Heterobifunctional linkers and bioconjugated molecules

Abstract

The present invention provides compounds according to formula (I): wherein A and Za are as defined in the specification. Use of compounds of formula (I) as heterobifunctional linkers is found. The present invention also provides compounds of formula (III) as defined in the description, which find their use as pharmaceuticals and / or diagnostic agents. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to compounds of formula (I), and their use as heterobifunctional linkers. The present invention also provides compounds of formula (III) obtainable from compounds of formula (I). Compounds of formula (III) are found to be useful as pharmaceuticals and / or diagnostic agents. Background Art

[0002] The listing or discussion of a previously published document in this specification should not necessarily be taken as an admission that the document is part of the state of the art or is common general knowledge.

[0003] The strategy of combining synthetic cargo with drug delivery systems or biomacromolecules is widely used in fields including but not limited to immunochemistry, drug development, polymer science and live cell imaging. The development of drug delivery systems such as monoclonal antibodies, peptides or liposomes enables clinical transformation of drug forms that cannot be administered alone (such as cytotoxins and small interfering ribonucleic acids (siRNA)). Cytotoxic drugs and toxins are attached to monoclonal antibodies that locate tumors, which is an immunotherapy method that has attracted much attention for many years. In addition to therapeutic agents, it is also crucial to produce stable and active enzyme-antigen conjugates in immunoassays.

[0004] In many fields such as biology or chemistry, it is increasingly necessary to obtain detailed information about living organisms, such as living cells or bacteria. However, this is a challenging task due to the enormous complexity of biological systems. Many efforts have been made to study the dynamics and functions of a wide variety of biomolecules in living systems, which are mainly based on spectroscopic measurements, which require the labeling of biomolecules.

[0005] Therefore, many reagents for preparing these conjugates have been reported. Such chemical cross-linking agents are designed to have specific reactivity with the functional groups contained in each reactant. Both homobifunctional reagents and heterobifunctional reagents are available, and the latter are more practical. Since heterobifunctional reagents have two selective reactive groups, coupling is allowed in a step-by-step manner, so better control of conjugation chemistry can be achieved. For example, part A can be activated, purified and characterized before combining with part B.

[0006] Common 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 enables chemical modification in aqueous media even at lower concentrations. Among the functional groups suitable for thiol conjugation, maleimide derivatives are the most commonly used. Although maleimide provides fast reaction kinetics and has been widely used, it still has some disadvantages.

[0007] First, the maleimide-thiol reaction generates two diastereomers, rather than a single product. The diastereomers produced complicate the spectrum of their chromatograms (Smyth, DGet al., Biochem. J., 1964, 91, 589), and are prone to isomerization with each other (Kuninori, T. & Nishiyama, J. Agric. Biol. Chem., 1985, 49, 2453). Secondly, it is reported that the sulfosuccinimide adducts produced are prone to deconjugation by retro-Michael reaction (Alley, SC et al., Bioconjug. Chem., 2008, 19, 759), or ring opening by hydrolysis (Lewis, MR & Shively, JE, Bioconjug. Chem., 1998, 9, 72). These shortcomings are particularly problematic in antibody-drug conjugate (ADC) applications. The reverse Michael reaction can lead to premature release of the drug in plasma. The regenerated maleimide linker-payload can then react rapidly with other thiol groups in the blood, such as human serum albumin (HSA) or glutathione (Shen, BQ et al., Nat. Biotechnol., 2012, 30, 184). This in vitro study was further supported by characterization studies conducted by Pfizer: trastuzumab-maleimide-drugs were incubated with human plasma for 144h, and almost 100% of drug loss occurred (Wei, C. et al., Anal. Chem., 2016, 88, 4979). This deconjugation and exchange reaction leads to off-target toxicity, reduced therapeutic efficacy, and less than the required amount of cytotoxins delivered to tumor cells.

[0008] Therefore, there remains a need for improved and / or alternative heterobifunctional linkers suitable for preparing biomacromolecules that exhibit beneficial properties. Summary of the invention

[0009] Aspects and embodiments of the invention are described in the following numbered clauses.

[0010] 1. A compound according to formula (I):

[0011]

[0012] in,

[0013] A chooses free-C 1-6 Alkylene-, -R a -(C 3-12 Carbon ring)-R a -and-R a -(5-10 membered heterocyclic ring)-R a - The groups formed;

[0014] The alkylene group is a straight chain alkylene group, and optionally one or more carbon atoms are replaced by a group independently selected from the group consisting of O, NH and S;

[0015] Wherein, the alkylene, carbocyclic ring and heterocyclic ring are optionally selected from OH, NH 2 , C 1-4 substituted with one or more groups of alkyl and halogen; and

[0016] Each R a independently a bond or -C 1-4 Alkylene-;

[0017] Z a is allenyl or according to formula (II)

[0018]

[0019] D a selected from the group consisting of a carrier entity, a biologically active entity, and a diagnostic entity;

[0020] or a pharmaceutically acceptable salt or solvate thereof.

[0021] 2. The compound according to clause 1, wherein A is -C 1-6 Alkylene- or -R a -(C 5-6 Carbon ring)-R a -, where each R a independently a bond or -C 1-4 Alkylene-.

[0022] 3. The compound according to clause 1 or clause 2, wherein A is -C 5 Alkylene- or -CH 2 -(C6 Carbon ring)-.

[0023] 4. The compound according to any one of clauses 1 to 3, wherein the carbocyclic ring is a non-aromatic carbocyclic ring, such as a cyclohexane group.

[0024] 5. The compound according to clause 1, wherein the compound is according to formula (Ia):

[0025]

[0026] 6. The compound according to clause 1, wherein the compound is according to formula (Ib):

[0027]

[0028] 7. A compound according to any one of clauses 1 to 6, wherein Z a It is an allenyl group.

[0029] 8. A compound according to any one of clauses 1 to 6, wherein Z a According to formula (II).

[0030] 9. The compound according to clause 8, wherein D a It is a biologically active entity.

[0031] 10. The compound according to clause 9, wherein D a It is maytansine, auristatin (eg monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin or a cytotoxic derivative thereof.

[0032] 11. A compound according to formula (III):

[0033]

[0034] in,

[0035] A is -C 1-6 Alkylene-, wherein the alkylene is a straight chain alkylene, and optionally one or more carbon atoms are replaced by a group independently selected from the group consisting of O, NH and S; and / or optionally selected from OH, NH 2 , C 1-4 Alkyl and halogen substitutions;

[0036] D a and D b One of the two is a small molecule drug and the other is an antibody or antibody fragment;

[0037] or a pharmaceutically acceptable salt or solvate thereof.

[0038] 12. The compound according to clause 11, wherein D a is a small molecule drug, and D b is an antibody or an antibody fragment.

[0039] 13. The compound according to clause 11 or 12, wherein D a It is maytansine, auristatin (eg monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin or a cytotoxic derivative thereof.

[0040] 14. A compound according to any one of clauses 11 to 13, wherein D b Selected from the group consisting of trastuzumab, humanized IgG1 anti-BCMA antibody and fragments thereof.

[0041] 15. A compound according to any one of clauses 11-14, wherein D a is a maytansine derivative, and D b It's trastuzumab.

[0042] 16. A compound according to any one of clauses 11 to 15 for use in therapy.

[0043] 17. A compound according to any one of clauses 13-15 for use in the treatment of cancer.

[0044] 18. Use of a compound according to any one of clauses 11 to 15 for the preparation of a medicament.

[0045] 19. Use of a compound according to any one of clauses 13 to 15 for the preparation of a medicament for the treatment of cancer.

[0046] 20. A method of treatment comprising the step of administering to a subject in need thereof a compound according to any one of clauses 11-15.

[0047] 21. The method according to clause 20, wherein the subject has cancer.

[0048] 22. The compound for use according to clause 17, the use according to clause 19 or the method according to clause 21, wherein the cancer is breast cancer or multiple myeloma.

[0049] 23. Use of a compound according to any one of clauses 1 to 10 in the preparation of an antibody-drug conjugate compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1An overview of the current invention is depicted.

[0051] Figure 2 Shown is the synthetic route to N-(ε-allylaminohexanoyloxy)pentafluorophenyl ester (EACF).

[0052] Figure 3 The synthetic route of (1r,4r)-4-(buta-2,3-dienamide-methyl)cyclohexane-1-carboxylic acid pentafluorophenyl ester (FACC) is shown.

[0053] Figure 4 The FDA-approved antibody-drug conjugate Kadcyla is shown TM Comparison with its allenamide analogs.

[0054] Figure 5 The FACC-DM1 adduct 1 H NMR spectrum of DM1 1 Comparison of H NMR spectra.

[0055] Figure 6 The FACC-DM1 adduct 1 H NMR spectrum (magnified sp 2 area).

[0056] Figure 7 The FACC-DM1 adduct 19 F NMR spectroscopy and FACC 19 Comparison of F NMR spectra.

[0057] Figure 8 Reaction schemes for comparing the reactivity and chemoselectivity of FACC and SMCC towards amines and thiols are shown.

[0058] Fig. 9 The monitoring of FACC with benzylamine (BnNH 2 ) and glutathione.

[0059] Fig.10 The results show that the SMCC and benzylamine (BnNH 2 ) and glutathione.

[0060] Fig.11 Reaction schemes for comparing the reactivity and chemoselectivity of EACF and EMCS towards amines and thiols are shown.

[0061] Fig.12 The results show that the EACF was monitored by benzylamine (BnNH 2 ) and glutathione.

[0062] Fig.13 shows the monitoring of EMCs with benzylamine (BnNH 2 ) and glutathione. DETAILED DESCRIPTION

[0063] The present invention provides compounds according to formula (I):

[0064]

[0065] or a pharmaceutically acceptable salt or solvate thereof.

[0066] In the compound of formula (I), A is selected from -C 1-6 Alkylene-, -R a -(C 3-12 Carbon ring)-R a -and-R a -(5-10 membered heterocyclic ring)-R a -consisting of a group; alkylene, carbocyclic or heterocyclic ring can be selected from OH, NH 2 , C 1-4 The alkylene group, carbocyclic ring or heterocyclic ring can be substituted by one or more groups selected from OH, NH 2 , C 1-4 The alkyl group is substituted with one or two groups of alkyl and halogen (eg, fluorine, chlorine, bromine or iodine).

[0067] In the compound of formula (I), when A is C 1-6 When A is C 1 Alkylene, straight chain C 2 Alkylene, straight chain C 3 Alkylene, straight chain C 4 Alkylene, straight chain C 5 Alkylene or straight chain C 6 In certain embodiments, A is a straight chain C 4 Alkylene or straight chain C 5 In certain exemplary embodiments, A is a linear C 5 Alkylene.

[0068] In the compound of formula (I), when A is C 1-6 When an alkylene group is present, one or more carbon atoms of the alkylene group may be replaced by a group independently selected from the group consisting of O, NH, and S. For example, one or two carbon atoms of the alkylene group may be replaced by a group independently selected from the group consisting of O, NH, and S. For example, A may be -C 5 Alkylene-.

[0069] In the compound of formula (I), when A is -R a -(C 3-12 Carbon ring)-R a -, the carbocyclic ring can be a non-aromatic carbocyclic ring. For example, it can be a cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane or cyclooctane group. In certain exemplary embodiments, the carbocyclic ring is a cyclohexane group.

[0070] In the compound of formula (I), when A is -R a -(5-10 membered heterocyclic ring)-R a -, the heterocycle can be an aromatic or non-aromatic heterocycle. For example, the heterocycle can be a 5-10 membered aromatic heterocycle or a 5-10 membered non-aromatic heterocycle containing one or more (e.g., one or two) heteroatoms independently selected from N, S, and O.

[0071] Each R a are independently selected from a bond or -C 1-4 In certain embodiments, R a One of them is a bond, and the other is a straight chain C 1-4 Alkylene-, for example, C 1 Alkylene or straight chain C 2 For example, when A is -R a -(C 3-12 Carbon ring)-R a -or-R a -(5-10 membered heterocyclic ring)-R a -, A can be -(C 3-12 Carbon ring)-CH 2 -、-CH 2 -(C 3-12 Carbocyclic)-、-(5-10 membered heterocyclic)-CH 2 -or-CH 2 -(5-10 membered heterocyclic)-. In certain exemplary embodiments, A may be -CH 2 -(C 6 Carbon ring)-.

[0072] In the compound of formula (I), Z a is an allenyl group or according to formula (II)

[0073]

[0074] in, represents the point of connection between the rest of the compound of formula (II) and formula (I).

[0075] In certain embodiments, the compound of formula (I) is according to formula (Ia):

[0076]

[0077] or a pharmaceutically acceptable salt or solvate thereof.

[0078] In certain other embodiments, the compound of formula (I) is according to formula (Ib):

[0079]

[0080] or a pharmaceutically acceptable salt or solvate thereof.

[0081] In certain embodiments, Z a In certain exemplary embodiments, the compound of formula (I) may be:

[0082]

[0083] N-(ε-Allenamidohexanoyloxy)pentafluorophenyl ester; or

[0084]

[0085] (1,4)-4-(Butane-2,3-dienamidomethyl)cyclohexane-1-carboxylic acid pentafluorophenyl ester.

[0086] For example, the compound of formula (I) may be:

[0087]

[0088] (1r,4r)-4-(Butane-2,3-dienamidomethyl)cyclohexane-1-carboxylic acid pentafluorophenyl ester.

[0089] The present inventors have found that, where Z a The compounds of formula (I) which are allenyl groups show excellent chemoselectivity for amine and thiol groups. The allenyl group has high reactivity and high selectivity for thiol groups, and the pentafluorophenoxy group has high reactivity for amine groups. a Compounds of formula (I) that are allenyl groups also exhibit good hydrolytic stability. In addition, conjugate molecules such as antibody-drug conjugates formed using such compounds exhibit good hydrolytic stability due to the stability of the bond linking the antibody and drug molecules together.

[0090] In certain embodiments, in the compounds of formula (I), Z a According to formula (II). In formula (II), D aIndependently selected from the group consisting of carrier entities, biologically active entities and diagnostic entities. The carrier entity, the biologically active entity and the diagnostic entity can each independently be a protein, a protein fragment, a peptide, a nanoparticle or a small molecule compound (e.g., a compound having an Mw of less than about 1500Da). Examples of carrier entities include, but are not limited to, antibodies, antibody fragments and albumin. Examples of biologically active entities include, but are not limited to, small molecule drugs, antibodies and antibody fragments (e.g., small molecule drugs, antibodies and antibody fragments that show biological activity such as cytotoxic activity). For the avoidance of doubt, an antibody or antibody fragment can serve as a carrier entity (e.g., it can transport other molecules such as small molecule drugs to the target antigen), or it can serve as a biologically active entity (i.e., it itself can directly cause a biological effect). In some cases, an antibody or antibody fragment can serve as a carrier entity and a biologically active entity at the same time. Examples of diagnostic entities include, but are not limited to, radioactive agents, enzymes, fluorescent compounds and electron transfer agents. In certain embodiments, D a It is a biologically active entity.

[0091] In certain embodiments, D a is an antibody, e.g., a monoclonal antibody. In certain embodiments, D a It is an antibody that can bind to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). a It can be an antibody that binds to human epidermal growth factor receptor-2 (HER2) or B cell maturation antigen (BCMA). a It can be a human, humanized or chimeric anti-HER2 antibody, or a human, humanized or chimeric anti-BCMA antibody. In certain embodiments, D a It is trastuzumab or humanized IgG1 anti-BCMA antibody.

[0092] In certain other embodiments, D a is a small molecule drug, such as a cytotoxic small molecule drug. In certain embodiments, D a It may be maytansine, auristatin (e.g. monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin or a cytotoxic derivative thereof. Suitable derivatives of such small molecule drugs include those that have been modified to include a linker connecting the derivative to the sulfur atom of formula (II). For example, when D a When it is a maytansine derivative, D a According to formula (IV):

[0093]

[0094] in, = represents the connection point between formula (IV) and formula (II). Formula (IV) can be derived from mertansine (DM1). For example, when Z a According to formula II and D a When the compound of formula (IV) is prepared, the compound of formula (I) can be prepared by reacting maytansinoids with Z a The compound of formula (I) having an allenyl group at the position is obtained by reaction.

[0095] In certain preferred embodiments, D a It 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):

[0096]

[0097] The stability of the thiol-alleneamide bond makes Z a Compounds of formula (I) according to formula (II) are particularly useful as intermediates in the preparation of bioconjugate molecules such as antibody-drug conjugates.

[0098] As disclosed herein, 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 are uses of compounds of formula (I) in the preparation of bioconjugate molecules such as antibody-drug conjugates. The Examples section disclosed herein provides exemplary methods for preparing compounds of formula (I) and their use in the preparation of bioconjugate molecules.

[0099] The present invention also provides compounds according to formula (III):

[0100]

[0101] or a pharmaceutically acceptable salt or solvate thereof.

[0102] In the compound of formula (III), A is a straight chain -C 1-6 Alkylene-. One or more carbon atoms of the alkylene group may be replaced by a group independently selected from the group consisting of O, NH, and S. For example, one or two carbon atoms of the alkylene group may be replaced by a group independently selected from the group consisting of O, NH, and S. For example, A may be -C in which one carbon atom is replaced by one O, NH, or S. 5 Alkylene-.

[0103] 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 ais maytansine, 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 It can be a human, humanized or chimeric anti-HER2 antibody, or a human, humanized or chimeric anti-BCMA antibody. In certain embodiments, D b It is trastuzumab or humanized IgG1 anti-BCMA antibody.

[0104] In certain embodiments, D a is a maytansine derivative, and D b is an antibody or a fragment thereof. For example, the compound of formula (III) can be according to formula (IIIa):

[0105]

[0106] Herein, Ab represents an antibody or a fragment thereof.

[0107] The present inventors expect that the compound of formula (III) exhibits enhanced stability in vivo compared to the corresponding compound comprising an antibody and a small molecule drug compound linked by a thioether bond. The latter is known to undergo a reverse Michael process that can lead to deconjugation of the small molecule drug from the compound.

[0108] As used herein, the term "alkyl" refers to both straight and branched saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, pentyl and hexyl groups. In the unbranched alkyl group, methyl, ethyl, n-propyl, isopropyl, n-butyl groups are preferred. In the branched alkyl group, tert-butyl, isobutyl, 1-ethylpropyl and 1-ethylbutyl groups may be mentioned.

[0109] As used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are particularly preferred.

[0110] The term "carbocycle" as used herein includes saturated, partially unsaturated or unsaturated monocyclic, bicyclic, tricyclic, polycyclic, spirocyclic, bridged and fused ring systems having 3 to 12 carbon atoms, wherein any substituted ring atom may be substituted by a substituent. The term includes aromatic carbocycles (e.g., phenyl groups) and non-aromatic carbocycles (e.g., cyclohexane groups).

[0111] For the avoidance of doubt, the term "heterocycle" as used herein encompasses any aromatic or non-aromatic cyclic group containing one or more heteroatoms (i.e., N, O, or S). Unless otherwise indicated, the term "heterocycle" as used herein encompasses bicyclic heterocyclic groups such as spirocyclic heterocycles, fused heterocycles, and bridged heterocycles. Heterocyclic groups containing suitable nitrogen atoms include corresponding N-oxides. Examples of 5-10 membered heterocyclic groups include pyrrolidine, piperidine, tetrahydrofuran, tetrahydropyran, pyridine, pyrimidine, indoline, pyrrole, pyrazole, imidazole, furan, guanidine-containing heterocycles, and quinazoline.

[0112] As described above, the present invention also provides compounds of the present invention as solvates and / or salts. Preferred solvates are solvates formed by incorporating molecules of nontoxic pharmaceutically acceptable solvents (hereinafter referred to as solvating solvents) into the solid state structure (e.g., crystal structure) of the compounds of the present invention. Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethyl sulfoxide. Solvates can be prepared by recrystallizing the compounds of the present invention with a solvent mixture or solvent containing a solvating solvent. In any given case, whether a solvate has been formed can be determined by subjecting the 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.

[0113] The solvate may be a stoichiometric or non-stoichiometric solvate. A particularly preferred solvate is a hydrate, and examples of the hydrate include hemihydrate, monohydrate and dihydrate.

[0114] Pharmaceutically acceptable salts include acid addition salts and base addition salts. Such salts can be formed by conventional means, for example, by making the free acid or free base form of the compound of the present invention react with one or more equivalents of appropriate acid or base in a solvent or medium in which the salt is insoluble, followed by standard techniques (e.g., by freeze drying or filtration in a vacuum) to remove the solvent or the medium. Salts can also be prepared by exchanging the counter ion of the compound of the formula of the present invention in the form of a salt with another counter ion, for example, using a suitable ion exchange resin.

[0115] Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral and organic acids, and salts derived from metals such as sodium, magnesium or, preferably, potassium and calcium.

[0116] Examples of acid addition salts include acid addition salts formed with acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acids (e.g., benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid (e.g., D-gluconic acid), glucuronic acid (

[0013] Examples of the invention include, but are not limited to, D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, isethionic acid, lactic acid (e.g., (+)-L-lactic acid and (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-)-L-malic acid), malonic acid, (±)-DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g., (+)-L-tartaric acid), thiocyanic acid, undecylenic acid, and valeric acid.

[0117] Specific examples of salts are those derived from mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid; organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, arylsulfonic acids; and metals such as sodium, magnesium or preferably potassium and calcium.

[0118] For a more detailed discussion of solvates and methods for preparing and characterizing them, see Bryn et al., Solid-State Chemistry of Drugs, 2nd edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0119] Those skilled in the art of organic chemistry will recognize that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are called "solvates." For example, a complex with water is called a "hydrate."

[0120] The compounds of the present invention may contain one or more asymmetric carbon atoms and may therefore exhibit optical and / or diastereoisomerism. Diastereomers may be separated using conventional techniques, such as chromatography or fractional crystallization. Various stereoisomers may be separated by separating the racemic or other mixtures of compounds using conventional techniques such as fractional crystallization or high performance liquid chromatography (HPLC). Alternatively, the desired optical isomers may be made by: reaction of appropriate optically active starting materials under conditions that do not cause racemization or epimerization (i.e., the 'chiral pool' method); reaction of appropriate starting materials with 'chiral auxiliary agents', which may then be removed at an appropriate stage; derivatization (i.e., resolution, including dynamic resolution), such as with a homochiral acid, followed by separation of diastereomeric derivatives by conventional means such as chromatography; or reaction with appropriate chiral reagents or chiral catalysts under conditions known to the technician. All stereoisomers and mixtures thereof are included within the scope of the present invention.

[0121] The compound of formula (III) may be in the form of a pharmaceutical composition. The pharmaceutical composition of the present invention may comprise a compound of formula (III) and one or more pharmaceutically acceptable excipients.

[0122] The pharmaceutical composition can 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 cutaneous, buccal, sublingual, and intraocular) administration, although the most appropriate route may depend on the characteristics of the subject being treated, such as species, age, weight, sex, and medical condition.

[0123] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injections, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation 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 provided in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions, requiring only the addition of sterile liquid carriers, such as saline or water for injection, immediately before use. Instant injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type described herein. Exemplary compositions for parenteral administration include injectable solutions or suspensions, which may include, for example, suitable non-toxic parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersants or wetting agents and suspending agents, including synthetic monoglycerides or diglycerides, and fatty acids, including oleic acid or Cremaphor.

[0124] Compositions for nasal, aerosol or inhaled administration include saline solutions which may 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.

[0125] Compositions for rectal administration may be presented as a suppository with a carrier such as cocoa butter, synthetic glycerides or polyethylene glycols. Such carriers are generally solid at ordinary temperatures but liquefy and / or dissolve in the rectal cavity to release the drug.

[0126] Compositions for topical administration in the mouth, e.g., buccal or sublingual, include lozenges comprising the active ingredient in a flavored matrix such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a matrix such as gelatin and glycerin or sucrose and acacia. Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).

[0127] 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 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.

[0128] The compound of formula (III) may also be present as a pill, granule or paste. The pharmaceutical composition may optionally be present in a form that provides sustained or controlled release of the compound of formula (III) after administration to a subject. Various pharmaceutically acceptable carriers and their formulations are described in standard formulation papers, for example, Remington's Pharmaceutical Sciences by EW Martin. See also, Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988.

[0129] 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 involved.

[0130] The compounds of formula (III) are found to be useful in treatment. In certain embodiments, when D a has cytotoxic activity (e.g., it is maytansine, auristatin, or a cytotoxic derivative thereof) and D bWhen the compound of formula (III) is an antibody that binds to a tumor associated antigen (TAA) or a tumor specific antigen (TSA), the compound of formula (III) may find use in treating cancer.

[0131] For example, when the compound of formula (III) is according to formula (IIIa), wherein Ab is trastuzumab, the compound of formula (III) is found to be useful for treating HER2-positive breast cancer. Alternatively, for example, when the compound of formula (III) is according to formula (IIIa), wherein Ab is a humanized IgG1 anti-BCMA antibody, the compound of formula (III) is found to be useful for treating multiple myeloma.

[0132] The compound of formula (III) will be administered to a subject in need thereof as a pharmaceutical preparation admixed with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected according to the intended route of administration and standard pharmaceutical practice. Such a pharmaceutically acceptable carrier may be chemically inert to the active compound and may have no harmful side effects or toxicity under the conditions of use. Suitable pharmaceutical preparations may be found in, for example, Remington: The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenterally acceptable aqueous solutions may be used which are pyrogen-free and have the necessary pH, isotonicity and stability. Suitable solutions will be well known to the technician, and many methods are described in the literature. A brief review of drug delivery methods may also be found in, for example, Langer, Science (1990) 249, 1527.

[0133] Additionally, the preparation of suitable formulations may be routinely accomplished by a skilled artisan using conventional techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0134] The amount of the compound of formula (III) in any pharmaceutical preparation used according to the present invention will depend on various factors, such as the severity of the condition to be treated, the specific patient to be treated and the (one or more) compounds employed. In any case, the amount of the compound of formula (III) in the preparation can be routinely determined by the technician.

[0135] For example, a solid oral composition such as a tablet or capsule may contain 1 to 99% (w / w) of active ingredient; 0 to 99% (w / w) of diluent or filler; 0 to 20% (w / w) of disintegrant; 0 to 5% (w / w) of lubricant; 0 to 5% (w / w) of glidant; 0 to 50% (w / w) of granulating agent or binder; 0 to 5% (w / w) of antioxidant and 0 to 5% (w / w) of pigment. A controlled release tablet may additionally contain 0 to 90% (w / w) of a controlled release polymer.

[0136] Parenteral formulations (such as solutions or suspensions for injection or solutions for infusion) may contain 1 to 50% (w / w) of active ingredient; and 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 buffers, antioxidants, suspension stabilizers, tonicity regulators and preservatives.

[0137] Depending on the condition and patient to be treated and the route of administration, the compound of formula (III) can be administered to a patient in need thereof at different therapeutically effective doses.

[0138] However, in the context of the present invention, the dose administered to a mammal, particularly a human, should be sufficient to produce a therapeutic response in the mammal within a reasonable time frame. Those skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be affected, inter alia, by 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, weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0139] Administration can be continuous or intermittent (e.g., by bolus). Dosage can also be determined by the timing and frequency of administration. In the case of oral or parenteral administration, the dosage of the compound of formula (III) can vary between about 0.01 mg to about 1000 mg per day.

[0140] In any case, a practicing physician or other technician will be able to routinely determine the actual dosage most suitable for an individual patient. The above dosages are examples of average cases; of course, there are individual cases where a higher or lower dosage range is required, and this is also within the scope of the present invention.

[0141] The compounds of formula (III) disclosed herein can be used in medicine. Therefore, in another aspect of the present invention, there is provided the use of an antibody-drug conjugate as described herein or a pharmaceutically acceptable salt or solvate thereof in medicine.

[0142] The various aspects of the invention described herein (e.g., the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in treating the conditions described herein, they may be more convenient for the physician and / or the patient, more effective, less toxic, have better selectivity, have a broader spectrum of activity, be more potent, produce fewer side effects, or may have other useful pharmacological properties compared to similar compounds, combinations, methods (treatments) or uses known in the prior art for treating these conditions or otherwise.

[0143] In the embodiments herein, the word "comprising" may be interpreted as requiring the features to be mentioned, but not limiting the presence of other features. Alternatively, the word "comprising" may also refer to situations where only the listed components / features are intended to be present (e.g., the word "comprising" may be replaced by the phrase "consisting of" or "consisting essentially of". It is expressly contemplated that both the broader and narrower interpretations may be applicable to all aspects and embodiments of the present invention. In other words, the word "comprising" and its synonyms may be replaced by the phrase "consisting of" or the phrase "consisting essentially of" or their synonyms, and vice versa.

[0144] The phrase "consisting essentially of..." and its synonyms may be interpreted herein to mean that a small amount of impurities may be present in the material. 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.

[0145] 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 a mixture of two or more such compositions, and the like.

[0146] Further aspects and embodiments of the invention will now be discussed with reference to the following non-limiting examples.

[0147] Example

[0148] The examples disclosed herein describe the development and use of heterobifunctional linkers bearing an allenamide on one side and a pentafluorophenyl ester on the other side. Figure 1 The use of heterobifunctional linkers for the preparation of bioconjugate molecules is outlined.

[0149] Material

[0150] 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 noted. Silica gel 60 (40-63 μm) from Merck was used for flash chromatography in the elution system. Analytical thin layer chromatography plates were purchased from Merck (aluminum sheets or glass backing, silica gel 60F 254 ) and visualized using UV light and / or by immersion in potassium permanganate solution. Silica gel 60F from Merck was used. 254 Preparative thin layer chromatography was performed on glass backing plates (layer thickness: 1.0 mm).

[0151] 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 in deuterated solvents at ambient temperature and 25°C. Chemical shifts are reported in ppm (d) using residual solvent as an internal standard. Peak multiplicities given in Hz are indicated as follows: 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, quintet; h, septet; m, multiplet; br s, broad singlet.

[0152] High resolution mass spectrometry (HRMS) was performed using a Waters G2-XS QTof spectrometer with positive electrospray ionization.

[0153] Example 1. Synthesis and Characterization of N-(ε-Allenamide Hexanoyloxy) Pentafluorophenyl Ester (EACF)

[0154] pass Figure 2 The reaction scheme shown prepares EACF. Further details of the synthesis of EACF are provided below.

[0155] a) Synthesis of 6-aminocaproic acid methyl ester

[0156]

[0157] 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 was added thionyl chloride (1.1 mL, 15 mmol, 1.5 eq) dropwise at 0°C. The resulting solution was warmed to room temperature and stirred overnight. After the reaction was complete, the volatiles were removed to give 2 as an off-white solid (1.80 g, >99%)

[0158] 1 H NMR (400 MHz, CDCl 3 )δ8.14(s,3H),3.64(s,3H),3.00(t,J=7.7Hz,2H),2.31(t,J=7.3Hz,2H),1.86–1.72(m,2H),1.69–1.55(m,2H),1.51–1.29(m,2H).

[0159] 13 C NMR (101 MHz, CDCl 3 )δ174.11,51.71,39.88,33.75,27.24,26.03,24.30.

[0160] HRMS: m / z calculated for C7H15NO2Na[M+Na]+: 168.1000, found: 168.1004.

[0161] b) Conversion into N-(ε-allylaminohexanoyloxy)methyl ester

[0162]

[0163] Anhydrous dichloromethane (8mL) was added to a 25mL Schlenk flask containing 3-butynoic acid (0.252g, 3.0mmol, 1.0 equivalent) and Mukaiyama reagent (1.15g, 4.5mmol, 1.5 equivalent) under an argon atmosphere. The resulting mixture was stirred at room temperature for 1h. Thereafter, a solution of triethylamine (0.61mL, 6.0mmol, 2.0 equivalent) and compound 2 in anhydrous dichloromethane (4mL) was added dropwise to the Schlenk flask. The reaction mixture was stirred at room temperature for another 30min. After the reaction was complete, dichloromethane was removed by 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 then dried over anhydrous sodium sulfate. The solvent was then evaporated in vacuo and the residue was purified by flash column chromatography on silica gel (40% ethyl acetate in hexanes) to give a white solid (mixture of isomers 3a and 3b, 0.456 g, 72%). Note: The homopropargyl isomer 3b can be converted to 3a with base.

[0164] Allene isomer (3a)

[0165] 1 H NMR (400 MHz, acetone-d 6 )δ7.07(s,1H),5.63(t,J=6.7Hz,1H),5.17(d,J=6.7Hz,2H),3.60(s,3H),3.28–3.14( m,2H),2.29(t,J=7.4Hz,2H),1.65–1.55(m,2H),1.55–1.45(m,2H),1.42–1.24(m,2H).

[0166] 13 C NMR (101 MHz, acetone-d 6 )δ213.22,174.15,164.43,91.19,79.76,51.58,39.95,34.35,30.23,27.17,25.44.

[0167] HRMS: C 11 H 18 NO 3 [M+H] + m / z calculated: 212.1287, found: 212.1283 Homopropargyl isomer (3b):

[0168] 1 H NMR (400 MHz, acetone-d 6 )δ7.22(s,1H),3.60(s,3H),3.20(td,J=7.1,5.8Hz,2H),3.12(d,J=2.8Hz,2H),2 .65(t,J=2.8Hz,1H),2.29(t,J=7.4Hz,2H),1.67–1.45(m,4H),1.40–1.26(m,2H).

[0169] 13 C NMR (101 MHz, acetone-d 6 )δ174.07,166.55,78.89,73.62,51.50,39.96,34.24,29.95,27.49,26.99,25.31.

[0170] HRMS: C 11 H 18 NO 3 [M+H] + m / z calculated: 212.1287, found: 212.1289

[0171] c) Deprotection of N-(ε-allylaminohexanoyloxy)methyl ester

[0172]

[0173] To a solution of compound 3a (56.3 mg, 0.267 mmol, 1.0 eq.) dissolved in tert-butyl alcohol / deionized water (2: 1, 1.0 mL) was added lithium hydroxide monohydrate (18.4 mg, 0.320 mmol, 1.2 eq.) at 0 ° C. The resulting mixture was stirred at 0 ° C for 2 h, then warmed to room temperature and stirred overnight. Once the reaction was complete, the reaction mixture was acidified with 1N HCl (aqueous solution) 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 saturated brine (x1). After the organic layer was dried over magnesium sulfate, the solvent was evaporated to give a white solid (32.1 mg, 61%).

[0174] 1 H NMR (400 MHz, acetone-d 6 )δ7.16(s,1H),5.67(t,J=6.7Hz,1H),5.19(d,J=6.7Hz,2H),3.31–3.15(m,2H),2.30(t,J=7.4Hz,2H),1.70–1.44(m,4H),1.44–1.32(m,2H).

[0175] 13 C NMR (101 MHz, acetone-d 6 )δ213.14,174.63,164.51,91.03,79.71,39.91,34.11,30.12,27.11,25.32.

[0176] HRMS: C 10 H 16 NO 3 [M+H] + Calculated m / z: 198.1130, found: 198.1132.

[0177] d) reacting N-(ε-allylamino)-n-hexanoic acid with pentafluorophenyl trifluoroacetate

[0178]

[0179] Compound 4 (0.0993 g, 0.503 mmol, 1.0 equivalent) was added to a 10 mL Schlenk flask under an argon atmosphere and dissolved in anhydrous dimethylformamide (2 mL). The solution was cooled to 0 ° C in an ice bath. Symmetric-trimethylpyridine (Sym-collidine) (0.25 mL, 1.86 mmol, 3.7 equivalents) was added, followed by dropwise addition of pentafluorophenyl trifluoroacetate (0.30 mL, 1.76 mmol, 3.5 equivalents). The reaction mixture was then warmed to room temperature and stirred for 4 h. Once the reaction was complete, the reaction was slowly quenched with deionized water and then transferred to a separatory funnel. The aqueous layer was extracted with ethyl acetate (x3). The organic layer was washed with 0.5 N HCl (aqueous solution), 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%). The total yield of EACF was 33%, and the 1 H and 19 The samples were characterized by F NMR spectroscopy.

[0180] 1 H NMR (400MHz, CD 3 CN)δ6.46(s,1H),5.59(t,J=6.7Hz,1H),5.19(d,J=6.7Hz,2H),3.19(td,J=6.7Hz,2 H), 2.71 (t, J = 7.4Hz, 2H), 1.85–1.62 (m, 2H), 1.58–1.46 (m, 2H), 1.46–1.35 (m, 2H).

[0181] 13 C NMR (101 MHz, CD 3 CN)δ213.09,170.70,164.91,90.98,80.18,39.77,33.64,29.78,26.66,25.04.

[0182] 19 F NMR (376MHz, CD 3 CN) δ -154.97 (d, J = 16.5Hz), -160.62 (t, J = 20.9Hz), -164.74 (dd, J = 21.1, 16.4Hz).

[0183] HRMS: C 16 H 15 NO 3 F 5 [M+H] + m / z calculated: 364.0972, found: 364.0973.

[0184] Example 2. Synthesis and Characterization of Pentafluorophenyl (1r, 4r)-4-(Butane-2,3-dienamidomethyl)cyclohexane-1-carboxylate (FACC)

[0185] pass Figure 3 The reaction scheme shown prepares FACC. Further details of the synthesis of FACC are provided below.

[0186] a) Introducing a protecting group into the carboxyl portion of tranexamic acid

[0187]

[0188] 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. Then thionyl chloride (1.1 mL, 15 mmol, 1.5 eq) was added dropwise to the mixture. Once the addition was complete, the reaction mixture was stirred at room temperature for 4 h. After the reaction was complete, the volatiles were removed by rotary evaporator to obtain an off-white solid as the product (2.07 g, >99%).

[0189] 1 H NMR (400 MHz, DMSO-d 6 )δ7.81(br s,1H),3.58(s,3H),2.63(d,J=6.9Hz,2H),2.25(tt,J=12.1,3.6Hz,1H),1.78-1.94(ddd,J=48.5 ,14.1,3.5Hz,4H),1.57-1.42(m,1H),1.29(qd,J=13.1,3.4Hz,2H),0.97(qd,J=13.1,3.5Hz,2H).

[0190] 13 C NMR (101 MHz, DMSO-d 6 )δ175.29,51.34,44.11,41.91,34.84,28.67,27.84.

[0191] HRMS: C 9 H 18 NO 2 [M+H] + Calculated m / z: 172.1338, found: 172.1338.

[0192] b) Conversion into (1r,4r)-4-(buta-2,3-dienamidomethyl)cyclohexane-1-carboxylic acid methyl ester

[0193]

[0194] 3-butynoic acid (0.0764g, 0.909mmol, 1.0 equivalent) and Mukaiyama reagent (0.348g, 1.36mmol, 1.5 equivalent) were added to a 25mL Schlenk flask. Anhydrous dichloromethane (3mL) was added, and the mixture was stirred at room temperature for 1h under a nitrogen atmosphere. Subsequently, a solution of compound 7 and triethylamine (0.38mL, 0.273mmol, 3.0 equivalent) in anhydrous dichloromethane (3mL) was added dropwise to the reaction mixture. The mixture was stirred for another 30min at room temperature. Once the reaction was complete, dichloromethane was removed by rotary evaporation, and ethyl acetate was added to the remaining residue. The mixture was transferred to a separating funnel, and the organic layer was washed with water (x2) and dried over anhydrous sodium sulfate. After this time, the solvent was removed in vacuo and the crude product was purified by flash column chromatography (40% ethyl acetate in hexanes) to give a white solid (mixture of allene and homopropargyl isomers, 0.108 g, 51%). Note: Homopropargyl isomer 9 can be converted to 8 with base.

[0195] Allene isomers (8):

[0196] 1 H NMR (400 MHz, DMSO-d 6 )δ7.88(t,J=6.0Hz,1H),5.74(t,J=6.6Hz,1H),5.27(d,J=6.6Hz,2H),3.57(s,3H),2.94(t,J=6.4Hz,2H),2.23(tt,J=12. 0,3.5Hz,1H),1.89(dd,J=13.5,3.4Hz,2H),1.71(dd,J=13.4,3.5Hz,2H),1.43–1.20(m,3H),0.91(qd,J=13.1,3.5Hz,2H).

[0197] 13 C NMR (101 MHz, DMSO-d 6 )δ212.50,175.52,163.52,89.98,79.64,51.31,44.83,42.33,36.93,29.31,28.21.

[0198] HRMS: C 13 H 20 NO 3 [M+H] + Calculated m / z: 238.1443, found: 238.1445.

[0199] Homopropargyl isomer (9):

[0200] 1 H NMR (400 MHz, DMSO-d 6 )δ7.91(t,J=5.7Hz,1H),3.57(s,3H),3.08(d,J=2.8Hz,2H),2.97(t,J=2.8Hz,1H),2.90(t,J=8.0Hz,2H),2.22(tt,J=12. 3,3.8Hz,1H),1.89(dd,J=13.8,3.3Hz,2H),1.71(dd,J=13.5,3.3Hz,2H),1.41–1.19(m,3H),0.91(qd,J=13.1,3.6Hz,2H).

[0201] 13 C NMR (100 MHz, DMSO-d 6 )δ175.47,166.04,78.94,73.45,51.29,44.82,42.30,36.80,29.21,28.18,26.40.

[0202] (c) Deprotection of methyl (1r,4r)-4-(buta-2,3-dienamidomethyl)cyclohexane-1-carboxylate

[0203]

[0204] Compound 8 (58.1 mg, 0.245 mmol, 1.0 equivalent) was dissolved in tert-butyl alcohol / deionized water (2: 1, 1 mL), and the reaction mixture was cooled to 0 ° C in an ice bath. Lithium hydroxide monohydrate (12.3 mg, 0.294 mmol, 1.2 equivalents) was added to a reaction flask, and the reaction mixture was stirred at 0 ° C for 2 h. Thereafter, the reaction mixture was warmed to room temperature and stirred overnight. Once the reaction was complete, the reaction mixture was acidified using 1N HCl (aqueous solution) 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), then dried over anhydrous magnesium sulfate. The solvent was then removed on a rotary evaporator to obtain a white solid as the product (0.0530 g, 97%).

[0205] 1 H NMR (400 MHz, DMSO-d 6)δ7.87(t,J=6.1Hz,1H),5.74(t,J=6.6Hz,1H),5.27(d,J=6.5Hz,2H),2.94(t,J=6.4Hz,2H),2.10(tt,J=12.8 ,4.2Hz,1H),1.88(dd,J=13.3,2.7Hz,2H),1.71(dd,J=13.2,2.7Hz,2H),1.29–1.20(m,3H),0.98–0.82(m,2H).

[0206] 13 C NMR (101 MHz, DMSO-d 6 )δ212.44,176.74,163.42,89.95,79.58,44.85,42.52,37.00,29.43,28.27.

[0207] HRMS: C 13 H 18 NO 3 [M+H] + Calculated m / z: 224.1287, found: 224.1287.

[0208] d) reacting (1r,4r)-4-(buta-2,3-dienylamidomethyl)cyclohexane-1-carboxylic acid with pentafluorophenyl trifluoroacetate

[0209]

[0210] Compound 10 (0.155 g, 0.692 mmol, 1.0 equivalent) was dissolved in anhydrous dimethylformamide (4 mL) in a 10 mL Schlenk flask under an argon atmosphere and cooled in an ice bath. Symmetrical-trimethylpyridine (0.46 mL, 3.46 mmol, 5.0 equivalent) was added to the solution, followed by dropwise addition of pentafluorophenyl trifluoroacetate (0.60 mL, 3.46 mmol, 5.0 equivalent). The reaction mixture was allowed to warm to room temperature and stirred for 6 h. After the reaction was complete, the reaction was slowly quenched with water, and the aqueous mixture was transferred to a separatory funnel. The aqueous mixture layer was extracted with ethyl acetate (x3), and the combined organic extracts were washed with 0.5 N HCl (aqueous solution), water and brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated. The crude product was purified by flash column chromatography (29% ethyl acetate in hexane) to give a white solid (0.115 g, 43%). A total yield of 21% FACC was obtained and 1 H and 19 F NMR characterization.

[0211] 1 H NMR (400MHz, CD2 Cl 2 )δ5.88(s,1H),5.59(t,J=6.7Hz,1H),5.23(d,J=6.7Hz,2H),3.16(t,J=6.5Hz,2H),2.64(tt,J=12.2,3.6Hz,1H ), 2.19 (dd, J=13.8, 3.7Hz, 2H), 1.89 (dd, J=13.7, 3.5Hz, 2H), 1.64–1.46 (m, 3H), 1.07 (qd, J=13.1, 3.5Hz, 2H).

[0212] 13 C NMR (101 MHz, CD 2 Cl 2 )δ212.21,172.24,164.74,91.26,80.66,45.85,43.28,37.99,30.06,28.95.

[0213] 19 F NMR (377MHz, CD 2 Cl 2 )δ-153.50–-154.21(m),-159.43(t,J=21.5Hz),-163.36–-163.59(m).

[0214] HRMS: C 18 H 17 NO 3 F 5 [M+H] + Calculated m / z: 390.1129, found: 390.1130.

[0215] Example 3. Synthesis and characterization of FACC-DM1 adducts

[0216] FACC-DM1 is a linker-drug compound (LDC) that generates the FDA-approved ADC Kadcyla TM (Ado-trastuzumab emtansine, T-DM1) is an allenamide analogue. Figure 4 )

[0217] Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate developed by Roche and approved by the FDA in 2013 for the treatment of advanced breast cancer and HER2-positive early breast cancer. It combines the anti-tumor properties of trastuzumab, a humanized anti-human epidermal growth factor receptor 2 (HER2) antibody, and DM1, a maytansinoid that acts as a potent microtubule disruptor. The two parts are connected by a heterobifunctional linker, SMCC.

[0218] The maleimide thioether bond is broken by the reverse Michael process, resulting in the deconjugation of DM1 from trastuzumab. A meta-analysis study showed that adverse events were mainly attributed to the premature release of DM1 in plasma (Shen, K. et al., Sci. Rep., 2016, 6, 1). The thiol-allenamide adducts disclosed herein are reliable and stable, and therefore, comparable to the FDA-approved Kadcyla TM In comparison, analogs produced with FACC linkers are expected to have superior therapeutic and safety properties.

[0219] Conjugation of FACC and DM1:

[0220]

[0221] Under argon atmosphere, anhydrous dichloromethane (0.5 mL) was added 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.). Once the solid was dissolved, triethylamine (3.5 μL, 0.025 mmol, 0.5 eq.) was added and the resulting mixture was stirred overnight at room temperature. The reaction mixture was then concentrated under reduced pressure. Using preparative thin layer chromatography (CH 2 Cl 2 The crude product was purified by HPLC-MS / MS (5% CO 2 / EtOAc / MeOH: 33:66:1) to give a white solid (36.1 mg, 64%).

[0222] The FACC-DM1 adduct was obtained with an overall yield of 13% and was synthesized by 1 H and 19 F NMR spectroscopy was used to characterize ( Figure 5-7 ). The NMR results were obtained following the protocol disclosed in Example 1. The FACC-DM1 adduct was also characterized by HRMS.

[0223] 1 H NMR (400 MHz, CDCl 3)δ6.83(s,1H),6.74–6.55(m,2H),6.43(dd,J=15.4,11.2Hz,1H),6.27(d,J=7.1Hz,1H),6.03(d,J=8.4Hz,1H),5.63(dd,J=1 5.3,9.0Hz,1H),5.34(dd,J=15.3,8.4Hz,1H),5.16(s,1H),4.93–4.70(m,2H),4.37–4.21(m,1H),3.98(s,3H),3.66(d,J=12 .8Hz,1H),3.49(d,J=9.0Hz,1H),3.35(d,J=1.4Hz,3H),3.21–2.92(m,11H),2.88(s,1H),2.84(s,2H),2.79–2.67(m,1H),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).

[0224] 19 F NMR (377 MHz, CDCl 3 )δ-152.95–-153.44(m),-157.96–-158.36(m),-162.20–-162.58(m).

[0225] HRMS for C 53 H 64 N 4 O 13 CIF 5 SNa[M+Na] + m / z calculated value: 1149.3697. Found value: 1149.3700.

[0226] Example 4: Comparison of reactivity and chemoselectivity of FACC and SMCC

[0227] like Figure 8 As shown, the reactivity and chemoselectivity of FACC and SMCC towards amines and thiols were compared. Benzylamine was used as a model for amine-containing compounds, and glutathione was used as a model for thiol-containing compounds. The reactions were monitored by analytical HPLC.

[0228] The reaction of FACC and SMCC with benzylamine was found to be complete in 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 for 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 rapidly with glutathione, but large amounts of it also reacted with benzylamine (see Fig.10 , especially peak E).

[0229] Example 5: Comparison of reactivity and chemoselectivity of EACF and EMCS (N-ε-maleimidocaproyl-oxysuccinimide ester)

[0230] like Fig.11 As shown, the reactivity and chemoselectivity of EACF and EMCS towards amines and thiols were compared. Benzylamine was used as a model for amine-containing compounds, and glutathione was used as a model for thiol-containing compounds. The reactions were monitored by analytical HPLC.

[0231] The reaction of EACF and EMCS with benzylamine was found to be complete in 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 for the reaction to complete). 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 terminus of EMCS was found to react rapidly with glutathione, but large amounts of it also reacted with benzylamine (see Fig.13 , especially peak J).

Claims

1. A compound according to formula (I): in, A chooses free-C 1-6 Alkylene-, -R a -(C 3-12 Carbon ring)-R a -and-R a -(5-10 membered heterocyclic ring)-R a - the groups formed; The alkylene group is a straight chain alkylene group, and optionally one or more carbon atoms are replaced by a group independently selected from the group consisting of O, NH and S; Wherein, the alkylene, carbocyclic ring and heterocyclic ring are optionally selected from OH, NH2, C 1-4 substituted with one or more groups of alkyl and halogen; and Each R a independently a bond or -C 1-4 Alkylene-; Z a is an allenyl group or according to formula (II) D a selected from the group consisting of a carrier entity, a biologically active entity, and a diagnostic entity; or a pharmaceutically acceptable salt or solvate thereof.

2. The compound according to claim 1, wherein A is -C 1-6 Alkylene- or -R a -(C 5-6 Carbon ring)-R a -, where each R a independently a bond or -C 1-4 Alkylene-.

3. The compound according to claim 1 or claim 2, wherein A is -C5 alkylene- or -CH2-(C6 carbocycle)-.

4. The compound according to any one of claims 1 to 3, wherein The carbocyclic ring is a non-aromatic carbocyclic ring, for example a cyclohexane group.

5. The compound according to claim 1, wherein The compound is according to formula (Ia): 。 6. The compound according to claim 1, wherein The compound is according to formula (Ib):

7. The compound according to any one of claims 1 to 6, wherein Z a It is an allenyl group.

8. The compound according to any one of claims 1 to 6, wherein Z a According to formula (II).

9. The compound according to claim 8, wherein D a It is a biologically active entity.

10. The compound according to claim 9, wherein D a It is maytansine, auristatin (eg monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin or a cytotoxic derivative thereof.

11. A compound according to formula (III): in, A is -C 1-6 Alkylene-, wherein the alkylene is a straight chain alkylene, and optionally one or more carbon atoms are replaced by a group independently selected from the group consisting of O, NH and S; and / or optionally selected from OH, NH2, C 1-4 Alkyl and halogen substitutions; D a and D b One of the two is an antibody or antibody fragment, while the other is a small molecule drug; or a pharmaceutically acceptable salt or solvate thereof.

12. The compound according to claim 11, wherein D a is a small molecule drug, and D b is an antibody or antibody fragment.

13. The compound according to claim 11 or 12, wherein D a It is maytansine, auristatin (eg monomethyl auristatin F and monomethyl auristatin E), 3-deazaneplanocin A, doxorubicin or a cytotoxic derivative thereof.

14. The compound according to any one of claims 11 to 13, wherein D b Selected from the group consisting of trastuzumab, humanized IgG1 anti-BCMA antibody and fragments thereof.

15. The compound according to any one of claims 11 to 14, wherein D a is a maytansine derivative, and D b It's trastuzumab.

16. A compound according to any one of claims 11 to 15 for use in therapy.

17. A compound according to any one of claims 13 to 15 for use in treating cancer.

18. Use of the compound according to any one of claims 11 to 15 in the preparation of a medicament.

19. Use of the compound according to any one of claims 13 to 15 in the preparation of a medicament for treating cancer.

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

21. The method according to claim 20, wherein: The subject suffers from cancer.

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

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

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