Field of Invention

The current invention relates to compounds of formula (I), and their use as heterobifunctional linkers. The present invention also provides compounds of formula (III) which can be obtained from a compound of formula (I). The compounds of formula (III) find use as medicaments and/or diagnostic agents.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

A strategy of binding synthetic cargoes to drug delivery system or biomacromolecules is popularly employed in fields including but not limited to immunochemistry, drug development, polymer science, and live-cell imaging. The development of drug delivery system such as monoclonal antibody, peptides, or liposomes has enabled the clinical translation of drug modalities that are unable to be administered on their own right, such as cytotoxins and small interfering ribonucleic acid (siRNA). The attachment of cytotoxic drugs and toxins to tumourlocalising monoclonal antibodies is an approach to immunotherapy that has received much attention over the years. Other than therapeutics, producing stable and active enzyme-antigen conjugates are also of paramount importance in immunoassays.

Obtaining detailed insights into living organisms, like living cells or bacteria, is increasingly necessary in many fields like biology or chemistry. However, it is a challenging task due to the vast complexity of the biological systems. Researchers have made many efforts to study the dynamics and functions of the huge diversity of biomolecules in living systems, mainly based on spectroscopic measurements, which require the biomolecules to be labelled.

As a result, many reagents for the preparation of these conjugates have been reported. Such chemical cross-linking reagents are designed to have specific reactivity with functional groups contained in each reactant. Both homo- and hetero-bifunctional reagents are available, with the latter having more utility. Since hetero-bifunctional reagents possess two selectively reactive groups that allow coupling to be carried out in a stepwise manner, better control of the conjugation chemistry is attainable. For instance, component A may be activated, purified and characterized, before combining with component B. Popular heterobifunctional linkers include SMCC (Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate, CAS No.: 64987-85-5), PMPI (N-(p- Maleimidophenyl isocyanate), CAS No.: 123457-83-0), and iodoacetamide-PEG3-azide (CAS No.: 1594986-04-5). Their high reactivity allows chemical modification even at lower concentrations in aqueous media. Among the functional groups suitable for thiol conjugation, maleimide derivatives are the most commonly applied. Even though it offers fast reaction kinetics and has been widely used, maleimides still suffer from some drawbacks.

Firstly, the maleimide-thiol reaction generates two diastereomers instead of a single product. The resulting diastereomers make the profile of their chromatograms complicated (Smyth, D. G. et al., Biochem. J., 1964, 91, 589), and readily undergo isomerisation into each other (Kuninori, T. & Nishiyama, J. Agric. Biol. Chem., 1985, 49, 2453). Secondly, the resulting thiosuccinimide adduct is reported to be prone to deconjugation via a retro-Michael reaction (Alley, S. C. et al., Bioconjug. Chem., 2008, 19, 759), or to undergo ring-opening by hydrolysis (Lewis, M. R. & Shively, J. E., Bioconjug. Chem., 1998, 9, 72). These drawbacks become particularly problematic in antibody-drug conjugate (ADC) applications. The retro-Michael reaction leads to premature drug release in blood plasma. Regenerated maleimide linkerpayload then can rapidly react with other thiol groups in the blood, such as human serum albumin (HSA) or glutathione (Shen, B. Q. et al., Nat. Biotechnol., 2012, 30, 184). The characterisation study conducted by Pfizer further supports this in-vitro study: trastuzumab- maleimide-drug was incubated with human plasma for 144 h, and almost 100% of the drug loss happened (Wei, C. et al., Anal.Chem., 2016, 88, 4979). Such deconjugation and exchange reaction results in off-target toxicity, reduced therapeutic efficacy, and less than desired amounts of cytotoxins get delivered to tumour cells.

Thus, there remains a need for improved and/or alternative heterobifunctional linkers suitable for the preparation of biomacromolecules that display beneficial properties.

Summary of Invention

Aspects and embodiments of the invention are described in the following numbered clauses.

1. A compound according to formula (I): wherein,

A is selected from the group consisting of -Ci-aalkylene-, -R ^a -(C3-i2carbocycle)-R ^a - and -R ^a -(5-10 membered heterocycle)- R ^a -; said alkylene being a linear alkylene and optionally having one or more carbon atoms replaced by a group independently selected from the group consisting of O, NH and S; wherein said alkylene, carbocycle and heterocycle are optionally substituted by one or more groups selected from OH, NH ₂ , Ci.4alkyl, and halogen; and each R ^a is independently a bond or -Ci.4alkylene-;

Z ^a is allenyl or according to formula (II)

D ^a is selected from the group consisting of a carrier entity, bioactive entity, and a diagnostic entity; or a pharmaceutically acceptable salt or solvate thereof. The compound of clause 1, wherein A is -Ci-ealkylene- or -R ^a -(C5-6carbocycle)-R ^a -, wherein each R ^a is independently a bond or -C- alkylene-. The compound of clause 1 or clause 2, wherein A is -Csalkylene- or -CH ₂ -(C6carbocycle)-. The compound of any one of clauses 1-3, wherein said carbocycle is a non-aromatic carbocycle, for example a cyclohexane group. The compound of clause 1, wherein the compound is according to formula (la): The compound of clause 1, wherein the compound is according to formula (lb): The compound of any one of clauses 1-6, wherein Z ^a is allenyl. The compound of any one of clauses 1-6, wherein Z ^a is according to formula (II). The compound of clause 8, wherein D ^a is a bioactive entity. The compound of clause 9, wherein D ^a is maytansine, an auristatin (e g. monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin, or a cytotoxic derivative thereof.

A compound according to formula (III): wherein,

A is -C-i-ealkylene-, said alkylene being a linear alkylene and optionally having one or more carbon atoms replaced by a group independently selected from the group consisting of O, NH and S; and/or optionally substituted by one or more groups selected from OH, NH2, Ci-4al kyl , and halogen; one of D ^a and D ^b is a small molecule drug, and the other is an antibody or antibody fragment; or a pharmaceutically acceptable salt or solvate thereof. 12. The compound of clause 11 , wherein D ^a is a small molecule drug and D ^b is an antibody or antibody fragment.

13. The compound of clause 11 or 12, wherein D ^a is maytansine, an auristatin (e.g. monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin, or a cytotoxic derivative thereof.

14. The compound of any one of clauses 11 to 13, wherein D ^b is selected from the group consisting of trastuzumab, a humanised lgG1 anti-BCMA antibody, and a fragment thereof.

15. The compound of any one of clauses 11-14, wherein D ^a is a maytansine derivative and D ^b is trastuzumab.

16. The compound of any one of clauses 11-15 for use in therapy.

17. The compound of any one of clauses 13-15 for use in the treatment of cancer.

18. Use of the compound of any one of clauses 11-15 in the manufacture of a medicament.

19. Use of the compound of any one of clauses 13-15 in the manufacture of a medicament for the treatment of cancer.

20. A method of treatment comprising a step of administering the compound of any one of clauses 11-15 to a subject in need thereof.

21 . The method of clause 20, wherein the subject has a cancer.

22. The compound for use according to clause 17, the use of clause 19, or the method of clause 21 , wherein the cancer is breast cancer or multiple myeloma.

23. Use of the compound of any one of clauses 1-10 in the preparation of an antibody-drug conjugate compound.

Drawings

FIG. 1 depicts a summary of the current invention. FIG. 2 shows the synthetic route of M-(£-allenamidocaproyloxy) pentafluorophenyl ester (EACF).

FIG. 3 shows the synthetic route of pentafluorophenyl (1r,4r)-4-(buta-2,3-dienamido- methyl)cyclohexane-1-carboxylate (FACC).

FIG. 4 shows a comparison of the FDA-approved antibody-drug conjugate Kadcyla™ and an allenamide analogue thereof.

FIG. 5 shows the comparison of ¹ H NMR spectrum of the FACC-DM1 adduct with ¹ H NMR spectrum of DM1

FIG. 6 shows the ¹ H NMR spectrum of the FACC-DM1 adduct (sp ² region zoomed).

FIG. 7 shows the comparison of ¹⁹ F NMR spectrum of the FACC-DM1 adduct with ¹⁹ F NMR spectrum of the FACC.

FIG. 8 shows a scheme of the reaction used to compare the reactivity and chemoselectivity of FACC and SMCC for amines and thiols.

FIG. 9 shows high performance liquid chromatography (HPLC) traces monitoring the reaction of FACC with benzylamine (BnNH ₂ ) and glutathione.

FIG. 10 shows HPLC traces monitoring the reaction of SMCC with benzylamine (BnNH ₂ ) and glutathione.

FIG. 11 shows a scheme of the reaction used to compare the reactivity and chemoselectivity of EACF and EMCS for amines and thiols.

FIG. 12 shows HPLC traces monitoring the reaction of EACF with benzylamine (BnNH ₂ ) and glutathione.

FIG. 13 shows HPLC traces monitoring the reaction of EMCS with benzylamine (BnNH ₂ ) and glutathione.

Detailed Description The present invention provides a compound according to formula (I): or a pharmaceutically acceptable salt or solvate thereof.

In the compound of formula (I), A is selected from the group consisting of Ci-salkylene, -R ^a - (C3-i2carbocycle)-R ^a - and -R ^a -(5-10 membered heterocycle)-R ^a -. The alkylene, carbocycle or heterocycle may be substituted by one or more groups selected from OH, NH2, Ci-4alkyl, and halogen. For example, the alkylene, carbocycle or heterocycle may be substituted by one or two groups selected from OH, NH2, Ci.4alkyl, and halogen (e.g. fluorine, chlorine, bromine or iodine).

In the compound of formula (I), when A is Ci-ealkylene, the alkylene is a linear alkylene. For example, A may be Cialkylene, a linear C2alkylene, a linear Csalkylene, a linear C4alkylene, a linear Csalkylene, or a linear Csalkylene. In certain embodiments, A is a linear C4alkylene or a linear Csalkylene. In certain exemplary embodiments, A is a linear Csalkylene.

In the compound of formula (I), when A is Ci-salkylene, the alkylene may have one or more carbon atoms replaced by a group independently selected from the group consisting of O, NH and S. For example, the alkylene may have one or two carbon atoms replaced by a group independently selected from the group consisting of O, NH and S. For example, A may be -Csalkylene- having one carbon atom replaced with one O, NH or S.

In the compound of formula (I), when A is -R ^a -(C3-i2carbocycle)-R ^a -, the carbocycle may be a non-aromatic carbocycle. For example, it may be a cyclopropane, a cyclobutane, a cyclopentane, cyclohexane, cycloheptane, or a cyclooctane group. In certain exemplary embodiments, the carbocycle is a cyclohexane group.

In the compound of formula (I), when A is -R ^a -(5-10 membered heterocycle)-R ^a -, the heterocycle may be an aromatic or non-aromatic heterocycle. For example, the heterocycle may be a 5-10 membered aromatic heterocycle or 5-10 membered non-aromatic heterocycle comprising one or more (e g. one or two) heteroatoms independently selected from N, S and O. Each R ^a is independently selected from a bond or -Ci-4alkylene-. In certain embodiments, one of R ^a is a bond and the other is a linear Ci.4alkylene-, for example, Cialkylene or a linear C2alkylene. For example, when A is -R ^a -(C3-i2carbocycle)-R ^a - or -R ^a -(5-10 membered heterocycle)-R ^a -, A may be -(C3-i2carbocycle)-CH ₂ -, -CH ₂ -(C3-i2carbocycle)-, -(5-10 membered heterocycle)-CH ₂ -, or -CH ₂ -(5-10 membered heterocycle)-. In certain exemplary embodiments, A may be -CH ₂ -(C6carbocycle)-.

In the compound of formula (I), Z ^a is allenyl or according to formula (II) wherein -'Wv indicates the point of connection between formula (II) and the rest of the compound of formula (I).

In certain embodiments, the compound of formula (I) is according to formula (la): or a pharmaceutically acceptable salt or solvate thereof.

In certain other embodiments, the compound of formula (I) is according to formula (lb): or a pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, Z ^a is allenyl. In certain exemplary embodiments, the compound of formula (I) may be:

/V-(E-allenamidocaproyloxy) pentafluorophenyl ester; or

Pentafluorophenyl (1 ,4)-4-(buta-2,3- dienamidomethyl)cyclohexane-1 -carboxylate.

For example, the compound of formula (I) may be:

Pentafluorophenyl (1 r,4r)-4-(buta-2,3- dienamidomethyl)cyclohexane-1 -carboxylate.

The present inventors have found that compounds of formula (I), wherein Z ^a is allenyl, display excellent chemoselectivity for amine and thiol groups. The allenyl group is highly reactive and highly selective for thiol groups, and the pentafluorophenoxy group is highly reactive towards amine groups. The compound of formula (I), wherein Z ^a is allenyl, also displays good hydrolytic stability. Furthermore, conjugate molecules such as antibody-drug conjugates formed using such compounds display good hydrolytic stability because of the stability of bonds connecting the antibody and drug molecules together.

In certain embodiments, in the compound of formula (I), Z ^a is according to formula (II). In formula (II), D ^a is independently selected from the group consisting of a carrier entity, bioactive entity, and a diagnostic entity. The carrier entity, bioactive entity, and diagnostic entity may each independently be a protein, a protein fragment, a peptide, a nanoparticle, or a small molecule compound (e.g. a compound having a Mw of less than about 1500 Da). Examples of carrier entities include, but are not limited to, antibodies, antibody fragments, and albumin. Examples of bioactive entities include, but are not limited to, small molecule drugs, antibodies, and antibody fragments (e.g. small molecule drugs, antibodies, and antibody fragments that display a biological activity, such as cytotoxic activity). For the avoidance of doubt, an antibody or antibody fragment may act as a carrier entity (e g. it may carry another molecule, such as a small molecule drug, to a target antigen) or it may act as a bioactive entity (i.e. it may directly cause a biological effect itself). In some circumstances, an antibody or antibody fragment may act as both a carrier entity and a bioactive entity. Examples of diagnostic entities include, but are not limited to, radioactive agents, enzymes, fluorescent compounds, and electron transfer agents. In certain embodiments, D ^a is a bioactive entity.

In certain embodiments, D ^a is an antibody, for example, a monoclonal antibody. In certain embodiments, D ^a is an antibody that is capable of binding to a tumour-associated antigen (TAA) or a tumour-specific antigen (TSA). For example, D ^a may be an antibody that binds to the Human epidermal growth factor receptor-2 (HER2) or the B-cell maturation antigen (BCMA). For example, D ^a may be a human, humanised or chimeric anti-HER2 antibody or a human, humanised or chimeric anti-BCMA antibody. In certain embodiments, D ^a is trastuzumab or a humanised lgG1 anti-BCMA antibody.

In certain other embodiments, D ^a is a small molecule drug, for example a cytotoxic small molecule drug. In certain embodiments, D ^a may be maytansine, an auristatin (e.g. monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin, or a cytotoxic derivative thereof. Suitable derivatives of such small molecules drugs include those that have been modified to include a linker that links the derivative to the sulphur atom of formula (II). For example, when D ^a is a maytansine derivative, D ^a may be according to formula (IV): wherein indicates the point of connection between formula (IV) and formula (II).

Formula (IV) may be derived from mertansine (DM1). For example, when Z ^a is according to formula II and D ^a is according to formula (IV), the compound of formula (I) may be obtained by reacting mertansine with a compound of formula (I) that has an allenyl group at the Z ^a position.

In certain preferred embodiments, D ^a may be maytansine or a derivative of maytansine, such as a derivative having a structure according to formula (IV). Thus, the compound of formula (I) may be according to formula (Ic):

The stability of the thiol-allenamide bond makes the compounds of formula (I), wherein Z ^a is according to formula (II), particularly useful as intermediates in the preparation of bioconjugate molecules, such as antibody-drug conjugates.

As disclosed herein, the compounds of formula (I) are effective as bifunctional linkers and/or intermediates in the preparation of bioconjugate molecules, such as antibody-drug conjugates. Thus, provided herein is the use of the compounds of formula (I) in the preparation of bioconjugate molecules, such as antibody-drug conjugates. Exemplary methods for the preparation of the compounds of formula (I) and their use in preparing bioconjugate molecules are provided in the Examples section disclosed herein.

The present invention also provides a compound according to formula (III): or a pharmaceutically acceptable salt or solvate thereof.

In the compound of formula (III), A is a linear -Cvealkylene-. The alkylene may have one or more carbon atoms replaced by a group independently selected from the group consisting of O, NH and S. For example, the alkylene may have one or two carbon atoms replaced by a group independently selected from the group consisting of O, NH and S. For example, A may be -Csalkylene- having one carbon atom replaced with one O, NH or S.

In the compound of formula (III), D ^a is a small molecule drug and D ^b is an antibody or antibody fragment. In certain embodiments, D ^a is maytansine, an auristatin (e g. monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin, or a cytotoxic derivative thereof, and D ^b is an antibody, or antibody fragment. For example, D ^b may be a human, humanised or chimeric anti-HER2 antibody or a human, humanised or chimeric anti-BCMA antibody. In certain embodiments, D ^b is trastuzumab or a humanised lgG1 anti-BCMA antibody.

In certain embodiments, D ^a is a maytansine derivative and D ^b is an antibody or fragment thereof. For example, the compound of formula (III) may be according to formula (Illa): wherein, Ab represents an antibody or a fragment thereof.

The present inventors expect that compounds of formula (III) display greater stability in vivo compared to corresponding compounds that comprise an antibody and a small molecule drug compound that are linked by a thioether bond. The latter is known to undergo a retro-Michael process that can lead to the deconjugation of the small molecule drug from the compound. As used herein, the term “alkyl” refers to both straight and branched chain saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, /so-propyl, n- butyl, t-butyl, /-butyl, sec-butyl, pentyl and hexyl groups. Among unbranched alkyl groups, there are preferred methyl, ethyl, n-propyl, /so-propyl, n-butyl groups. Among branched alkyl groups, there may be mentioned f-butyl, /-butyl, 1-ethylpropyl and 1 -ethylbutyl groups.

As used herein, the term “halogen” refers to fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are particularly preferred.

The term ’’carbocycle” as used herein includes saturated, partially unsaturated or unsaturated monocyclic, bicyclic, tricyclic, polycyclic, spirocyclic, bridged and fused ring system having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent. The term includes aromatic carbocycles (e g., a benzene group) and non-aromatic carbocycles (e g. a cyclohexane group).

For the avoidance of doubt, the term “heterocycle” as used herein encompasses any aromatic or non-aromatic cyclic group comprising one or more heteroatoms (i.e. N, O or S). The term “heterocycle” as used herein encompasses bicyclic heterocycle groups such as spirocyclic heterocycles, fused heterocycle and bridged heterocycles, unless otherwise stated. Heterocycle groups containing a suitable nitrogen atom include the corresponding N-oxides. Examples of 5-10 membered heterocycle groups include pyrrolidine, piperidine, tetrahydrofurane, tetrahydropyrane, pyridine, pyrimidine, indoline, pyrrole, pyrazole, imidazole, furane, guanidine-containing heterocycles, and quinazoline.

As mentioned above, the present invention also provides the compounds of the present invention as solvates and/or 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 include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. 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 (TGE), differential scanning calorimetry (DSC) and X-ray crystallography. The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

Pharmaceutically acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of the present invention 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 of formula the present invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

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, or preferably, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-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, methanesulphonic, 1- hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

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.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”.

Compounds of the present invention may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or high performance liquid chromatography (HPLC), techniques. Alternatively, the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

Compounds of formula (III) may be present in form of a pharmaceutical composition. Pharmaceutical compositions of the present invention may comprise the compound of formula (III) and one or more pharmaceutically acceptable excipients.

The pharmaceutical composition may be any pharmaceutical composition suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration, although the most suitable route may depend upon the characteristics of the subject under treatment, for example the species, age, weight, sex, and medical conditions. Pharmaceutical compositions suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The composition may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze- dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water- for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind described herein. Exemplary compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water, Ringer’s solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor. Compositions for nasal, aerosol or inhalation administration include solutions in saline, which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.

Compositions for rectal administration may be presented as a suppository with carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol. Such carriers are typically solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

Compositions for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerine or sucrose and acacia. Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).

Pharmaceutical compositions suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The compound of formula (III) may also be presented as a bolus, electuary or paste. The pharmaceutical compositions may optionally be present in a form that provides slow or controlled release of the compound of formula (III) once administered to a subject. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

It should be understood that in addition to the ingredients particularly mentioned above, the compositions for use in this invention may include other agents conventional in the art having regard to the type of composition in question.

Compounds of formula (III) find use in therapy. In certain embodiments, when D ^a has cytotoxic activity (e.g. it is maytansine, an auristatin, or a cytotoxic derivative thereof) and D ^b is an antibody that binds to a tumour-associated antigen (TAA) or a tumour-specific antigen (TSA), the compound of formula (III) may find use in the treatment of cancer.

For example, when the compound of formula (III) is according to formula (Illa), wherein Ab is trastuzumab, the compound of formula (III) finds utility in the treatment of HER2-positive breast cancer. Or, for example, when the compound of formula (III) is according to formula (Illa) wherein Ab is a humanised lgG1 anti-BCMA antibody, the compound of formula (III) finds utility in the treatment of multiple myeloma.

Compounds of formula (III) will generally be administered to a subject in need thereof as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington: The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527. Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

The amount of compound of formula (III) in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula (III) in the formulation may be determined routinely by the skilled person.

For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer.

A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula (III) may be administered at varying therapeutically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease. Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula (III).

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The compounds of formula (III) disclosed herein may be used in medicine. Thus, in a further aspect of the invention, there is provided a use of an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof, in medicine.

The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.

The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, and the like.

Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

Examples

The examples disclosed herein describe the development and use of heterobifunctional linkers that bear an allenamide on one side and a pentafluorophenyl ester on the other side. The use of the heterobifunctional linkers for the preparation of bioconjugate molecules is summarised in FIG. 1.

Materials

All reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, Tokyo Chemical Industry, Fluorochem, BLD Pharmatech, Alfa Aesar, Apollo Scientific and Combi- Blocks) and used without further purification, unless otherwise stated. Flash chromatography was carried out using silica gel 60 (40-63 pm) from Merck in the stated eluent system. Analytical thin layer chromatography plates were purchased from Merck (aluminium sheets or glass-backed plates, silica gel 60 F254) and visualised using ultraviolet light and/or by submersion in potassium permanganate solution. Preparative thin layer chromatography was carried out using silica gel 60 F254 glass-backed plates (layer thickness: 1.0 mm) from Merck.

¹ H, ¹³ C and ¹⁹ F Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker Avance 400 MHz or JEOL ECA 400 MHz spectrometer at ambient temperature in deuterated solvents at 25 °C. Chemical shifts are reported in ppm (d) using the residual solvent as internal standard. Peak multiplicities given in Hz are expressed as follow: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; t, triplet; dt, doublet of triplets; q, quartet; dq, doublet of quartets; p, pentet; h, heptet; m, multiplet; br s, broad singlet.

High resolution mass spectrometry (HRMS) was carried out using a Waters G2-XS QTof spectrometer with positive electrospray ionization. Example 1. Synthesis and Characterisation of A/-(E-Allenamidocaproyloxy) Pentafluorophenyl Ester (EACF)

EACF was prepared via the reaction scheme shown in FIG. 2. Further details of the synthesis of EACF are provided below. a) Synthesis of methyl 6-aminohexanoate 6-aminohexaiioic acid (1)

To a Schlenk flask containing a mixture of 6-aminohexanoic acid (1.31 g, 10 mmol, 1.0 eq.) in anhydrous methanol (15 mL) under argon atmosphere, thionyl chloride (1.1 mL, 15 mmol, 1.5 eq.) was added dropwise at 0°C. The resulting solution was allowed to warm to room temperature and stirred overnight. Upon reaction completion, volatiles were removed to provide 2 as an off-white solid (1 .80 g, >99%)

¹ H NMR (400 MHz, CDCI ₃ ) 6 8.14 (s, 3H), 3.64 (s, 3H), 3.00 (t, J = 7.7 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.86 - 1.72 (m, 2H), 1.69 - 1.55 (m, 2H), 1.51 - 1.29 (m, 2H).

¹³ C NMR (101 MHz, CDCI ₃ ) 6 174.11, 51.71, 39.88, 33.75, 27.24, 26.03, 24.30.

HRMS: m/z calculated for C7H15NO2Na [M+Na]+: 168.1000, found: 168.1004. b) Conversion into N-(£-A!lenamidocaproyloxy) Methyl Ester

To a 25 mL Schlenk flask containing 3-butynoic acid (0.252 g, 3.0 mmol, 1.0 eq.) and Mukaiyama reagent (1.15 g, 4.5 mmol, 1.5 eq.) under argon atmosphere, anhydrous dichloromethane (8 mL) was added. The resulting mixture was stirred at room temperature for 1 h. After which, a solution of triethylamine (0.61 mL, 6.0 mmol, 2.0 eq.) and compound 2 in anhydrous dichloromethane (4 mL) was added dropwise to the Schlenk flask. The reaction mixture was left to stir at room temperature for another 30 min. Upon reaction completion, dichloromethane was removed via rotary evaporation. Ethyl acetate was added to the resulting residue and the mixture was transferred to a separatory funnel. The organic layer was washed twice with water and later dried over anhydrous sodium sulfate. The solvent was then evaporated in vacuo and the residue was purified via flash column chromatography on silica gel (40% ethyl acetate in hexane) to provide a white solid (a mixture of isomers 3a and 3b, 0.456 g, 72%). Note: homopropargylic isomer 3b can be converted to 3a using a base.

Allenic isomer (3a)

¹ H NMR (400 MHz, Acetone-cfe) 5 7.07 (s, 1 H), 5.63 (t, J = 6.7 Hz, 1 H), 5.17 (d, J = 6.7 Hz, 2H), 3.60 (s, 3H), 3.28 - 3.14 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.65 - 1.55 (m, 2H), 1.55 - 1.45 (m, 2H), 1.42 - 1.24 (m, 2H).

¹³ C NMR (101 MHz, Acetone-cfe) 6 213.22, 174.15, 164.43, 91.19, 79.76, 51.58, 39.95, 34.35, 30.23, 27.17, 25.44.

HRMS: m/z calculated for CnHi ₈ NO ₃ [M+H] ⁺ : 212.1287, found: 212.1283

Homopropargylic isomer (3b):

¹ H NMR (400 MHz, Acetone-cfe) 6 7.22 (s, 1H), 3.60 (s, 3H), 3.20 (td, J = 7.1 , 5.8 Hz, 2H), 3. 12 (d, J = 2.8 Hz, 2H), 2.65 (t, J = 2.8 Hz, 1 H), 2.29 (t, J = 7.4 Hz, 2H), 1 .67 - 1 .45 (m, 4H), 1.40 - 1.26 (m, 2H).

¹³ C NMR (101 MHz, Acetone-cfe) 6 174.07, 166.55, 78.89, 73.62, 51.50, 39.96, 34.24, 29.95, 27.49, 26.99, 25.31.

HRMS: m/z calculated for CUHI ₈ NO ₃ [M+H] ⁺ : 212.1287, found: 212.1289

To a solution of compound 3a (56.3 mg, 0.267 mmol, 1.0 eq.) dissolved in tert- butanol/deionized water (2:1 , 1.0 mL), lithium hydroxide monohydrate (18.4 mg, 0.320 mmol, 1.2 eq.) was added at 0°C. The resulting mixture was left to stir at 0°C for 2 h before it was warmed to room temperature and stirred overnight. Once the reaction is complete, the reaction mixture was acidified using 1N HCI (aq) and transferred to a separatory funnel. The aqueous layer was extracted with ethyl acetate (x3). The combined organic extract was washed with water (x1) and saturated brine (x1). After the organic layer was dried over magnesium sulfate, the solvent was evaporated to provide a white solid (32.1 mg, 61%). ¹ H NMR (400 MHz, Acetone-c/ _s ) 6 7.16 (s, 1H), 5.67 (t, J = 6.7 Hz, 1 H), 5.19 (d, J = 6.7 Hz, 2H), 3.31 - 3.15 (m, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.70 - 1.44 (m, 4H), 1.44 - 1.32 (m, 2H).

¹³ C NMR (101 MHz, Acetone-cfe) 6 213.14, 174.63, 164.51 , 91.03, 79.71 , 39.91 , 34.11 , 30.12, 27.11 , 25.32.

HRMS: m/z calculated for CI ₀ HI ₆ NO ₃ [M+H] ⁺ : 198.1130, found: 198.1132.

To a 10 mL Schlenk flask, compound 4 (0.0993 g, 0.503 mmol, 1.0 eq.) was added and dissolved in anhydrous dimethylformamide (2 mL) under argon atmosphere. The solution was cooled to 0°C in an ice-bath. Sym-collidine (0.25 mL, 1.86 mmol, 3.7 eq.) was added, followed by pentafluorophenyl trifluoroacetate (0.30 mL, 1.76 mmol, 3.5 eq.) dropwise. The reaction mixture was then warmed to room temperature and stirred for 4 h. Once the reaction is complete, the reaction was quenched with deionized water slowly before it was transferred to a separatory funnel. The aqueous layer was extracted with ethyl acetate (x3). The organic layer was washed with 0.5N HCI (aq), water and brine and dried over anhydrous sodium sulfate. The organic layer was then concentrated, and the remaining residue was purified by flash column chromatography (26% ethyl acetate in hexane) to give a white solid (0.135 g, 74%). EACF was obtained with an overall yield of 33% and was characterised by ¹ H and ¹⁹ F

NMR spectroscopy.

¹ H NMR (400 MHz, CD ₃ CN) 6 6.46 (s, 1 H), 5.59 (t, J = 6.7 Hz, 1 H), 5.19 (d, J = 6.7 Hz, 2H),

3.19 (td, J = 6.7 Hz, 2H), 2.71 (t, J = 7.4 Hz, 2H), 1.85 - 1.62 (m, 2H), 1.58 - 1.46 (m, 2H), 1.46 - 1.35 (m, 2H).

¹³ C NMR (101 MHz, CD ₃ CN) 5 213.09, 170.70, 164.91 , 90.98, 80.18, 39.77, 33.64, 29.78, 26.66, 25.04.

¹⁹ F NMR (376 MHz, CD ₃ CN) <5 -154.97 (d, J = 16.5 Hz), -160.62 (t, J = 20.9 Hz), -164.74 (dd, J = 21.1, 16.4 Hz). HRMS: m/z calculated for C16H15NO3F5 [M+H] ⁺ : 364.0972, found: 364.0973.

Example 2. Synthesis and Characterisation of Pentafluorophenyl (1r,4r)-4-(Buta-2,3- dienamidomethyl)cyclohexane-1 -carboxylate (FACC)

FACC was prepared via the reaction scheme shown in FIG. 3. Further details of the synthesis of FACC are provided below.

Anhydrous methanol (12 mL) was added to tranexamic acid (1.57 g, 10 mmol, 1.0 eq.) in a Schlenk flask and the mixture was cooled to 0°C in an ice bath. Thionyl chloride (1.1 mL, 15 mmol, 1 .5 eq.) was then added dropwise to the mixture. Once the addition was complete, the reaction mixture was allowed to stir for 4 h at room temperature. Upon reaction completion, the volatiles were removed via rotary evaporator to give an off-white solid as the product (2.07 g, > 99%).

¹ H NMR (400 MHz, DMSO-cfe) 6 7.81 (br s, 1H), 3.58 (s, 3H), 2.63 (d, J = 6.9 Hz, 2H), 2.25 (tt, J = 12.1, 3.6 Hz ,1 H), 1.78-1.94 (ddd, J = 48.5, 14.1, 3.5 Hz, 4H), 1.57-1.42 (m, 1 H), 1.29 (qd, J = 13.1 , 3.4 Hz, 2H), 0.97 (qd, J = 13.1 , 3.5 Hz, 2H).

¹³ C NMR (101 MHz, DMSO-cfe) 6 175.29, 51.34, 44.11, 41.91, 34.84, 28.67, 27.84.

HRMS: m/z calculated for C ₉ Hi ₈ NO2 [M+H] ⁺ : 172.1338, found: 172.1338.

To a 25 ml_ Schlenk flask, 3-butynoic acid (0.0764 g, 0.909 mmol, 1.0 eq.) and Mukaiyama reagent (0.348 g, 1.36 mmol, 1.5 eq.) were added. Anhydrous dichloromethane (3 mL) was added, and the resulting mixture was stirred at room temperature for 1 h under nitrogen atmosphere. Subsequently, a solution of compound 7 and triethylamine (0.38 mL, 0.273 mmol, 3.0 eq.) in anhydrous dichloromethane (3 mL) was added to the reaction mixture dropwise. The resulting mixture was stirred for another 30 min at room temperature. Once the reaction is complete, dichloromethane was removed via rotary evaporation and ethyl acetate was added to the remaining residue. The mixture was transferred to a separatory funnel and the organic layer was washed with water (x2) and dried over anhydrous sodium sulfate. After which, the solvent was removed in vacuo and the crude product was purified using flash column chromatography (40% ethyl acetate in hexane) to give a white solid (a mixture of allenic and homopropargylic isomers, 0.108 g, 51%). Note: homopropargylic isomer 9 can be converted to 8 using a base.

Allenic isomer (8):

¹ H NMR (400 MHz, DMSO-cfe) 6 7.88 (t, J = 6.0 Hz, 1 H), 5.74 (t, J = 6.6 Hz, 1 H), 5.27 (d, J = 6.6 Hz, 2H), 3.57 (s, 3H), 2.94 (t, J = 6.4 Hz, 2H), 2.23 (tt, J = 12.0, 3.5 Hz, 1 H), 1 .89 (dd, J = 13.5, 3.4 Hz, 2H), 1.71 (dd, J = 13.4, 3.5 Hz, 2H), 1.43 - 1.20 (m, 3H), 0.91 (qd, J = 13.1 , 3.5 Hz, 2H).

¹³ C NMR (101 MHz, DMSO-cfe) 6 212.50, 175.52, 163.52, 89.98, 79.64, 51.31 , 44.83, 42.33, 36.93, 29.31 , 28.21.

HRMS: m/z calculated for C13H20NO3 [M+H] ⁺ : 238.1443, found: 238.1445.

Homopropargylic isomer (9):

¹ H NMR (400 MHz, DMSO-cfe) 6 7.91 (t, J= 5.7 Hz, 1 H), 3.57 (s, 3H), 3.08 (d, J = 2.8 Hz, 2H), 2.97 (t, J = 2.8 Hz, 1 H), 2.90 (t, J = 8.0 Hz, 2H), 2.22 (tt, J = 12.3, 3.8 Hz, 1 H), 1.89 (dd, J = 13.8, 3.3 Hz, 2H), 1.71 (dd, J = 13.5, 3.3 Hz, 2H), 1.41 - 1.19 (m, 3H), 0.91 (qd, J = 13.1 , 3.6 Hz, 2H).

¹³ C NMR (100 MHz, DMSO-cfe) 6 175.47, 166.04, 78.94, 73.45, 51.29, 44.82, 42.30, 36.80, 29.21 , 28.18, 26.40.

Compound 8 (58.1 mg, 0.245 mmol, 1.0 eq.) was dissolved in fert-butanol/deionised water (2:1 , 1 mL) and the reaction mixture was cooled in an ice bath to 0°C. Lithium hydroxide monohydrate (12.3 mg, 0.294 mmol, 1 .2 eq.) was added to the reaction flask and the reaction mixture was left to stir at 0°C for 2 h. After which, the reaction mixture was warmed to room temperature and stirred overnight. Once the reaction was complete, the reaction mixture was acidified with 1N HCI (aq) and transferred to a separatory funnel. The aqueous layer was extracted with ethyl acetate (x3). The combined organic extracts were washed with water (x1) and brine (x1) before it was dried over anhydrous magnesium sulfate. The solvent was then removed on the rotary evaporator to provide a white solid as the product (0.0530 g, 97%).

¹ H NMR (400 MHz, DMSO-cfe) 6 7.87 (t, J = 6.1 Hz, 1 H), 5.74 (t, J = 6.6 Hz, 1 H), 5.27 (d, J = 6.5 Hz, 2H), 2.94 (t, J = 6.4 Hz, 2H), 2.10 (tt, J = 12.8, 4.2 Hz, 1H), 1.88 (dd, J = 13.3, 2.7 Hz, 2H), 1.71 (dd, J = 13.2, 2.7 Hz, 2H), 1.29 - 1.20 (m, 3H), 0.98 - 0.82 (m, 2H).

¹³ C NMR (101 MHz, DMSO-cfe) 6 212.44, 176.74, 163.42, 89.95, 79.58, 44.85, 42.52, 37.00, 29.43, 28.27.

HRMS: m/z calculated for CI ₃ HI ₈ NO ₃ [M+H] ⁺ : 224.1287, found: 224.1287. d) Reacting (1r,4r)-4-(Buta-2,3-dienamidomethyl)cyclohexane-1 -carboxylate acid with pentafluorophenyl trifluoroacetate

Compound 10 (0.155g, 0.692 mmol, 1.0 eq.) was dissolved in anhydrous dimethylformamide (4 mL) in a 10 ml_ Schlenk flask under argon atmosphere and cooled in an ice-bath. Sym- collidine (0.46 mL, 3.46 mmol, 5.0 eq.) was added to the solution, followed by pentafluorophenyl trifluoroacetate (0.60 mL, 3.46 mmol, 5.0 eq.) dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 6 h. Upon reaction completion, the reaction was quenched with water slowly and the aqueous mixture was transferred to a separator funnel. The aqueous mixture layer was extracted with ethyl acetate (x3) and the combined organic extract was washed with 0.5N HCI (aq), water and brine. The organic layer was dried over anhydrous sodium sulfate and later concentrated. The crude product was purified via flash column chromatography (29% ethyl acetate in hexane) to give a white solid (0.115 g, 43%). FACC was obtained with an overall yield of 21% and was characterised by ¹ H and ¹⁹ F NMR.

¹ H NMR (400 MHz, CD ₂ CI ₂ ) 6 5.88 (s, 1 H), 5.59 (t, J = 6.7 Hz, 1 H), 5.23 (d, J = 6.7 Hz, 2H), 3.16 (t, J = 6.5 Hz, 2H), 2.64 (tt, J = 12.2, 3.6 Hz, 1H), 2.19 (dd, J = 13.8, 3.7 Hz, 2H), 1.89 (dd, J = 13.7, 3.5 Hz, 2H), 1.64 - 1.46 (m, 3H), 1.07 (qd, J = 13.1 , 3.5 Hz, 2H).

¹³ C NMR (101 MHz, CD ₂ CI ₂ ) 6 212.21 , 172.24, 164.74, 91.26, 80.66, 45.85, 43.28, 37.99, 30.06, 28.95.

¹⁹ F NMR (377 MHz, CD ₂ CI ₂ ) 6 -153.50 - -154.21 (m), -159.43 (t, J = 21.5 Hz), -163.36 - - 163.59 (m).

HRMS: m/z calculated for Ci8Hi ₇ NO ₃ F5 [M+H] ⁺ : 390.1129, found: 390.1130.

Example 3. Synthesis and characterisation of FACC-DM1 adduct

FACC-DM1 is a linker-drug compound (LDC) that is able to generate an allenamide analogue of FDA-approved ADC, Kadcyla™ (Ado-trastuzumab emtansine, T-DM1). (FIG. 4)

Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate developed by Roche and it received an FDA approval in 2013 to treat late-stage breast cancer and HER2-positive early breast cancer. It combines the antitumor properties of trastuzumab, which is the humanized anti-human epidermal growth factor receptor 2 (HER2) antibody, and DM1 , the maytansinoid which acts as a potent microtubule-disrupting agent. These two components are joined by a heterobifunctional linker SMCC.

The maleimide thioether bond breaks via retro-Michael process, leading to the deconjugation of DM1 from trastuzumab. A meta-analysis study has shown that adverse events were mainly attributed to the prematurely release of DM1 in blood plasma (Shen, K. et al., Sei. Rep., 2016, 6, I). The thiol-allenamide adduct disclosed herein is robust and stable, as such the analogue created with the FACC linker is expected to have a superior therapeutic and safety profile as compared to the FDA-approved Kadcyla™.

Coupling of FACC with DM1:

To a 5 mL Schlenk flask containing DM1 (36.9 mg, 0.050 mmol, 1.0 eq.) and FACC (21.0 mg, 0.055 mmol, 1.1 eq.), anhydrous dichloromethane (0.5 mL) was added under argon atmosphere. Once the solids were dissolved, triethylamine (3.5 pL, 0.025 mmol, 0.5 eq.) was added and the resulting mixture was stirred at room temperature overnight. The reaction mixture was then concentrated under reduced pressure. The crude product was purified using preparative thin layer chromatography (CH2Cl2/EtOAc/MeOH: 33:66:1) to provide a white solid (36.1 mg, 64%).

FACC-DM1 adduct was obtained with an overall yield of 13% and was characterised by ¹ H and ¹⁹ F NMR spectroscopy (FIG. 5-7). NMR results were obtained by following the protocol disclosed in Example 1. The FACC-DM1 adduct was also characterized by HRMS.

¹ H NMR (400 MHz, CDCh) 6 6.83 (s, 1 H), 6.74 - 6.55 (m, 2H), 6.43 (dd, J = 15.4, 11.2 Hz, 1H), 6.27 (d, J = 7.1 Hz, 1 H), 6.03 (d, J = 8.4 Hz, 1 H), 5.63 (dd, J = 15.3, 9.0 Hz, 1 H), 5.34 (dd, J = 15.3, 8.4 Hz, 1 H), 5.16 (s, 1 H), 4.93 - 4.70 (m, 2H), 4.37 - 4.21 (m, 1 H), 3.98 (s, 3H), 3.66 (d, J = 12.8 Hz, 1 H), 3.49 (d, J = 9.0 Hz, 1H), 3.35 (d, J = 1.4 Hz, 3H), 3.21 - 2.92 (m, 11 H), 2.88 (s, 1 H), 2.84 (s, 2H), 2.79 - 2.67 (m, 1 H), 2.67 - 2.51 (m, 3H), 2.26 - 2.07 (m, 4H), 1.96 - 1.77 (m, 3H), 1.69 - 1.40 (m, 8H), 1.40 - 1.14 (m, 8H), 1.14 - 0.91 (m, 2H), 0.80 (s, 3H).

¹⁹ F NMR (377 MHz, CDCh) 6 -152.95 - -153.44 (m), -157.96 - -158.36 (m), -162.20 - -162.58 (m).

HRMS calculated for C ₅₃ H64N ₄ OI ₃ CIFsSNa [M+Na] ⁺ m/z 1149.3697, found 1149.3700.

Example 4: A comparison of the reactivity and chemoselectivity of FACC and SMCC

The reactivity and chemoselectivity of FACC and SMCC for amines and thiols were compared as shown in FIG. 8. Benzylamine was used as a model amine-containing compound, and glutathione was used as a model thiol-containing compound. The reactions were monitored by analytical HPLC.

The reactions of FACC and SMCC with benzylamine were found to be completed within a very short time (about 1 to 3 min). The reaction of FACC with glutathione was much slower than the reaction of SMCC with glutathione (90 min vs 5 min reaction completion). However, the reaction of FACC was found to be highly selective for glutathione in the presence of excess amine and pentafluorophenol (see FIG. 9). The maleimide end of SMCC was found to react quickly with glutathione, but a significant amount of it also reacted with benzylamine (see FIG. 10, in particular peak E).

Example 5: A comparison of the reactivity and chemoselectivity of EACF and EMCS (N- E-malemidocaproyl-oxysuccinimide ester)

The reactivity and chemoselectivity of EACF and EMCS for amines and thiols were compared as shown in FIG. 11. Benzylamine was used as a model amine-containing compound, and glutathione was used as a model thiol-containing compound. The reactions were monitored by analytical HPLC.

The reactions of EACF and EMCS with benzylamine were found to be completed within a very short time (about 1 to 3 min). The reaction of EACF with glutathione was much slower than the reaction of EMCS with glutathione (90 min vs 5 min reaction completion). However, the reaction of EACF was found to be highly selective for glutathione in the presence of excess amine and pentafluorophenol (see FIG. 12). The maleimide end of EMCS was found to react quickly with glutathione, but a significant amount of it also reacted with benzylamine (see FIG. 13, in particular peak J).