BEJOT ROMAIN (GB)
LIANG GUYAN (US)
HAJI ABDUL (GB)
BARNES COLIN (GB)
RAHMAN SABITUR (GB)
BEJOT ROMAIN (GB)
LIANG GUYAN (US)
HAJI ABDUL KARIM (GB)
BARNES COLIN (GB)
WO2019020831A1 | 2019-01-31 | |||
WO2021005125A1 | 2021-01-14 | |||
WO2021005131A1 | 2021-01-14 | |||
WO2019083990A2 | 2019-05-02 | |||
WO2019154886A1 | 2019-08-15 | |||
WO2018111989A1 | 2018-06-21 | |||
WO2019020831A1 | 2019-01-31 | |||
WO2020157184A1 | 2020-08-06 | |||
WO2019083990A2 | 2019-05-02 | |||
WO2019154886A1 | 2019-08-15 | |||
WO2018111989A1 | 2018-06-21 | |||
WO2021005125A1 | 2021-01-14 | |||
WO2020157177A1 | 2020-08-06 | |||
WO2021005131A1 | 2021-01-14 | |||
WO2019020831A1 | 2019-01-31 |
EP2020052268W | 2020-01-30 |
ZHANG, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 132, 2010, pages 12711 - 12716
BRYN ET AL.: "Solid-State Chemistry of Drugs", 1999, SSCI, INC
L LOVKOVA ET AL., CHEM. EUR. J., vol. 15, 2009, pages 2140 - 2147
JANSEN ET AL., ACS MED. CHEM. LETT., vol. 4, 2013, pages 491 - 496
KARTAL O ET AL., SLAS DISCOV., vol. 26, no. 8, September 2021 (2021-09-01), pages 995 - 1003
Claims 1. A ligand-SIFA conjugate, comprising, within in a single molecule two separate moieties: (a) one or more ligands which are capable of binding to Fibroblast Activation Protein (FAP), and (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom and which SIFA is optionally labelled with 18F; or a pharmaceutically or diagnostically acceptable salt or solvate thereof. 2. The conjugate according to claim 1, further comprising: (c) one or more chelating moieties, optionally containing a chelated nonradioactive or radioactive cation. 3. The conjugate according to claim 1 or 2, wherein the one or more ligands which are capable of binding to Fibroblast Activation Protein (FAP) each independently comprise one or more heterocyclic group(s), selected from optionally substituted pyrrolidinyl, quinolinyl, isoquinolinyl, quinoxalinyl, phthalazinyl, quinazolinyl, cinnolinyl and naphthyridinyl. 4. The conjugate according to claim 2, which is a conjugate of formula (4), (4a) or (4b): or a salt thereof, wherein X1, X2 and X3 represent divalent linking groups, and where X1, X2 and X3 together with the groups to which they are attached comprise one or more amide bonds; FAP represents a ligand which is capable of binding to Fibroblast activation protein (FAP); L is an optionally substituted linker group; SIFA represents the silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom; and CM represents a chelating moiety, optionally containing a chelated nonradioactive or radioactive cation. 5. The conjugate according to claim 4, wherein X1 is an optionally substituted 10-20 atom linker comprising 1 or more amide bonds, wherein the optional substituent is selected from - X3-FAP, CO2H and CH2OH; X2 is an optionally substituted 1-5 atom linker comprising 1 or more amide bonds, where in compounds of formula (4) or (4b), X2 may also be -NH- or represent a bond; X3 is an optionally substituted 10-20 atom linker comprising 1 or more amide bonds, wherein the optional substituent is selected from CO2H and CH2OH. 6. The conjugate according to any one of claims 1 to 5, wherein the silicon-fluoride acceptor (SIFA) moiety comprises the structure represented by formula (3): erein R1S wh and R2S are independently a linear, branched or cyclic C3 to C10 alkyl group; R3S is a C1 to C20 hydrocarbon group comprising one or more aromatic and/or aliphatic units and/or up to 3 heteroatoms selected from O and S; and wherein the SIFA moiety is attached to the remainder of the conjugate via the bond marked by . 7. The conjugate according to claim 6, wherein the silicon-fluoride acceptor (SIFA) moiety comprises the structure represented by formula (3a): wherein t-Bu indicates a tert-butyl group. 8. The conjugate according to any one of claims 1 to 7, wherein the chelating moiety comprises at least one of: (i) a macrocyclic ring structure with 8 to 20 ring atoms of which 2 or more are heteroatoms selected from oxygen atoms and nitrogen atoms; (ii) an acyclic, open chain chelating structure with 8 to 20 main chain atoms of which 2 or more are heteroatoms selected from oxygen atoms and nitrogen atoms; or (iii) a branched chelating structure containing a quaternary carbon atom. 9. The conjugate according to claim 8, wherein the chelating moiety is selected from bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2- diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4- oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11- tetraazabicyclo[6.6.2]hexadecane (DO2A) 1,4,7,10-tetracyclododecan-N,N',N'',N'''-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10 tetraazacyclododecane N, N’, N’’, N’’’ 1,4,7,10-tetra(methylene) phosphonic acid (DOTMP), N,N'-dipyridoxylethylendiamine-N,N'-diacetate-5,5'- bis(phosphate) (DPDP), diethylene triamine N,N’,N’’ penta(methylene) phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-Ν,Ν'-tetraacetic acid (EDTA), ethyleneglycol-O,O-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N- bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate (HP-DOA3), 6- hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra 3-hydroxy-N-methyl-2-pyridinone chelators (4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4- carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4- carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), abbreviated as Me-3,2-HOPO, 1,4,7-triazacyclononan-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1- carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7- triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11- tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11- tetraacetic acid (TETA), tris(hydroxypyridinone) (THP), terpyridin- bis(methyleneamintetraacetic acid (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2- carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-tetraazacyclotridecan-N,N',N'',N'''-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazonan-1- yl]methyl-hydroxy-phosphoryl]propanoic acid, and triethylenetetraaminehexaacetic acid (TTHA). 10. The conjugate according to claim 9, wherein the chelating moiety is 1,4,7,10- tetracyclododecan-N,N',N'',N'''-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA) or 1,4,7-triazacyclononane- 1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP). 11. The conjugate according to claim 9 or claim 10, wherein the chelating moiety contains a chelated cation selected from the cations of 43Sc, 44Sc, 47Sc, 61Cu, 64Cu, 67Cu, 67Ga, 68Ga, 90Y, 111In, 149Tb, 152Tb, 155Tb, 161Tb, 166Ho, 177Lu, 186Re, 188Re,212Pb, 212Bi, 213Bi, 225Ac, and 227Th or a cationic molecule comprising 18F. 12. The conjugate according to any one of claims 1 to 11, wherein the SIFA fluorine atom is 18F. 13. The conjugate according to any one of claims 1 to 12, wherein the FAP binding moiety comprises a substituted pyrrolidine ring. 14. The conjugate according to any one of claims 1 to 13, wherein the FAP binding moiety comprises a moiety of formula (2): wherein, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from H, OH, B(OH)2, CO2H, CN, halo, C1-6 alkyl and –O-C1-6 alkyl; and R9 and R10 are independently H or C1-6 alkyl. 15. The conjugate according to claim 13, comprising a moiety of formula (2a): wherein, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from H, OH, B(OH)2, CO2H, CN, halo, C1-6 alkyl and -O-C1-6 alkyl; R9 and R10 are independently H or C1-6 alkyl; n is 0 to 3; and X is a 5 to 10-membered N-containing monocyclic or bicyclic heterocycle which optionally further comprises 1, 2 or 3 heteroatoms selected from O, N and S and is optionally substituted with 1 to 3 substituents selected from C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl and -NR20R21, where R20 and R21 are independently selected from H and C1-6 alkyl. 16. The conjugate according to claim 15, wherein n is 0. 17. The conjugate according to claim 15 or 16, wherein X is selected from: ; ; ; and 18. The conjugate according to any one of claims 14 to 17, wherein: R3 and R4 are F; R7 is CN; and R1, R2, R5, R6, R8, R9 and R10 are H. 19. The conjugate according to any one of claims 1 to 12, wherein the FAP binding moiety comprises a moiety which is selected from the group consisting of: ; ; where m is 0 to 10. 20. The conjugate according to any one of claims 1 to 12, wherein the FAP binding moiety comprises a cyclic peptide. 21. The conjugate according to any one of claims 1 to 12, wherein the FAP binding moiety comprises a moiety selected from the group consisting of: 22. The conjugate according to claim 1, which is selected from the group consisting of: 23. A pharmaceutical or diagnostic composition comprising or consisting of one or more conjugates or compounds according to any one of claims 1 to 22. 24. A conjugate, compound or composition according to any one of claims 1 to 23 for use in medicine. 25. A conjugate, compound or composition according to any one of claims 1 to 24 for use as a cancer diagnostic or imaging agent. 26. A method of imaging and/or diagnosing cancer comprising administering a conjugate, compound or composition according to any one of claims 1 to 25 to a patient in need thereof. 27. A conjugate, compound or composition according to any one of claims 1 to 24 for use in the treatment of cancer. 28. A conjugate, compound or composition according to any one of claims 1 to 25 for use in the diagnosis or treatment of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodelling and keloid disorder. 29. The conjugate, compound or composition for use according to claim 28, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymus carcinoma, desmoid tumors, glioma, astrocytoma, cervix carcinoma and prostate cancer. |
Particular chelating moieties (CM) include: Particular chelating moieties (CM) include: Among the above exemplary chelating agents, particular preference is given to a chelating moiety selected from TRAP, DOTA and DOTAGA. The chelating moiety (CM) may be 1,4,7,10-tetracyclododecan-N,N',N'',N'''-tetraacetic acid (DOTA): ; or α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (DOTAGA): The chelating moiety (CM) may be selected from: wherein M represents a chelated metal cation. Metal- or cation-chelating macrocyclic and acyclic compounds are well-known in the art and available from a number of manufacturers. While the chelating moiety in accordance with the present invention is not particularly limited, it is understood that numerous moieties can be used in an off-the-shelf manner by a skilled person without further ado. The chelating moiety may comprise a chelated cation which may be radioactive or non- radioactive, preferably a chelated metal cation which may be radioactive or non-radioactive. The chelating moiety may comprise a chelated cation which is radioactive. The chelating moiety may comprise a chelated cation which is non-radioactive. Especially preferred is that CM represents a chelating moiety selected from DOTA and DOTAGA bound with one of its carboxylic groups via an amide bond to the remainder of the conjugate. Preferred examples of cations that may be chelated by the chelating group are the radioactive or non-radioactive cations of Sc, Cr, Mn, Co, Fe, Ni, Cu, Ga, Zr, Y, Tc, Ru, Rh, Pd, Ag, In, Sn, Te, Pr, Pm, Tb, Sm, Gd, Tb, Ho, Dy, Er, Yb, Tm, Lu, Re, Pt, Hg, Au, Pb, Bi, Ra, Ac, Th; more preferably the cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Th and Er. The cation may be Ga. The cation may be Lu. The chelating moiety may contain a chelated cation or cationic species selected from the cations of 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 58 Co, 52 Fe, 56 Ni, 57 Ni, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga 68 Ga, 89 Zr, 90 Y, 89 Y, <Tc, 99m Tc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 165 Dy, 169 Er, 169 Yb, 175 Yb, 172 Tm, 177 Lu, 186 Re, 188 Re, 191 Pt, 197 Hg, 198 Au, 199 Au, 212 Pb, 203 Pb, 211 At, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, a cationic molecule comprising 18 F or 211 At, or a cation such as 18 F- [AlF] 2+ ; more preferably the cations of 44 Sc, 47 Sc, 61 Cu, 64 Cu, 67 Cu, 68 Ga, 90 Y, 111 In, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th or a cationic molecule comprising 18 F. The chelating moiety may contain a chelated cation selected from the cations of 43 Sc, 44 Sc, 47 Sc, 61 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 90 Y, 111 In, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 166 Ho, 177 Lu, 186 Re, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th or a cationic molecule comprising 18 F. The chelating moiety may contain a chelated cation selected from the cations of 68 Ga or 177 Lu. The chelating moiety may contain a chelated 68 Ga cation. The chelating moiety may contain a chelated 177 Lu cation. M may be selected from the cations of 43 Sc, 44 Sc, 47 Sc, 61 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 90 Y, 111 In, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 166 Ho, 177 Lu, 186 Re, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th. M may be selected from the cations of 68 Ga, 64 Cu, 177 Lu, 90 Y and 225 Ac. M may be selected from the cations of 68 Ga and 177 Lu. M may be a chelated 68 Ga cation. M may be a chelated 177 Lu cation. M may be a chelated 64 Cu cation. M may be a chelated 90 Y cation. M may be a chelated 225 Ac cation. In the compounds herein, the chelated nonradioactive or radioactive cation of the chelating moiety may be chelated to one or more COO- groups. The chelated nonradioactive or radioactive cation of the chelating moiety may be chelated to one or more N atoms. The chelated nonradioactive or radioactive cation of the chelating moiety may be chelated to one or more N atoms or one or more COO- groups. The chelated nonradioactive or radioactive cation of the chelating moiety may be chelated to one or more N atoms and one or more COO- groups. In the structures provided herein, where chelated nonradioactive or radioactive cations are shown, the groups to which they are chelated are merely representatively shown. For example, disclosure of a compound comprising the chelating moiety shown below includes within its scope all complexes or modes of chelation that are chemically possible between the Ga 3+ cation and the conjugate as a whole: A key aspect of the invention is the combination, within a single molecule, of a silicon fluoride acceptor and a chelating group (chelator) or a chelate. These two structural elements, SIFA and the chelator, exhibit a spatial proximity. Preferably, the shortest distance between two atoms of the two elements is less or equal 25 Å, more preferably less than 20 Å and even more preferably less than 15 Å. Alternatively or in addition, it is preferred that not more than 25 covalent bonds separate an atom of the SIFA moiety and an atom the chelator, preferably not more than 20 chemical bonds and even more preferably not more than 15 chemical bonds. Suitably, the cation is a radioactive or non-radioactive cation. It is preferably a radioactive or non-radioactive metal cation, and more preferably a radioactive metal cation. Examples are given further below. As a consequence, conjugates fall under the terms of the first aspect which are radioactively labelled at both the SIFA moiety and the chelating group, molecules which are radioactive labelled at only one of the two sides, as well as molecules which are not radiolabelled at all. In the latter case, the chelating group may be either a complex of a cold (non-radioactive) ion or may devoid of any ion. The placement of the silicone fluoride acceptor in the neighbourhood of a hydrophilic chelator such as, but not limited to, DOTAGA or DOTA, may shield or compensate efficiently the lipophilicity of the SIFA moiety to an extent which shifts the overall hydrophobicity of the radio- therapeutic or -diagnostic compound in a range which renders the compound suitable for in- vivo administration. In addition, the combination of the use of a chelator and an isotopic exchange on SIFA by means of 18 F-fluoride also results in “paired” diagnostic tracers that can either be used as [ 18 F][ nat Ion]tracers at centers with onsite cyclotron or centers that obtain 18 F-fluoride by shipment from cyclotron centers, whereas in centers, that do not have access to 18 F-fluoride but have access to radioisotope generators, such as a Ge-68/Ga-68 generator, the corresponding versions, e.g. [ nat F][ 68 Ga]tracers can be used. Importantly, in both cases, the chemically identical radiopharmaceutical is injected, and thus no differences in the in vivo behavior are expected. Whereas currently, due to chemical differences, the clinical data of a 18 F-labelled compound provided by a patient cohort at one site cannot be directly compared with the clinical data of a 68 Ga-analogue provided by another group at another site, radiopharmaceuticals and/or diagnostics according to the invention can be directly compared and thus will allow to link such data (e.g. data from a center in Europe working with F-18 and another center in India working with Ga-68). Furthermore, when suitably selected, the chelate can also be used for labelling with a therapeutic isotope, such as the beta-emitting isotopes Lu-177, Y-90, or the alpha emitting isotope Ac-225, thus allowing to expand the concept of “paired” tracers to bridge diagnostic ([ 18 F][ nat Lu]tracers) and therapeutic radiopharmaceuticals ([ nat F][ 177 Lu]. Also provided is a pharmaceutical imaging composition comprising or consisting of one or more conjugates of the invention as disclosed herein. Also provided is a diagnostic composition comprising or consisting of one or more conjugates of the invention as disclosed herein. Also provided is a therapeutic composition comprising or consisting of one or more conjugates of the invention as disclosed herein. The pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected in different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection and/or delivery, e.g., to a site in the pancreas or into a brain artery or directly into brain tissue. The compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the pancreas or brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Pharmaceutically active matter may be present in an effective therapeutic amount, which may be between 0.1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Also provided is one or more conjugates, compounds or compositions of the invention as disclosed herein for use in diagnostic medicine. The conjugates of the invention may be useful in the treatment or diagnosis of medical indications associated with elevated FAP expression in human tissue. The conjugates of the invention may be useful in the treatment or diagnosis of cancer. The conjugates of the invention may be useful in (i) the detection of smaller primary tumors, thus allowing earlier diagnosis, (ii) the detection of smaller metastasis, thus affording a better assessment of tumor stage, (iii) providing precise intra-operative guidance facilitating complete surgical removal of tumor tissue, (iv) providing better differentiation between inflammation and tumor tissue, (v) providing more precise staging of patients with tumors, (vi) providing better follow up of tumor lesions after antitumor therapy, and (vii) as theranostic agents for diagnosis and therapy. Furthermore, the molecules can be used for the diagnosis and treatment of non-malignant diseases such as chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorders. The conjugates of the invention may be for use in the diagnosis or treatment of a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal or a human subject. The disease characterized by overexpression of fibroblast activation protein (FAP) may be selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodelling and keloid disorder. The cancer may be selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymus carcinoma, desmoid tumors, glioma, astrocytoma, cervix carcinoma and prostate cancer. Preferred uses in medicine are in nuclear medicine such as nuclear diagnostic imaging, also named nuclear molecular imaging, and/or targeted radiotherapy of diseases associated with an overexpression, of FAP on the diseased tissue. Also provided is a conjugate, compound or composition of the invention as defined herein for use in a method of diagnosing and/or staging cancer. The term “treatment”, in relation to the uses of any of the conjugates or compounds described herein, is used to describe any form of intervention where a compound is administered to a subject suffering from, or at risk of suffering from, or potentially at risk of suffering from the disease or disorder in question. Thus, the term “treatment” covers both preventative (prophylactic) treatment and treatment where measurable or detectable symptoms of the disease or disorder are being displayed. The term “effective therapeutic amount” (for example in relation to methods of treatment of a disease or condition) refers to an amount of the compound which is effective to produce a desired therapeutic effect. Terms such as “alkyl”, “hydrocarbon” and “cycloalkyl” are all used in their conventional sense (e.g. as defined in the IUPAC Gold Book), unless indicated otherwise. “Optionally substituted” as applied to any group means that the said group may if desired be substituted with one or more substituents, which may be the same or different. To the extent that any of the compounds described have chiral centers, the present invention extends to all optical isomers of such compounds, whether in the form of racemates or resolved enantiomers. The invention described herein relates to all crystal forms, solvates and hydrates of any of the disclosed compounds however so prepared. To the extent that any of the compounds disclosed herein have acid or basic centers such as carboxylates or amino groups, then all salt forms of said compounds are included herein. In the case of pharmaceutical uses, the salt should be seen as being a pharmaceutically acceptable salt. Salts or pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts as well as salt forms arising due to the presence of the chelated nonradioactive or radioactive cation. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound in the form of a salt with another counter-ion, for example using a suitable ion exchange resin. Further to the suitable chelated nonradioactive or radioactive cations described herein above, further examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, potassium and calcium. Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulfonic acids (e.g. benzenesulfonic, naphthalene-2- sulfonic, naphthalene-1,5-disulfonic and p-toluenesulfonic), ascorbic (e.g. L-ascorbic), L- aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)- camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, tartaric (e.g.(+)-L- tartaric), thiocyanic, undecylenic and valeric acids. Also encompassed are any solvates of the conjugates or compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents may include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulfoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray crystallography. The solvates can be stoichiometric or non-stoichiometric solvates. Particular solvates may be hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates. For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al, Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3. The conjugates of the invention may contain one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes within its scope 1 H, 2 H (D), and 3 H (T). Similarly, references to carbon and oxygen include within their scope respectively 12 C, 13 C and 14 C and 16 O and 18 O. In an analogous manner, a reference to a particular functional group also includes within its scope isotopic variations, unless the context indicates otherwise. For example, a reference to an alkyl group such as an ethyl group or an alkoxy group such as a methoxy group also covers variations in which one or more of the hydrogen atoms in the group is in the form of a deuterium or tritium isotope, e.g. as in an ethyl group in which all five hydrogen atoms are in the deuterium isotopic form (a perdeuteroethyl group) or a methoxy group in which all three hydrogen atoms are in the deuterium isotopic form (a trideuteromethoxy group). The isotopes may be radioactive or non-radioactive. Selected Embodiments Some embodiments of the invention include: 1. Ligand-SIFA conjugates comprising, within a single molecule, two separate moieties (a) and (b), wherein: (a) is one or more ligands which are capable of binding to Fibroblast Activation Protein (FAP); and, (b) is a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom and which is optionally labelled with 18 F; and, wherein said ligand-SIFA conjugate optionally includes within said single molecule: (c) one or more chelating moieties (CM), optionally containing a chelated nonradioactive cation or radioactive cation; or, (d) one or more ligands which are capable of binding to prostate- specific membrane antigen (PSMA); or, (e) a combination of both (c) said one or more chelating moieties (CM) and (d) said one or more PSMA ligands; or a pharmaceutically or diagnostically acceptable salt or solvate thereof. 2. Ligand-SIFA conjugates according to embodiment 1 comprising, within a single molecule, two separate moieties: (a) one or more ligand(s) which is capable of binding to Fibroblast Activation Protein (FAP); and, (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom and which is optionally labelled with 18 F; or a pharmaceutically or diagnostically acceptable salt or solvate thereof. 3. Ligand-SIFA conjugates according to embodiment 1 comprising, within a single molecule, three separate moieties: (a) one or more ligands which are capable of binding to Fibroblast Activation Protein (FAP); (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom and which is optionally labelled with 18 F; and (c) one or more chelating moieties (CM), optionally containing a chelated nonradioactive or radioactive cation; or a pharmaceutically or diagnostically acceptable salt or solvate thereof. 4. Ligand-SIFA conjugates according to embodiment 1 comprising, within a single molecule three separate moieties: (a) one or more ligands which are capable of binding to Fibroblast Activation Protein (FAP); (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom and which is optionally labelled with 18 F; and (d) one or more ligands which are capable of binding to prostate-specific membrane antigen (PSMA); or a pharmaceutically or diagnostically acceptable salt or solvate thereof. 5. Ligand-SIFA conjugates according to embodiment 1 comprising, within a single molecule four separate moieties: (a) one or more ligands which are capable of binding to Fibroblast Activation Protein (FAP); (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom and which is optionally labelled with 18 F; (c) one or more chelating moieties (CM), optionally containing a chelated nonradioactive or radioactive cation; and (d) one or more ligands which are capable of binding to prostate-specific membrane antigen (PSMA); or a pharmaceutically or diagnostically acceptable salt or solvate thereof. 6. Ligand-SIFA conjugates according to embodiments 1 to 5 wherein the one or more FAP ligand(s) is as defined in any one of claims 3, and 13 to 21. 7. Ligand-SIFA conjugates according to embodiments 1 to 6 wherein the SIFA moiety is as defined in any one of claims 6, 7 and 12. 8. Ligand-SIFA conjugates according to embodiments 1, 3, 5, 6 and 7 wherein the one or more chelating moieties (CM) is as defined in any one of claims 8, 9, 10 and 11. 9. Ligand-SIFA conjugates according to embodiments 1, 4, 5, 6, 7 and 8 wherein the one or more PSMA ligand(s) is selected from a structure of formulae PSMA 1, PSMA 2, PSMA 3, PSMA 4 and PSMA 5 as identified herein. 10. A pharmaceutical or diagnostic composition comprising or consisting of one or more conjugates or compounds according to any one of embodiments 1 to 9. 11. Ligand-SIFA conjugates according to any one of embodiments 1 to 10 for use in medicine. 12. Ligand-SIFA conjugates according to any one of embodiments 1 to 10 for use as a cancer diagnostic or imaging agent. 13. A method of imaging and/or diagnosing cancer comprising administering a ligand-SIFA conjugate according to any one of embodiments 1 to 10 to a patient in need thereof. 14. Ligand-SIFA conjugates according to any one of embodiments 1 to 10 for use in the treatment of cancer. 15. Ligand-SIFA conjugates according to any one of embodiments 1 to 10 for use in the diagnosis or treatment of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodelling and keloid disorder. 16. Ligand-SIFA conjugates according to any one of embodiments 1 to 10 for use in the diagnosis or treatment of cancer wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymus carcinoma, desmoid tumors, glioma, astrocytoma, cervix carcinoma and prostate cancer. Highly preferred conjugates of the invention include the conjugates of Example 1 to 13a shown in Table 1 below. Examples and Preparations The following non-restrictive examples illustrate the invention. The synthesis of the conjugates of some embodiments of the invention and of the intermediates for use therin are illustarted by the following non-limiting Examples and Preparations. Examples of conjugates of the invention include those shown in Table 1 below. The conjugate of the invention may be selected from any one of Example 1 to 13a shown in Table 1. Table 1 - Example Conjugates
General Method Certain conjugates of the invention may be prepared in accordance with the general scheme below, where FAP comprises an FAP binding moiety, PSMA comprises a PSMA binding moiety, L represents a linker moiety, SIFA represents a SIFA-containing moiety, CM comprises a chelating moiety and CM(M) comprises a chelating moiety with a chelated metal cation. Materials and Methods Where no preparative routes are included, the relevant intermediate is commercially available Commercial grade reagents were utilized without further purification, unless otherwise stated. The purity of compounds was determined by HPLC, and all final target compounds had purities of >95%, unless otherwise stated. Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded in the deuterated solvents specified on a Varian 400 spectrometer operating at 400 MHz. Mass spectra were determined by using Shimadzu LCMS 2020 with N- Series DUIS (ESI) system using positive-negative switching. HPLC spectra were determined by using Agilent 1200 series. Room temperature includes 20 to 25°C. Synthesis of the silicon-fluoride acceptor reagent (SiFA-BA) The silicon-fluoride acceptor reagent used herein was 4-(di-tert-butylfluorosilyl)benzoic acid (SiFA-BA): SiFA-BA was synthesized according to a previously published procedure (L. Iovkova et al. Chem. Eur. J.2009, 15, 2140 – 2147) as depicted in the below scheme. All reactions were carried out in dried reaction vessels under argon using a vacuum gas manifold. Synthesis of SiFA-BA: a) TBDMSCl, imidazole (DMF); b) tBuLi, di-tert-butyldifluorosilane (THF); c) HCl (MeOH); d) pyridinium chlorochromate (DCM); e) KMnO 4 (DCM, tert-butanol, NaH 2 PO 4 buffer). Synthesis of conjugates of Example 1 and Example 2 There is shown in the scheme below an exemplary synthetic procedure to obtain the conjugates of the invention of Example 1 and Example 2, as detailed in Table 1.
Synthesis of Intermediates Preparation 1 6-Hydroxyquinoline-4-carboxylic acid (2) 6-methoxyquinoline-4-carboxylic acid (5 g, 0.0246 mol; BLD-pharma) and solution of HBr (48% in water, 75 ml) in a seal tube was heated to 140 °C for 4 h with vigorous stirring. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis. After completion, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water (20 ml) and resulting solution was basified (pH= 7.5) using 4 N aqueous sodium hydroxide (NaOH) solution. The precipitate formed was removed by filtration and the filtrate was concentrated to dryness under reduced pressure. The residue was triturated with methanol (20 ml x 3 and diethyl ether (20 ml). The resulting slurry was dissolved in acetonitrile (20 ml) and water (60 ml). The resulting mixture was lyophilized to obtain the title compound as a light brown solid (yield: 4.0 g, 86%). The title compound was characterized by LC-MS and 1 H NMR analysis. LC-MS, C 10 H 7 NO 3 calculated 189.04; observed 190.25 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 8.46 (d, J= 4.0 Hz, 1H), 8.10 (s, 1H), 7.70 (d, J= 9.2 Hz, 1H), 7.32 (d, J= 4.0 Hz, 1H), 7.17-7.14 (m, 1H). Preparation 2 (S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-6-hydroxyquino line-4-carboxamide (4) N, N-Diisopropylethylamine (3.5 ml, 0.0198 mol), 1-hydroxy-7-azabenzotriazole (4.0 g, 0.264 mol; Spectrochem) and 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium (6.0 g, 0.0158 mol; Spectrochem) were added to a stirred solution of the title compound of Preparation 1 (2.5 g, 0.0132 mol) and (S)-1-glycylpyrrolidine-2-carbonitrile (3.0 g, 0.0198 mol, BLD-pharma) in dry N,N-dimethyl formamide (DMF, 50 ml) under nitrogen. The resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis. After completion of the reaction, water (30 ml) was added, and the resulting mixture was extracted with ethyl acetate (50 ml x 2). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified by silica-gel (230-400 silica) column chromatography using 5% methanol in dichloromethane to afford the title compound as a yellow solid (yield: 1.9 g, 45%). The title compound was characterized by LC-MS analysis. LC-MS, C 17 H 16 N 4 O 3 calculated 324.12; observed 325.30 [M+H] + . Preparation 3 tert-Butyl-(S)-(3-((4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethy l)carbamoyl)quinolin-6- yl)oxy)propyl)carbamate (6) tert-Butyl(3-bromopropyl)carbamate (0.87 g, 0.0037 mol) was added portion wise to a solution of the title compound of Preparation 2 (1 g, 0.0031 mol) and potassium carbonate (0.51 g, 0.0037 mol) in N,N-dimethyl formamide (DMF, 10 ml) under nitrogen. The resulting mixture was stirred at 60 °C for 16 h. The progress of the reaction was monitored by TLC analysis. After completion, the reaction mixture was diluted with water (50 ml) and extracted with ethyl acetate (30 ml x 3). The combined organic layer was washed with brine and dried over anhydrous sodium sulphate. The solution was filtered, and the filtrate concentrated under reduced pressure to afford a light brown crude compound. The crude compound was purified by silica gel (230-400 mesh) eluting with 0-5% methanol in dichloromethane to afford the title compound as a yellow solid (yield: 0.75 g, 50 %). The title compound was characterized by LC-MS and 1 H NMR analysis. LC-MS, C 25 H 31 N 5 O 5 calculated 481.23; observed 482.40 [M+H] + . 1 H NMR (400 MHz, CDCl 3 ): δ 8.79 (d, J= 4.4 Hz, 1H), 7.99 (d, J= 9.2 Hz, 1H), 7.62 (bs, 1H), 7.51 (d, J= 4.0 Hz, 1H), 7.38-7.35 (m, 1H), 7.30 (bs, 1H), 5.17 (bs, 1H), 4.80 (bs, 1H), 4.46-4.40 (m, 1H), 4.31-4.27 (m, 1H), 4.17 (m, 2H), 3.73 (bs, 1H), 3.58-3.54 (m, 1H), 3.38-3.37 (m, 2H), 2.37-2.26 (m, 4H), 2.05 (bs, 2H), 1.44 (s, 9H). Preparation 4 (S)-6-(3-Aminopropoxy)-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoet hyl)quinoline-4-carboxamide (7) Trifluoroacetic acid (0.25 ml, 0.0032 mol) was added to a solution of the title compound of Preparation 3 (0.75 g, 0.0016 mol) in dichloromethane (7.5 ml) under nitrogen. The resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis. The reaction mixture was then concentrated under reduced pressure and the residue was co-distilled (3 times) with fresh dichloromethane. Finally, the resulting residue was triturated firstly with diethyl ether (10 ml) and then with n- pentane (10 ml), and then dried under vacuo to afford the title compound as a light brown solid in quantitative yield. The crude title compound was used without further purification. The title compound was characterized by LC-MS and 1 H NMR analysis. LC-MS, C 20 H 23 N 5 O 3 calculated 381.18; observed 382.35 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 9.09 (d, J= 5.2 Hz, 1H), 8.84 (d, J= 4.0 Hz, 1H), 8.02 (d, J= 9.2 Hz, 1H), 7.96-7.87 (m, 4H), 7.56-7.49 (m, 2H), 4.82 (d, J= 4.0 Hz, 1H), 4.23 (s, 4H), 3.74 (bs, 1H), 3.59-3.53 (m, 1H), 3.04-3.03 (m, 2H), 2.23-2.09 (m, 5H). Preparation 5 (9H-Fluoren-9-yl)methyl((R)-1-((3-((4-((2-((S)-2-cyanopyrrol idin-1-yl)-2- oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-3-((1-(4, 4-dimethyl-2,6- dioxocyclohexylidene)ethyl)amino)-1-oxopropan-2-yl)carbamate (8) 2,4,6-Collidine (1.65 mL, 12.30 mmol), 1-hydroxy-7-azabenzotriazole (0.51 g, 3.674 mmol) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (1.18 g, 3.674 mmol) were added to a stirred solution of the title compound of Preparation 4 (0.7 g, 1.84 mmol) and N-alpha-(9-fluorenylmethyloxycarbonyl)-N-beta-[(4,4-dimethyl -2,6-dioxocyclohex- 1-ylidene)ethyl]-D-2,3-diaminopropionic acid (0.99 g, 2.02 mmol, ACT-China) in dry N,N- dimethyl formamide (DMF, 14 ml) under nitrogen. The resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC analysis. After completion of the reaction, water (30 ml) was added, and the resulting mixture was extracted with ethyl acetate (50 ml x 2). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude title compound was purified by silica-gel (230-400 silica) column chromatography using 5% methanol in dichloromethane to afford the title compound as an off-white solid (yield: 0.90 g, 51%). The title compound was characterized by LC-MS analysis. LC-MS, C 48 H 51 N 7 O 8 calculated 853.38; observed 854.50 [M+H] + . Preparation 6 (9H-Fluoren-9-yl)methyl-((R)-3-amino-1-((3-((4-((2-((S)-2-cy anopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-1-oxoprop an-2-yl)carbamate (9) Hydrazine hydrate (25%, 1.3 ml) was added to a stirred solution of the title compound of Preparation 5 (2.6 g, 0.0027 mol) in ethanol (25 ml) and the resulting mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis. After completion of the reaction, water (20 ml) was added, and the resulting mixture was extracted with dichloromethane (100 ml x 3). The combined organic layers were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude title compound was purified by silica-gel (230-400 silica) column chromatography using 10 % methanol in dichloromethane to afford the title compound as an off-white solid (yield: 1.50 g, 78%). The title compound was characterized by LC-MS and 1 H NMR analysis. LC-MS, C 38 H 39 N 7 O 6 calculated 689.30; observed 690.45 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 9.03 (t, J= 5.6 Hz, 1H), 8.81 (d, J= 4.4 Hz, 1H), 8.12 (bs, 1H), 7.97 (d, J= 8.8 Hz, 1H), 7.89-7.88 (m, 3H), 7.68 (d, J= 7.6 Hz, 2H), 7.51 (d, J= 4.4 Hz, 1H), 7.46-7.39 (m, 3H), 7.32 (t, J= 7.2 Hz, 3H), 4.83-4.81 (m, 1H), 4.30-4.17 (m, 8H), 3.72 (bs, 4H), 3.57-3.51 (m, 1H), 3.06-2.99 (m, 2H), 2.22-2.16 (m, 2H), 2.09-2.05 (m, 2H), 1.97-1.94 (m, 3H). Preparation 7 (9H-Fluoren-9-yl)methyl-((R)-1-((3-((4-((2-((S)-2-cyanopyrro lidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-3-(4-(di- tert-butylfluorosilyl)benzamido)- 1-oxopropan-2-yl)carbamate (10) N,N-Diisopropylethylamine (1.8 ml, 9.79 mmol), 1-hydroxy-7-azabenzotriazole (0.44 g, 3.27 mmol) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (1.0 g, 3.27 mmol) were added to a stirred solution of the title compound of Preparation 6 (1.5 g, 2.18 mmol) and 4-(di-tert-butylfluorosilyl)benzoic acid (0.92 g, 3.27 mmol) in dry N,N-dimethyl formamide (DMF, 15 ml) under nitrogen. The resulting mixture was stirred at room temperature for 24 h and the progress of reaction monitored by thin-layer chromatography (TLC_ analysis. After completion of the reaction, water (100 ml) was added, and the resulting mixture was extracted with ethyl acetate (300 ml x 2). The combined organic layers were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude title compound was purified by silica-gel (230-400 silica) column chromatography using 1-10% methanol in dichloromethane to afford the title compound as an off-white solid (yield: 1.2 g, 60%). The title compound was characterized by LC-MS analysis. LC-MS, C 53 H 60 FN 7 O 7 S calculated 953.43; observed 954.65 [M+H] + . Preparation 8 6-(3-((R)-2-Amino-3-(4-(di-tert-butylfluorosilyl)benzamido)p ropanamido)propoxy)-N-(2-((S)-2- cyanopyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide (11) (Example 12) A solution of piperidine (0.15 ml, 1.51 mmol) in N,N-dimethyl formamide (DMF, 1 ml) was added dropwise to a stirred solution of the title compound of Preparation 7 (1.2 g, 1.26 mmol) in N,N-dimethyl formamide (DMF, 12 ml). The resulting mixture was stirred at room temperature for 1 h and the reaction monitored by TLC analysis. After completion of the reaction, water (100 ml) was added, and the resulting mixture was extracted with ethyl acetate (70 ml x 3). The combined organic layers were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude title compound was purified by silica-gel (230-400 silica) column chromatography using 5-12% methanol in dichloromethane to afford the title compound as an off-white solid (yield: 0.90 g, 98%). The title compound was characterized by LC-MS and 1 H NMR analysis. LC-MS, C 38 H 50 FN 7 O 5 Si calculated 731.36; observed: 732.45 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 9.06 (t, J= 6.0 Hz, 1H), 8.81-8.77 (m, 2H), 8.26 (t, J= 4.8 Hz, 1H), 8.03-7.95 (m, 3H), 7.87 (bs, 1H), 7.67 (d, J= 7.6 Hz, 2H), 7.51 (d, J= 4.4 Hz, 1H), 7.45 (d, J= 8.8 Hz, 1H), 6.01 (bs, 2H), 4.84 (bs, 1H), 4.60 (bs, 1H), 4.25-4.16 (m, 4H), 3.74 (bs, 1H), 3.57-3.37 (m, 1H), 3.17-3.14 (m, 2H), 3.13-3.08 (m, 2H), 3.06-3.02 (m, 1H), 2.50 (s, 8H), 2.30-1.94 (m, 6H), 1.30-1.14 (m, 1H), 1.08 (s, 18 H). Preparation 9 tri-tert-Butyl 2,2',2''-(10-((R)-1-(tert-butoxy)-5-(((R)-1-((3-((4-((2-((S) -2-cyanopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-3-(4-(di- tert-butylfluorosilyl)benzamido)- 1-oxopropan-2-yl)amino)-1,5-dioxopentan-2-yl)-1,4,7,10-tetra azacyclododecane-1,4,7- triyl)triacetate (12) N,N-Diisopropylethylamine (0.70 ml, 3.69 mmol) was added to a stirred solution of the title compound of Preparation 8 (0.90 g, 1.23 mmol) and 5-(tert-butoxy)-5-oxo- 4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraaza cyclododecan-1-yl) pentanoic acid (0.86 g, 1.23 mmol, Argonix Reagents & Intermediates) in dichloromethane (18 ml) under nitrogen. This was followed by an addition of 1-propanephosphonic anhydride solution (50 % in ethyl acetate, 1.2 ml, 3.69 mmol). The resulting mixture was stirred at room temperature for 3 h and the reaction monitored by TLC analysis. After completion of the reaction, water (50 ml) was added, and the resulting mixture was extracted with dichloromethane (100 mL x 3). The combined organic layers were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude title compound was purified by silica-gel (230-400 silica) column chromatography using 5-14% methanol in dichloromethane to afford the title compound as an off-white solid (yield: 1.0 g, 57%). The product (12) was taken forward to the next step without further characterisation. Synthesis of Conjugates of Examples 1 and 2 Preparation 10 – Compound of Example 1 2,2',2''-(10-((R)-1-Carboxy-4-(((R)-1-((3-((4-((2-((S)-2-cya nopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-3-(4-(di- tert-butylfluorosilyl) benzamido)- 1-oxopropan-2-yl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclodo decane-1,4,7-triyl) triacetic acid (13) A solution of the title compound of Preparation 9 (1.0 g, 0.71 mmol) in trifluoroacetic acid: triisopropyl silane:water (95:2.5:2.5, 15 ml) was stirred at room temperature for 16 h under nitrogen. The progress of the reaction was monitored by LC-MS analysis. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain a sticky liquid residue. The residue was triturated with methyl tert-butyl ether (10 x3 ml times) followed with n-pentane (10 ml) to produce a solid. The solid was dried under vacuum to afford crude title compound as an off-white solid (yield: 0.83 g, 99%). The crude title compound (70 mg) was purified by reverse phase high performance liquid chromatography (HPLC) (Mobile phase A: 0.1% Formic acid in water; and Mobile phase B: acetonitrile - column: Inertsil ODS3V 250×20 mm, 5.0μm) to produce 20 mg of the title compound as a pale-yellow solid. LC-MS, C 57 H 80 FN 11 O 14 Si calculated 1189.56; observed 1188.35 [M-H]-. 1 H NMR (400 MHz, DMSO): δ 11.80 (bs, 4H), 9.03 (bs, 1H), 8.80 (s, 1H), 8.47-8.33 (m, 2H), 8.14 (bs, 1H), 7.96-7.85 (m, 4H), 7.64-7.62 (m, 2H), 7.51-7.44 (m, 1H), 4.83 (bs, 1H), 4.44 (bs, 2H), 4.21-4.16 (m, 4H), 3.72 (bs, 1H), 3.53-3.40 (m, 10H), 3.10-2.80 (m, 13H), 2.33-1.71 (m, 14H), 1.02 (s, 18H). High performance liquid chromatography (HPLC): 10.361 min, 98.46 %, (Column : Inertsil ODS 3V- C18 4.6×250 mm, 5 μm, Mobile phase A: 0.1% Formic acid in water; Mobile phase B: Acetonitrile). Preparation 11 – Compound of Example 2 Gallium(I) 2-(16-((R)-1-carboxy-4-(((R)-1-((3-((4-((2-((S)-2-cyanopyrro lidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-3-(4-(di- tert-butylfluorosilyl)benzamido)- 1-oxopropan-2-yl)amino)-4-oxobutyl)-3,6-dioxo-4,5-dioxa-1,8, 11,16- tetraazabicyclo[6.5.5]octadecan-11-yl)acetate (14, Ga-(S,R,R)-SiFA-FAP-1) Gallium (III) nitrate (0.236 g, 0.925 mmol) was added to a stirred solution of the title compound of Preparation 10 (0.55 g, 0.462 mmol) in tert-butanol:water (3:1, 20 ml) under nitrogen and the resulting mixture was heated at 75 °C for 3h. The progress of the reaction was monitored by LC-MS analysis. After completion, the reaction was cooled to room temperature and water (20 ml) added. The resulting mixture was filtered through a micro-filter and the filtrate concentrated under reduced pressure. The residue obtained was triturated with methyl tert- butyl ether (5 x 3 ml) and diethyl ether (10 ml) to produce a sticky solid which was dried under vacuum to afford the crude tile compound as a pale-yellow solid. The crude title compound was purified by reverse phase HPLC (Mobile phase A: 0.1% Formic acid in water; and Mobile phase B: acetonitrile - column: Inertsil ODS3V 250×20 mm, 5 μm) to produce the title compound as an off-white solid (yield: 78 mg, 13.4%). LC-MS, C 57 H 77 FGaN 11 O 14 Si calculated 1255.47; observed 1254.40 [M-H]-. 1 H NMR (400 MHz, DMSO): δ 9.03 (bs, 1H), 8.80 (d, J= 4.0 Hz, 1H), 8.59 (bs, 1H), 8.12 (bs, 2H), 7.97-7.85 (m, 4H), 7.67 (d, J= 7.6 Hz, 2H), 7.51 (d, J= 4.4 Hz, 1H), 7.46-7.44 (m, 1H), 4.84 (d, J= 4.8 Hz, 1H), 4.56 (bs, 1H), 4.47 (bs, 1H), 4.21- 4.16 (m, 4H), 3.75 (bs, 1H), 3.56-3.41 (m, 14H), 3.20-2.70 (m, 16H), 2.17 (bs, 1H), 2.07 (bs, 1H), 1.95 (bs, 2H), 1.07 (s, 18H). Synthesis of conjugates of Example 3 and Example 4 There is shown in the below scheme an exemplary synthetic procedure to obtain the conjugates of Example 3 and Example 4, as detailed in Table 1. Synthesis of Intermediates Preparation 1 4-Hydroxyquinazolin-6-yl acetate (2) A mixture of quinazoline-4,6-diol (5 g, 30.86 mmol; Combi-blocks), acetic anhydride (46.6 mL, 49.38 mmol; Spectrochem) and pyridine (8 mL; Spectrochem) was heated at 100 o C under nitrogen for 2 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (5% methanol in dichloromethane). After completion, the reaction was cooled to room temperature and the reaction mixture was poured on to crushed ice (50 mL). This resulted in pale-yellow precipitation. The solid was collected by filtration, washed with fresh ice-cold water (200 mL) and dried under vacuum to afford the desired compound as a pale- yellow solid. The title compound was characterized by LC-MS and 1 H NMR analysis. Yield: 6.3 g (100 %). LC-MS C 10 H 8 N 2 O 3 calculated 204.05; observed 205.25 [M+H] + . 1 H NMR (400 MHz, CDCl 3 ): δ 10.74 (bs, 1H), 8.06-8.02 (m, 2H), 7.80 (d, J=8.8 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H), 2.37 (s, 3H). Preparation 2 4-Chloroquinazolin-6-yl acetate (3) To a mixture of 4-hydroxyquinazolin-6-yl acetate (6.3 g, 30.86 mmol) and thionyl chloride (43.05 g, 26.25 mL, 361.8 mmol; Spectrochem) was added catalytic amount of dry N,N- dimethyl formamide (DMF, 0.3 mL) under nitrogen. The resulting mixture was heated at 90 °C for 3 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (5% methanol in dichloromethane). After completion, the reaction was cooled to room temperature and the reaction mixture concentrated under reduced pressure. The residue was azeotroped with toluene (50 mL) and dried under vacuum to afford the desired compound as a pale-yellow solid. The title compound was characterized by LC-MS and 1 H NMR analysis. Yield: 6.8 g (100 %). LC-MS C 10 H 7 ClN 2 O 2 calculated 222.02; observed 223.15 [M+H] + . 1 H NMR (400 MHz, CDCl 3 ): δ 9.08 (s, 1H), 8.17 (d, J=9.2 Hz, 1H), 8.04 (s, 1H), 7.76 (d, J=9.2 Hz, 1H), 2.42 (s, 3H). Preparation 3 4-Chloroquinazolin-6-ol (4) A mixture of 4-chloroquinazolin-6-yl acetate (19 g, 85.30 mmol) and ammonia (7 N in methanol; 400 mL; Hychem laboratories) was stirred at room temperature for 1h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (5% methanol in dichloromethane). After completion, the reaction mixture concentrated under reduced pressure. The residue was triturated with diethyl ether (10 mL) and dried under vacuum to afford the desired compound as a dark brown solid. It was taken in next step without further purification. The title compound was characterized by LC-MS analysis. Yield: 13.3 g (86.6 %). LC-MS C 8 H 5 ClN 2 O calculated 180.01; observed: 181.20 [M+H] + . Preparation 4 Methyl 6-hydroxyquinazoline-4-carboxylate (5) A stirred solution of 4-chloroquinazolin-6-ol (13.3 g, 73.6 mmol), triethyl amine (30.6 mL, 220.9 mmol; Spectrochem) in dry methanol (200 mL) was purged with nitrogen for 20 min at room temperature. To this was added PdCl 2 dppf (5.3 g, 7.36 mmol; Chempure) and the resulting mixture was heated at 60 o C under CO pressure (5 kg). The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (5% methanol in dichloromethane). After completion, the reaction mixture was cooled to room temperature and filtered through a celite bed. The filtrated was concentrated under reduced pressure to get crude compound. The crude was subjected to silica gel (100-200) column chromatography using 10-50 % ethyl acetate in n-hexane to afford the desired compound as a pale-yellow solid. The title compound was characterized by LC-MS and 1 H NMR analysis. Yield: 8.6 g (57.3 %). LC-MS C 10 H 8 N 2 O 3 calculated 204.05; observed 205.25 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 10.76 (s, 1H), 9.21 (s, 1H), 8.01 (d, J=9.2 Hz, 1H), 7.72 (s, 1H), 7.66 (d, J=9.2 Hz, 1H), 4.02 (s, 3H). Preparation 5 6-Hydroxyquinazoline-4-carboxylic acid (6) To a stirred solution of methyl 6-hydroxyquinazoline-4-carboxylate (8.6 g, 42.1 mmol) in tetrahydrofuran:methanol:water (6:1:0.5; 75 mL) was added sodium hydroxide (4.2 g, 105.3 mmol) and the mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC analysis (5 % methanol in dichloromethane). After completion, the reaction mixture was concentrated under reduced pressure to one third of the volume. The pH of the solution was adjusted to 7 using concentrated hydrochloric acid (HCl). Initially formed solid was removed by filtration and the filtrated was further acidified to pH=1 using concentrated HCl. The yellow solid formed was collected by filtration, washed with fresh water, and dried under vacuum to afford the desired compound as a pale-yellow solid. The title compound was characterized by LC-MS and 1 H NMR analysis. Yield: 5.3 g (66.6 %). LC-MS C 9 H 6 N 2 O 3 calculated 190.04; observed 191.20 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 14.12 (bs, 1H), 10.69 (s, 1H), 9.19 (s, 1H), 7.99 (d, J=9.2 Hz, 1H), 7.72 (s, 1H), 7.64 (d, J=9.6 Hz, 1H). Preparation 6 (S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-6-hydroxyquina zoline-4-carboxamide (8) To a stirred solution of HBTU (9.09 g, 24.0 mmol; Spectrochem) in dry N,N-dimethyl formamide (DMF, 70 mL) were added 6-hydroxyquinazoline-4-carboxylic acid (3.8 g, 20.0 mmol), hydroxybenzotriazole (HOBt, 6.1 g, 40.0 mmol; Spectrochem) and diisopropyl ethyl amine (8.9 mL, 50.0 mmol; Spectrochem). This was followed by an addition of (S)-1- glycylpyrrolidine-2-carbonitrile (4.8 g, 30.0 mmol) and the resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (10 % methanol in dichloromethane). After completion of the reaction, water (100 mL) was added, and the mixture was extracted with ethyl acetate (500 mL x 3). The combined organic layer was given brine (200 mL) wash and dried over anhydrous sodium sulphate. The solvent was removed under reduced pressure and the crude compound was subject to silica gel (230-400) column chromatography using 5-10% methanol in dichloromethane to afford the desired compound as a pale-yellow solid. The title compound was characterized by LC-MS analysis. Yield: 3.2 g (49.0 %). LC-MS C 16 H 15 N 5 O 3 calculated 325.12; observed 326.05 [M+H] + . Preparation 7 tert-Butyl (S)-(3-((4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamoyl )quinazolin-6- yl)oxy)propyl)carbamate (10) To a solution of (S)-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-6-hydroxyquina zoline-4- carboxamide (3.2 g, 9.8 mmol) in dry DMF (32 mL) under nitrogen was added K 2 CO 3 (1.6 g, 11.81 mmol; Spectrochem) and tert-butyl (3-bromopropyl)carbamate (2.8 g, 11.81 mmol; BLD- pharma). The resulting mixture was stirred at 60 °C for 24 h. The progress of the reaction was monitored by TLC analysis (10% methanol in dichloromethane). After completion, the reaction was cooled to room temperature and water (100 mL) was added. The resulting mixture was extracted with ethyl acetate (200 mL x 3). The combined organic layer was given brine wash and dried over anhydrous sodium sulphate. The solution was filtered and concentrated under reduced pressure. The crude compound was purified by flash silica-gel (230-400) column chromatography using 5-6% methanol in dichloromethane to afford the desired compound as a pale-yellow solid. The desired compound was characterized by LC-MS and 1 H NMR analysis. Yield: 3.4 g (72.0 %). LC-MS C 24 H 30 N 6 O 5 calculated 482.23; observed 481.15 [M-H]- . 1 H NMR (400 MHz, DMSO): δ 9.30-9.28 (m, 2H), 8.44 (s, 1H), 8.03 (d, J=9.6 Hz, 1H), 7.73 (d, J=8.8 Hz, 1H), 6.96 (s, 1H), 4.83 (bs, 1H), 4.25 (d, J=5.2 Hz, 2H), 4.15 (s, 2H), 3.74 (bs, 1H), 3.58-3.54 (m, 1H), 3.14 (d, J=5.6 Hz, 2H), 2.19-2.07 (m, 4H), 1.95-1.92 (m, 2H), 1.37 (s, 9 H). Preparation 8 (S)-6-(3-aminopropoxy)-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoet hyl)quinazoline-4-carboxamide (11) To a solution of tert-butyl (S)-(3-((4-((2-(2-cyanopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)carbamate (3.4g, 7.0 mmol) in dichloromethane (DCM, 34 mL) under inert atmosphere added trifluoracetic acid (2.1 mL, 28.2 mmol; Spectrochem). The resulting mixture was stirred at room temperature for 24 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (15% methanol in dichloromethane). After completion, the reaction mixture was concentrated under reduced pressure and the residue was co-distilled with fresh dichloromethane (20 mL x 3). Finally, the residue was triturated with diethyl ether (20 mL) and dried under vacuo to afford the desired compound as a light brown solid in quantitative yield. The crude compound was taken for the next step without further purifications. The desired compound was characterized by LC-MS analysis. LC-MS C 19 H 22 N 6 O 3 calculated 382.18; observed 383.35 [M+H] + . Preparation 9 (9H-Fluoren-9-yl)methyl ((R)-1-((3-((4-((2-((S)-2-cyanopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-3-((1-( 4,4-dimethyl-2,6- dioxocyclohexylidene)ethyl)amino)-1-oxopropan-2-yl)carbamate (12) To stirred solution of (S)-6-(3-aminopropoxy)-N-(2-(2-cyanopyrrolidin-1-yl)-2- oxoethyl)quinazoline-4-carboxamide (1.8 g, 4.71 mmol) and Fmoc-(n-beta-1-(4,4-dimethyl- 2,6-dioxocyclohex-1-ylidene)ethyl)-l-alpha, beta-diamino propionic acid (2.5 g, 5.1 mmol; Argonix Reagents & Intermediates) in dry N,N-dimethylformamide (DMF, 20 mL) were added 2,4,6-collidine (4.2 mL, 31.4 mmol; Spectrochem), 1-hydroxy-7-azabenzotriazole (HOAt, 1.2 g, 9.4 mmol; Spectrochem) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU, 3.0 g, 9.4 mmol; Spectrochem) undernitrogen. The resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by thin- layer chromatography (TLC) analysis (15% methanol in dichloromethane). After completion of the reaction, ice cold water (100 mL) was added, and the resulting mixture was extracted with ethyl acetate (200 mL x 3). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified by silica-gel (230-400 silica) column chromatography using 5-10% methanol in dichloromethane to afford the desired compound as an off-white solid. The desired compound was characterized by LC-MS and 1 H NMR analysis. Yield: 1.3 g (30%). %). LC-MS C 47 H 50 N 8 O 8 calculated 854.38; observed 855.65 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 13.48 (d, J=7.6 Hz, 1H), 9.27 (s, 1H), 8.55-8.46 (m, 2H), 7.99 (d, J=8.8 Hz, 1H), 7.86 (d, J=7.6 Hz, 2H), 7.71- 7.62 (m, 4H), 7.40-7.37 (m, 2H), 7.30-7.28 (m, 2H), 4.81 (bs, 1H), 4.58 (bs, 1H), 4.28-4.18 (m, 8H), 3.71 (bs, 1H), 3.53-3.51 (m, 1H), 3.37 (bs, 1H), 2.46 (s, 3H), 2.22-1.98 (m, 11H), 1.09 (t, J=6.4 Hz, 2H), 0.91 (s, 6H). Preparation 10 (9H-Fluoren-9-yl)methyl ((R)-3-amino-1-((3-((4-((2-((S)-2-cyanopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-1-oxopr opan-2-yl)carbamate (13) To a stirred solution of (9H-fluoren-9-yl)methyl ((R)-1-((3-((4-((2-((S)-2-cyanopyrrolidin-1-yl)- 2-oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-3-((1 -(4,4-dimethyl-2,6- dioxocyclohexylidene)ethyl)amino)-1-oxopropan-2-yl)carbamate (0.62 g, 0.65 mmol) in ethanol (6 mL) was added hydrazine hydrate (25%, 0.3 mL; Spectrochem). The resulting mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (15% methanol in dichloromethane). After completion of the reaction, water (30 mL) was added, and the resulting mixture was extracted with dichloromethane (100 mL x 3). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified by silica-gel (100-200 silica) column chromatography using 5-10 % methanol in dichloromethane to afford the desired compound as an off-white solid. The desired compound was characterized by LC-MS and 1 H NMR analysis. Yield: 0.42 g (100%). LC-MS C 37 H 38 N 8 O 6 calculated 690.29; observed 691.50 [M+H] + . 1 H NMR (400 MHz, ): δ 9.29-9.27 (m, 2H), 8.45 (s, 1H), 8.11 (bs, 1H), 8.01 (d, J=8.8 Hz, 1H), 7.87 (d, J=7.2 Hz, 2H), 7.73-7.66 (m, 3H), 7.40- 7.31 (m, 4H), 4.82 (bs, 1H), 4.27-4.17 (m, 8H), 3.72 (bs, 1H), 3.53-3.52 (m, 1H), 3.29-3.27 (m, 4H), 3.02 (bs, 1H), 2.17-1.91 (m, 6H), 1.09 (t, J=6.0 Hz, 2H). Preparation 11 (9H-Fluoren-9-yl)methyl ((R)-1-((3-((4-((2-((S)-2-cyanopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-3-(4-(d i-tert- butylfluorosilyl)benzamido)-1-oxopropan-2-yl)carbamate (14) (Example 13) To stirred solution of (9H-fluoren-9-yl)methyl ((R)-3-amino-1-((3-((4-((2-((S)-2-cyanopyrrolidin- 1-yl)-2-oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino) -1-oxopropan-2-yl)carbamate (0.42 g, 0.61 mmol) and 4-(di-tert-butylfluorosilyl)benzoic acid (SiFA-BA, 0.25 g, 0.89 mmol) in dry N,N-dimethylformamide (DMF, 5 mL) were added N,N-diisopropylethylamine (DIPEA, 0.72 mL, 4.0 mmol; Spectrochem), 1-hydroxy-7-azabenzotriazole (0.122 g, 0.9 mmol; Spectrochem) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU, 0.28 g, 0.9 mmol; Spectrochem) under nitrogen. The resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC analysis (10% methanol in dichloromethane). After completion of the reaction, water (30 mL) was added, and the resulting mixture was extracted with ethyl acetate (100 mL x 2). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified by silica-gel (230-400 silica) column chromatography using 1-10% methanol in dichloromethane to afford the desired compound as an off-white solid. The desired compound was characterized by LC-MS analysis. Yield: 0.4 g (70%). LC-MS C 52 H 59 FN 8 O 7 Si calculated 954.43; observed 955.65 [M+H] + . Preparation 12 6-(3-((R)-2-amino-3-(4-(di-tert-butylfluorosilyl)benzamido)p ropanamido)propoxy)-N-(2-((S)-2- cyanopyrrolidin-1-yl)-2-oxoethyl)quinazoline-4-carboxamide (15) To a stirred solution of (9H-fluoren-9-yl)methyl ((R)-1-((3-((4-((2-((S)-2-cyanopyrrolidin-1-yl)- 2-oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-3-(4- (di-tert- butylfluorosilyl)benzamido)-1-oxopropan-2-yl)carbamate (0.4 g, 0.41 mmol) in dimethylformamide (DMF, 3 mL) was dropwise added a solution of piperidine (0.04 mL, 0.50 mmol; Spectrochem) in N,N-dimethylformamide (1 mL). The resulting mixture was stirred at room temperature for 1 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) analysis (15% methanol in dichloromethane). After completion of the reaction, water (50 mL) was added, and the resulting mixture was extracted with ethyl acetate (70 mL x 3). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified by silica-gel (230- 400 silica) column chromatography using 5-12% methanol in dichloromethane to afford the desired compound as an off-white solid. The desired compound was characterized by LC-MS and 1 H NMR analysis. Yield: 0.22 g (73%). LC-MS C 37 H 49 FN 8 O 5 Si calculated 732.36; observed 733.65 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 9.31-9.26 (m, 2H), 8.84 (bs, 1H), 8.45 (s, 1H), 8.30 (s, 1H), 8.01 (d, J=7.6 Hz, 3H), 7.74-7.64 (m, 3H), 7.10 (bs, 3H), 4.82 (bs, 1H), 4.66 (bs, 1H), 4.25-4.13 (m, 4H), 3.73 (bs, 1H), 3.53-3.52 (m, 1H), 3.35-3.09 (m, 3H), 2.50-2.07 (m, 6H), 1.23 (s, 18H). Preparation 13 tri-tert-Butyl 2,2',2''-(10-((R)-1-(tert-butoxy)-5-(((R)-1-((3-((4-((2-((S) -2-cyanopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-3-(4-(d i-tert- butylfluorosilyl)benzamido)-1-oxopropan-2-yl)amino)-1,5-diox opentan-2-yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetate (16) To a stirred solution of 6-(3-((R)-2-amino-3-(4-(di-tert- butylfluorosilyl)benzamido)propanamido)propoxy)-N-(2-((S)-2- cyanopyrrolidin-1-yl)-2- oxoethyl)quinazoline-4-carboxamide (0.22 g, 0.30 mmol) and 1,4,7,10- tetraazacyclodocecane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA(tBu) 4 ,0.21 g, 0.30 mmol; Argonix Reagents & Intermediates) in dichloromethane (4 mL) was added N,N- diisopropylethylamine (DIPEA, 0.16 mL, 0.90 mmol; Spectrochem) under nitrogen. This was followed by an addition of 1-propanephosphonic anhydride (50 % in ethyl acetate, 0.28 mL, 0.90 mmol: Spectrochem). The resulting mixture was stirred at room temperature for 3 h. The progress of the reaction was monitored by TLC analysis (15% methanol in dichloromethane). After completion of the reaction, water (30 mL) was added, and the resulting mixture was extracted with dichloromethane (50 mL x 3). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified by silica-gel (230-400 silica) column chromatography using 10-15% methanol in dichloromethane to afford the desired compound as an off-white solid. The desired compound was characterized by LC-MS and 1 H NMR analysis. Yield: 0.29 g (69.0 %). %). LC-MS C 72 H 111 FN 12 O 14 Si calculated 1414.81; observed 1413.85 [M-H]-. 1 H NMR (400 MHz, DMSO): δ 9.31-9.28 (m, 2H), 8.56-8.47 (m, 2H), 8.16 (bs, 1H), 8.09 (bs, 1H), 8.02-7.90 (m, 3H), 7.73- 7.63 (m, 3H), 4.81 (bs, 1H), 4.47 (bs, 1H), 4.24-4.13 (m, 5H), 3.72 (bs, 1H), 3.54-3.52 (m, 2H), 3.30 (bs, 3H), 3.16 (d, J=5.2 Hz, 2H), 3.03-3.00 (m, 5H), 2.85-2.70 (m, 4H), 2.33-1.83 (m, 21H), 1.40 (s, 36H), 1.08 (s, 18H). Synthesis of Conjugates of Examples 3 and 4 Preparation 14 – Compound of Example 3 2,2',2''-(10-((R)-1-carboxy-4-(((R)-1-((3-((4-((2-((S)-2-cya nopyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-3-(4-(d i-tert- butylfluorosilyl)benzamido)-1-oxopropan-2-yl)amino)-4-oxobut yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid (17) A solution of tri-tert-butyl 2,2',2''-(10-((R)-1-(tert-butoxy)-5-(((R)-1-((3-((4-((2-((S) -2- cyanopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinazolin-6-yl)o xy)propyl)amino)-3-(4-(di-tert- butylfluorosilyl)benzamido)-1-oxopropan-2-yl)amino)-1,5-diox opentan-2-yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetate (0.29 g, 0.20 mmol) in trifluoroacetic acid (TFA): triisopropylsilane (TIS):water (95:2.5:2.5, 22 mL; Spectrochem) was stirred at room temperature for 36 h under nitrogen. The progress of the reaction was monitored by LC-MS analysis. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain sticky liquid. The residue was triturated with methyl tert-butyl ether (20 mL x 3) followed by with n-pentane (10 mL) resulted in solid formation. The solid was dried under vacuum to afford the desired compound as a light brown solid. The desired compound was characterized by LC-MS analysis. Yield: 0.19 g (77.8%, crude). LC-MS C 56 H 79 FN 12 O 14 Si calculated 1191.56; observed 1189.70 [M-H]-. Preparation 15 – Compound of Example 4 Gallium(I) 2-(16-((R)-1-carboxy-4-(((R)-1-((3-((4-((2-((S)-2-cyanopyrro lidin-1-yl)-2- oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino)-3-(4-(d i-tert- butylfluorosilyl)benzamido)-1-oxopropan-2-yl)amino)-4-oxobut yl)-3,6-dioxo-4,5-dioxa- 1,8,11,16-tetraazabicyclo[6.5.5]octadecan-11-yl)acetate (18, Ga-(S,R,R)-SiFA-FAP-2) To a stirred solution of 2,2',2''-(10-((R)-1-carboxy-4-(((R)-1-((3-((4-((2-((S)-2-cya nopyrrolidin- 1-yl)-2-oxoethyl)carbamoyl)quinazolin-6-yl)oxy)propyl)amino) -3-(4-(di-tert- butylfluorosilyl)benzamido)-1-oxopropan-2-yl)amino)-4-oxobut yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid (crude, 0.19 g, 0.16 mmol) in tert-butanol: water (3:1, 7.3 mL) under nitrogen was added gallium (III) nitrate (0.082 g, 0.32 mmol; Sigma Aldrich) and the resulting mixture was heated at 75 o C for 3h. The progress of the reaction was monitored by LC-MS analysis. After completion, the reaction was cooled to room temperature and added water (10 mL). The resulting mixture was filtered through micro filter and the filtrate was concentrated under reduced pressure. The residue obtained was triturated with methyl tert-butyl ether (10 mL x 3) and diethyl ether (10 mL). The sticky residue was dried under high vacuum to afford the pale-yellow solid. The crude compound was purified by reverse phase HPLC; Mobile phase A: A: 0.1% Formic acid in water and Mobile phase B: Acetonitrile; Column: Waters XBridge Prep C18 250×19 mm, 5 μm to afford the desired compound as an off-white solid. Yield: 13 mg (6.5%). LC-MS C 56 H 76 FGaN 12 O 14 Si calculated 1256.46; observed 1257.80 [M+H] + . 1 H NMR (400 MHz, DMSO): δ 9.33-9.27 (m, 2H), 8.61 (m, 1H), 8.46 (bs, 1H), 8.16 (bs, 2H), 8.02-7.91 (m, 3H), 7.75-7.65 (m, 3H), 4.84 (bs, 1H), 4.57 (bs, 1H), 4.45 (bs, 1H), 4.25-4.17 (m, 4H), 3.73 (bs, 1H), 3.48 (m, 4H), 3.30-2.70 (m, 23H), 2.18-1.83 (m, 11H), 1.02 (s, 18H); HPLC: 7.730 min; 90.6%, Column : XBridge C18250×4.6, 5 μm, Mobile phase A : 0.1% formic acid in H 2 O; Mobile phase B : Acetonitrile. Synthesis of conjugates of Examples 5 to 10 The conjugates of Examples 5 to 10 may be prepared in accordance with the below schemes. Steps 1-5 above can be conducted according to published methods (WO2019/020831): Step 1: a) 20% piperidine, (DMF); b) (tBuO)EuE(OtBu) 2 , HOBt, TBTU, DIPEA, (DMF); Step 2: a) 2% hydrazine (DMF); b) succinic anhydride, DIPEA, (DMF); c) Fmoc-D-Lys-OAII-HCI, HOBt, TBTU, DIPEA, (DMF); Step 3: a) 20% piperidine, (DMF); b) Fmoc-D-Dap(Dde)-OH, HOBt, TBTU, DIPEA, (DMF); c) imidazole, hydroxylamine hydrochloride, (NMP, DMF); Step 4: a) SIFA-BA (WO2019/020831), HOBt, TBTU, DIPEA (DMF); b) 20% piperidine, (DMF); Step 5: DOTAGA anhydride, DIPEA, (DMF) Example 5 Example 6 Example 7 Synthesis of (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile (step 6 above) is Step 6: a) HATU, DIPEA, (DMF); b) cleavage: TFA TIS, water; c) Final deprotection: TFA. Example 8 & 8a
Example 9 & 10 Synthesis of (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile (step 6) is conducted according to the method described by Jansen et al. (ACS Med. Chem. Lett. 2013, 4, 491– 496).
Radiolabelling General Information No-carrier-added fluorine-18 was produced by Curium Pharma, via the [ 18 O(p, n) 18 F] nuclear reaction by irradiation of a 2.8 mL > 97%-enriched [ 18 O]H 2 O target (Bruce Technology) on a PETtrace cyclotron (16 MeV proton beam, GE healthcare). Radio-instant thin-layer chromatography (radio-ITLC) analyses were measured on a mini GITA Dual radio-TLC instrument (Elysia-Raytest) using silica gel-impregnated chromatography paper (Varian Inc.) eluted for 4 minutes with an aqueous Na 2 CO 3 0.1 M solution. Analytical HPLC measurements were performed on a system consisting of an Agilent HP series 1100 (Hewlett Packard, Les Ulis, France) combined with a Flo-one A500 Radiomatic detector (Packard, Canberra, Australia). The separation was carried out on a C-18 column (Kinetex ® EVO C18, 5 μm, 4.6 x 150 mm, 100 Å, equipped with a guard column) using the following solvent conditions: water containing 0.1% of trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% of trifluoroacetic acid (solvent B); 0 to 10 min: gradient elution 99% - 0% A with a flow rate of 1 mL/min, λ = 254 and 214 nm. Preparation of anhydrous the K[ 18 F]F-K222-carbonate complex was performed using a SynChrom R&D EVOI synthesis module (Raytest). Oasis HLB Plus LP cartridge (60 μm), Sep-Pak ® Light Accell Plus QMA carbonate cartridges (46 mg, 45 μm) and Sep-Pak ® light C18 Plus cartridges (130 mg, 55-105 μm) were purchased from Waters. All radiolabelled compounds were compared by TLC or analytical HPLC to the authentic non- radioactive material and to be free of significant UV-absorbing chemical and radiochemical impurities. Radiosynthesis of Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) On a SynChrom R&D EVOI synthesis module, the aqueous solution of [ 18 F]F- in [ 18 O]H 2 O (2.67 GBq at 12h16) was passed through an anion exchange resin (Sep-Pak ® Light Accell Plus QMA carbonate cartridge 46 mg) preconditioned with deionised water (10 mL) and air (10 mL). Then a solution of potassium carbonate (2.8 mg) and Kryptofix (K222, 21 mg) in a mixture of water (200 μL) and acetonitrile (700 μL) was passed through the cartridge to elute the radioactivity to the reactor. After 30 s of helium bubbling, the azeotropic drying of the mixture was performed under vacuum and helium flow at 100 °C for 3 minutes. After cooling to 30 °C, acetonitrile (1 mL) was added to the reactor. After 30 s of helium bubbling, the reaction mixture was evaporated to dryness under vacuum and helium flow at 110 °C for 3 min. After cooling to 30 °C, a freshly prepared solution of the compound of Example 2 (66 μg, 52 nmol) and acetic acid (9 μL) in anhydrous DMSO (500 μL) was added to the dry reactor. The solution was stirred for 10 s and transferred in a closed glass vial containing a magnetic stirrer. The reaction mixture was then stirred at room temperature for 10 min, diluted with water (20 mL) and passed through an Oasis HLB Plus cartridge. The latter was washed with water (10 mL), dried with air (20 mL) and the radioactivity was recovered from the cartridge using absolute ethanol (2 mL) and air (3 mL). After evaporation under vacuum, the final product was formulated in saline (0.9% NaCl, 0.5 mL). Total synthesis time: 61 min. Radiochemical yield decay-corrected: 57%. The radiochemical purity of the radiotracer was verified using analytical radio RP-HPLC measurements at the end of radiosynthesis (>99%, Figure 1). BIOLOGICAL ACTIVITY Studies relating to in vitro binding affinity and in vivo biodistribution were conducted. In vivo PET imaging and Biodistribution The human glioblastoma U87-MG cell line was purchased from ATCC. Cells were cultured in EMEM (supplemented with 2 mM L-glutamine, with 10% fetal bovine serum and 0.1 mM NEAA) at 37°C in a humidified atmosphere (5% CO2, 95% air). All animal experiments were conducted in accordance with the Federation for Laboratory Animal Science Associations guidelines. Healthy female Swiss nude (Crl:NU(Ico)-Foxn1nu) mice (5-6 weeks old) were purchased from Charles River. Mice were irradiated 24-72 hours prior to tumour cell inoculation (whole-body irradiation, 2 Gy/mouse), and U87-MG cells (1X10 7 in 200 μL of RPMI 1640) were then injected subcutaneously into the right shoulder flank. Female Swiss nude (Crl:NU(Ico)-Foxn1nu) mice each bearing a subcutaneous U87-MG tumour in the right flank (n=4) were administered with radiolabelled compound of Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) (8 ± 1.5 MBq, 100 μL per mouse) intravenously into the tail vein (approximately 3 weeks post inoculation of U87-MG cells). Static PET imaging was performed 60 minutes post-radiotracer administration under anaesthesia (2% isoflurane) using a small animal PET scanner (eXploreVISTA, GE) (n=3). Whole-body PET acquisitions were performed over two bed positions for 20 minutes. Regions of interest were generated, and radiotracer uptake in tissues was calculated as % injected dose per region of interest volume (%ID/cm 3 ). Immediately post-PET imaging, mice (n=4) were sacrificed and select tissues (blood, tumor, heart, lung, liver, kidneys, spleen, pancreas, intestine, bone, muscle, tail) were harvested and weighed. Radioactivity in the collected samples was determined using γ-counting (Packard). Radiotracer uptake was calculated as % injected dose per gram of tissue (%ID/g). Results Tumor uptake of the radiolabelled compound of Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) was observed using both PET imaging (Table 2, Figure 2) and biodistribution analyses by γ- counting excised tissues (Table 3a and 3b, Figure 3). PET imaging demonstrated radiotracer uptake greater than or equal to 4-fold higher in tumor compared to muscle (Table 2, Figure 2). Biodistribution analyses demonstrated radiotracer uptake 8-fold higher in tumor compared to muscle (Table 3a and 3b, Figure 3). Table 2 - Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) PET Imaging Uptake Table 3a - Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) Biodistribution (%ID/g) Table 3b - Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) Biodistribution - Tumor to muscle ratio (T:M) In vitro FAP binding affinity The binding affinity of non-radiolabelled compounds Example 2 (Ga-(S,R,R)-SiFA-FAP-1) and Example 1 ((S,R,R)-SiFA-FAP-1) was assessed in vitro using Grating-Coupled Interferometry (GCI) and waveRAPID kinetics assay (Kartal Ö et al. SLAS Discov. 2021 Sep; 26(8): 995- 1003). FAP was immobilized on a streptavidin sensor chip in order to assess target binding of compounds. Briefly, the streptavidin chip was conditioned with borate buffer and activated with EDC-NHS solution. Immobilization was achieved via amine coupling to the surface on an amine reactive sensor chip (NeutrAvidin sensor chip) and unused amine reactive groups were deactivated with ethanolamine. FAP was immobilized via via NeutrAvidin capturing of the biotinylated FAP and remaining NeutrAvidin quenched with biocytin. Binding of the compounds to immobilized FAP was assessed using the waveRAPID kinetics assay, using different concentrations of each compound in 1xPBS pH 7.4, 1 mM DTT, 0.005% Tween, 2% DMSO. Binding affinity to FAP was reported as K D . Results The binding affinity for both compounds was in the pM range (Table 4). Table 4 - Binding affinity Brief Description of the Figures Figure 1: Quality control of Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) at the end of radiosynthesis. Figure 2: Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) PET imaging uptake of U87-MG tumor- bearing mice (Table 2). Mice were injected with [ 18 F]-Ga-(S,R,R)-SiFA-FAP-1 (8 ± 1.5 MBq) and imaged 60 minutes post radiotracer administration. Average radiotracer uptake in muscle and tumor (%ID/cm 3 ), mean ± SD is shown (n=3). Radiotracer uptake was >4-fold higher in tumor compared to muscle. Figure 3: Biodistribution of Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA-FAP-1) in U87-MG tumor- bearing mice (Tables 3a and 3b). Mice were injected with Example 11 ([ 18 F]-Ga-(S,R,R)-SiFA- FAP-1) (8 ± 1.5 MBq). Radioactivity in tissues was measured by J-counting 80 minutes post- radiotracer injection. Radiotracer biodistribution (%ID/g), mean ± SD is shown (n=4). Biodistribution analyses demonstrated radiotracer uptake 8-fold higher in tumor compared to muscle.