), 7.513 (2.11), 7.533 (1.95), 7.570 (2.38), 7.591 (2.05), 7.967 (0.54), 8.355 (0.43).  Intermediate 221

tri-tert-butyl (3R,10S,14S)-3-({3-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2- yl}methyl)-1,4,12-trioxo-1-[(1r,4S)-4-({2-[4,7,10-tris(2-ter t-butoxy-2-oxoethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexyl]-2,5,1 1,13-tetraazahexadecane- 10,14,16-tricarboxylate 

)

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (66.3 mg, 116 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (43.4 mg, 114 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (15 µl, 87 µmol) were stirred in DMF (3.0 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-({3-[(t ert- butoxycarbonyl)(methyl)amino]naphthalen-2-yl}methyl)-1,4,12- trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (30.0 mg, 92 % purity, 29.0 µmol) was added and the mixture was stirred for 3h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 42.0 mg (96 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.36 min; MS (ESIpos): m/z = 1509 [M+H] ⁺   Example 39-B

(3R,10S,14S)-3-{[3-(methylamino)naphthalen-2-yl]methyl}-1 ,4,12-trioxo-1-[(1r,4S)-4-({2- [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-  

yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadec ane-10,14,16-tricarboxylic

acid 

( ₎ y y

tri-tert-butyl (3R,10S,14S)-3-({3-[(tert-butoxycarbonyl)(methyl)amino]napht halen-2-yl}methyl)- 1,4,12-trioxo-1-[(1r,4S)-4-({2-[4,7,10-tris(2-tert-butoxy-2- oxoethyl)-1,4,7,10- tetraazacyclododecan-1-yl]acetamido}methyl)cyclohexyl]-2,5,1 1,13-tetraazahexadecane- 10,14,16-tricarboxylate (46.0 mg, 30.5 µmol) was solubilised in DCM (3.0 ml), TFA (700 µl, 9.15 µmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 4.10 mg (90 % purity, 11 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.78 min; MS (ESIpos): m/z = 1072 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.833 (0.52), 0.852 (0.46), 1.232 (1.64), 1.308 (0.59), 1.353 (0.92), 1.605 (0.59), 1.639 (0.59), 1.672 (0.59), 1.706 (0.59), 1.907 (1.70), 2.085 (0.39), 2.210 (0.52), 2.231 (0.72), 2.238 (0.66), 2.260 (0.46), 2.337 (1.38), 2.518 (16.00), 2.523 (10.69), 2.674 (3.02), 2.679 (1.44), 2.828 (5.64), 2.983 (2.43), 3.090 (0.98), 3.430 (3.08), 3.976 (0.46), 3.989 (0.46), 4.066 (0.46), 4.078 (0.39), 4.877 (0.39), 4.903 (0.46), 6.294 (0.66), 6.685 (2.03), 7.067 (0.46), 7.084 (0.85), 7.104 (0.52), 7.154 (0.39), 7.206 (0.46), 7.223 (0.59), 7.236 (0.66), 7.256 (0.85), 7.276 (0.46), 7.411 (1.51), 7.516 (0.85), 7.535 (0.72), 7.574 (0.92), 7.594 (0.79), 7.941 (0.59), 8.084 (0.52), 8.137 (0.79), 8.452 (0.66), 8.467 (0.59), 9.203 (0.59). #40 Linker Intermediate 222

tri-tert-butyl (5S,12S,16S)-1-{(1r,4S)-4-[(tert-butoxycarbonyl)amino]cycloh exyl}-5- [(naphthalen-2-yl)methyl]-3,6,14-trioxo-2,4,7,13,15-pentaaza octadecane-12,16,18- tricarboxylate 

 

tert-butyl [(1r,4r)-4-(aminomethyl)cyclohexyl]carbamate (5.87 mg, 25.7 µmol) was solubilised in DCM (450 µl, 7.0 mmol), N,N-diisopropylethylamine (20 µl, 120 µmol) and tri-tert-butyl (3S,10S,14S)-3-[(naphthalen-2-yl)methyl]-1-(4-nitrophenoxy)- 1,4,12-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (19.9 mg, 23.4 µmol) were added. The mixture was stirred under argon over the weekend at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 17.0 mg (90 % purity, 70 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.55 min; MS (ESIpos): m/z = 940 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.232 (0.54), 1.352 (2.17), 1.367 (7.47), 1.380 (16.00), 1.382 (14.02), 2.331 (0.51), 2.518 (2.65), 2.523 (1.76), 2.673 (0.51), 7.621 (0.42), 7.780 (0.54).  Example 40-A

(5S,12S,16S)-1-[(1r,4S)-4-aminocyclohexyl]-5-[(naphthalen -2-yl)methyl]-3,6,14-trioxo- 2,4,7,13,15-pentaazaoctadecane-12,16,18-tricarboxylic acid 

 

tri-tert-butyl (5S,12S,16S)-1-{(1r,4S)-4-[(tert-butoxycarbonyl)amino]cycloh exyl}-5-[(naphthalen- 2-yl)methyl]-3,6,14-trioxo-2,4,7,13,15-pentaazaoctadecane-12 ,16,18-tricarboxylate (16.0 mg, 17.0 µmol) was solubilised in DCM (540 µl), TFA (19.4 mg, 170 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 10.0 mg (95 % purity, 83 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.71 min; MS (ESIpos): m/z = 671 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.813 (0.39), 0.844 (1.27), 0.875 (1.40), 0.909 (0.61), 1.170 (1.97), 1.202 (2.45), 1.224 (3.28), 1.232 (3.50), 1.249 (2.05), 1.269 (1.05), 1.286 (1.62), 1.303 (1.62), 1.432 (0.66), 1.450 (0.83), 1.468 (0.70), 1.571 (0.61), 1.584 (0.87), 1.605 (1.44), 1.634 (1.53), 1.688 (0.61), 1.705 (0.70), 1.722 (0.92), 1.734 (0.79), 1.738 (0.70), 1.845 (1.62), 1.864 (1.75), 1.877 (1.84), 1.898 (0.96), 1.907 (0.83), 2.216 (1.22), 2.225 (1.31), 2.237 (1.75), 2.242 (1.79), 2.263 (1.05), 2.298 (0.66), 2.331 (1.79), 2.336 (0.83), 2.518 (11.41), 2.522 (7.13), 2.539 (16.00), 2.546 (4.81), 2.673 (1.84), 2.678 (0.87), 2.727 (1.75), 2.762 (1.53), 2.777 (2.62), 2.793 (1.53), 2.866 (1.44), 2.888 (3.19), 2.900 (1.44), 2.921 (1.09), 2.979 (1.27), 2.993 (1.70), 3.007 (1.44), 3.021 (1.66), 3.035 (1.40), 3.055 (1.01), 3.067 (0.96), 3.090 (0.48), 3.725 (0.39), 3.738 (0.44), 3.985 (0.57), 3.997 (0.74), 4.005 (1.14), 4.018 (1.18), 4.038 (0.70), 4.063 (1.09), 4.078 (1.09), 4.098 (0.52), 4.380 (0.57), 4.400 (1.14), 4.415 (1.09), 4.435 (0.52), 6.144 (0.61), 6.200 (0.87), 6.283 (2.45), 6.303 (2.36), 7.326 (1.92), 7.330 (1.88), 7.348 (1.97), 7.351 (2.01), 7.423 (0.66), 7.427 (0.83), 7.440 (1.97), 7.444 (1.88), 7.452 (2.05), 7.457 (3.76), 7.463 (2.19), 7.471 (1.84), 7.476 (1.92), 7.488 (0.83), 7.492 (0.61), 7.634 (3.72), 7.783 (4.46), 7.796 (2.19), 7.803 (3.58), 7.840 (1.88), 7.845 (1.92), 7.863 (1.79), 7.935 (0.92), 7.949 (1.88), 7.962 (0.83).  #41 Linker Intermediate 223  

1-[(4-chlorophenyl)sulfanyl]propan-2-one 

To a solution of 4-chlorobenzene-1-thiol (10.0 g, 69.1 mmol) and 1-chloropropan-2-one (6.72 g, 72.6 mmol) in N,N-dimethylformamide (100 ml) was added potassium carbonate (19.1 g, 138 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 hours. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 30: 1 to 5: 1) to give 1-[(4-chlorophenyl)sulfanyl]acetone (5.00 g, 99% purity) and 1-[(4- chlorophenyl)sulfanyl]acetone (7.00 g, 83% purity) as yellow oil.

LC-MS (Method C): R _t = 0.875 min; MS (ESIpos): m/z = 200.9 [M+H] ⁺ .

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 7.27 (s, 4H), 3.65 (s, 2H), 2.28 (s, 3H). Intermediate 224

5-chloro-3-methyl-1-benzothiophene 

To a solution of 1-[(4-chlorophenyl)sulfanyl]propan-2-one (7.00 g, 83% purity, 29.0 mmol) in toluene (100 ml) was added polyphosphoric acid (29.3 g, 86.9 mmol) at room temperature. The reaction mixture was refluxed for 16 hours. The mixture (combined with another batch) was concentrated, dissolved in water and extracted with ethyl acetate. The organic phase was washed with sodium hydroxide (1M), brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue. The residue was purified by column chromatography (1000 mesh, petroleum ether) to give 5-chloro-3-methyl-1-benzothiophene (7.00 g) as yellow oil.

1H NMR (400 MHz, CDCl ₃ ) d [ppm] = 7.76 (d, 1H), 7.70 (d, 1H), 7.32 (dd, 1H), 7.14 (s, 1H), 2.43 (s, 3H). Intermediate 225

3-(bromomethyl)-5-chloro-1-benzothiophene 

 

To a solution of 5-chloro-3-methyl-1-benzothiophene (6.00 g, 32.8 mmol) in carbon tetrachloride (60 ml, 620 mmol) were added N-bromosuccinimide (5.85 g, 32.8 mmol) and dibenzoyl peroxide (159 mg, 0.657 mmol) at room temperature. After stirring at 80 °C for 12 hours, the reaction mixture was cooled to room temperature.and diluted with water. The mixture was extracted with dichloromethane. The organic layer was evaporated under reduced pressure and triturated with methyl tert-butyl ether to give 3-(bromomethyl)-5-chloro-1-benzothiophene (5.60 g, 65% yield) as a light yellow solid.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 8.09-8.04 (m, 2H), 8.00 (d, 1H), 7.45 (dd, 1H), 5.04

(s, 2H). Intermediate 226

 

(5-chloro-1-benzothiophen-3-yl)mEtOAc 

To a solution of 3-(bromomethyl)-5-chloro-1-benzothiophene (5.60 g, 21.4 mmol) in N,N- dimethylformamide (80 ml) were added sodium acetate (5.27 g, 64.2 mmol) and potassium iodide (711 mg, 4.28 mmol) at room temperature. After stirring at 50 °C for 12 hours, the reaction mixture was cooled to room temperature and diluted with water. The mixture was extracted with ethyl acetate. The organic layer was evaporated under reduced pressure to give (5-chloro-1- benzothiophen-3-yl)methyl acetate (5.10 g, 99% yield) as yellow oil.

1H NMR (400 MHz, DMSO-d ₆ ) d = 8.06 (d, 1H), 7.97-7.93 (m, 2H), 7.44 (dd, 1H), 5.32 (s, 2H),

2.06 (s, 3H). Intermediate 227

 

(5-chloro-1-benzothiophen-3-yl)MeOH 

 

To a solution of (5-chloro-1-benzothiophen-3-yl)methyl acetate (5.10 g, 21.2 mmol) in methanol (80 ml) was added potassium carbonate (5.86 g, 42.4 mmol) at room temperature. After stirring at room temperature for 12 hours, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was evaporated under reduced pressure and purified by chromatography (100 mesh, petroleum ether: ethyl acetate = 1: 0, then 100: 1, then 20: 1) to give (5-chloro-1-benzothiophen-3-yl)methanol (4.00 g, 95% yield) as a yellow solid.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 8.01 (d, 1H), 7.92 (d, 1H), 7.67 (s, 1H), 7.39 (dd, 1H), 5.29 (t, 1H), 4.72 (dd, 2H). Intermediate 228

5-chloro-1-benzothiophene-3-carbaldehyde 

To a solution of (5-chloro-1-benzothiophen-3-yl)methanol (4.00 g, 20.1 mmol) in acetonitrile (50 ml) was added manganese(IV) oxide (8.75 g, 101 mmol) at room temperature. The reaction mixture was heated to 80 °C and stirred at 80 °C for 12 hours. The reaction mixture was cooled to room temperature and filtered through celite. The filtrate was evaporated under reduced pressure to give 5-chloro-1-benzothiophene-3-carbaldehyde (4.00 g) as a yellow solid.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 10.10 (s, 1H), 9.06 (s, 1H), 8.50 (d, 1H), 8.16 (d, 1H), 7.54 (dd, 1H), 5.30 (t, 1H), 4.71 (dd, 2H). Intermediate 229

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(5-chloro-1-benzothiop hen-3-yl)prop-2- enoate 

 

1,8-Diazabicyclo(5.4.0)undec-7-ene was added dropwise to a solution of methyl (2R)- {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (8.09 g, 24.4 mmol) in dichloromethane (50 mL). After stirring at room temperature for 10 minutes, a solution of 5- chloro-1-benzothiophene-3-carbaldehyde (4.00 g, 20.3 mmol) in dichloromethane (30 ml) was added. The reaction was stirred at room temperature for 2 hours. The reaction solvent was removed under reduced pressure to give a residue. The residue was diluted with ethyl acetate (40 mL). The solution was washed with 1M HCl and brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by column chromatography (1000 mesh, petroleum ether: ethyl acetate = 1: 0, then 100: 1, then 70:1, then 40: 1, then 10: 1) to give methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(5-chloro-1- benzothiophen-3-yl)acrylate as a white solid.

LC-MS (Method D): R _t = 0.921 min; MS (ESIpos): m/z = 402.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 9.32 (s, 1H), 8.26 (s, 1H), 8.10 (d, 1H), 8.04 (d, 1H), 7.54-7.45 (m, 2H), 7.43-7.22 (m, 5H), 5.11 (s, 2H), 3.76 (s, 3H). Intermediate 230

methyl N-[(benzyloxy)carbonyl]-3-(5-chloro-1-benzothiophen-3-yl)-D- alaninate 

 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(5-chloro-1-benzothiop hen-3- yl)prop-2-enoate (3.90 g, 9.70 mmol) in methanol (150 ml) were added (R)- [Rh(COD)(MaxPhos)]BF ₄ (0.06 eq) (345 mg, 0.582 mmol). After stirring at room temperature for 16 hours under hydrogen atmosphere (50 psi), the reaction mixture (combined with another batch) was evaporated under reduced pressure to give a residue. The residue was dissolved in dichloromethane and filtered through silica gel (200-300 mesh). The filtrate was evaporated under reduced pressure to give methyl N-[(benzyloxy)carbonyl]-3-(5-chloro-1-benzothiophen-3- yl)-L-alaninate (4.00 g, 97 % purity, 99% yield) as a yellow oil.

LC-MS (Method D): R _t = 0.928 min; MS (ESIpos): m/z = 404.0 [M+H] ⁺ . Intermediate 231

N-[(benzyloxy)carbonyl]-3-(5-chloro-1-benzothiophen-3-yl) -D-alanine 

To a solution of methyl N-[(benzyloxy)carbonyl]-3-(5-chloro-1-benzothiophen-3-yl)-D- alaninate (3.80 g, 97% purity, 9.13 mmol) in tetrahydrofuran (70 ml) was added lithium hydroxide (2M in water) (15 ml, 2.0 M, 30 mmol) at 0 °C. After stirring at room temperature for 12 hours, the pH of the mixture was adjusted to 5~6 with formic acid. The mixture was concentrated and purified  

by preparative HPLC (Instrument: Shimadzu LC-20AP; Column: Phenomenex luna C18 250*100mm*10 µm; eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-20 min 40-75% B; flow 100 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give N-[(benzyloxy)carbonyl]-3-(5-chloro-1-benzothiophen-3-yl)-L- alanine (3.18 g, 99% purity, 88% yield) as a light yellow solid.

LC-MS (Method D): R _t = 0.857 min; MS (ESIpos): m/z = 411.9 [M+23] ⁺ .

1H NMR (400MHz, DMSO-d ₆ ) d [ppm] = 8.01 (d, 1H), 7.91 (d, 1H), 7.72 (d, 1H), 7.57 (s, 1H), 7.40 (dd, 1H), 7.35-7.21 (m, 5H), 4.96 (s, 2H), 4.36-4.25 (m, 1H), 3.28-3.25 (m, 1H), 3.13 (dd, 1H). Intermediate 232

tri-tert-butyl (5R,12S,16S)-5-[(5-chloro-1-benzothiophen-3-yl)methyl]-3,6,1 4-trioxo-1- phenyl-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarbox ylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (876 mg, 1.80 mmol) and N-[(benzyloxy)carbonyl]-3-(5-chloro-1-benzothiophen-3-yl)-D- alanine (700 mg, 1.80 mmol) were solubilised in DMF (14 ml), 4-methylmorpholine (490 µl, 4.5 mmol, CAS-RN:

109-02-4) and HATU (956 mg, 2.51 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 559 mg (36 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.61 min; MS (ESIpos): m/z = 861 [M+H] ⁺   Intermediate 233

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(5-chloro-1-benzothiophen- 3- yl)propanoyl]amino}-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl} amino)pentanedioate 

 

tri-tert-butyl (5R,12S,16S)-5-[(5-chloro-1-benzothiophen-3-yl)methyl]-3,6,1 4-trioxo-1-phenyl-2- oxa-4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate (559 mg, 98 % purity, 637 µmol) was solubilised in MeOH (5.2 ml), Palladium on carbon (67.8 mg, 10 % purity, 63.7 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 3.5h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 24.8 mg (5 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.24 min; MS (ESIpos): m/z = 726 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.107 (2.19), 1.382 (15.28), 1.388 (16.00), 2.327 (0.53), 2.518 (2.18), 2.522 (1.36), 2.669 (0.55), 7.550 (0.87), 7.892 (0.60), 7.897 (0.59), 7.980 (0.67), 8.002 (0.63). Intermediate 234

tri-tert-butyl (3R,10S,14S)-3-[(5-chloro-1-benzothiophen-3-yl)methyl]-1-{(1 r,4S)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,1 2-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

 

(1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (13.8 mg, 36.4 µmol) was solubilised in DMF (510 µl), 4-methylmorpholine (11 µl, 99 µmol, CAS- RN: 109-02-4) and HATU (13.8 mg, 36.4 µmol) were added, stirred for 20min at rt, di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(5-chloro-1-benzothiophen- 3-yl)propanoyl]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (24.0 mg, 33.1 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was filtered and evaporated to give 35.0 mg (15 % purity, 15 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.67 min; MS (ESIpos): m/z = 1087 [M+H] ⁺   Intermediate 235

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(5-chl oro-1- benzothiophen-3-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazah exadecane-10,14,16- tricarboxylate 

 

tri-tert-butyl (3R,10S,14S)-3-[(5-chloro-1-benzothiophen-3-yl)methyl]-1-{(1 r,4S)-4-[({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,1 2-trioxo-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (35.0 mg, 32.2 µmol) was solubilised in DMF (500 µl), piperidine (64 µl, 640 µmol) was added and the mixture was stirred under argon for 2h at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 12.0 mg (95 % purity, 41 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.28 min; MS (ESIpos): m/z = 866 [M+H] ⁺   Example 41-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (5-chloro-1-benzothiophen-3- yl)-D-alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(5-chl oro-1-benzothiophen- 3-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,1 4,16-tricarboxylate (5.00 mg, 5.78 µmol) was solubilised in DCM (190 µl), TFA (89 µl, 1.2 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 4.00 mg (95 % purity, 94 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.76 min; MS (ESIpos): m/z = 697 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.828 (0.73), 0.851 (2.19), 0.888 (2.39), 0.913 (1.20), 1.082 (0.60), 1.113 (1.33), 1.146 (1.59), 1.168 (1.13), 1.197 (1.53), 1.230 (3.39), 1.249 (3.52), 1.265 (3.39), 1.310 (1.86), 1.328 (1.99), 1.345 (1.99), 1.362 (1.86), 1.378 (1.53), 1.395 (1.53), 1.414 (1.86), 1.430 (2.26), 1.446 (2.39), 1.465 (1.86), 1.497 (1.79), 1.527 (1.39), 1.601 (2.06), 1.614 (2.79), 1.634 (2.79), 1.648 (2.19), 1.669 (3.32), 1.703 (2.59), 1.760 (2.06), 1.780 (2.26), 1.794 (2.59), 1.815 (1.66), 1.831 (1.06), 1.850 (0.60), 1.904 (2.19), 2.037 (1.06), 2.067 (1.86), 2.073 (4.05), 2.097 (1.00), 2.126 (0.86), 2.139 (1.06), 2.145 (1.06), 2.162 (1.93), 2.176 (1.93), 2.181 (1.86), 2.195 (1.33), 2.216 (1.46), 2.238 (2.46), 2.252 (1.20), 2.259 (1.53), 2.274 (1.39), 2.295 (0.73), 2.332 (2.92), 2.336 (1.39), 2.352 (0.60), 2.518 (16.00), 2.523 (11.09), 2.539 (0.93),  

2.552 (0.66), 2.584 (2.59), 2.599 (3.39), 2.673 (3.05), 2.678 (1.53), 2.692 (0.60), 2.710 (0.60), 2.727 (0.66), 2.888 (1.13), 2.956 (1.99), 2.972 (2.52), 2.993 (3.65), 3.004 (3.05), 3.017 (3.59), 3.030 (3.78), 3.053 (4.25), 3.077 (4.05), 3.095 (3.92), 3.110 (3.72), 3.155 (5.98), 3.168 (6.71), 3.192 (7.24), 3.204 (7.77), 3.912 (2.59), 3.932 (4.12), 3.944 (4.12), 4.483 (1.20), 4.497 (1.33), 4.506 (2.06), 4.520 (2.06), 4.528 (1.33), 4.542 (1.13), 6.140 (1.26), 6.431 (1.26), 7.362 (4.32), 7.366 (4.05), 7.383 (4.12), 7.388 (4.51), 7.533 (10.36), 7.970 (11.22), 7.974 (8.56), 7.979 (7.77), 7.991 (9.23), 8.019 (1.53), 8.250 (2.32).  #42 Linker Intermediate 236

methyl isoquinoline-7-carboxylate 

To a solution of 7-bromoisoquinoline (15.0 g, 72.1 mmol) in MeOH (150 ml) were added 1,1'- bis(diphenylphosphino)ferrocenepalladium(II) chloride (5.28 g, 7.21 mmol; CAS-RN:[72287-26- 4]) and trimethylamine (20 ml, 140 mmol). The reaction mixture was stirred at 70 °C for 16 h under carbon monoxide atmosphere (50 Psi). The precipitate was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-20%) to give 11.5 g (85 % yield) of the target compound.

1H NMR (400 MHz, CDCl ₃ ): d [ppm] = 9.35 (s, 1H), 8.72 (s, 1H), 8.62 (dd, 1H), 8.28 (d, 1H), 7.86 (d, 1H), 7.69 (d, 1H), 4.01 (s, 3H). Intermediate 237

(isoquinolin-7-yl)MeOH 

To a mixture of methyl isoquinoline-7-carboxylate (8.50 g, 45.4 mmol) in THF (85 ml) was lithium aluminum hydride (1.72 g, 45.4 mmol; CAS-RN:[16853-85-3]) at 0 °C. The reaction mixture was stirred at rt for 2 h. EtOAc and sodium potassium tartrate aqueous solution were added to the reaction mixture. The mixture was stirred at rt for overnight. The mixture was extracted with EtOAc. The organic phase was dried and concentrated under reduced pressure. The residue  

was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 20%-50%) to give 3.90 g (54 % yield) of the target compound. Intermediate 238

isoquinoline-7-carbaldehyde 

To a solution of (isoquinolin-7-yl)MeOH (3.90 g, 24.5 mmol) in acetonitrile (50 ml) was added manganese(IV) oxide (10.6 g, 122 mmol). The reaction was stirred at 80 °C for 16 h. The solid was removed by filtration. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 20%-50%) to give 2.41 g (63 % yield) of the target compound.

1H NMR (400 MHz, CDCl ₃ ): d [ppm] = 10.23 (s, 1H), 9.47 (s, 1H), 8.72 (d, 1H), 8.53 (s, 1H), 8.22 (dd, 1H), 7.98 (d, 1H), 7.78 (d, 1H). Intermediate 239

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-7-yl)prop -2-enoate 

1,8-Diazabicyclo(5.4.0)undec-7-ene (6.0 ml, 40 mmol) was added dropwise to a solution of methyl {[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetate (13.4 g, 40.5 mmol) in DCM

(100 ml). After stirring at rt for 10 min, a solution of isoquinoline-7-carbaldehyde (5.30 g, 33.7 mmol) in DCM (50 ml) was added. The reaction was stirred at rt for 2 h. The reaction solvent was removed under reduced pressure. The residue was diluted with EtOAc. The solution was washed with 1M HCl and brine. The organic phase was dried. The filtrate was concentrated and purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-50%) to give 8.50 g (70 % yield) of the target compound.

LC-MS (Method D): R _t = 0.608 min; MS (ESIpos): m/z =363.1 [M+H] ⁺ .

 

Intermediate 240

methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-7-yl)prop anoate 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-7-yl)prop -2-enoate (3.20 g, 8.83 mmol) in MeOH were added (R)-[Rh(COD)(MaxPHOS)]BF4 (150 ml). The reaction mixture was stirred at rt for 96 h under hydrogen atmosphere (1 MPa). The reaction mixture was evaporated under reduced pressure. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-50%) to give 1.20 g (91 % purity, 34 % yield) and 500 mg (50 % purity, 8 % yield) of the target compound.

LC-MS (Method D): R _t = 0.623 min; MS (ESIpos): m/z =365.0 [M+H] ⁺ . Intermediate 241

(2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-7-yl)p ropanoic acid 

To a cooled solution (0 °C) of methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-7- yl)propanoate (3.30 g, 9.06 mmol) in THF (30 ml) was added lithium hydroxide (5.4 ml, 2.0 M, 11 mmol; CAS-RN:[1310-65-2]). The reaction mixture was stirred at rtfor 16 h. The reaction solution was concentrated under reduced pressure and dissolved in MeOH. The pH of the mixture was adjusted to 5 with formic acid. The mixture was concentrated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.00 g (63 % yield) of the target compound.

 

LC-MS (Method C): R _t = 0.685 min; MS (ESIpos): m/z =351.1 [M+H] ⁺ .

1H-NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.23 (s, 1H), 8.48 (d, 1H), 7.96 (s, 1H), 7.90 (d, 1H), 7.83-7.68 (m, 3H), 7.29-7.07 (m, 5H), 4.94 (s, 2H), 4.39-4.31 (m, 1H), 3.27-3.22 (m, 1H), 3.07 (dd, 1H). Intermediate 242

tri-tert-butyl (5R,12S,16S)-5-[(isoquinolin-7-yl)methyl]-3,6,14-trioxo-1-ph enyl-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (1.11 g, 2.28 mmol) and (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-7-yl)prop anoic acid (800 mg, 2.28 mmol) were solubilised in DMF (18 ml), 4-methylmorpholine (630 µl, 5.7 mmol, CAS- RN: 109-02-4) and HATU (1.22 g, 3.20 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 460 mg (98 % purity, 24 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.28 min; MS (ESIpos): m/z = 821 [M+H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.107 (10.01), 1.381 (16.00), 1.384 (15.19), 2.518 (1.60), 2.522 (1.00), 2.539 (0.89), 4.190 (0.78), 4.885 (0.48), 4.908 (0.53), 7.155 (0.46), 7.159 (0.45), 7.210 (0.53), 7.226 (0.63), 7.795 (0.42), 7.868 (0.41), 7.925 (0.42), 8.453 (0.82), 8.468 (0.71), 9.203 (0.73). Intermediate 243

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(isoquinolin-7-yl)propanoy l]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate   

tri-tert-butyl (5R,12S,16S)-5-[(isoquinolin-7-yl)methyl]-3,6,14-trioxo-1-ph enyl-2-oxa-4,7,13,15- tetraazaoctadecane-12,16,18-tricarboxylate (460 mg, 561 µmol) was solubilised in MeOH (4.5 ml), Palladium on carbon (59.7 mg, 10 % purity, 56.1 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred for 8h at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated to give 330 mg (91 % purity, 78 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.29 min; MS (ESIpos): m/z = 686 [M+H] ⁺   Intermediate 244

tri-tert-butyl (3R,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(isoquinolin -7-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

 

(1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (183 mg, 482 µmol) was solubilised in DMF (6.7 ml), 4-methylmorpholine (140 µl, 1.3 mmol, CAS-RN: 109-02-4) and HATU (183 mg, 482 µmol) were added, stirred for 20min at rt, di-tert- butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(isoquinolin-7-yl)propanoy l]amino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (330 mg, 91 % purity, 438 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 150 mg (95 % purity, 31 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.42 min; MS (ESIpos): m/z = 1049 [M+H] ⁺   Intermediate 245

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-7- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

 

tri-tert-butyl (3R,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(isoquinolin -7-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (150 mg, 95 % purity, 136 µmol) was solubilised in DMF (2.1 ml), piperidine (270 µl, 2.7 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 84.0 mg (98 % purity, 73 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.96 min; MS (ESIpos): m/z = 826 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.381 (12.36), 1.387 (16.00), 2.518 (1.73), 2.522 (1.09), 7.872 (0.71), 8.419 (0.59), 8.431 (0.59), 8.446 (0.52), 9.190 (0.61).  Example 42-A

(3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(is oquinolin-7-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-7-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (10.0 mg, 98 % purity, 11.9 µmol) was solubilised in DCM (380 µl), TFA (180 µl, 2.4 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 4.00 mg (95 % purity, 49 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.45 min; MS (ESIpos): m/z = 658 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.832 (2.25), 0.862 (2.50), 0.895 (1.07), 0.987 (0.61), 1.018 (1.38), 1.049 (1.28), 1.076 (0.51), 1.142 (0.61), 1.175 (1.38), 1.207 (1.74), 1.231 (2.96), 1.248 (3.17), 1.266 (3.42), 1.302 (1.79), 1.319 (1.84), 1.339 (1.84), 1.354 (2.15), 1.374 (2.20), 1.412 (2.56), 1.431 (3.32), 1.624 (3.42), 1.645 (4.55), 1.732 (1.53), 1.762 (1.79), 1.799 (1.48), 1.817 (1.28), 1.834 (0.97), 2.029 (1.12), 2.058 (1.84), 2.073 (2.15), 2.133 (0.77), 2.147 (1.02), 2.169 (1.79), 2.183 (1.79), 2.201 (1.23), 2.222 (1.33), 2.244 (2.25), 2.265 (1.38), 2.279 (1.28), 2.301 (0.72), 2.322 (2.45), 2.326 (3.17), 2.331 (2.40), 2.522 (9.66), 2.572 (3.83), 2.587 (3.99), 2.664 (2.40), 2.669 (3.22), 2.673 (2.40), 2.944 (2.35), 2.977 (3.99), 3.002 (3.78), 3.059 (2.91), 3.074 (3.32), 3.091 (3.12), 3.108 (2.91), 3.154 (4.81), 3.166 (5.32), 3.188 (5.62), 3.200 (5.83), 3.364 (16.00), 3.938 (3.78), 3.949 (3.68), 4.503 (1.07), 4.516 (1.28), 4.528 (1.84), 4.538 (1.89), 4.550 (1.23), 4.562 (1.02), 6.154 (1.28), 6.425 (1.38), 7.658 (2.96), 7.662 (3.07), 7.679 (3.58), 7.683 (3.58), 7.758 (3.37), 7.772 (3.42), 7.847 (4.91), 7.868 (3.99), 7.895 (5.98), 7.967 (1.74), 8.238 (1.48), 8.429 (3.17), 8.443 (3.02), 9.192 (4.60).  Intermediate 246

tri-tert-butyl (3R,10S,14S)-3-[(isoquinolin-7-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2- [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

 

(

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (136 mg, 238 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (89.0 mg, 235 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (30 µl, 180 µmol) were stirred in DMF (6.2 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-7-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (49.0 mg, 59.4 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 70.0 mg (95 % purity, 81 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.26 min; MS (ESIpos): m/z = 1381 [M+H] ⁺   Example 42-B

(3R,10S,14S)-3-[(isoquinolin-7-yl)methyl]-1,4,12-trioxo-1 -[(1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3R,10S,14S)-3-[(isoquinolin-7-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate (4.00 mg, 2.90 µmol) was solubilised in DCM (1.0 ml), TFA (510 µl) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.50 mg (90 % purity, 74 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.48 min; MS (ESIpos): m/z = 1042 [M-H] ⁺  

1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.788 (0.96), 0.818 (1.08), 0.852 (1.02), 1.044 (0.45), 1.075 (0.45), 1.233 (3.00), 1.296 (1.27), 1.316 (1.53), 1.333 (1.47), 1.352 (1.21), 1.438 (1.34), 1.453 (1.21), 1.592 (1.59), 1.685 (0.76), 1.705 (0.64), 1.726 (0.76), 1.741 (0.57), 1.881 (0.70), 1.899 (0.70), 1.986 (0.51), 2.005 (0.76), 2.038 (0.83), 2.074 (0.70), 2.212 (1.08), 2.227 (1.53), 2.245 (1.53), 2.263 (0.96), 2.322 (2.87), 2.327 (3.82), 2.332 (2.87), 2.518 (16.00), 2.522 (10.07), 2.642 (2.10), 2.660 (2.61), 2.664 (3.82), 2.669 (4.65), 2.673 (3.44), 2.895 (1.91), 2.980 (5.16), 3.005 (5.04), 3.088 (2.29), 3.140 (1.53), 3.152 (1.72), 3.173 (1.72), 3.185 (1.72), 3.334 (10.01), 3.958 (0.51), 3.979 (1.02), 3.991 (1.02), 4.011 (0.51), 4.052 (0.51), 4.071 (0.96), 4.084 (0.96), 4.104 (0.45), 4.488 (0.45), 4.511 (0.76), 4.523 (0.83), 4.547 (0.45), 6.300 (1.47), 6.319 (1.66), 6.332 (1.15), 7.656 (1.40), 7.678 (1.66), 7.758 (2.10), 7.772 (2.23), 7.851 (2.49), 7.873 (2.04), 7.888 (2.68), 7.979 (0.89), 8.072 (1.47), 8.138 (2.49), 8.428 (4.08), 8.442 (3.63), 9.202 (4.53).  

Example 42-B 227Th

(3R,10S,14S)-3-[(isoquinolin-7-yl)methyl]-1,4,12-trioxo-1 -{(1r,4S)-4-[(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ² N ¹ ,N ⁴ }acetamido)methyl]cyclohexyl}-2,5,11,13-tetraazahexade cane-10,14,16- tricarboxylato(3-)( ²²⁷ Th)thorium

(3R,10S,14S)-3-[(isoquinolin-7-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction.. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 96.1% by iTLC.

#43 Linker Intermediate 247  

tri-tert-butyl (3S,10S,14S)-3-[(1-benzothiophen-3-yl)methyl]-1-{(1r,4S)-4-[ ({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate 

(1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (57.4 mg, 151 µmol) was solubilised in DMF (2.1 ml), 4-methylmorpholine (45 µl, 410 µmol, CAS- RN: 109-02-4) and HATU (57.5 mg, 151 µmol) were added, stirred for 20min at rt, di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(1-benzothiophen-3-yl)prop anoyl]amino}-1-tert-butoxy-1- oxohexan-2-yl]carbamoyl}amino)pentanedioate (95.0 mg, 138 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 40.0 mg (95 % purity, 26 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.64 min; MS (ESIpos): m/z = 1053 [M+H] ⁺   Intermediate 248

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1-ben zothiophen-3- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

 

tri-tert-butyl (3S,10S,14S)-3-[(1-benzothiophen-3-yl)methyl]-1-{(1r,4S)-4-[ ({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-2 ,5,11,13-tetraazahexadecane- 10,14,16-tricarboxylate (40.0 mg, 38.0 µmol) was solubilised in DMF (580 µl), piperidine (75 µl, 760 µmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 27.0 mg (96 % purity, 82 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.20 min; MS (ESIpos): m/z = 831 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.370 (0.78), 1.383 (16.00), 1.387 (11.91), 1.391 (11.33), 2.518 (1.65), 2.522 (0.95), 7.374 (0.86), 8.429 (0.47).  Example 43-A

N ⁶ -{N-[(1r,4S)-4-(aminomethyl)cyclohexane-1-carbonyl]-3- (1-benzothiophen-3-yl)-L- alanyl}-N ² -{[(1S)-1,3-dicarboxypropyl]carbamoyl}-L-lysine 

tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(1-ben zothiophen-3- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate (11.6 mg, 14.0 µmol) was solubilised in DCM (450 µl), TFA (220 µl, 2.8 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative

 

HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 7.10 mg (95 % purity, 73 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.70 min; MS (ESIpos): m/z = 663 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (3.63), 0.884 (3.48), 0.911 (1.72), 1.066 (0.86), 1.083 (1.16), 1.131 (3.13), 1.149 (3.68), 1.208 (2.83), 1.232 (8.18), 1.246 (6.71), 1.255 (6.46), 1.268 (5.30), 1.295 (3.79), 1.315 (3.13), 1.331 (7.27), 1.352 (3.28), 1.373 (2.37), 1.382 (2.42), 1.389 (2.37), 1.433 (3.38), 1.451 (3.48), 1.470 (2.78), 1.517 (2.47), 1.549 (2.22), 1.613 (2.57), 1.632 (2.68), 1.687 (5.50), 1.702 (4.74), 1.761 (4.14), 1.780 (3.94), 1.795 (3.48), 1.988 (0.45), 2.005 (0.45), 2.046 (1.46), 2.076 (2.47), 2.106 (1.41), 2.157 (1.36), 2.178 (2.68), 2.198 (3.28), 2.226 (3.53), 2.247 (2.07), 2.263 (1.46), 2.285 (0.86), 2.297 (2.12), 2.323 (2.47), 2.326 (3.18), 2.332 (2.47), 2.539 (1.77), 2.609 (4.59), 2.664 (2.52), 2.669 (3.23), 2.673 (2.47), 2.728 (8.73), 2.817 (1.06), 2.888 (10.40), 2.975 (3.38), 3.002 (4.64), 3.025 (4.19), 3.038 (4.90), 3.062 (5.20), 3.086 (3.74), 3.103 (3.23), 3.181 (5.96), 3.193 (6.56), 3.216 (6.51), 3.230 (6.66), 3.385 (9.89), 3.578 (3.99), 3.946 (5.45), 3.960 (5.25), 4.519 (1.46), 4.541 (2.57), 4.554 (2.62), 4.576 (1.36), 6.174 (1.92), 6.412 (2.27), 6.430 (2.22), 7.126 (0.40), 7.160 (0.81), 7.180 (0.91), 7.207 (0.76), 7.230 (0.40), 7.254 (0.76), 7.273 (0.76), 7.335 (2.12), 7.352 (4.74), 7.370 (4.04), 7.386 (4.14), 7.402 (16.00), 7.420 (2.32), 7.889 (5.25), 7.908 (4.79), 7.932 (6.16), 7.951 (7.92), 7.970 (3.13), 8.189 (2.17), 8.211 (2.27), 8.227 (2.52).  #44 Linker Intermediate 249

methyl isoquinoline-6-carboxylate 

To a solution of isoquinoline-6-carboxylic acid (8.50 g, 49.1 mmol) in MeOH (100 ml) was added thionyl chloride (11 ml, 150 mmol) at rt. The reaction mixture was stirred at 65 °C for 12 h. The mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 5%-33%) to give 8.40 g (91 % yield) of the target compound.

1H NMR (400 MHz, CDCl ₃ ): d [ppm] = 9.33 (s, 1H), 8.61 (d, 1H), 8.57 (s, 1H), 8.18 (d, 1H), 8.03 (d, 1H), 7.75 (d, 1H), 4.01 (s, 3H). Intermediate 250

(isoquinolin-6-yl)MeOH   

To a solution of methyl isoquinoline-6-carboxylate (8.40 g, 44.9 mmol) in THF (150 ml) was added lithium aluminum hydride (2.2 ml, 10 M, 22 mmol; CAS-RN:[16853-85-3]) at 0 °C. The reaction mixture was stirred at 25 °C for 1 h. The mixture was quenched by saturated ammonium chloride and extracted with EtOAc. The organic phase was washed with brine, dried and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-50%) to give 6.40 g (90 % yield) of the target compound.

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.26 (s, 1H), 8.47 (d, 1H), 8.06 (d, 1H), 7.85 (s 1H), 7.80 (d, 1H), 7.60 (d, 1H), 5.49 (t, 1H), 4.72 (d, 2H). Intermediate 251

isoquinoline-6-carbaldehyde 

To a solution of (isoquinolin-6-yl)MeOH (6.40 g, 40.2 mmol) in DCM (130 ml) was added 3,3,3- triacetoxy-3-iodophthalide (22.2 g, 52.3 mmol; CAS-RN:[87413-09-0]) at rt. The reaction mixture was stirred at 25 °C for 1 h. The mixture was filtered through a pad of celite and the filtrate was concentrated to give a residue. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-50%) to give 6.70 g (90 % purity, 95 % yield) of the target compound.

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 10.22 (s, 1H), 9.46 (s, 1H), 8.66 (d, 1H), 8.62 (s, 1H), 8.28 (d, 1H), 8.08-8.04 (m, 2H). Intermediate 252

methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)prop -2-enoate 

o

 

To a solution of methyl (2S)-{[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetat e (16.5 g, 49.9 mmol) in DCM (100 ml) was added 1,8-diazabicyclo(5.4.0)undec-7-ene (8.6 ml, 58 mmol) at rt. After stirred for 0.5 h, a solution of isoquinoline-6-carbaldehyde (6.70 g, 90 % purity, 38.4 mmol) in DCM (50 ml) was added and the reaction mixture was stirred at rt for 2 h. The mixture was quenched with saturated ammonium chloride and extracted with EtOAc. The organic phase was washed with brine, dried and concentrated to give a residue. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 10%-33%) to give 2.80 g (96 % purity, 21 % yield) and 1.20 g (87 % purity, 8 % yield) of the target compound.

LC-MS (Method D): R _t = 0.600 min; MS (ESIpos): m/z = 363.1 [M+H] ⁺ . 

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.40 (br.s, 1H), 9.31 (s, 1H), 8.53 (d, 1H), 8.18 (s, 1H), 8.11 (d, 1H), 7.92 (d, 1H), 7.75 (d, 1H), 7.45-7.30 (m, 6H), 5.12 (s, 2H), 3.75 (s, 3H). Intermediate 253

methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)prop anoate 

To a solution of methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)prop -2-enoate (2.80 g, 96% purity, 7.42 mmol) in methanol (100 ml) were added (R)-[Rh(COD)(MaxPhos)]BF ₄ (220 mg, 0.371 mmol). The reaction mixture was stirred at room temperature for 96 hours under hydrogen atmosphere (2.5 MPa). The mixture was concentrated and filtered through silica gel (200-300 mesh). The filtrate was concentrated and purified by preparative HPLC (Instrument:

Shimadzu LC-20AP; Column: Phenomenex Synergi Max-RP 250*50mm*10 µm; eluent A:

0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-30 min 5-40% B; flow 100 ml/min;

temperature: room temperature; Detector: UV 220/254 nm) to give methyl (2S)-2- {[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)propanoate (1.10 g, 41% yield) as yellow oil and methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)acry late (0.8 g) as yellow oil. Intermediate 254

(2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)p ropanoic acid 

 

To a solution of methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)prop anoate (1.10 g, 3.02 mmol) in pyridine (20 ml) was added lithium iodide anhydrous (1.21 g, 9.06 mmol) at room temperature. The reaction mixture was stirred at 105 °C for 12 hours. The mixture (combined with another batch starting with 0.80g methylester) was concentrated and purified by preparative HPLC (Instrument: Shimadzu LC-20AP; Column: Phenomenex Synergi Max-RP

250*50mm*10 µm; eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-24 min 8-38% B; flow 100 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give (2S)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)prop anoic acid as a brown solid.

The product (combined with another batch starting with 0.80g methylester) was dissloved in a mixed solvent of acetonitrile and water, lyophilized to give (2S)-2-{[(benzyloxy)carbonyl]amino}- 3-(isoquinolin-6-yl)propanoic acid (1.17 g) a brown solid.  

LC-MS (Method C): R _t = 0.636 min; MS (ESIpos): m/z = 351.0 [M+H] ⁺ .  

1H NMR (400 MHz, DMSO-d ₆ ): d [ppm] = 9.51 (s, 1H), 8.55 (d, 1H), 8.21 (d, 1H), 8.02 (d, 1H), 7.95 (s, 1H), 7.77 (d, 1H), 7.26-7.19 (m, 5H), 4.93 (s, 2H), 4.42-4.36 (m, 1H), 3.38-3.34 (m, 1H), 3.14-3.08 (m, 1H). Intermediate 255

tri-tert-butyl (5R,12S,16S)-5-[(isoquinolin-6-yl)methyl]-3,6,14-trioxo-1-ph enyl-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (660 mg, 1.35 mmol) and (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(isoquinolin-6-yl)prop anoic acid (474 mg, 1.35 mmol) were solubilised in DMF (10 ml), 4-methylmorpholine (370 µl, 3.4 mmol, CAS- RN: 109-02-4) and HATU (720 mg, 1.89 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 456 mg (90 % purity, 37 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.25 min; MS (ESIpos): m/z = 821 [M+H] ⁺   Intermediate 256

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(isoquinolin-6-yl)propanoy l]amino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5R,12S,16S)-5-[(isoquinolin-6-yl)methyl]-3,6,14-trioxo-1-ph enyl-2-oxa-4,7,13,15- tetraazaoctadecane-12,16,18-tricarboxylate (456 mg, 90 % purity, 500 µmol) was solubilised in  

MeOH (4.1 ml), Palladium on carbon (53.3 mg, 10 % purity, 50.0 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred at rt under hydrogen atmosphere.

The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 130 mg (95 % purity, 36 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 687 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.382 (16.00), 1.387 (7.54), 2.298 (0.73), 3.331 (0.66), 8.440 (0.50), 8.454 (0.45), 9.242 (0.56).  Intermediate 257

tri-tert-butyl (3R,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(isoquinolin -6-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate� �

,

(1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (79.1 mg, 208 µmol) was solubilised in DMF (2.9 ml), 4-methylmorpholine (63 µl, 570 µmol, CAS- RN: 109-02-4) and HATU (79.3 mg, 208 µmol) were added, stirred for 20min at rt, di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(isoquinolin-6-yl)propanoy l]amino}-1-tert-butoxy-1-oxohexan- 2-yl]carbamoyl}amino)pentanedioate (130 mg, 190 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 195 mg (98 % yield) of the target compound.

 

LC-MS (Method 1): R _t = 1.38 min; MS (ESIpos): m/z = 1048 [M+H] ⁺   Intermediate 258

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-6- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

tri-tert-butyl (3R,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(isoquinolin -6-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (200 mg, 191 µmol) was solubilised in DMF (2.9 ml), piperidine (380 µl, 3.8 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 43.0 mg (95 % purity, 26 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 826 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.381 (13.30), 1.386 (16.00), 2.327 (0.47), 2.518 (1.96), 2.523 (1.22), 2.669 (0.49), 7.718 (0.45), 7.996 (0.44), 8.017 (0.50), 8.411 (0.81), 8.440 (0.55), 8.454 (0.50), 9.238 (0.64).  Example 44-A

(3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(is oquinolin-6-yl)methyl]-1,4,12- trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-6-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (6.80 mg, 8.24 µmol) was solubilised in DCM (270 µl), TFA (130 µl, 1.6 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 1.00 mg (90 % purity, 17 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.44 min; MS (ESIpos): m/z = 657 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.834 (1.93), 0.862 (2.18), 1.026 (1.17), 1.053 (1.01), 1.144 (0.75), 1.168 (1.17), 1.234 (3.43), 1.258 (2.85), 1.275 (2.93), 1.432 (2.68), 1.454 (2.09), 1.605 (3.02), 1.640 (3.18), 1.767 (1.34), 1.808 (1.09), 1.987 (0.67), 2.020 (1.01), 2.050 (1.42), 2.074 (1.93), 2.141 (0.84), 2.162 (1.34), 2.176 (1.42), 2.229 (1.01), 2.251 (1.59), 2.271 (1.09), 2.284 (0.92), 2.518 (16.00), 2.523 (11.98), 2.539 (7.46), 2.573 (2.68), 2.945 (1.76), 2.978 (2.85), 3.004 (3.02), 3.089 (2.60), 3.104 (2.51), 3.157 (4.02), 3.169 (4.61), 3.950 (2.93), 4.533 (1.42), 4.544 (1.51), 6.140 (1.01), 6.442 (0.92), 7.561 (2.76), 7.582 (2.93), 7.696 (3.18), 7.711 (3.27), 7.747 (4.94), 7.959 (1.09), 7.992 (4.19), 8.013 (3.60), 8.233 (0.75), 8.280 (0.67), 8.437 (3.94), 8.452 (3.60), 9.234 (5.78).  Intermediate 259

tri-tert-butyl (3R,10S,14S)-3-[(isoquinolin-6-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2- [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (38.9 mg, 67.9 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (25.4 mg, 67.0 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (8.7 µl, 51 µmol) were stirred in DMF (1.8 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3-[(isoqu inolin-6-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylate (14.0 mg, 17.0 µmol) was added and the mixture was stirred overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 17.0 mg (30 % purity, 22 % yield) of the target compound.

LC-MS (Method 1): Rt = 1.23 min; MS (ESIpos): m/z = 1380 [M+H] ⁺ Example 44-B

(3R,10S,14S)-3-[(isoquinolin-6-yl)methyl]-1,4,12-trioxo-1 -[(1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

 

tri-tert-butyl (3R,10S,14S)-3-[(isoquinolin-6-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate (4.00 mg, 2.90 µmol) was solubilised in DCM (1.0 ml), TFA (510 µl) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.50 mg (95 % purity, 79 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.46 min; MS (ESIneg): m/z = 1042 [M-H]- 

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.784 (0.76), 0.814 (0.83), 1.228 (1.45), 1.319 (1.18), 1.352 (1.18), 1.463 (0.90), 1.594 (1.25), 1.722 (0.62), 1.738 (0.48), 1.907 (2.08), 2.032 (0.69), 2.209 (0.76), 2.224 (1.25), 2.246 (1.25), 2.263 (0.69), 2.318 (1.45), 2.322 (3.05), 2.326 (4.02), 2.331 (3.05), 2.336 (1.52), 2.518 (16.00), 2.522 (9.77), 2.659 (2.49), 2.664 (3.88), 2.669 (4.71), 2.673 (3.53), 2.678 (1.87), 2.729 (1.59), 2.889 (2.84), 2.979 (4.09), 3.095 (1.66), 3.152 (1.66), 3.185 (1.80), 3.436 (6.03), 3.987 (0.69), 4.000 (0.69), 4.073 (0.76), 4.086 (0.69), 4.534 (0.62), 6.298 (0.97), 6.317 (1.25), 6.333 (0.83), 7.555 (1.18), 7.576 (1.25), 7.699 (1.66), 7.714 (1.73), 7.731 (2.08), 7.993 (2.08), 8.015 (1.66), 8.075 (1.11), 8.136 (0.90), 8.437 (3.19), 8.451 (2.70), 9.233 (3.74). Example 44-B 227Th

 

(3R,10S,14S)-3-[(isoquinolin-6-yl)methyl]-1,4,12-trioxo-1 -{(1r,4S)-4-[(2-{4,7,10- tris[(carboxy-kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1 -yl- kappa ² N ¹ ,N ⁴ }acetamido)methyl]cyclohexyl}-2,5,11,13-tetraazahexade cane-10,14,16- tricarboxylato(3-)( ²²⁷ Th)thorium

(3R,10S,14S)-3-[(isoquinolin-6-yl)methyl]-1,4,12-trioxo-1-[( 1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 97.8% by iTLC.

#45 Linker Intermediate 260

Methyl quinoline-7-carboxylate 

 

0 To a solution of 7-bromoquinoline (40.0 g, 192 mmol) in MeOH (400 ml) were added 1,1'- bis(diphenylphosphino)ferrocenepalladium(II) chloride (14.1 g, 19.2 mmol; CAS-RN:[72287-26- 4]) and trimethylamine (54 ml, 380 mmol) at rt. The reaction mixture was stirred at 70 °C for 12 h under carbon monoxide (50 Psi). The reaction mixture was cooled to rt. The mixture was added to water. The mixture was extracted with EtOAc. The organic layer was evaporated under reduced pressure to give a residue. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 0%-10%) to give 40.0 g of the target compound.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 9.00 (dd, 1H), 8.56 (s, 1H), 8.43 (dd, 1H), 8.10-8.00 (m, 2H), 7.64 (dd, 1H), 3.92 (s, 3H). Intermediate 261

(quinolin-7-yl)MeOH  To a solution of methyl quinoline-7-carboxylate (40.0 g, 214 mmol) in THF (400 ml) was added lithium aluminum hydride (8.11 g, 214 mmol; CAS-RN:[16853-85-3]) at 0 °C under nitrogen atmosphere. The reaction was stirred at rt for 2 h under nitrogen atmosphere. Saturated sodium potassium tartrate tetrahydrate was added to the reaction mixture. The mixture was stirred at rt for 12 h. The mixture was extracted with ethyl actate. The organic layer was evaporated under reduced pressure. The residue was purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 5%-75%) to give 25.8 g (76 % yield) of the target compound.

LC-MS (Method D): R _t = 0.205 min; MS (ESIpos): m/z = 160.0 [M+H] ⁺ . Intermediate 262

Quinoline-7-carbaldehyde 

To a solution of (quinolin-7-yl)MeOH (25.8 g, 162 mmol) in acetonitrile (250 ml) was added manganese(IV) oxide (70.4 g, 810 mmol) at rt. The reaction mixture was stirred at 80 °C for 12 h. The reaction mixture was cooled to rt and then filtered through celite. The organic layer was  

evaporated under reduced pressure to give 11.1 g (98 % purity, 43 % yield) of the target compound.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 10.24 (s, 1H), 9.04 (dd, 1H), 8.61 (s, 1H), 8.46 (d, 1H), 8.12 (d, 1H), 7.97 (d, 1H), 7.68 (dd, 1H). Intermediate 263

Methyl (2Z)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-7-yl)prop-2- enoate  1,8-Diazabicyclo(5.4.0)undec-7-ene (14 ml, 92 mmol) was added dropwise to a solution of methyl (2S)-{[(benzyloxy)carbonyl]amino}(dimethoxyphosphoryl)acetat e (30.3 g, 91.6 mmol) in DCM (200 ml). After stirring at rt for 10 min, a solution of quinoline-7-carbaldehyde (12.0 g, 76.3 mmol) in DCM (100 ml) was added. The reaction was stirred at rt for 2 h. The reaction solvent was removed under reduced pressure. The residue was diluted with EtOAc. The solution was washed with 1M HCl and brine. The organic phase was dried. The filtrate was concentrated and purified by flash chromatography (SiO2, petroleum ether / EtOAc gradient 2%-33%) and trituration with methyl tert-butyl ether to give 12.5 g (45 % yield) of the target compound.

LC-MS (Method D): R _t = 0.639 min; MS (ESIpos): m/z = 363.1 [M+H] ⁺ . Intermediate 264

Methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-7-yl)propano ate 

To a solution of (E)-1-(((benzyloxy)carbonyl)amino)-2-(quinolin-7-yl)vinyl acetate (12.5 g, 34.5 mmol) in methanol (520 ml) was added (R)-[Rh(COD)(MaxPhos)]BF ₄ (1.02 g, 1.72 mmol). The reaction mixture was stirred at room temperature for 96 hours under hydrogen atmosphere (50 psi). The reaction mixture was evaporated under reduced pressure to give a residue. The residue was dissolved in dichloromethane/methanol (10/1) and filtered through silica gel (200-300 mesh). The filtrate was evaporated under reduced pressure and purified by preparative-HPLC  

(Instrument: Shimadzu LC-20AP; Column: Phenomenex Synergi Max-RP 250*50 mm*10 mm;

eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-33 min 15-45% B; flow

100 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give methyl (2S)-2- {[(benzyloxy)carbonyl]amino}-3-(quinolin-7-yl)propanoate (4.80 g, 87% purity, 33% yield) as yellow oil.

LC-MS (Method C): R _t = 0.786 min; MS (ESIpos): m/z = 365.2 [M+H] ⁺ . Intermediate 265

(2R)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-7-yl)prop anoic acid 

To a cooled solution (0 °C) of methyl (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-7- yl)propanoate (4.60 g, 12.6 mmol) in THF (70 ml) was added lithium hydroxide (15 ml, 2.0 M, 30 mmol; CAS-RN:[1310-65-2]). The reaction mixture was stirred at rtfor 1 h. The reaction mixture was cooled to rt and evaporated under reduced pressure. The residue was dissolved in MeOH, the solution was adjusted to pH 5-6 through 1 M HCl and evaporated under reduced pressure.

The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 4.30 g (99 % purity, 96 % yield) of the target compound.

LC-MS (Method C): R _t = 0.704 min; MS (ESIpos): m/z = 351.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d = 12.84 (br.s, 1H), 8.88 (dd, 1H), 8.33 (dd, 1H), 7.97-7.85 (m, 2H), 7.76 (d, 1H), 7.54 (dd, 1H), 7.51-7.48 (m, 1H), 7.32-7.09 (m, 5H), 4.98 (s, 2H), 4.40-4.28 (m, 1H), 3.32-3.28 (m, 1H), 3.07 (dd, 1H). Intermediate 266

tri-tert-butyl (5R,12S,16S)-3,6,14-trioxo-1-phenyl-5-[(quinolin-7-yl)methyl ]-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate 

 

di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (974 mg, 2.00 mmol) and (2R)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-7-yl)propano ic acid (700 mg, 2.00 mmol) were solubilised in DMF (15 ml), 4-methylmorpholine (550 µl, 5.0 mmol, CAS-RN:

109-02-4) and HATU (1.06 g, 2.80 mmol) were added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 410 mg (98 % purity, 25 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m/z = 821 [M+H] ⁺   Intermediate 267

di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(quinolin-7-yl)propanoyl]a mino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate 

tri-tert-butyl (5R,12S,16S)-3,6,14-trioxo-1-phenyl-5-[(quinolin-7-yl)methyl ]-2-oxa-4,7,13,15- tetraazaoctadecane-12,16,18-tricarboxylate (410 mg, 500 µmol) was solubilised in MeOH (4.1 ml), Palladium on carbon (53.2 mg, 10 % purity, 50.0 µmol) was added and the mixture was purged with hydrogen. The mixture was stirred at rt under hydrogen atmosphere. The mixture was filtered over Celite, washed with MeOH and evaporated. The residue was purified by

 

preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 151 mg (97 % purity, 43 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 687 [M+H] ⁺   Intermediate 268

tri-tert-butyl (3R,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(quinolin-7-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate 

(1r,4r)-4-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl ]cyclohexane-1-carboxylic acid (91.9 mg, 242 µmol) was solubilised in DMF (3.4 ml), 4-methylmorpholine (73 µl, 660 µmol, CAS- RN: 109-02-4) and HATU (92.1 mg, 242 µmol) were added, stirred for 20min at rt, di-tert-butyl (2S)-2-({[(2S)-6-{[(2R)-2-amino-3-(quinolin-7-yl)propanoyl]a mino}-1-tert-butoxy-1-oxohexan-2- yl]carbamoyl}amino)pentanedioate (151 mg, 220 µmol) was added and the mixture was stirred under argon overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 200 mg (97 % purity, 84 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.49 min; MS (ESIpos): m/z = 1048 [M+H] ⁺   Intermediate 269

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3- [(quinolin-7-yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate 

 

tri-tert-butyl (3R,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-1,4,12-trioxo-3 -[(quinolin-7-yl)methyl]-2,5,11,13- tetraazahexadecane-10,14,16-tricarboxylate (200 mg, 191 µmol) was solubilised in DMF (2.9 ml), piperidine (380 µl, 3.8 mmol) was added and the mixture was stirred under argon for 2h at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 26.0 mg (96 % purity, 16 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 826 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.211 (0.42), 1.230 (0.59), 1.364 (0.45), 1.382 (16.00), 1.387 (13.94), 1.390 (13.47), 2.518 (2.72), 2.523 (1.90), 7.473 (0.64), 7.846 (0.85), 7.865 (0.51), 8.387 (0.46), 8.839 (0.42), 8.843 (0.43), 8.850 (0.43).  Example 45-A

(3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12 -trioxo-3-[(quinolin-7- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ic acid 

tri-tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-7- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate (6.80 mg, 8.24 µmol) was solubilised in DCM (270 µl), TFA (130 µl, 1.6 mmol) was added and the mixture was stirred under argon overnight at rt. The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 2.20 mg (95 % purity, 39 % yield) of the target compound.  

LC-MS (Method 1): R _t = 0.48 min; MS (ESIpos): m/z = 657 [M+H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.835 (0.44), 0.852 (0.68), 0.868 (0.48), 1.233 (1.76), 1.353 (0.44), 1.452 (0.48), 1.606 (0.44), 2.518 (10.59), 2.523 (6.90), 2.540 (16.00), 2.556 (0.44), 2.568 (0.48), 3.166 (0.44), 3.200 (0.44), 3.929 (0.40), 7.444 (0.52), 7.454 (0.52), 7.464 (0.52), 7.475 (0.52), 7.487 (0.44), 7.512 (0.48), 7.847 (1.12), 7.867 (0.68), 8.286 (0.48), 8.303 (0.44), 8.841 (0.56), 8.845 (0.56), 8.851 (0.56), 8.856 (0.52).  Intermediate 270

tri-tert-butyl (3R,10S,14S)-1,4,12-trioxo-3-[(quinolin-7-yl)methyl]-1-[(1r, 4S)-4-({2-[4,7,10- tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan -1- yl]acetamido}methyl)cyclohexyl]-2,5,11,13-tetraazahexadecane -10,14,16-tricarboxylate 

[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacycl ododecan-1-yl]acetic acid (53.3 mg, 93.1 µmol; CAS-RN:[137076-54-1]), [(1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium hexafluoridophosphate(1-) (34.9 mg, 91.9 µmol; CAS-RN:94790-37-1) and N,N-diisopropylethylamine (12 µl, 70 µmol) were stirred in DMF (2.4 ml) for 10min at rt. tri- tert-butyl (3R,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-1,4,12-tr ioxo-3-[(quinolin-7- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate (19.2 mg, 23.3 µmol) was added and the mixture was stirred overnight at rt. The mixture was filtered and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 26.0 mg (90 % purity, 73 % yield) of the target compound.

LC-MS (Method 1): R _t = 1.28 min; MS (ESIpos): m/z = 1381 [M+H] ⁺  

 

Example 45-B

(3R,10S,14S)-1,4,12-trioxo-3-[(quinolin-7-yl)methyl]-1-[( 1r,4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid 

tri-tert-butyl (3R,10S,14S)-1,4,12-trioxo-3-[(quinolin-7-yl)methyl]-1-[(1r, 4S)-4-({2-[4,7,10-tris(2- tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]a cetamido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (4.00 mg, 2.90 µmol) was solubilised in DCM (1.0 ml), TFA (510 µl) was added and the mixture was stirred under argon overnight at rt.

The mixture was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 1.00 mg (70 % purity, 23 % yield) of the target compound.

LC-MS (Method 1): R _t = 0.48 min; MS (ESIpos): m/z = 1042 [M-H] ⁺  

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.815 (1.04), 0.851 (0.70), 0.999 (0.52), 1.015 (0.58), 1.044 (0.58), 1.074 (0.64), 1.230 (2.55), 1.306 (1.51), 1.321 (1.91), 1.339 (1.68), 1.439 (1.45), 1.456 (1.33), 1.603 (1.86), 1.640 (1.33), 1.687 (1.04), 1.703 (0.87), 1.724 (0.99), 1.739 (0.75), 1.876 (0.81), 1.894 (0.81), 2.004 (0.75), 2.033 (0.99), 2.073 (1.22), 2.211 (1.45), 2.232 (1.86), 2.240 (1.91), 2.261 (1.22), 2.322 (2.67), 2.326 (3.48), 2.331 (2.67), 2.336 (1.39), 2.518 (16.00), 2.522 (9.62), 2.539 (7.01), 2.644 (2.49), 2.659 (2.90), 2.664 (3.83), 2.669 (4.41), 2.673 (3.42), 2.892 (2.55), 2.958 (5.86), 2.991 (5.74), 3.013 (5.62), 3.086 (3.48), 3.150 (3.30), 3.173 (3.54), 3.185 (3.65), 3.433 (14.61), 3.957 (0.81), 3.977 (1.33), 3.990 (1.39), 4.011 (0.75), 4.050 (0.75),  

4.070 (1.28), 4.084 (1.28), 4.104 (0.70), 4.532 (0.87), 6.307 (2.09), 6.327 (2.03), 7.445 (2.09), 7.455 (1.97), 7.466 (1.97), 7.476 (2.61), 7.504 (1.74), 7.849 (6.03), 7.869 (2.67), 7.978 (1.16), 8.073 (1.62), 8.140 (2.38), 8.287 (1.68), 8.304 (1.62), 8.427 (0.52), 8.441 (0.52), 8.845 (2.20), 8.849 (2.26), 8.855 (2.20), 8.860 (1.91), 9.201 (0.52). Example 45-B 227Th

(3R,10S,14S)-1,4,12-trioxo-3-[(quinolin-7-yl)methyl]-1-{( 1r,4S)-4-[(2-{4,7,10-tris[(carboxy- kappaO)methyl]-1,4,7,10-tetraazacyclododecan-1-yl- kappa ² N ¹ ,N ⁴ }acetamido)methyl]cyclohexyl}-2,5,11,13-tetraazahexade cane-10,14,16- tricarboxylato(3-)( ²²⁷ Th)thorium

(3R,10S,14S)-1,4,12-trioxo-3-[(quinolin-7-yl)methyl]-1-[(1r, 4S)-4-({2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceta mido}methyl)cyclohexyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid was dissolved as 10mM solution in DMSO and diluted to a 250µM solution in 400 mM sodium acetate buffer (pH 5) containing 0.5 mg/mL pABA.1,07 µl of this solution was used in a 40µl labeling reaction. Thorium-227 in 400 mM sodium acetate buffer (pH 5) plus 20% EtOH was added giving a specific activity of 0.375 MBq/nmol and a RAC of 2.5 MBq/mL. The mixture was incubated at 95 °C for 60 min. The labelling efficiency was determined to be 89.9% by iTLC.

 

#46 Linker Intermediate 271

Trifluoroacetic acid—tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3- [(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahe xadecane-10,14,16- tricarboxylate (1/1) 

tri-tert-butyl (3S,10S,14S)-1-{(1r,4S)-4-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (182 mg, 174 µmol) was stirred in piperidine (20%, in DMF) for 30min at rt. The mixture was diluted with tert-butyl methyl ether /petrolether (9:1) and washed 2 times with sodium hydrogen carbonate solution. The organic layer was dried and evaporated. The residue was treated with acetonitrile, filtered and washed with acetonitrile. The filtrate was evaporated and purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 141.0 mg (100 % purity, 87 % yield) of the target compound.

LC-MS (Method M): Rt = 3.93 min; MS (ESIpos): m/z = 825 [M+H] ⁺   Intermediate 272

N ² -[4-(4-{3-(bis{2-[(3-hydroxy-1-methyl-2-oxo-1,2-dihydr opyridine-4- carbonyl)amino]ethyl}amino)-2-[(bis{2-[(3-hydroxy-1-methyl-2 -oxo-1,2-dihydropyridine- 4-carbonyl)amino]ethyl}amino)methyl]propyl}anilino)-4-oxobut anoyl]-N ⁵ -[N-(2,2,4,6,7- pentamethyl-2,3-dihydro-1-benzofuran-5-sulfonyl)carbamimidoy l]-D-ornithyl-N ⁵ -[N- (2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-sulfonyl)c arbamimidoyl]-D-ornithine 

 

Fmoc-D-Arg(Pbf)-2-CT (CT = chlorotrityl resin) resin (0,378 mmol/g, 273.0 mg, 103 µmol) was mixed with piperidine 20% in DMF (6 mL) in a 10 mL filter syringe body and shaken for 30

minutes after which the supernatant solvent was removed by filtration and washed with DMF.

The procedure was repeated one more time.

To the resulting H-D-Arg(Pbf)-2-CT was then added a solution of Fmoc-D-Arg(Pbf)-OH (134

mg, 206 µmol), ) N,N'-diisopropylcarbodiimide (32 µl, 210 µmol) and ethyl cyanoglyoxylate-2- oxime (29.3 mg, 206 µmol; CAS-RN [3849-21-6]) in DMF (4 mL), then shaken for 2 hours.

Afterwards the supernatant solution was removed and the resin washed with several time with

DMF. The coupling procedure was repeated 1x.

The resin was then mixed with piperidine 20% in DMF (6 mL) and then shaken for 30 minutes for the deprotection step; then the supernatant solvent was subsequently removed and the

resin was washed with DMF. The deprotection procedure was repeated 1x.

The resulting H-D-Arg(Pbf)-D-Arg(Pbf)-2-CT was mixed mith 3 mL of DCM, to which then was added PYAOP (80.7 mg, 155 µmol in 1ml DMF; CAS-RN:[156311-83-0]), 4-methylmorpholine

(34 µl, 310 µmol) and then 4-(4-{3-(bis{2-[(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridin e-4- carbonyl)amino]ethyl}amino)-2-[(bis{2-[(3-hydroxy-1-methyl-2 -oxo-1,2-dihydropyridine-4- carbonyl)amino]ethyl}amino)methyl]propyl}anilino)-4-oxobutan oic acid (109 mg, 103 µmol,

solid) under an argon counter-stream. The mixture was shaken for 2 h, the supernatant

solution was removed, and then the resin washed with DCM. The coupling procedure was

repeated 2x, and completion of reaction was monitored by test cleavage / analytical HPLC.  

Cleavage of the target comound was facilitated by adding 1,1,1,3,3,3-hexafluoro-2-propanol

(20% in DCM, 6 mL), shaking the mixture for 30 minutes at rt and then removing the

supernatant solution by filtration. The procedure was repeated twice. The combined filtrates were then concentrated, the residue taken up in a mixture of acetonitrile and water (8 mL + 2 mL), and the the solution was lyophilized to to afford 103 mg (88% purity, 46% yield) of the

target compound.

LC-MS (Method N): R _t = 10.53 min; MS (ESI pos): m/z = 937 [M+2H]2 ⁺  

Intermediate 273

tri-tert-butyl (3S,10S,14S)-1-{(1R,4S)-4-[({(2R)-2-({(2R)-2-[4-(4-{3-(bis{2 -[(3-hydroxy-1- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl)amino]ethyl}amin o)-2-[(bis{2-[(3-hydroxy- 1-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl)amino]ethyl}amino)methyl]propyl}anilino)-4-oxobutan amido]-5-[N'-(2,2,4,6,7- pentamethyl-2,3-dihydro-1-benzofuran-5-sulfonyl)carbamimidam ido]pentanoyl}amino)- 5-[N'-(2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5- sulfonyl)carbamimidamido]pentanoyl}amino)methyl]cyclohexyl}- 3-[(naphthalen-2- yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14, 16-tricarboxylate 

N ² -[4-(4-{3-(bis{2-[(3-hydroxy-1-methyl-2-oxo-1,2-dihydr opyridine-4- carbonyl)amino]ethyl}amino)-2-[(bis{2-[(3-hydroxy-1-methyl-2 -oxo-1,2-dihydropyridine-4- carbonyl)amino]ethyl}amino)methyl]propyl}anilino)-4-oxobutan oyl]-N ⁵ -[N-(2,2,4,6,7- pentamethyl-2,3-dihydro-1-benzofuran-5-sulfonyl)carbamimidoy l]-D-ornithyl-N ⁵ -[N-(2,2,4,6,7- pentamethyl-2,3-dihydro-1-benzofuran-5-sulfonyl)carbamimidoy l]-D-ornithine (101 mg, 53.9  

µmol), trifluoroacetic acid—tri-tert-butyl (3S,10S,14S)-1-[(1r,4S)-4-(aminomethyl)cyclohexyl]-3- [(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahe xadecane-10,14,16-tricarboxylate

(1/1) (75.9 mg, 80.9 µmol) and PYAOP (56.2 mg, 107.9 µmol; CAS-RN:[156311-83-0]) were solubilised in DCM (6.0 µl) and DMF (0.50 µl), 4-methylmorpholine (21 µl, 187 µmol; CAS- RN:[109-02-4]) was added and the mixture was stirred for at rt and overnight at 7°C (fridge). TFA

(21 µl, 270 µmol) in acetonitrile (3.0 ml) was added and the DCM was evaporated. The residue was purified by preparative HPLC (C18, acetonitrile/water with 0.1% formic acid) to give 77.0 mg (68 % purity, 36,5 % yield) of the target compound.

LC-MS (Method N): R _t = 13,67 min; MS (ESI pos): m/z = 1339.65 [M+2H]2 ⁺  

  Example 46-E

(3S,10S,14S)-1-[(1R,4S)-4-({[(2R)-2-({(2R)-2-[4-(4-{3-(bi s{2-[(3-hydroxy-1-methyl-2-oxo- 1,2-dihydropyridine-4-carbonyl)amino]ethyl}amino)-2-[(bis{2- [(3-hydroxy-1-methyl-2- oxo-1,2-dihydropyridine-4-carbonyl)amino]ethyl}amino)methyl] propyl}anilino)-4- oxobutanamido]-5-carbamimidamidopentanoyl}amino)-5- carbamimidamidopentanoyl]amino}methyl)cyclohexyl]-3-[(naphth alen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylic acid 

tri-tert-butyl (3S,10S,14S)-1-{(1R,4S)-4-[({(2R)-2-({(2R)-2-[4-(4-{3-(bis{2 -[(3-hydroxy-1-methyl- 2-oxo-1,2-dihydropyridine-4-carbonyl)amino]ethyl}amino)-2-[( bis{2-[(3-hydroxy-1-methyl-2-oxo- 1,2-dihydropyridine-4-carbonyl)amino]ethyl}amino)methyl]prop yl}anilino)-4-oxobutanamido]-5- [N'-(2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5- sulfonyl)carbamimidamido]pentanoyl}amino)-5-[N'-(2,2,4,6,7-p entamethyl-2,3-dihydro-1- benzofuran-5-sulfonyl)carbamimidamido]pentanoyl}amino)methyl ]cyclohexyl}-3-[(naphthalen- 2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,1 4,16-tricarboxylate (101 mg, 37.7 µmol) was solubilised in DCM (1,5 mL), a TFA solution (1,3 mL in 1,5 mL DCM) was added under argon and the reaction mixture was stirred for 40 min in a sealed vessel. The mixture was  

evaporated at 30°C and purified by preparative HPLC (C18, acetonitrile/water with 0.1% TFA) to give 28.0 mg (95 % purity, 68 % yield) of the target compound.

LC-MS (Method N): R _t = 7.89 min; MS (ESIpos): m/z = 1003 [M+2H]2 ⁺   Example 46-E ²²⁷ Th

(3S,10S,14S)-1-[(1R,4S)-4-({[(2R)-2-{[(2R)-2-{4-[4-(3-[bi s(2-{[3-(hydroxy-kappaO)-1- methyl-2-oxo-1,2-dihydropyridine-4-carbonyl]amino}ethyl)amin o]-2-{[bis(2-{[3-(hydroxy- kappaO)-1-methyl-2-oxo-1,2-dihydropyridine-4- carbonyl]amino}ethyl)amino]methyl}propyl)anilino]-4-oxobutan amido}-5- carbamimidamidopentanoyl]amino}-5- carbamimidamidopentanoyl]amino}methyl)cyclohexyl]-3-[(naphth alen-2-yl)methyl]- 1,4,12-trioxo-2,5,11,13-tetraazahexadecane-10,14,16-tricarbo xylato(4-)( ²²⁷ Th)thorium

(3S,10S,14S)-1-[(1R,4S)-4-({[(2R)-2-({(2R)-2-[4-(4-{3-(bi s{2-[(3-hydroxy-1-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl)amino]ethyl}amino)-2-[(bis{2-[(3- hydroxy-1-methyl-2-oxo-1,2- dihydropyridine-4-carbonyl)amino]ethyl}amino)methyl]propyl}a nilino)-4-oxobutanamido]-5- carbamimidamidopentanoyl}amino)-5-carbamimidamidopentanoyl]a mino}methyl)cyclohexyl]-3- [(naphthalen-2-yl)methyl]-1,4,12-trioxo-2,5,11,13-tetraazahe xadecane-10,14,16-tricarboxylic acid was dissolved in 30 mM citrate buffer (pH 5.5) containing 50 mg/mL sucrose, 2 mM EDTA and 0.5 mg/mL pABA was added thorium-227 dissolved in 30 mM citrate buffer (pH 5.5)

resulting in a specific activity of 0.3 MBq/nmol and a RAC of 4.6 MBq/mL. The mixture was

adjusted to pH 8 by adding 1M carbonate buffer. The mixture was incubated at room

temperature for 60 min. The labelling efficiency was determined to be 98.9% by iTLC. #47 Linker Intermediate 274

di-tert-butyl N-[(4-nitrophenoxy)carbonyl]-L-glutamate

 

A mixture of di-tert-butyl L-glutamate—hydrogen chloride (2.00 g, 6.76 mmol), 4-nitrophenyl carbonochloridate (1.36 g, 6.76 mmol), N,N-diisopropylethylamine (2.5 ml, 14 mmol), and THF

(55mL) was stirred at 30°C for 3h. After that the mixture was cooled to r.t., separated between ethyl acetate and 1N aqueous sodium hydroxide solution. The organic phase was washed 3x with 1N aqueous sodium hydroxide solution and once with brine and filtered through a phase separator. After concentration under reduced pressure the crude product was directly used in the next step. Intermediate 275

(2S)-2-({[(2S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl]ca rbamoyl}amino)-6-({[(9H-fluoren- 9-yl)methoxy]carbonyl}amino)hexanoic acid

To a solution of N ⁶ -{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-lysine (2.49 g, 6.76 mmol) in DCM

(10 mL) was added at r.t. N,N-diisopropylethylamine (1.3 ml, 7.4 mmol) and di-tert-butyl N-[(4-  

nitrophenoxy)carbonyl]-L-glutamate (2.87 g, 6.76 mmol). The mixture stirred for 14h at r.t.. After that the mixture was filtered and the residue washed with DCM. The filtrate was concentrated under reduced pressure and the crude product purified by column chromatography (SiO ₂ , DCM/EtOH gradient) and thereafter by recrystallzation from ethyl acetate/hexane. The solids were filtered off, washed with ethyl acetate, and dried under to give the title compound (1.72 g, 92 % purity, 36 % yield).

LC-MS (Method 1): R _t = 1.42 min; MS (ESIpos): m/z = 1308 [M+H]+

1H-NMR (400 MHz, DMSO-d6) delta [ppm]: 1.154 (0.42), 1.172 (0.84), 1.190 (0.41), 1.263 (0.21), 1.281 (0.20), 1.368 (0.57), 1.388 (16.00), 1.987 (1.49), 2.198 (0.16), 2.204 (0.20), 2.209 (0.18), 2.227 (0.19), 2.518 (0.31), 2.523 (0.20), 2.728 (0.28), 2.888 (0.32), 2.944 (0.28), 2.959 (0.27), 4.017 (0.44), 4.026 (0.25), 4.035 (0.56), 4.047 (0.25), 4.053 (0.23), 4.199 (0.21), 4.215 (0.18), 4.273 (0.61), 4.290 (0.38), 6.252 (0.27), 6.272 (0.26), 6.303 (0.24), 6.324 (0.23), 7.280 (0.24), 7.305 (0.33), 7.308 (0.34), 7.324 (0.80), 7.327 (0.77), 7.343 (0.52), 7.345 (0.51), 7.390 (0.39), 7.409 (0.62), 7.428 (0.27), 7.672 (0.55), 7.691 (0.49), 7.877 (0.65), 7.895 (0.59). Intermediate 276

2-Chlorotrityl chloride (CTC) resin (1.58mmol/g, 98.0 mg, 150 µmol) were mixed with DCM (2mL) and filled into a 2mL syringe body and shaken for 1h after which the supernatant solvent was removed. After that (2S)-2-({[(2S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl]carba moyl}amino)-6- {[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexanoic acid (198 mg, 303 µmol) and 4-methyl morpholine (100 µl, 910 µmol) were dissolved in DCM (2mL) and added to the resin. The mixture was shaken for 14h at r.t. After that the supernatant was removed and 4-methylmorpholine (6.7 µl, 61 µmol) dissolved in methanol (0.25mL) was added to the resin. After 15min the superpatent was removed and the resin was washed with DCM (5x 2mL), DMF (5x2mL), DMF/methanol (1:1, 5x2mL), methanol (3x2mL), and DMF (5x2 mL). Then a mixture of piperidine in DMF (1:4, 2mL) was added to the resin twice and the mixture was shaken for 5 min each. After that the supernatant was removed and the resin was washed with DMF (3x2mL) and DCM (3x2mL).  

Intermediate 277

The resin-bound starting material from the previous step was mixed with a solution of (2S)-2- ({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(naphthalen-2- yl)propanoic acid (262 mg, 600 µmol), HATU (222 mg, 585 µmol), and 4-methylmorpholine (130 µl, 1.2 mmol) in DMF (1.5 mL) and the mixture was shaken at r.t. for 1h. After that the resin was washed with DMF (5x2mL) and DCM (5x2mL). Intermediate 278

The resin-bound starting material from the previous step was washed with DMF (3x2mL) and then a mixture of DMF/piperidine (4:1, 2x2mL) was added and the mixture was shaken at r.t. for 15min each. Then the resin was washed with DMF (5x2mL) and DCM (3x2mL).  

Intermediate 279

The resin-bound starting material from the previous step was mixed with a solution of 6-({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)spiro[3.3]heptane-2-carb oxylic acid (110 mg, 293 µmol), HATU (111 mg, 293 µmol), and 4-methylmorpholine (66 µl, 600 µmol) in DMF (1.1 mL) and the mixture was shaken at r.t. for 1h. After that the resin was washed with DMF (5x2mL) and DCM

(5x2mL). Intermediate 280

(2S)-6-{[(2S)-2-[(6-aminospiro[3.3]heptane-2-carbonyl)ami no]-3-(naphthalen-2- yl)propanoyl]amino}-2-({[(2S)-1,5-di-tert-butoxy-1,5-dioxope ntan-2- yl]carbamoyl}amino)hexanoic acid

 

The resin-bound starting material from the previous step was washed with DMF (3x2mL) and then a mixture of DMF/piperidine (4:1, 2x2mL) was added and the mixture was shaken at r.t. for 15min each. Then the resin was washed with DMF (5x2mL) and DCM (3x2mL). After that the product was released from the resin by adding a mixture of HFIP/TIPS/water (95:2.5:2.5) (3x2mL, 15min each). The combined solutions were concentrated under reduced pressure and the crude product was purified by preparative HPLC (C18, acetonitrile/1% formic acid) to give the title compound (10 mg, 85% purity).

LC-MS (Method 1): R _t = 1.07 min; MS (ESIpos): m/z = 766 [M+H] ⁺ . Example 47-A

(3S,10S,14S)-1-(6-aminospiro[3.3]heptan-2-yl)-3-[(naphtha len-2-yl)methyl]-1,4,12-trioxo- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid

 

A mixture of (2S)-6-{[(2S)-2-[(6-aminospiro[3.3]heptane-2-carbonyl)amino] -3-(naphthalen-2- yl)propanoyl]amino}-2-({[(2S)-1,5-di-tert-butoxy-1,5-dioxope ntan-2- yl]carbamoyl}amino)hexanoic acid (10.0 mg, 13.1 µmol) and hydrogenchloride (4M in dioxane, 33µL) was stirred for 14h at r.t. After that the mixture was concentrated under reduced pressure to give the title compound (8.0 mg, 95% purity, 89% yield).

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 654 [M+H] ⁺ .

¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.40), 1.232 (2.88), 1.295 (1.36), 1.313 (1.36), 1.352 (0.64), 1.460 (0.72), 1.481 (0.56), 1.579 (0.72), 1.600 (0.56), 1.682 (0.48), 1.696 (0.64), 1.717 (0.80), 1.731 (0.56), 1.815 (0.80), 1.848 (1.12), 1.870 (1.36), 1.900 (1.36), 1.919 (1.36), 1.939 (1.12), 2.004 (1.60), 2.026 (2.00), 2.050 (2.32), 2.073 (1.60), 2.093 (0.64), 2.211 (1.04), 2.227 (1.68), 2.249 (1.76), 2.268 (1.20), 2.289 (0.88), 2.331 (3.52), 2.518 (16.00), 2.522 (10.72), 2.539 (1.68), 2.673 (3.44), 2.859 (0.96), 2.879 (1.28), 2.900 (1.44), 2.922 (0.88), 2.970 (0.72), 2.987 (0.72), 3.015 (0.72), 3.031 (0.80), 3.047 (0.64), 3.064 (0.96), 3.100 (0.56), 3.426 (0.40), 3.483 (0.40), 3.983 (0.48), 4.004 (0.88), 4.018 (0.96), 4.038 (0.48), 4.067 (0.56), 4.089 (1.12), 4.101 (1.12), 4.122 (0.48), 4.514 (0.80), 4.526 (0.80), 6.276 (1.44), 6.297 (1.52), 6.312 (1.68), 6.332 (1.52), 7.373 (1.44), 7.395 (1.60), 7.426 (0.64), 7.440 (1.52), 7.443 (1.44), 7.457 (2.72), 7.462 (1.68), 7.472 (1.36), 7.488 (0.56), 7.668 (2.72), 7.778 (3.20), 7.797 (3.20), 7.840 (1.84), 7.859 (1.60), 7.901 (0.96), 7.912 (0.96), 7.923 (0.96), 7.933 (0.80), 7.993 (1.12), 8.133 (0.40). #48 Linker Intermediate 290

Methyl (2E)-3-(quinolin-6-yl)prop-2-enoate

To a solution of 6-bromoquinoline (20.0 g, 96.1 mmol) and methyl prop-2-enoate (24.8 g, 288 mmol) in N,N-dimethylformamide (200 ml) were added dichlorobis(triphenylphosphine) palladium(II) (3.37 g, 4.81 mmol) and potassium carbonate (33.2 g, 240 mmol) at room temperature. The reaction mixture was heated to 100 °C and stirred at 100 °C for 12 hours. The reaction mixture was poured to water (1000 ml) and the mixture was extracted with ethyl acetate.

The organic layer was evaporated under reduced pressure to give a residue. The residue was purified by chromatography (200 - 300 mesh, petroleum ether: ethyl acetate = 10: 1, then 5: 1, then 2: 1) to give methyl 3-(quinolin-6-yl)acrylate (11.1 g, 54% yield) as yellow oil.

 

¹ H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 8.91 (dd, J = 4.4, 2.0 Hz, 1H), 8.37-8.31 (m, 1H), 8.24 (d, J = 1.6 Hz, 1H), 8.11 (dd, J = 8.8, 1.6 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 16.0 Hz, 1H), 7.55 (dd, J = 8.0, 4.0 Hz, 1H), 6.78 (d, J = 16.0 Hz, 1H), 3.74 (s, 3H). Intermediate 291

Methyl 3-(1,2,3,4-tetrahydroquinolin-6-yl)propanoate

To a solution of methyl (2E)-3-(quinolin-6-yl)prop-2-enoate (11.1 g, 52.1 mmol) in methanol (100 ml) was added palladium on carbon (2.77 g, 10% purity, 2.60 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 hours under hydrogen atmosphere. The reaction mixture was filtered through celite. The filtrate was evaporated under reduced pressure to give a residue. The residue was purified by chromatography (200 - 300 mesh, petroleum ether: ethyl acetate = 10: 1, then 5: 1, then 2: 1) to give methyl 3-(1,2,3,4-tetrahydroquinolin-6- yl)propanoate (10.1 g, 88% yield)) as a yellow oil. Intermediate 292

Methyl 3-(quinolin-6-yl)propanoate

To a solution of methyl 3-(1,2,3,4-tetrahydroquinolin-6-yl)propanoate (10.1 g, 46.1 mmol) in a mixed solvent of 1,4-dioxane (100 ml) and water (5.0 ml) was added sodium periodate (19.7 g, 92.1 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was heated to 80 °C and stirred at 80 °C for 48 hours. The reaction mixture was cooled to room temperature.

Saturated sodium thiosulfate and ethyl acetate were added to the reaction mixture. After separated, the organic layer was evaporated unde reduced pressure to give a residue. The residue was purified by chromatography (200 - 300 mesh, petroleum ether: ethyl acetate = 9: 1, then 4: 1, then 2: 1) to give methyl 3-(quinolin-6-yl)propanoate (5.20 g, 52% yield) as brown oil.

 

¹ H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 8.84 (dd, J = 1.6, 4.0 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 0.8 Hz, 1H), 7.65 (dd, J = 8.4, 2.0 Hz, 1H), 7.49 (dd, J = 8.4, 4.4 Hz, 1H), 3.58 (s, 3H), 3.05 (t, J = 7.6 Hz, 2H), 2.75 (t, J = 7.6 Hz, 2H). Intermediate 293

3-(quinolin-6-yl)propanoic acid

To a solution of methyl 3-(quinolin-6-yl)propanoate (5.20 g, 24.2 mmol) in a mixed solvent of tetrahydrofuran (10 ml) and water (2.0 ml) was added lithium hydroxide (1.16 g, 48.3 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was adjusted to pH = 3~4 through hydrochloric acid (1M). The mixture was extracted with ethyl acetate. The organic layer was evaporated under reduced pressure to give 3-(quinolin-6-yl)propanoic acid (3.60 g, 74% yield) as a yellow solid.

LC-MS (Method D): R _t = 0.486 min; MS (ESIpos): m/z = 202.0 [M+H] ⁺ . Intermediate 294

3-(quinolin-6-yl)propanoyl chloride

To a solution of 3-(quinolin-6-yl)propanoic acid (3.60 g, 17.9 mmol) in dichloromethane (50 ml) was added oxalyl chloride (3.1 ml, 36 mmol) at room temperature under nitrogen atmosphere.

After stirring at room temperature for 2 hours under nitrogen atmosphere, the reaction mixture was evaporated under reduced pressure to give 3-(quinolin-6-yl)propanoyl chloride (3.93 g, 100% yield) as a brown solid. Intermediate 295

(4S)-4-benzyl-3-[3-(quinolin-6-yl)propanoyl]-1,3-oxazolid in-2-one

 

To a stirred solution of (4S)-4-benzyl-1,3-oxazolidin-2-one (3.17 g, 17.9 mmol) in tetrahydrofuran (90 ml) was added n-butyllithium (2.5 M in hexane, 11 ml, 27 mmol) under an atmosphere of nitrogen at -78 °C. After stirring at -78 °C for 30 min, 3-(quinolin-6-yl)propanoyl chloride (3.93 g, 17.9 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The organic layer was evaporated under reduced pressure to give a residue. The residue was purified by chromatography (200 - 300 mesh, petroleum ether: ethyl acetate = 20: 1, then 10: 1, then 4: 1, then 2: 1) to give (4S)-4-benzyl-3- [3-(quinolin-6-yl)propanoyl]-1,3-oxazolidin-2-one (2.90 g, 45% yield) as yellow oil.

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 8.85 (dd, J = 4.0, 1.6 Hz, 1H), 8.31 (dd, J = 7.6, 0.8 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.86-7.78 (m, 1H), 7.71 (dd, J = 8.4, 2.0 Hz, 1H), 7.50 (dd, J = 8.0, 4.4 Hz, 1H), 7.26-7.20 (m, 3H), 7.16-7.08 (m, 2H), 4.71-4.62 (m, 1H), 4.35-4.15 (m, 2H), 3.42- 3.27 (m, 2H), 3.19-3.08 (m, 2H), 3.02-2.86 (m, 2H). Intermediate 296

(4S)-3-[(2S)-2-azido-3-(quinolin-6-yl)propanoyl]-4-benzyl -1,3-oxazolidin-2-one

A solution of (4S)-4-benzyl-3-[3-(quinolin-6-yl)propanoyl]-1,3-oxazolidin- 2-one (1.90 g, 5.27 mmol) in tetrahydrofuran (10 ml) was added to a solution of lithium bis(trimethylsilyl)amide (5.8 ml, 1.0 M, 5.8 mmol) in tetrahydrofuran (20 ml) at -70 °C. After stirring at -70 °C for 30 minutes, a solution of 2,4,6-tri(propan-2-yl)benzene-1-sulfonyl azide (1.96 g, 6.33 mmol) in tetrahydrofuran (10 ml) was added at the same temperature. The reaction mixture was stirred at -70 °C for 2 minutes. Then acetic acid was added to the reaction mixture at -70 °C. The reaction mixture was warmed to room temperature and stirred at room temperature for 12 hours.

Saturated ammonium chloride was added to the reaction mixture. The solution was extracted with ethyl acetate. The organic layer was evaporated under reduced pressure to give a residue.  

The residue was purified by preparative HPLC (Instrument: Shimadzu LC-20AP; Column:

Phenomenex Synergi Max-RP 250*50 mm*10 mm; eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-35 min 25-55% B; flow 100 ml/min; temperature: room temperature;

Detector: UV 220/254 nm) to give (4S)-3-[(2S)-2-azido-3-(quinolin-6-yl)propanoyl]-4-benzyl-1, 3- oxazolidin-2-one (1.10 g, 52% yield) as a light yellow solid.

LC-MS (Method C): R _t = 0.695 min; MS (ESIpos): m/z = 401.9 [M+H] ⁺ . 

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 8.98 (dd, J = 4.4, 1.6 Hz, 1H), 8.55 (d, J = 7.6 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 1.2 Hz, 1H), 7.82 (dd, J = 8.8, 2.0 Hz, 1H), 7.66 (dd, J = 8.4, 4.8 Hz, 1H), 7.36-7.21 (m, 5H), 5.35-5.27 (m, 1H), 4.77-4.69 (m, 1H), 4.39 (t, J = 8.8 Hz, 1H), 4.29 (dd, J = 8.8, 2.8 Hz, 1H), 3.51-3.45 (m, 1H), 3.18-3.06 (m, 2H), 3.05-2.97 (m, 1H). Intermediate 297

(2S)-2-azido-3-(quinolin-6-yl)propanoic acid

To a solution of (4S)-3-[(2S)-2-azido-3-(quinolin-6-yl)propanoyl]-4-benzyl-1, 3-oxazolidin-2-one (900 mg, 2.24 mmol) in a mixed solvent of tetrahydrofuran (9.0 ml) and water (2 ml) was added hydrogen peroxide (1.5 ml, 30% purity, 15 mmol) dropwise at 0 °C. After stirring at 0°C for 10 minutes, a solution of lithium hydroxide (118 mg, 4.93 mmol) in water (1 ml) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours. Saturated sodium sulfite and water was added to the reaction mixture. The mixture was washed with ethyl acetate. The aqueous layer was adjusted to pH = 4~5 with hydrochloric acid (1M) and extracted with ethyl acetate. The organic layer was evaporated under reduced pressure to give (2S)-2- azido-3-(quinolin-6-yl)propanoic acid (430 mg, 79% yield) as light yellow oil.

LC-MS (Method D): R _t = 0.526 min; MS (ESIpos): m/z = 243.0 [M+H] ⁺ .  Intermediate 298 (2S)-2-amino-3-(quinolin-6-yl)propanoic acid

To a solution of (2S)-2-azido-3-(quinolin-6-yl)propanoic acid (400 mg, 1.65 mmol) in a mixed solvent of tetrahydrofuran (2.0 ml) and hydrochloric acid (2.0 ml, 1M) was added  

triphenylphosphine (455 mg, 1.73 mmol) at room temperature. After stirring at room temperature for 12 hours, the reaction mxiture was used to next step without further purification.

LC-MS (Method C): R _t = 0.122 min; MS (ESIpos): m/z = 216.9 [M+H] ⁺ .  Intermediate 299

(2S)-2-amino-3-(quinolin-6-yl)propanoic acid

To a solution of (2S)-2-azido-3-(quinolin-6-yl)propanoic acid (5.20 g, 89% purity, 19.1 mmol) in a mixed solvent of tetrahydrofuran (20 ml) and hydrochloric acid (20 ml, 1M in water) was added triphenylphosphine (5.51 g, 21.0 mmol) at room temperature. After stirring at room temperature for 12 hours, the reaction mxiture was washed with ethyl acetate. The aqueous layer was lyophilized to give (2S)-2-amino-3-(quinolin-6-yl)propanoic acid (5.10 g, 80% purity, 99% yield) as a yellow solid.

LC-MS (Method A): R _t = 0.137 min; MS (ESIpos): m/z = 216.9 [M+H] ⁺ . Intermediate 300

(2S)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-6-yl)prop anoic acid

To a solution of (2S)-2-amino-3-(quinolin-6-yl)propanoic acid (350 mg, 1.62 mmol) in tetrahydrofuran (6.8 ml) were added sodium hydrogen carbonate (272 mg, 3.24 mmol) and benzyl chloroformate (230 µl, 1.6 mmol) at 14 °C. The reaction mixture was stirred at 14 °C for 16 hours. The reaction mixture was warmed to 28 °C and stirred at 28 °C for 16 hours. Saturated sodium hydrogen carbonate was added to the reaction mixure. The mixture was washed with ethyl acetate. The aqueous layer was adjusted to pH = 4~5 by hydrochloric acid (1M) and extracted with ethyl acetate. The oraganic layer was evaporated under reduced pressiure to give a crude product. The crude product was purified by preparative HPLC (Instrument: Gilson-281;

Column: Phenomenex Synergi C18 150*25mm*10 µm; eluent A: 0.225% formic acid in water,

 

eluent B: acetonitrile; gradient: 0-10 min 7-37% B; flow 25 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give the product (82% LCMS purity). The product was purified again by preparative HPLC (Instrument: Gilson-281; Column: Phenomenex Synergi C18 150*25mm*10 µm; eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient:

0-10 min 3-33% B; flow 25 ml/min; temperature: room temperature; Detector: UV 220/254 nm) to give (2S)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-6-yl)propano ic acid (4.00 mg, 98% purity, 1% yield) as a white solid.

LC-MS (Method C): R _t = 0.532 min; MS (ESIpos): m/z = 350.9 [M+H] ⁺ . 

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 8.85 (dd, J = 4.0, 1.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.79 (s, 1H), 7.67 (dd, J = 8.4, 1.6 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.50 (dd, J = 8.4, 4.4 Hz, 1H), 7.28-7.08 (m, 5H), 4.94 (s, 2H), 4.34-4.24 (m, 1H), 3.27-3.25 (m, 1H), 3.08-3.02 (m, 1H). Intermediate 301

(2S)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-6-yl)prop anoic acid

To a solution of (2S)-2-amino-3-(quinolin-6-yl)propanoic acid (1.90 g, 80% purity, 7.03 mmol) in a mixed solvent of tetrahydrofuran (10 ml) and water (10 ml) were added sodium hydrogen carbonate (1.77 g, 21.1 mmol) and N-(benzyloxycarbonyloxy)succinimide (1.75 g, 7.03 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was evaporated under reduced pressure. The resulting suspension was filtered.

The filter cake was washed with water and methanol to give (2S)-2- {[(benzyloxy)carbonyl]amino}-3-(quinolin-6-yl)propanoic acid (1.44 g, 98% purity, 99% ee%, 57% yield) as a white solid. The filtrate was purified by preparative HPLC (Instrument: Shimadzu LC-20AP; Column: Phenomenex Synergi Max-RP 250*80 mm*10 mm; eluent A: 0.225% formic acid in water, eluent B: acetonitrile; gradient: 0-20 min 20-48% B; flow 160 ml/min; temperature:

room temperature; Detector: UV 220/254 nm) to give (2S)-2-{[(benzyloxy)carbonyl]amino}-3- (quinolin-6-yl)propanoic acid (660 mg, 96% purity, 82% ee%, 25% yield) as a light yellow solid.

LC-MS (Method A): R _t = 0.645 min; MS (ESIpos): m/z = 351.0 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) d [ppm] = 12.82 (br. s, 1H), 8.86 (dd, J = 4.0, 1.6 Hz, 1H), 8.26  

(d, J = 7.6 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.81 (s, 1H), 7.77-7.64 (m, 2H), 7.51 (dd, J = 8.4, 4.0 Hz, 1H), 7.29-7.15 (m, 5H), 4.94 (s, 2H), 4.39-4.29 (m, 1H), 3.28-3.25 (m, 1H), 3.11-3.00 (m, 1H). Intermediate 302

tri-tert-butyl (5S,12S,16S)-3,6,14-trioxo-1-phenyl-5-[(quinolin-6-yl)methyl ]-2-oxa- 4,7,13,15-tetraazaoctadecane-12,16,18-tricarboxylate

A mixture of di-tert-butyl N-{[(2S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl]carbamoyl}-L- glutamate (1.95 g, 4.00 mmol), (2S)-2-{[(benzyloxy)carbonyl]amino}-3-(quinolin-6-yl)propano ic acid (1.40 g, 4.00 mmol), 4-methylmorpholine (1.3 ml, 12 mmol), HATU (2.28 g, 5.99 mmol), and DMF (31 ml, 400 mmol) was stirred for 3h at r.t. After that the mixture was separated between DCM and water. The organic phase was filtered through a phase separator and concentrated under reduced pressure. The crude product was redissolved in DCM, filtered, and the filtrate concentrated under reduced pressure and purified by preparative HPLC (C18, acetonitrile/1%formic acid) to give the title compound (1.28g, 1.25mmol, 31% yield). Intermediate 303

di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-6-yl)propanoyl]a mino}-1-tert- butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate

 

To a solution of tri-tert-butyl (5S,12S,16S)-3,6,14-trioxo-1-phenyl-5-[(quinolin-6- yl)methyl]-2-oxa-4,7,13,15-tetraazaoctadecane-12,16,18-trica rboxylate (1.28 g, 1.56 mmol) in ethanol (10mL) was added palladium (10% on charcoal, 166 mg, 156 µmol) an the mixture was stirred under a hydrogen atmosphere at 50°C for 5h. After cooling to r.t. the mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC (C18, acetonitrile/1%ammonia) to give the title compound (550 mg, 80 % purity, 41 % yield).

LC-MS (OA01b02): R _t = 1.27 min; MS (ESIpos): m/z = 687 [M+H] ⁺ . Intermediate 304

tri-tert-butyl (3S,10S,14S)-1-[trans-4-({[(benzyloxy)carbonyl]amino}methyl) cyclohexyl]- 1,4,12-trioxo-3-[(quinolin-6-yl)methyl]-2,5,11,13-tetraazahe xadecane-10,14,16- tricarboxylate

 

A mixture of di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-6-yl)propanoyl]a mino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (105 mg, 80 % purity, 122 µmol), (1r,4r)-4-({[(benzyloxy)carbonyl]amino}methyl)cyclohexane-1- carboxylic acid (39.3 mg, 135 µmol; CAS-RN:[27687-12-3]), HATU (55.9 mg, 147 µmol), 4-methylmorpholine (34 µl, 310 µmol), and DMF (1.9 mL) was stirred at r.t. for 3h. After that the mixture was separated between DCM and water and the organic phase was filtered through a phase separator and concentrated under reduced pressure. The crude product was purified by preparative HPLC (C18, acetonitrile/1%TFA gradient) to give the title compound (130 mg, 90 % purity, 100 % yield).

LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 959 [M+H] ⁺ . Intermediate 305

tri-tert-butyl (3S,10S,14S)-1-[trans-4-(aminomethyl)cyclohexyl]-1,4,12-trio xo-3-[(quinolin- 6-yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarbox ylate

 

To a solution of tri-tert-butyl (3S,10S,14S)-1-[4- ({[(benzyloxy)carbonyl]amino}methyl)cyclohexyl]-1,4,12-triox o-3-[(quinolin-6-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (130 mg, 136 µmol) in ethanol (2 mL) was added palladium (10% on charcoal, 14.4 mg, 13.6 µmol) an the mixture was stirred under a hydrogen atmosphere at r.t. for 3h. After that the mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC

(C18, acetonitrile/1%ammonia) to give the title compound (14 mg, 95 % purity, 12 % yield).

LC-MS (Method 2): R _t = 1.38 min; MS (ESIneg): m/z = 824 [M-H]- Example 48-A

(3S,10S,14S)-1-[trans-4-(aminomethyl)cyclohexyl]-1,4,12-t rioxo-3-[(quinolin-6- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ic acid

A mixture of tri-tert-butyl (3S,10S,14S)-1-[trans-4-(aminomethyl)cyclohexyl]-1,4,12-trio xo-3- [(quinolin-6-yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate (15.0 mg, 18.2 µmol) and hydrogenchloride (4M in dioxane, 91µL) was stirred for 14h at r.t. After that the mixture was concentrated under reduced pressure and the crude product was purified by preparative HPLC (C18, acetonitrile/1% TFA) to give the title compound (5.2 mg, 95% purity, 41% yield).

LC-MS (Method 1): R _t = 0.48 min; MS (ESIpos): m/z = 657 [M+H] ⁺ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.830 (0.46), 0.861 (0.68), 0.890 (0.51), 1.194 (0.54), 1.231 (1.71), 1.248 (1.23), 1.306 (0.78), 1.325 (0.79), 1.458 (0.74), 1.479 (0.63), 1.577 (0.44), 1.638 (0.73), 1.667 (0.64), 1.714 (0.61), 1.735 (0.73), 1.752 (0.61), 2.077 (0.50), 2.214 (0.85), 2.233 (1.41), 2.253 (0.66), 2.322 (0.54), 2.326 (0.70), 2.331 (0.53), 2.518 (4.04), 2.523 (16.00), 2.539 (3.74), 2.589 (1.59), 2.602 (1.34), 2.664 (0.55), 2.669 (0.73), 2.673 (0.55), 2.950 (0.50), 2.959 (0.58), 2.984 (0.66), 3.014 (0.75), 3.128 (0.58), 3.140 (0.65), 3.162 (0.63), 3.174 (0.56), 3.990 (0.51), 4.003 (0.54), 4.051 (0.51), 4.066 (0.51), 4.550 (0.45), 4.564 (0.45), 6.291 (0.58),  

6.311 (0.58), 6.326 (0.75), 6.347 (0.68), 7.471 (0.91), 7.481 (0.90), 7.491 (0.85), 7.502 (0.90), 7.634 (0.79), 7.639 (0.84), 7.656 (0.88), 7.660 (0.96), 7.740 (1.46), 7.890 (1.44), 7.912 (1.20), 7.991 (0.46), 8.005 (0.83), 8.019 (0.48), 8.051 (0.83), 8.072 (0.76), 8.233 (0.89), 8.252 (0.81), 8.826 (1.00), 8.831 (1.03), 8.837 (0.99), 8.841 (0.95). #49 Linker Intermediate 306

tri-tert-butyl (3S,10S,14S)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-1,4,12-trioxo -3-[(quinolin-6-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate

A mixture of di-tert-butyl (2S)-2-({[(2S)-6-{[(2S)-2-amino-3-(quinolin-6-yl)propanoyl]a mino}-1- tert-butoxy-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate (225 mg, 80 % purity, 262 µmol), 5-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]pyridin e-2-carboxylic acid (98.3 mg, 262 µmol), HATU (120 mg, 315 µmol), 4-methylmorpholine (72 µl, 660 µmol), and DMF (4 mL) was stirred at r.t. for 3h. After that the mixture was separated between DCM/water and the organic phase was filtered through a phase separator. The filtrate was concentrated under reduced pressure and the residue purified by preparative HPLC (C18, acetonitrile/1%TFA) to give the title compound (175 mg, 91 % purity, 58 % yield).

LC-MS (Method 1): R _t = 1.49 min; MS (ESIpos): m/z = 1043 [M+H] ⁺

Intermediate 307

tri-tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 -[(quinolin-6- yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,16-tricarboxyl ate

 

A mixture of tri-tert-butyl (3S,10S,14S)-1-{5-[({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)methyl]pyridin-2-yl}-1,4,12-trioxo -3-[(quinolin-6-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylate (170 mg, 163 µmol), piperidine (160 µl, 1.6 mmol), and DMF (2.5mL) was stirred at r.t. for 3h. After that the mixture was concentrated under reduced pressure and the residue purified by preparative HPLC (C18, acetonitrile/1% ammonia) to give the title compound (65.0 mg, 97 % purity, 47 % yield).

LC-MS (Method 2): R _t = 1.27 min; MS (ESIpos): m/z = 820.9 [M+H]+.

Example 49-A

(3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-triox o-3-[(quinolin-6-yl)methyl]- 2,5,11,13-tetraazahexadecane-10,14,16-tricarboxylic acid

 

A mixture of tri-tert-butyl (3S,10S,14S)-1-[5-(aminomethyl)pyridin-2-yl]-1,4,12-trioxo-3 - [(quinolin-6-yl)methyl]-2,5,11,13-tetraazahexadecane-10,14,1 6-tricarboxylate—(16.0 mg, 19.1 µmol) and hydrogen chloride (4M in dioxane, 96µL) was stirred at r.t. for 14h. After that the mixture was concentrated under reduced pressure and the crude product was purified by preparative HPLC (C18, acetonitrile/1% TFA) to give the title compound (3.8 mg, 30% yield).

LC-MS (Method 1): R _t = 0.50 min; MS (ESIpos): m/z = 652 [M+H] ⁺ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.237 (2.92), 1.258 (2.26), 1.333 (2.32), 1.352 (2.92), 1.425 (0.93), 1.444 (1.20), 1.462 (1.00), 1.546 (0.86), 1.559 (1.20), 1.579 (1.00), 1.660 (0.60), 1.674 (1.06), 1.693 (1.39), 1.708 (1.26), 1.727 (1.33), 1.746 (1.53), 1.765 (1.59), 1.781 (1.06), 1.799 (0.73), 2.074 (0.40), 2.137 (0.53), 2.151 (0.73), 2.174 (1.66), 2.194 (1.93), 2.204 (1.86), 2.223 (2.52), 2.244 (1.33), 2.261 (1.00), 2.282 (0.53), 2.327 (4.05), 2.331 (2.92), 2.518 (16.00), 2.523 (10.76), 2.669 (4.18), 2.674 (3.05), 2.835 (0.93), 3.019 (2.52), 3.036 (3.52), 3.052 (3.85), 3.069 (3.45), 3.088 (2.85), 3.103 (2.92), 3.117 (2.72), 3.215 (5.11), 3.249 (7.50), 3.271 (8.43), 3.287 (9.23), 3.300 (10.09), 3.320 (10.36), 3.334 (10.95), 3.385 (12.08), 3.727 (2.06), 3.961 (3.39), 3.971 (3.12), 4.021 (7.97), 4.797 (0.93), 4.818 (1.73), 4.832 (1.73), 4.852 (0.86), 6.154 (1.33), 6.170 (1.26), 6.249 (0.46), 6.269 (0.40), 6.363 (2.46), 6.383 (2.32), 6.650 (1.06), 7.451 (2.79), 7.461 (2.79), 7.471 (2.79), 7.482 (2.79), 7.614 (2.39), 7.619 (2.39), 7.636 (2.66), 7.641 (2.72), 7.759 (4.45), 7.868 (4.25), 7.889 (3.52), 7.920 (1.39), 7.940 (5.84), 7.947 (3.92), 7.973 (0.86), 7.987 (1.26), 8.187 (1.26), 8.201 (2.46), 8.217 (3.39), 8.238 (2.52), 8.515 (0.46), 8.655 (4.12), 8.729 (2.92), 8.750 (2.79), 8.814 (3.32), 8.817 (3.39), 8.824 (3.32), 8.828 (2.99).

Example 50

Conjugation of carboxy-HOPO chelator to mABs

2,2',2'',2'''-(propane-1,3-diylbis{nitrilobis[(ethane-2,1 -diyl)carbamoyl(3-hydroxy-6-methyl-2- oxopyridine-4,1(2H)-diyl)]})tetraacetic acid (Example A) (24.23 mg) was dissolved in DMA to 30 mg/mL. TSTU (3.4 mg) was dissolved in DMA to 40 mg/mL and added to the carboxy-HOPO solution. 2,4,6-trimethylpyridine (14.8 µL) was added and the solution mixed for 30 min at r.t..

The solution was then added to a solution of mAb (50 mg) in PBS (7 mg mAb/mL) droplet by droplet. The mixture was mixed for 30 min at r.t..

Purification using Äkta FPLC (running buffer: 30 mM citrate pH 5.5, flow rate: 1 mL/min, column:

HiLoad 10/300 Superdex 200 pg, detection: UV 280/335 nm) of the crude reaction mixture afforded pure mAb-carboxy-HOPO conjugate.

The pure product was analyzed by analytical SEC (running buffer: 10% DMSO in PBS, flow rate:

0.3 mL/min, column: Acuity UPLC Protein BEH SEC, 1.7 µm, 200 Å, 300 x 4.6 mm, detection:  

UV 280/335 nm) determining the monomeric purity and CAR. Table 2 list the purity obtained for various mAb-carboxy-HOPO conjugates.

Example 51: Th-227 labelling of mAb-carboxy-HOPO conjugates

Each mAb-carboxy-HOPO conjugate was dissolved in 30 mM citrate buffer (pH 5.5) containing 50 mg/ml sucrose, 2 mM EDTA and 0.5 mg/mL pABA and 0.075% PS80 was mixed with Th-227 in 0.5M HCl at 2 kBq/µg specific activity at room temperature for 90 min. The labelling efficiently (RCP) was determined by iTLC (2 µL sample was applied on an iTLC strip at 1 cm and the strip developed to 8 cm, then cut at 4 cm and each section counted on a HPGe detector calibrated for Th-227). Table 2 list the RCP obtained for various Th-227 labelled mAb-carboxy-HOPO conjugates.

Example 52: Zr-89 labelling of HER2 mAb-carboxy-HOPO conjugate

89Zr-was neutralized in 2 M Na ₂ CO ₃ . TBS buffer pH 6 and 5 mg/ml gentisic acid were added to a final volume of 1 ml. 50 MBq for each labeling and 250 µg HER2 mAb HOPO chelator conjugate (0.1 mg/ml) was used for each labelling. Labelling samples were incubated at 37 °C for 1 h, then purified by PD10 SEC gravity columns eluting with 10 mM sodium acetate, 240 mM sucrose and 0.02 % PS80. Final products were analysed by radio SEC and results are listed in table 1.

Table 1 Purity of Zr-89 labelled HER2 chelator conjugates

Example 5: Determination of antigen binding capacity by IRF of radiolabelled antibody carboxy-HOPO chelator conjugates  

IRF experiments were performed using PSMA antigen coated magnetic beads. The beads were placed in tubes and diluted to 0.625 x 10 ⁶ beads/µl in IRF assay buffer. The diluted beads were divided into two groups: Blocked (BL) and unblocked (UBL). 10 µl naked PSMA antibody was added to the BL group while for the UBL group, just 10 µl of IRF buffer was added. Both groups were incubated for 2 h at r.t. with shaking at 750 rpm. Th-227 labelled antibody carboxy-HOPO chelator conjugates were prepared as described in example 3 and diluted to 5 Bq/µl in IRF assay buffer. The dilutions were confirmed using HPGe detector. At the end of the incubation step, 10 µl of the Th-227 labelled conjugates (~50 Bq) were added to all the beads (both groups) and incubated for another 2 h at r.t. with shaking at 750 rpm. Then, IRF assay buffer was added to all beads to 1 mL total volume. Tubes filled with samples were placed on a magnet for 2 min. In this way, the samples were divided into 0.5 mL“beads + supernatant” and 0.5 mL“supernatant”.

All the samples were counted for 5 min using the HPGe detector and the measured Th-227 activity was used to calculate the % total binding (100 x activity on UBL beads / total activity UBL sample), the % nonspecific binding (100 x activity on BL beads / total activity BL sample) and the % specific binding (% total binding - % nonspecific binding). IRF results are listed in Table 2 for PSMA antibody carboxy-HOPO chelator conjugates.

Table 2 Analytical results for mAb-chelator conjugate

 

EXPERIMENTAL SECTION– BIOLOGICAL ASSAYS Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein

● the average value, also referred to as the arithmetic mean value, represents the sum of

the values obtained divided by the number of times tested, and

^ the median value represents the middle number of the group of values when ranked in

ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values.

Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch.

 

Competition assay For binding competition assay 7.5x10^5 LNCaP cells were seeded in 500 µl growth medium

(RPMI1640 (Biochrom #FG1215), 10% FCS Superior (Biochrom #S0615)) in 24well plates

(#3526, Corning). Cells were incubated at 37°C and 5%CO2 over night. On the following day

400µl of the media were removed and a total of 200µl growth medium added supplemented

with 3H-PSMA-617 (926.6 GBq/mmol produced in Bayer laboratories) in the presence or

absence of competing compounds in triplicates. The final concentration of 3H-PSMA-617 in

the test wells was 10nM, the competing non-radiolabelded PSMA ligands were tested at final concentrations of 10 and 100 nM. After addition of the compounds in growth media the cells

were swiveled slightly on a shaker mixing for 10 min followed by incubation for 2h at 37°C, 5%

CO2. Following the incubation time the supernatant was removed by pipetting and cells were washed twice with ice cold PBS (total of 800µl). Cells were lysed in 500 µl of 0.3M NaOH

(PanReac AppliChem ITW Reagents) by vigorous shaking for 15 min and the cell lysated

pipetted into scintillator tubes (S207 - SNAPTWIST® Scintillation Vials 6.5 ml #S207,

Simport). In the following the wells were washed with 500µl PBS and the washing PBS added to the corresponding samples in the sincitallator tubes. To each sample in the scintillator tubes

4 mL of Ultima Gold liquid scintillation cocktail (Sigma-Aldrich #L8286) was added and the

samples let stand for at least 30 min. Thereafter radioactivity was counted in a Tri-Carb Liquid

Scinzillation Analyzer (Perkin Elmer). Non-specific binding was assessed by measureing

remaining binding in the presence of unlabeled PSMA-617 at 5000 nM concentration (500 fold excess) and was found to be below 5%. The percentage of remaining 3H-PSMA-617 bound in the presence of the test compounds compared to total binding of 3H-PSMA-617 alone was

calculated for each compound. Result from triplicate values are reported. By comparing examples 1-B, 1-C monomer, 1-C dimer, 1-C trimer, 1-C tetramer, 1-E, 1-F, 1- PEG and 46-E it can be seen that the compounds–C bearing the HOPO chelator Example A surprisingly show an improved competition against 3H-PSMA-617 on PSMA-expressing

LNCaP cells. By comparing quinoline isomer examples 17-B, 36-B, 44-B, 42-B and 45-B it can be seen that surprisingly the 2-quinolinyl isomer example 17-B shows the best competition amongst all

quinoline isomer examples against 3H-PSMA-617 on PSMA-expressing LNCaP cells. By comparing examples 25-A, 25-B, 26-A, 26-B, 1-A and 1-B it can be seen that surprisingly the pyridine linker examples 25-A, 25-B, 26-A, and 26-B show improved competition in comparison to the cyclohexyl examples 1-A and 1-B against 3H-PSMA-617 on PSMA-expressing LNCaP cells.

 

By comparing examples 34-A, 34-B, 1-A, and 1-B it can be seen that surprisingly examples 34- A and 34-B bearing the 2-quinolinyl-pyridine linker show an improved competition in comparison to the cyclohexyl examples 1-A and 1-B against 3H-PSMA-617 on PSMA-expressing LNCaP cells.

 

 

Cell proliferation assay Inhibition of cell proliferation of Thorium-227 labeled PMSA SMOL ligands was tested different prostate cancer cell lines, namely 22Rv1, VCaP, C4-2 (all from ATCC, Manassas, VA, USA) and LNCaP (DMSZ, Braunschweig). Cells were seeded using the following conditions in 384 well plates (Corning) in 30 µl of growth media at 4000 cells/well for VCaP cells and 1000

cells/well for 22Rv1, C4-2 and LNCaP cells. The growth media for the cell lines were as

follows: - 22Rv1: RPMI1640 (Biochrom #FG1215),

- C4-2: DMEM/Ham´s F12 (Biochom #FG4815), 5µg/ml human recombinant Insulin (Sigma

#I9278), 0,25µg/ml Biotin (Sigma #B4639), 25µg/ml Adenine Hydrochlorid Hydrate (Sigma

#A9795), 5µg/ml Transferrin (Roche #10652202001), 13,6pg/ml Triiodothyronine (3,3,- 5Triodo-L-Thyronine Sodium, Sigma #T6397)

- LNCaP: RPMI1640 (Biochrom #FG1215), 10% FCS Superior (Biochrom #S0615)

- VCaP: DMEM (high glucose with stable glutamine; Biochrom #FG 0445), 10% FCS

Superior (Biochrom #S0615)

Cells were cultivated an incubator at 37°C and 5% CO2 over night. On the following days cells were treated in triplicates with serial dilutions of PSMA-SMOLs labeled with Th-227 at a

specific activity of 375 kBq/nmol (RCP>70% as assessed by iTLC analysis; compounds where

RCP was between 50-70% in individual experiments are marked with an * in the table) using a

D300e Digital Dispenser (Tecan). In sister plates cell viability at treatment start (d=0) was

determined using CellTiter-Glo® assay (CTG, Promega, Madison, WI, USA), by adding 30µl per well CTG solution, incubation for 10 min and reading of Luminescence in a PheraStarFSX instrument (BMG Labtech). After a 5-day exposure in the presenced of test compounds, cell viability of treated or untreated control cells was determined again using CellTiter-Glo® assay

(Promega, Madison, WI, USA). Dose response curves and calculation of IC50 values (50%

inhibition of proliferation) were generated using BELLA-Dose Response Curve (DRC)

spreadsheets. The DRC software is a Biobook Spreadsheet that was developed by Bayer AG and Bayer Business Services on the IDBS E-Workbook Suite platform (IDBS: ID Business

Solutions Ltd., Guildford, UK). Results show mean IC50 from 1 to 6 independent experiments.

  Proliferation

Proliferation

Proliferation Proliferation assay on assay on cancer

assay on cancer assay on cancer cancer cell lines cell lines by

cell lines by TTCs cell lines by TTCs by TTCs TTCs Default

Default Pivot #3 Default Pivot #3 Default Pivot #3 Pivot #3

Patent examples IC50 [kBq/mL] IC50 [kBq/mL] IC50 [kBq/mL]

IC50 [kBq/mL]

C4-2 l OOOcells 22Rv1 l OOOcells LnCap

VCap 4000cells

l OOOcells

375kBq/nmol 375kBq/nmol

375kBq/nmol

(mean) (mean) 375kBq/nmol

(mean)

(mean)

*radiochemical purity as detected by iTLC was between 50 and 70% only in individual experiments, whereas it was >70% in all other experiments

It can be seen that surprisingly the 227Th-labelled compounds of the invention examples 1-C monomer, 1-C dimer, 1-C trimer, 1-C tetramer, 17-C monomer, 22-C monomer, 22-C dimer, 22- C trimer, 34-C monomer, 34-C dimer, 34-C trimer, 34-C tetramer and 37-C dimer bearing the HOPO chelator Example A show high antiproliferative activity on VCap, C4-2, 22Rv1 and LnCap cells. Even more surprisingly the 227Th-labelled compounds of the invention examples 34-C monomer, 34-C dimer, 34-C trimer and 34-C tetramer show an improved antiproliferative activity on VCap and C4-2 cells in comparison to 227Th-labelled compounds examples 1-C monomer, 1-C dimer, 1-C trimer and 1-C tetramer. By comparing examples 1-C-monomer-227Th, 1-PEG- 227Th and 46-E 227Th it can be seen that surprisingly example 1-C-monomer-227 bearing the HOPO chelator Example A shows a superior antiproliferative activity compared to 1-PEG-227Th and 46-E 227Th on LnCap cells.

Internalization assay For internalization assays 24well plates (#3526, Corning) were coated with 200µl Poly-L- Lysine (0.1 mg/ml, #3438-100-01, Fa. Cultrex) per well for 2h at 37°C. After 2 washes with

growth medium, 7.5x10^5 LNCaP cells were seeded in 500 µl growth medium

(RPMI1640 (Biochrom #FG1215), 10% FCS Superior (Biochrom #S0615)) in the coated

wells. Cells were incubated at 37°C and 5%CO2 over night. On the following day 400µl of the media were removed and a total of 200µl growth medium added supplemented with PSMA- SMOLs labeled with Th-227 at 375 kBq/nmol in triplicates. Radiochemical purity of the labeling was analyzed by instant thin layer chromatography and found to be above 70% for all

compounds used. The final concentration of radiolabeled SMOLs was in the test wells was

10nM. Cells were incubated for 30min or 2h. After the incubation time supernatants were taken off and cells washed with PBS. Thereafter cells were washed twice with 0.2ml 50mM Glycine- HCl buffer (# 55097, Sigma Aldrich) and once with PBS and the washing solutions combined in counting vials (#TK75-018, Fa. Corning) and subjected to gamma counting (1470 Wizard

automatic gamma counter, Wallac). The resulting counts were attributed to membrane bound radioactivity. Cells were then lysed with 500µl 0.3N NaOH (#181691.1211, Fa. PanReac

AppliChem ITW Reagents) followed by another wash with 500µl PBS. The NaOH lysate as

well as the final wash were combined in vials and subjected to gamma counting as described above. Measured radioactivity was attributed to internalized fraction. Results were calculated as fmol of Th-227 per/10^cells in the membrane bound and internalized fractions from

triplicate values. To check for specificity of binding/internalization cells were incubated with the

  radiolabeled compounds in the presence of 100fold excess of non-radiolabeled compound and observed cell binding/internalization was found to be below 10% of the non-competed value.

It can be seen that the compound 1-C monomer-227Th bearing the HOPO chelator Example A surprisingly shows an increased intracellular radioactivity after 30 min and 2h on PSMA- 5 expressing LNCaP cells compared to the compound 1-B-227Th bearing a DOTA chelator.

3. Biological in vivo Experiments:

The LNCaP human prostate cancer model was used for the human xenograft study to evaluate the efficacy or biodistribution of the PSMAsmol compounds.

3.1 Test system, study design and methods

Experiments were initiated after an acclimatization period of at least 7 days. Animals were kept in a 12 hours light/dark cycle, food and water was available ad libitum and housing temperature 15 was 23 °C. All animal experiments were conducted in accordance with the German animal welfare law and approved by local authorities.

Study design treatment study

SCID mice were randomly assigned to experimental groups, ten animals per group (efficacy) 20 or n=3-5 per time point of biodistribution study. At the initiation of the study, a testosterone pellet was subcutaneously transplanted, the animals were shaved, tattooed and each cage was labeled with the cage number, study number and number of animals per cage.

Methods and parameters

LNCaP human prostate cancer cells were cultured as described according to the supplier´s protocols. Cells were harvested for transplantation in a subconfluent (70%) state. Animals were injected with 5 x 10 ⁶ LNCaP cells suspended in 100% Matrigel subcutaneously into the flank of the mice.

Efficacy study: When tumors reached a predetermined size in mice of 35-50 mm ² animals were randomized into treatment and control groups (n=10 animals/group) and treatment with the defined radioactive dose was started. The i.v. application volume was 100 µl/mice.

Tumor response was assessed by determination of the tumor area (product of the longest diameter and its perpendicular) using a caliper. The animal body weight was monitored as a measure for treatment-related toxicity. Measurement of tumor area and body weight was performed two to three times weekly.

Animals were sacrificed when showing signs of toxicity (>20% body weight loss) or when tumors reached a size of approximately 200 mm².

Tumor growth inhibition is presented as T/C ratio (Treatment / Control) calculated with tumor areas when the vehicle control group had to be closed.

Biodistribution study: When tumors reached a predetermined size in mice of ~50 mm ² animals were randomized into groups (n=3 animals/time point) and the radioactive compound was once applied intraveneously. The i.v. application volume was 100 µl/mice.

At the predetermined time point, n=3 animals were sacrificed, the organs removed, weight and measured in a Germanium detector to count the Th227 in the sample. 3.2 Results Example I

In a study using 1-F-227Th at a dosing of 2x400 kBq/kg weekly followed by 2x750 kBq/kg weekly a T/C _area of 1.03 was reached after 27 days and after an additional treatment with 750 kBq/kg a T/C _area of 0.82 was reached after 37 days. For 34-C monomer 227Th with a single treatment with 750 kBq/kg a T/C _area of 0.37 was already reached after 27 days.

 

Example II

In this study the following treatment groups were included: vehicle control, 1-B-227Th, 1-C monomer-227Th, 1-C dimer-227Th. 1-C monomer-227Th and 1-C dimer-227Th were included as two groups with a dosis of 750 kBq/kg resp.375 kBq/kg each. For 1-B-227Th was only one treatment group with 750 kBq/kg. Vehicle control was citrate buffer only. Treatment for all groups was started at a tumor size of approx. 42-49 mm², at day 14 after tumor cell inoculation.

Treatment was weekly for 5 weeks with 750 kBq/kg or 375 kBq/kg and some of the animals were followed until day 131. T/C _area was determined on day 33 after start of therapy. The groups with the compounds 1-B-227Th 750 kBq/kg and 1-C monomer-227Th 750 kBq/kg were continued until day 120, were both groups were terminated. Mean tumor size for 1-B-227Th 750 kBq/kg was measured at 20 mm ² and 25 mm ² for 1-C monomer-227Th 750 kBq/kg, which was counted as full response for both groups and none of the tumors showed regrowth.

Example III

In this study the following treatment groups were included: vehicle control, 1-B-227Th, 22-C dimer, 34-B 227Th and 1-C tetramer. Treatment dosis for all groups was a single treatment of 1.5 MBq/kg. Vehicle control was 0.9 NaCl only. Treatment for all groups except for group 2 was started at a tumor size of approx.49 mm², at day 14 after tumor cell inoculation. Group 2 started one week later due to labeling problems at first. T/C _area was determined on day 64 after start of therapy.  

Example IV

In this study the following treatment groups were included: vehicle control, 1-B-D-227Th, 1-C monomer-227Th, 22-C dimer-227Th, 22-C trimer-227Th, 34-B 227Th, 34-C monomer 227Th, 34-C dimer 227Th, 34-C trimer 227Th, 1-C monomer-227Thand 1-C dimer-227Th. Treatment for all groups started at a tumor size of approx. 58 mm², at day 20 after tumor cell inoculation.

Treatment was once with 1 MBq/kg and the animals were followed until day 47. T/C _area was determined on day 36 after start of therapy.

Example V

In this study the following treatment groups were included: vehicle control, 1-B-227Th, 1-B-D- 227Th, 34-C monomer 227Th, 34-C dimer 227Th, 34-C trimer 227Th and 17-C monomer-227Th.

Treatment for all groups started at a tumor size of approx. 48 mm², at day 21 after tumor cell inoculation. Treatment was once with 750 kBq/kg. and the animals were followed until day 29.

T/C _area was determined on day 27 after start of therapy.

 

Example VI

In this biodistribution study the compounds 1-F, 1-B and 1-PEG-227Th were applied with 500 kBq/kg per animal. At the corresponding time point 3-5 animals were sacrificed, organs removed and measured in the Germanium detector. Values below are given at %ID Th-227/g organ.

Example VI

In this biodistribution study the compounds 1-C monomer-227Th and 1-C dimer-227Th were applied with 500 kBq/kg per animal. At the corresponding time point 3 animals were sacrificed, organs removed and measured in the Germanium detector. Values below are given at %ID Th- 227/g organ.  

Example VII

In this biodistribution study the compounds 1-B-227Th, 1-B-D-227Th, 22C dimer-227Th, 34-B- 227Th, 1C-Tetramer-227Th were applied with 1500 kBq/kg per animal. At the corresponding time point 3 animals were sacrificed, organs removed and measured in the Germanium detector.

Values below are given at %ID Th-227/g organ.

 

The compound 34-B-227Th showed a good and stable accumulation in the tumor and after a 1.5 MBq/kg treatment had a T/C _area of 0.23 (example III) resp. T/C _area of 0.66 (example IV) after 1 MBq/kg treatment. The compound 34-C monomer-227Th and compound 34-C dimer-227Th both showed after 750 kBq/kg treatment T/C _area of 0.37 resp.0.38 (example V). Example VII

Comparing the percent of injected dose per gram tissue between 1-F-Th227, 1-PEG-227Th, 1- C-monomer-227Th and 1-C-Dimer-227Th, 1-F-227Th shows the highest background which can been seen by the highest values in blood, liver and spleen. Adding the PEG-linker as in compound 1-PEG-227Th reduced the overall background of the compound leading to low values in blood, liver and spleen, but also in low tumor accumulation. 1-C-monomer-227Th and 1-C-

 

Dimer-227Th show lower background compared to 1-F-227Th in blood, liver and spleen but much higher accumulation in the tumor.

Example VIII Cell cytotoxicity assay to determine the potency and the specificity of radiolabelled antibody carboxy-HOPO chelator conjugates Cells were harvested and seeded at 500 cells/wells (30 µl) in 384 well plates and placed in the incubator at 37°C and 5% CO2 overnight. The cells were treated with antibody carboxy-HOPO chelator conjugates radiolabelled with Th-227 at 20 kBq/µg using a half dilution series starting at 50 kBq/mL over 14 points using a TECAN D300e dispenser. The treated cells were incubated at 37°C with 5% CO ₂ for 5 days. Then, the cells were added 30 µl cell titer glow, incubated 10 min and luminescence was measured in the wells using a microplate reader. The % viability was calculated relatively to the luminescence displayed by untreated cells. The specificity ratio was calculated using statistical analysis and by dividing the IC50 of the isotype non-binding control to the IC50 of the corresponding sample. A high specificity ratio indicates specificity. Table 3 list specific ratio for various Th-227 labelled antibody carboxy-HOPO chelator conjugates. Table 3 Cell cytotoxicity results

Example IX: Biodistribution of radiolabelled antibody carboxy-HOPO chelator conjugates Carboxy-HOPO chelator (designated AGC0047 in Figure 2) offers altered biodistribution profile for antibody conjugates compared to more hydrophobic HOPO chelates. This is evidenced by the lower liver uptake for this new chelating system and is driven by the increased negative charge (hydrophilicity) of antibody conjugate compared to the antibody conjugated with the 3,2- HOPO (produced according to the methods described in WO2016/096843 and designated  

AGC0019 in Figure 2). By altering liver uptake we also anticipate that more thorium-227 will be delivered to the tumor compared to the more hydrophobic HOPO chelates. This is evidenced by the lower liver uptake for this new chelating system and is driven by the increased negative charge (hydrophilicity) of antibody conjugate compared to the antibody conjugated with the 3,2- HOPO. By altering liver uptake we also anticipate that more thorium-227 will be delivered to the target site in the tumor. The results are shown in Figure 2.