NOVEL CRYPTOPHYCIN COMPOUNDS AND CONJUGATES, THEIR PREPARATION

AND THEIR THERAPEUTIC USE

The present invention relates to new cryptophycin compounds, to new cryptophycin payloads, to new cryptophycin conjugates, to compositions containing them and to their therapeutic use, especially as anticancer agents. The invention also relates to the process for preparing these conjugates.

Cryptophycins are secondary metabolites belonging to the class of depsipeptide macrocycles produced by cyanobacteria of the genus Nostoc. The first representative of this class of molecules, cryptophycin- 1 (C-1 ), was isolated in 1990 from cyanobacterium Nostoc sp (ATCC 53789; see EiBler S., et ai, Synthesis 2006, 22, 3747-3789). Cryptophycins C-1 and C-52, which are characterized by an epoxide function, were discovered to have in vitro and in vivo antitumor activity in the early 1990s. The chlorohydrins of these, C-8 and C-55, were markedly more active but could not be formulated as stable solutions (Bionpally R.R., et ai., Cancer Chemother Pharmacol 2003, 52, 25-33). Their higher activity was correlated by their putative ability to generate the corresponding epoxides in cells. With no method to adequately stabilize the chlorohydrins at the time, cryptophycin C-52 (LY355703) entered clinical trials, produced marginal antitumor activity in two phase II lung cancer trials with 30-40% stable disease and was thus discontinued (Edelman M.J., et ai, Lung Cancer 2003, 39, 197-99 and Sessa C, et ai, Eur J Ca -96).

R = Me, C-52

Considering their high potency and common mechanism of action with maytansinoids and auristatins, the two cytotoxic molecules validated in the clinic for antibody-drug conjugates (ADC), this series was considered as a potential tubulin binder for ADC. Therefore conjugates in the cryptophycin series were developped starting from derivatization at the para-benzylic position of the macrocycle (Al-Awar R.S., et ai, J Med Chem 2003, 46, 2985-3007).

WO2011/001052 describes cryptophycin antibody-drug conjugates for which the cytotoxic moiety is linked to the antibody through the para-benzylic position using various kinds of linkers. They might be cleavable, disulfide or protease-sensitive, or non-cleavable.

Further optimization of cryptophycin antibody-drug conjugates described in WO2011/001052 led to potent cryptophycin conjugates which displayed promising antitumor activity but were found unstable in the plasma of mice while being stable in the plasma of non-rodent species. Therefore, there was a need for cryptophycin conjugates exhibiting improved stability properties.

The purpose of this invention is that of proposing new cryptophycin macrocycles which once conjugated to an antibody are stable in mice plasma and new cryptophycin conjugates composed of those stable macrocycles.

Summary of the invention

In this respect, the inve (I):

wherein:

■ Ri represents a (CrC6)alkyl group;

■ R2 and R3 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group; or alternatively R2 and R3 form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl or a (C3-C6)heterocycloalkyl group;

■ R4 and R5 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group or a (Ci-C6)alkyl-N H(R12) group or a (CrC6)alkyl-OH group or a (CrC6)alkyl-SH group or a (C C6)alkyl-C02H group;

or alternatively R4 and R5 form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl or a (C3-C6)heterocycloalkyl group;

■ R6 represents a hydrogen atom or a (CrC6)alkyl group;

■ R7 and R8 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group or a (Ci-C6)alkyl-C02H group or a (Ci-C6)alkyl-N(Ci-C6)alkyl2 group;

or alternatively R7 and Re form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl group or a (C3-C6)heterocycloalkyl group;

■ Rg represents one or more substituents of the phenyl nucleus chosen, independently of each other, from: a hydrogen atom, -OH, (Ci-C4)alkoxy, a halogen atom, -N H2, -N H(Ci-C6)alkyl, - N(C C6)alkyl2, -N H(C C6)cycloalkyl or (C3-C6)heterocycloalkyl;

■ R10 represents at least one substituent of the phenyl nucleus chosen from a hydrogen atom and a group (CrC4)alkyl;

■ W represents

• (Ci-C6)alkyl-N H(Rn ), more particularly (CH2)nNH Rn ;

• (C C6)alkyl-OH, more particularly (CH2)nOH;

• (C C6)alkyl-SH, more particularly (CH2)nSH;

• C02H or C(=0)NH2;

• (CrC6)alkyl-C02H or (C C6)alkyl-C(=0)NH2; or

• (CrC6)alkyl-N3.

W being positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus;

■ Rii and R12 represent, independently of each other, a hydrogen atom or (Ci-C6)alkyl, more particularly a methyl group;

■ n represents an integer between 1 and 6.

The invention furthe

■ Ri, R2, R3, R4, R5, R6, R7, Re, R9 and R10 are as defined in formula (I);

■ Y represents (Ci-C6)alkyl-NRn or (C C6)alkyl-0 or (C C6)alkyl-S;

or alternatively Y represents C(=0)0, C(=0)NH, (C C6)alkyl-C(=0)0 or (C C6)alkyl-C(=0)NH;

or alternatively Y represents (CrC6)alkyl-triazole- like
;

Y being positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus;

■ R11 represents a hydrogen atom or a (CrC6)alkyl group;

■ L represents a linker;

■ RCG1 represents a reactive chemical group present at the end of the linker, RCG1 being reactive towards a reactive chemical group present on an antibody.

The invention also relates to conjugates of

formula (III):

wherein:

■ Ri, R2, R3, R4, R5, R6, R7, Re, R9 and R10 are as defined in formula (I);

■ Y and L are as defined in formula (II);

■ G represents the product of reaction between RCG1 , a reactive chemical group present at the end of the linker and RCG2, an orthogonal reactive chemical group present on the antibody (Ab);

■ Ab represents an antibody.

Each substituent Ri to Rn may also adopt one of the spatial configurations (e.g. R or S or alternatively Z or E) as described in the examples.

The compounds of formula (I), (II) and (III) may contain one or more asymetric carbon atoms. They may therefore exist in the form of enantiomers or diastereoisomers. These enantiomers and diastereoisomers, and also mixtures thereof, including racemic mixtures, form part of the invention.

The compounds of formula (I), including those that are illustrated, may exist in the form of bases or of acid-addition salts, especially of pharmaceutically acceptable acids.

Definitions

In the context of the present invention, certain terms have the following definitions:

• alkenyl group: a hydrocarbon group obtained by removing one hydrogen atom from an alkene. The alkenyl group may be linear or branched. Examples that may be mentionned include ethenyl (-CH=CH2, also named vinyl) and propenyl (-CH2-CH=CH2, also named allyl).

• alkoxy group: a group -O-alkyl, in which the alkyl group is as defined below;

· alkyl group: a linear or branched saturated aliphatic hydrocarbon-based group obtained by removing a hydrogen atom from an alkane. Examples that may be mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fert-butyl, pentyl, neopentyl, 2,2-dimethylpropyl and hexyl groups;

• alkylene group: a saturated divalent group of empirical formula -CnH2n-, obtained by removing two hydrogen atoms from an alkane. The alkylene group may be linear or branched. Examples that may be mentioned include methylene (-CH2-), ethylene (-CH2CH2-), propylene (- CH2CH2CH2-), butylene (-CH2CH2CH2CH2-) and hexylene (-CH2CH2CH2CH2CH2CH2-) groups or

^

the following branched groups , ' , " , 7 ; preferably, the alkylene group is of the formula -(CH2)n-, n representing an integer; in the ranges of values, the limits are included (e.g. a range of the type "n ranging from 1 to 6" or "between 1 and 6" includes limits 1 and 6);

• antibody: an antibody with affinity for a biological target, more particularly a monoclonal antibody. The function of the antibody is to direct the biologically active compound as a cytotoxic compound towards the biological target. The antibody may be monoclonal, polyclonal or multispecific; it may also be an antibody fragment; it may also be a murine, chimeric, humanized or human antibody. An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond (also referred to as a "full-length antibody"). The terms "conventional (or full-length) antibody" refers both to an antibody comprising the signal peptide (or pro-peptide, if any), and to the mature form obtained upon secretion and proteolytic processing of the chain(s). There are two types of light chain, lambda (I) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. As used herein, the term "antibody" denotes both conventional (full-length) antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies. Fragments of (conventional) antibodies typically comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody, and retain the biological function of the conventional antibody. Examples of such fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2 and diabodies.

The function of the antibody is to direct the biologically active compound as a cytotoxic compound towards the biological target.

• aryl group: a cyclic aromatic group containing between 5 to 10 carbon atoms. Examples of aryl groups include phenyl, tolyl, xylyl, naphtyl;

• biological target: an antigen (or group of antigens) preferably located at the surface of cancer cells or stromal cells associated with this tumour; these antigens may be, for example, a growth factor receptor, an oncogene product or a mutated "tumor suppressant" gene product, an angiogenesis-related molecule or an adhesion molecule;

• conjugate: an antibody-drug conjugate or ADC, i.e. an antibody to which is covalently attached via a linker at least one molecule of a cytotoxic compound;

• cycloalkyl group: a cyclic alkyl group comprising between 3 and 6 carbon atoms engaged in the cyclic structure. Examples that may be mentioned include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups;

• DAR (drug-to-antibody ratio): an average number of cytotoxic molecules attached via a linker to an antibody;

• halogen: any of the four elements fluorine, chlorine, bromine and iodine;

· heteroaryl group: an aryl group containing between 2 to 10 carbon atoms and between 1 to 5 heteroatoms such as nitrogen, oxygen or sulphur engaged in the ring and connected to the carbon atoms forming the ring. Examples of heteroaryl groups include pyridyl, pyrimidyl, thienyl, imidazolyl, triazolyl, indolyl, imidazo-pyridyl, pyrazolyl;

• heterocycloalkyl group: a cycloalkyl group containing between 2 to 8 carbon atoms and between 1 to 3 heteroatoms, such as nitrogen, oxygen or sulphur engaged in the ring and connected to the carbon atoms forming the ring. Examples include aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, azetidinyl, oxetanyl and pyranyl;

• linker: a group of atoms or a single bond that can covalently attach a cytotoxic compound to an antibody in order to form a conjugate;

• fragment: simpler molecules that allow the total synthesis of cryptophycin compounds;

• payload: a cytotoxic compound to which is covalently attached a linker;

• reactive chemical group: group of atoms that can promote or undergo a chemical reaction.

Abbreviations

ADC: antibody-drug conjugate; AcOH: acetic acid; AIBN: azobisisobutyronitrile; ALK: (d-Ci2)alkylene group, more particularly (CrC6)alkylene, more particularly of the form -(CH2)n-, n being an integer from 1 to 12 and preferably from 1 to 6; aq.: aqueous; Ar: argon ; AUC: area under the curve; BEMP: 2-fert-butylimino-2-diethylamino-1 ,3-dimethylperhydro-1 ,3,2-diazaphosphorine; BF3: boron trifluoride; B0C2O: di-fert-butyl-dicarbonate; BuLi: butyl lithium; CAN: eerie ammonium nitrate; Cbz: carboxybenzyl; CHCI3: chloroform; CH3CN: acetonitrile; CH3I: methyl iodide; CO2: carbon dioxide; CL: clearance; m-CPBA: m-chloroperbenzoic acid; CR: complete response; crypto denotes the unit of formula

, crypto especially denoting one of the D1-D19 cryptophycin compounds described later or a cryptophycin compound of an example; D1 refers to an ADC with a DAR of 1 , D2 to an ADC with a DAR of 2, etc.; d: day; DAR : drug-to-antibody ratio; DBU: 8-diazabycyclo[5.4.0]undec-7-ene; DCC: Ν,Ν'-dicyclohexylcarbodiimide; DCE: dichloroethane; DCM: dichloromethane; DDQ: 2,3-dichloro-5,6-dicyano-1 ,4-benzoquinone; DIC:

N,N'-diisopropylcarbodiimide; DIAD: diisopropylazodicarboxylate; DIEA: N,N-diisopropylethylamine; DMA: dimethylacetamide; DMAP : 4-(dimethylamino)pyridine ; DME: dimethoxyethane; DMEM: Dulbecco's Modified Eagle Medium; DMEM/F12: Dulbecco's Modified Eagle Medium Nutrient Mixture F-12; DMF: dimethylformamide; DMSO: dimethylsulfoxide; DPBS: Dulbecco's phosphate-buffered saline; DPPA: diphenylphosphorazide (PhO)2P(=0)N3; DSC: Ν,Ν'-disuccinimidyl carbonate; EDA: ethylenediamine; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; EDTA: ethylenediaminetetraacetic acid; EEDQ: N-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline; ELSD: evaporating light scattering detector; eq.: equivalent; ES: electrospray; EtOAc: ethyl acetate; Et20: diethyl ether; EtOH: ethanol; Ex.: example; FCS: foetal calf serum; Fmoc: 9-fluorenylmethoxycarbonyl; FmocOSu: N-(9-fluorenylmethoxycarbonyloxy) succinimide; Gl: electroinductive group; Grubbs I: benzylidene-bis(tricyclohexylphosphine)dichlororuthenium; Grubbs II: (1 ,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(phenylmethylene)(tricyclohexylphosphine)ruthenium; h: hour; H20: water; Hal: halogen; HATU: 1-[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; HCI: chlorohydric acid; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HIC: hydrophobic interaction chromatography; HOAt: 1-hydroxy-7-azabenzotriazole; HOBt: 1-hydroxy-benzotriazole; HPLC: high performance liquid chromtography; HRMS: high resolution mass spectrometry; IC5o: median inhibitory concentration; i.e.: id est meaning that is; IEC: ion exchange chromatography; iPrOH: 2-propanol; iPr20: diisopropyl ether; i.v.: intravenous; K2COs: potassium carbonate; LDA: lithium diisopropylamide; LiOH: lithium hydroxide; marg.: marginally; MeOH: methanol; MeTHF: 2-methyl-tetrahydrofuran; MgS04: magnesium sulfate; min: minute; MNBA : 2-methyl-6-nitrobenzoic anhydride ; MTBE: methyl tert-butyl ether; MTD: maximum tolerated dose; NaH: sodium hydride; NaHS04: sodium bisulfate; Na2S203: sodium thiosulfate; NaCI: sodium chloride; NaHCOs: sodium hydrogen carbonate; NaHSOs: sodium bisulfite; NaHS04: sodium hydrogen sulfate; NaOH: sodium hydroxide; NH4CI: ammonium chloride; NHS: N-hydroxysuccinimide; NMP: 1-methyl-2-pyrrolidone; NMR: nuclear magnetic resonance; P2-Et: 1 -ethyl-2, 2,4,4, 4-pentakis(dimethylamino)-2A5,4A5-catenadi(phosphazene); PBS: phosphate-buffered saline; PEG: polyethylene glycol; PK: pharmacokinetics; PMB: pa ra-methoxy benzyl; PNGase F: Peptide-N-Glycosidase F; PPh3:

triphenylphosphine; ppm: parts per million; PR: partial response; QS: quantum satis meaning the amount what is needed; Q-Tof: quadrupole time-of-flight; quant.: quantitative yield; RCG: reactive chemical group; RT: room temperature; sat.: saturated; s.c: subcutaneously; SCID: severe combined immunodeficiency; SEC: size exclusion chromatography; TBAF: tetrabutylammonium fluoride; tBuOK: potassium fert-butoxide; TCEP: tris(2-carboxyethyl)phosphine hydrochloride; TEA: triethylamine; TEMPO: (2,2,6,6-tetramethylpiperidin-1-yl)oxyl; TFA: trifluoroacetic acid; TFS: tumor-free survivor; THF: tetrahydrofuran; THP: tetrahydropyran; TLC: thin layer chromatography; t /2: half-life; tR: retention time; p-TsOH: para-toluenesulfonic acid; UPLC: Ultra Performance Liquid Chromatography; UV: ultra-violet; Vss: apparent volume of distribution at steady state.

Figures

Figure 1 : High resolution mass spectrum of Ex.3

Figure 2: High resolution mass spectrum of Ex.7

Figure 3: High resolution mass spectrum of Ex.10

Figure 4: High resolution mass spectrum of Ex.14

Figure 5: High resolution mass spectrum of Ex.20

Figure 6: High resolution mass spectrum of Ex.23

Figure 7: In vivo efficacy of Ex.3 against MDA-MB-231 xenograft in SCID mice

Figure 8: In vivo efficacy of Ex.7 against MDA-MB-231 xenograft in SCID mice

Figure 9: In vivo efficacy of Ex.10 against MDA-MB-231 xenograft in SCID mice

Figure 10: In vivo efficacy of Ex.14 against MDA-MB-231 xenograft in SCID mice

Figure 1 1 : In vivo efficacy of Ex.20 against MDA-MB-231 xenograft in SCID mice

Figure 12: In vivo efficacy of Ex.23 against MDA-MB-231 xenograft in SCID mice

Figure 13: In vivo PK profile of ADC1 following a single i.v. administration in SCID mice

Figure 14: In vivo PK profile of ADC1 following a single i.v. administration in non-rodent species Figure 15: In vivo PK profile of Ex.3 following a single i.v. administration in SCID mice

Figure 16: In vivo PK profile of Ex.7 following a single i.v. administration in SCID mice

Figure 17: HRMS spectrum of deglycosylated ADC1 at 96 h following a single i.v. administration in SCID mice

Figure 18: HRMS spectrum of ADC2 light chain at 96 h following a single i.v. administration in SCID mice

Figure 19: HRMS spectrum of deglycosylated ADC1 at 6 d following a single i.v. administration in non-rodent species

Figure 20: HRMS spectrum of Ex.3 at 96 h following a single i.v. administration in SCID mice Figure 21 : HRMS spectrum of Ex.7 at 96 h following a single i.v. administration in SCID mice Figure 22: HRMS spectrum of Ex.10 at 96 h following a single i.v. administration in SCID mice Figure 23: HRMS spectrum of Ex.14 at 96 h following a single i.v. administration in SCID mice Figure 24: HRMS spectrum of Ex.20 at 96 h following a single i.v. administration in SCID mice Figure 25: HRMS spectrum of Ex.23 at 96 h following a single i.v. administration in SCID mice

Detailed description of the invention

The invention relates to

wherein:

■ Ri represents a (CrC6)alkyl group;

■ R2 and R3 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group; or alternatively R2 and R3 form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl or a (C3-C6)heterocycloalkyl group;

■ R4 and R5 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group or a (Ci-C6)alkyl-N H(R12) group or a (CrC6)alkyl-OH group or a (CrC6)alkyl-SH group or a (C C6)alkyl-C02H group;

or alternatively R4 and R5 form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl or a (C3-C6)heterocycloalkyl group;

■ R6 represents a hydrogen atom or a (CrC6)alkyl group;

■ R7 and R8 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group or a (Ci-C6)alkyl-C02H group or a (Ci-C6)alkyl-N(Ci-C6)alkyl2 group;

or alternatively R7 and Re form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl group or a (C3-C6)heterocycloalkyl group;

■ Rg represents one or more substituents of the phenyl nucleus chosen, independently of each other, from: a hydrogen atom, -OH , (Ci-C4)alkoxy, a halogen atom, -N H2, -N H(Ci-C6)alkyl or - N(CrC6)alkyl2 or -N H(CrC6)cycloalkyl or (C3-C6)heterocycloalkyl group;

■ R10 represents at least one substituent of the phenyl nucleus chosen from a hydrogen atom and a group (CrC4)alkyl;

■ W represents

• (Ci-C6)alkyl-N H(Rn ), more particularly (CH2)nNH Rn ;

• (C C6)alkyl-OH , more particularly (CH2)nOH ;

• (C C6)alkyl-SH , more particularly (CH2)nSH ;

· C02H or C(=0)N H2;

• (Ci-C6)alkyl-C02H or (C C6)alkyl-C(=0)N H2; or

• (CrC6)alkyl-N3.

W is positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus;

■ Rii and R12 represent, independently of each other, a hydrogen atom or a group (CrC6)alkyl, more particularly a methyl group;

■ n represents an integer between 1 and 6.

The cryptophycin compound may be one of the following D D19 :

■ W and Ri2 are as defined in formula (I);

Or the cryptophycin compound may be an equivalent unit described in one of the examples.

Among the compounds of formula (I) that are subject matter of the invention, a first group of compounds is composed of the compounds for which Ri represents a (CrC6)alkyl, in particular a methyl group.

Among the compounds of formula (I) that are subject matter of the invention, a second group of compounds is composed of the compounds for which each of R2 and R3 represents a hydrogen atom.

Among the compounds of formula (I) that are subject matter of the invention, a third group of compounds is composed of the compounds for which one of R2 and R3 represents a (CrC6)alkyl, in particular a methyl group, and the other one represents a hydrogen atom.

Among the compounds of formula (I) that are subject matter of the invention, a fourth group of compounds is composed of the compounds for which R2 and R3 form together with the carbon atom to which they are attached a (C3-C6)cycloalkyl group, in particular a cyclopropyl group.

Among the compounds of formula (I) that are subject matter of the invention, a fifth group of compounds is composed of the compounds for which each of R4 and R5 represents a (Cr C6)alkyl, in particular a methyl group.

Among the compounds of formula (I) that are subject matter of the invention, a sixth group of compounds is composed of the compounds for which R6 represents a hydrogen atom.

Among the compounds of formula (I) that are subject matter of the invention, a seventh group of compounds is composed of the compounds for which R7 and Re represent independently of each other an hydrogen atom or a (CrC6)alkyl group.

Among the compounds of formula (I) that are subject matter of the invention, an eighth group of compounds is composed of the compounds for which R9 represents two substituents selected from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-CI and 4-methoxy.

Among the compounds of formula (I) that are subject matter of the invention, a ninth group of compounds is composed of the compounds for which R 0 represents a hydrogen atom.

Among the compounds of formula (I) that are subject matter of the invention, a tenth group of compounds is composed of the compounds for which W is positioned in the para position of the phenyl nucleus.

Among the compounds of formula (I) that are subject matter of the invention, a eleventh group of compounds is composed of the compounds for which W is selected from (Ci-C6)alkyl-NHRn, in particular (Ci-CsJalkyl-NHR-n, particularly a CH2-NH2 group, (CrC6)alkyl-OH, in particular (d-C3)alkyl-OH, particularly a CH2-OH group, (C C6)alkyl-SH, in particular (C C3)alkyl-SH and (C C6)alkyl-C02H, in particular (Ci-C3)alkyl-C02H.

Among the compounds of formula (I) that are subject matter of the invention, a twelfth group of compounds is composed of the compounds for which Ri represents a (CrC6)alkyl, in particular a methyl group, each of R2 and R3 represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, more particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-CI and 4-methoxy and R10 represents a hydrogen atom.

Among the compounds of formula (I) that are subject matter of the invention, a thirteenth group of compounds is composed of the compounds for which Ri represents a (Ci-Ce)alkyl, in particular a methyl group, R2 and R3 represents a (Ci-Ce)alkyl, in particular a methyl group, and the other one represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, more particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-CI and 4-methoxy and R10 represents a hydrogen atom.

Alternatively, W is selected from (CH2)nNHRn, (CH2)nSH from (CH2)nOH, wherein n represents an integer between 1 and 6.

Among the compounds of formula (I) that are subject matter of the invention, a fourteenth group of compounds is composed of the compounds of the following structure (beta epoxide configuration):

All these sub-groups taken alone or in combination are part of the invention.

Among the compounds of formula (I) that are the subject matter of the invention, mention may be made in particular of the following compounds:



The invention furth

wherein:

■ Ri , R2, R3, R4, R5, R6, R7, R7, R9 and R10 are as defined in formula (I);

■ Y represents (Ci-C6)alkyl-NRn or (C C6)alkyl-0 or (C C6)alkyl-S;

or alternatively Y represents C(=0)0, C(=0)NH, (C C6)alkyl-C(=0)0 or (C C6)alkyl-C(=0)NH;

or alternatively Y represents (CrC6)alkyl-triazole- like
;

Y is positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus;

■ R11 represents a hydrogen atom or a (CrC6)alkyl group;

■ L represents a linker;

■ RCG1 represents a reactive chemical group present at the end of the linker, RCG1 being reactive towards a reactive chemical group present on an antibody.

Among the compounds of formula (II) that are subject matter of the invention, a first group of compounds is composed of the compounds for which Ri represents a (Ci-Ce)alkyl, in particular a methyl group.

Among the compounds of formula (II) that are subject matter of the invention, a second group of compounds is composed of the compounds for which each of R2 and R3 represents a hydrogen atom.

Among the compounds of formula (II) that are subject matter of the invention, a third group of compounds is composed of the compounds for which one of R2 and R3 represents a (CrC6)alkyl group, in particular a methyl group and the other one represents a hydrogen atom.

Among the compounds of formula (II) that are subject matter of the invention, a fourth group of compounds is composed of the compounds for which R2 and R3 form together with the carbon atom to which they are attached a (C3-C6)cycloalkyl group, in particular a cyclopropyl group.

Among the compounds of formula (II) that are subject matter of the invention, a fifth group of compounds is composed of the compounds for which each of R4 and R5 represents a (CrC6)alkyl group, in particular a methyl group.

Among the compounds of formula (II) that are subject matter of the invention, a sixth group of compounds is composed of the compounds for which R6 represents a hydrogen atom.

Among the compounds of formula (II) that are subject matter of the invention, a seventh group of compounds is composed of the compounds for which R7 and Re represent independently of each other an hydrogen atom or a (CrC6)alkyl group.

Among the compounds of formula (II) that are subject matter of the invention, an eighth group of compounds is composed of the compounds for which R9 represents two substituents selected from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-CI and 4-methoxy.

Among the compounds of formula (II) that are subject matter of the invention, a ninth group of compounds is composed of the compounds for which R 0 represents a hydrogen atom.

Among the compounds of formula (II) that are subject matter of the invention, a tenth group of compounds is composed of the compounds for which Y is positioned in the para position of the phenyl nucleus.

Among the compounds of formula (II) that are subject matter of the invention, an eleventh group of compounds is composed of the compounds for which Y represents (CrC6)alkyl-NRn in particular (CrC3)alkyl-NRn more particularly CH2-NH.

Among the compounds of formula (I) that are subject matter of the invention, a twelfth group of compounds is composed of the compounds for which Ri represents a (Ci-Ce)alkyl, in particular a methyl group, each of R2 and R3 represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, more particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-CI and 4-methoxy and Rio represents a hydrogen atom.

Among the compounds of formula (I) that are subject matter of the invention, a thirteenth group of compounds is composed of the compounds for which Ri represents a (CrC6)alkyl, in particular a methyl group, R2 and R3 represents a (CrC6)alkyl, in particular a methyl group, and the other one represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, more particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-CI and 4-methoxy and R10 represents a hydrogen atom.

Among the compounds of formula (II) that are subject matter of the invention, a fourteenth group of compounds is composed of the compounds of the following structure (beta epoxide configuration):

All these sub-groups taken alone or in combination are part of the invention.

Among the compounds of formula (II) that are the subject matter of the invention, mention may be made in particular of the following compounds:

The invention also relates to conjugates of formula (III):

wherein:

■ Ri, R2, R3, R4, R5, R6, R7, Re, R9 and R10 are as defined in formula (I);

■ Y and L are as defined in formula (II);

■ G represents the product of reaction between RCG1 , a reactive chemical group present at the end of the linker and RCG2, an orthogonal reactive chemical group present on the antibody (Ab);

■ Ab represents an antibody.

Among the compounds of formula (III) that are subject matter of the invention, a first group of compounds is composed of the compounds for which Ri represents a (CrC6)alkyl, in particular a methyl group.

Among the compounds of formula (III) that are subject matter of the invention, a second group of compounds is composed of the compounds for which each of R2 and R3 represents a hydrogen atom.

Among the compounds of formula (III) that are subject matter of the invention, a third group of compounds is composed of the compounds for which one of R2 and R3 represents a (CrC6)alkyl group, in particular a methyl group and the other one represents a hydrogen atom.

Among the compounds of formula (III) that are subject matter of the invention, a fourth group of compounds is composed of the compounds for which R2 and R3 form together with the carbon atom to which they are attached a (C3-C6)cycloalkyl group, in particular a cyclopropyl group.

Among the compounds of formula (III) that are subject matter of the invention, a fifth group of compounds is composed of the compounds for which each of R4 and R5 represents a (CrC6)alkyl group, in particular a methyl group.

Among the compounds of formula (III) that are subject matter of the invention, a sixth group of compounds is composed of the compounds for which R6 represents a hydrogen atom.

Among the compounds of formula (III) that are subject matter of the invention, a seventh group of compounds is composed of the compounds for which R7 and Re represent independently of each other an hydrogen atom or a (CrC6)alkyl group.

Among the compounds of formula (III) that are subject matter of the invention, an eighth group of compounds is composed of the compounds for which R9 represents two substituents selected from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-CI and 4-methoxy.

Among the compounds of formula (III) that are subject matter of the invention, a ninth group of compounds is composed of the compounds for which R 0 represents a hydrogen atom.

Among the compounds of formula (II) that are subject matter of the invention, a tenth group of compounds is composed of the compounds for which Y is positioned in the para position of the phenyl nucleus.

Among the compounds of formula (III) that are subject matter of the invention, an eleventh group of compounds is composed of the compounds for which Y represents (CrC6)alkyl-NRn in particular (CrC3)alkyl-NRn more particularly CH2-NH.

Among the compounds of formula (I) that are subject matter of the invention, a twelfth group of compounds is composed of the compounds of the following structure (beta epoxide configuration):

All these sub-groups taken alone or in combination are part of the invention.

Among the compounds of formula (III) that are the subject matter of the invention, mention may be made in particular of the following compounds:

The attachment between the cryptophycin payload of formula (II) and the antibody, in order to obtain the conjugate of formula (III), is produced by means of a reactive chemical group RCG1 present on the payload that is reactive towards a reactive group RCG2 present on the antibody. The reaction between RCG1 and RCG2 ensures the attachment of the compound of formula (II) to the antibody by formation of a covalent bond. In the conjugate of formula (III), parts of RCG1 and RCG2 can remain forming the attachment between the linker and the antibody.

Examples of RCG1 that may be mentioned include:

(i) the -C(=0)-ZaRa reactive group for which Za represents a single bond, O or NH, more particularly O, and Ra represents a hydrogen atom or a (CrC6)alkyl, (C3-C7)cycloalkyl, alkenyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl group. The aryl group may be substituted by 1 to 5 groups chosen from halogen, in particular F, alkyl, alkoxy, nitro and cyano groups;

(ii) one of the following reactive groups: the maleimido
group; the haloacetamido

group with R13 representing a hydrogen atom or a (CrC6)alkyl group, more particularly M -CI; -N3; -OH; -SH; -NH2; -C≡CH or an activated C≡C such as a cyclooctyne

moiety like
; an O-alkyl hydroxylamine or a Pictet-Spengler reaction

substrate such as
described in Agarwal P., et al., Bioconjugate Chem 2013, 24, 846-851 .

More particularly, -ZaRa may represent -OH, -OCH3, -OCH2CH=CH2,
(O-NHS) or

where cation represents for example sodium, potassium or cesium or

group in which Gl represents at least one electroinductive group such as -NO2 or -Hal, especially -F. They may be, for example, the following groups:

Another type of -C(=0)ZaRa group is the

follo
wing: More particularly, RCG1 may be chosen from one of those described in the examples.

Examples of RCG2 that may be mentioned include (Garnett M.C., et al., Advanced Drug Delivery Reviews 2001 , 53, 171-216):

(i) ε-amino groups of lysines borne by the side chains of the lysine residues that are present at the surface of an antibody;

(ii) a-amino groups of N-terminal amino acids of antibody heavy and light chains;

(iii) the saccharide groups of the hinge region;

(iv) the thiols of cysteines generated by reducing intra-chain disulfide bonds or the thiols of engineered cysteines;

(v) amide groups borne by the side chains of some glutamine residues that are present at the surface of an antibody;

(vi) aldehyde groups introduced using formylglycine generating enzyme.

More recently, other conjugation approaches have been considered, for instance the introduction of cysteines by mutation (Junutula J.R., et al., Nature Biotechnology 2008, 26, 925-932), the introduction of unnatural amino acids allowing other types of chemistry (Axup J.Y., et al., PNAS 2012, 109, 40, 16101-16106) or the conjugation on antibody glycans (Zhou Q., et al., Bioconjugate Chem. 2014, 25, 510-520). Another approach for site-specific modifications of antibodies is based on enzymatic labeling using for example bacterial transglutaminase (Jeger S., et al., Angew. Chem. Int. Ed. 2010, 49, 9995-9997; Strop P., et al., Chem. Biol. 2013, 20, 161-167) or formylglycine generating enzyme (Hudak J.E., et al., Angew. Chem. Int. Ed. 2012, 51, 4161-4165). For a review of site-specific conjugation strategies, see Agarwal P. and Bertozzi C.R., Bioconjugate Chem 2015, 26, 176-192. These conjugation technologies may also be applied to cryptophycin payloads described in the present invention.

It is also possible to chemically modify the antibody so as to introduce novel reactive chemical groups RCG2. Thus, it is well known to those skilled in the art how to modify an antibody with the aid of a modifying agent introducing for example activated disulfide, thiol, maleimido or haloacetamido groups (see especially WO2005/077090 page 14 and WO2011/001052). The modification makes it possible to improve the conjugation reaction and to use a wider variety of groups RCG1.

More particularly, in the case where RCG1 is of the type (ii) above, it is possible to chemically modify the antibody using an adequate modifying agent or to introduce one or more unnatural amino acids so as to introduce the adequate functions RCG2. For example:

- when RCG1 represents a N-hydroxysuccinimidyl ester, RCG2 represents a -NH2 group;

- when RCG1 represents a maleimido or haloacetamido function or a -CI group, RCG2 may be a -SH group;

- when RCG1 represents a -N3 group, RCG2 may be a -C≡CH group or an activated C≡C such as a cyclooctyne moiety;

- when RCG1 represents a -OH or -NH2 group, RCG2 may be a carboxylic acid or amide function;

- when RCG1 represents a -SH group, RCG2 may be a maleimido or haloacetamido function;

- when RCG1 represents a -C≡CH function or an activated C≡C, RCG2 may be a -N3 group;

- when RCG1 represents a -O-alkyl hydroxylamine function or a Pictet-Spengler reaction substrate, RCG2 may be an aldehyde or ketone function.

Examples of G that may be mentioned include:

The invention rela of formula (III):

in which the linker L is of formula (IV):

in which:

■ L1 represents

■ a single bond or a NR16(hetero)aryl-CRi5 i4-0-C(=0) group if Y = (C C6)alkyl- N(Rii);

■ a NR18-(C2-C6)alkyl-NR17-C(=0) group or a NR16(hetero)aryl-CRi5Ri4-0- C(=0)NR18-(C2-C6)alkyl-NR17-C(=0) group if Y = (C C6)alkyl- O- or (C C6)alkyl- S;

■ a NR16(hetero)aryl-CRi5Ri4 group if Y = C(=0)0, C(=0)NH, (C C6)alkyl-C(=0)0 or (CrC6)alkyl-C(=0)NH;

■ Rii , Ri4, i5, Ri6, Ri7 and Ri8 represent, independently of each other, H or a (CrC6)alkyl group;

■ (AA)W represents a sequence of w amino acids AA connected together via peptide bonds; ■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ L2 represents a single bond or a (CrC6)alkyl group or a (Ci-C6)alkyl-(OCH2CH2)i group or a (Ci-C6)alkyl-(OCH2CH2)rO(Ci-C6)alkyl group or a (CH2CH20)i(Ci-C6)alkyl group or a CH(S03H)-(CrC6)alkyl group or a (Ci-C6)alkyl-CH(S03H) group or a (C C6)alkyl-cyclohexyl group or a NR19-(CrC6)alkyl group or a NR2o-(CH2CH20)i(Ci-C6)alkyl group or a NR21-aryl group or a NR21-heteroaryl group or a (Ci-C6)alkyl-NR22C(=0)-(Ci-C6)alkyl group or a(C C6)alkyl-NR22C(=0)-(Ci-C6)alkyl-(OCH2CH2)i group. More particularly L2 represents a (C1- C6)alkyl group or a (C1-C6)alkyl-(OCH2CH2)i group or a CH(S03H)-(C1-C6)alkyl group; ■ Rig, R2o, R21 and R22 represent, independently of each other, H or a (CrC6)alkyl group;

■ i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50).

AA denotes a natural or unnatural amino acid, of configuration D or L, more particularly chosen from: alanine (Ala), β-alanine, γ-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), a.a-dimethyl-y-aminobutyric acid, β,β-dimethyl-γ-aminobutyric acid, glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), lysine (Lys), ε-acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val). More particularly, AA is chosen from alanine (Ala), citrulline (Cit), glutamine (Gin), glycine (Gly), ε-acetyl-lysine (AcLys), valine (Val).

The sequence (AA)W has the formula:

H 0

•+ ALKJL t..

23 in which R23 represents the side chain of one of the amino acids described above. Examples of sequences are as follows: Gly-Gly, Phe-Lys, Val-Lys, Val-AcLys, Val-Cit, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Ala, Phe-Cit, Leu-Cit, lle-Cit, Trp-Cit, Phe-Ala, Ala-Phe, Gly-Gly-Gly, Gly-Ala-Phe, Gly-Val-Cit, Gly-Phe-Leu-Cit, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu.

For Y = (CrC6)alkyl-N(Rn) and more particularly CH2NH, RCG1-L may be one of the following (IV1-17):

■ (AA)W represents a sequence of w amino acids AA connected together via peptide bonds as described above;

■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50);

■ Mi, M2, M3 and M4 are chosen, independently of each other, from CR24 and N;

■ Rig, R2o, R21 , R22 and R24 represent, independently of each other, a hydrogen atom or a (Cr Ce)alkyl group, more particularly a hydrogen atom or a methyl group.

For Y = (C C6)alkyl-0 or (C C6)alkyl-S and more particularly CH20 or CH2S, RCG1 -L may be one of the following (IV18-34):

in which:

■ (AA)W represents a sequence of w amino acids AA connected together via peptide bonds as described above;

■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50);

■ Mi, M2, M3 and M4 are chosen, independently of each other, from CR24 and N;

■ Ri7, 18, Ri9, R20, R21 , R22 and R24 represent, independently of each other, a hydrogen atom or a (C1-C6)alkyl group, more particularly a hydrogen atom or a methyl group.

For Y = (Ci-C6)alkyl-N(Rn) and more particularly CH2NH, RCG1-L may also be one of the following (IV35-51 ):

in which:

■ (AA)W represents a sequence of w amino acids AA connected together via peptide bonds as described above;

■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50);

■ Ji , J2, J3 and J4 are chosen, independently of each other, from CR24 and N;

■ Mi , M2, M3 and M4 are chosen, independently of each other, from CR24 and N;

■ Ri4, Ri5, R16, Ri9, R20, R21 , R22 and R24 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, more particularly a hydrogen atom or a methyl group.

For Y = (C C6)alkyl-0 or (C C6)alkyl-S and more particularly CH20 or CH2S, RCG1 -L may also be one of the following (IV52-68):

in which:

■ (AA)W represents a sequence of w amino acids AA connected together via peptide bonds as described above;

■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50);

■ Ji , J2, J3 and J4 are chosen, independently of each other, from CR24 and N;

■ Mi , M2, M3 and M4 are chosen, independently of each other, from CR24 and N;

■ Ri4, Ri5, R16, Ri7, R18, Ri9, R20, R21 , R22 and R24 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, more particularly a hydrogen atom or a methyl group.

For Y = C(=0)0, C(=0)NH, (C C6)alkyl-C(=0)0 or (C C6)alkyl-C(=0)NH and more particularly C(=0)0 or CH2-C(=0)0, RCG1-L may be one of the following (IV69-85):

in which:

■ (AA)W represents a sequence of w amino acids AA connected together via peptide bonds as described above;

■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50);

■ Ji , J2, J3 and J4 are chosen, independently of each other, from CR24 and N;

■ Mi , M2, M3 and M4 are chosen, independently of each other, from CR24 and N;

■ Ri4, Ri5, R16, Ri9, R20, R21 , R22 and R24 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, more particularly a hydrogen atom or a methyl group.

For Y = C(=0) or (CrC6)alkyl-C(=0), the invention also relates to cryptophycin payloads of formula (II) and to conjugates of formula (III):

in which the linker L is of formula (V):

+L3^NR2 AA)'w+ (v)

in which:

■ (AA)'W represents a sequence of w amino acids AA connected together via peptide bonds;

■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ R25 represents a hydrogen atom or a (CrC6)alkyl group;

■ L3 represents a (CrC6)alkyl group or a (CH2CH20)i-(Ci-C6)alkyl group or a (CrC6)alkyl- (OCH2CH2)i group or a CH(S03H)-(CrC6)alkyl group or or a (Ci-C6)alkyl-(OCH2CH2)rO(Ci- C6)alkyl group or a NR19-(CrC6)alkyl group or a NR2o-(CH2CH20)iCH2CH2 group or a (C C6)alkyl-cyclohexyl-C(=0)NR19-(Ci-C6)alkyl group or a (Ci-C6)alkyl-cyclohexyl-C(=0)NR2o- (CH2CH20)i-CH2CH2 group or a NR2i-aryl-C(=0)NR19-(Ci-C6)alkyl group or a NR2 heteroaryl-C(=0)NR19-(Ci-C6)alkyl group or a (Ci-C6)alkyl-NR22C(=0)(CH2CH20)r(Ci- C6)alkyl group;

■ Rig, R2o, R21 , R22 and R25 represents a hydrogen atom or a (CrC6)alkyl group;

■ RCG1 represents a reactive chemical group that is reactive towards a reactive chemical group present on the antibody, as defined above;

■ Ri , R2, R3, R4, R5, R6, R7, Re, R9 and R10 are as defined above.

AA denotes a natural or unnatural amino acid, of configuration D or L, more particularly chosen from: alanine (Ala), β-alanine, γ-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), a.a-dimethyl-y-aminobutyric acid, β,β-dimethyl-γ-aminobutyric acid, glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), lysine (Lys), ε-acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val). More particularly, AA is chosen from alanine (Ala), citrulline (Cit), glutamine (Gin), glycine (Gly), ε-acetyl-lysine (AcLys), valine (Val)

The sequence (AA)'W has the formula:

in which R26 represents the side chain of one of the amino acids described above. Examples of sequences are as follows: Gly-Gly, Lys-Phe, Lys-Val, AcLys-Val, Cit-Val, Lys-Phe-Phe, Lys-Phe-DPhe, Lys-Phe-Gly, Lys-Ala, Ala-Val, Cit-Phe, Cit-Leu, Cit-lle, Cit-Trp, Ala-Phe, Phe-Ala, Gly-Gly-Gly, Phe-Ala-Gly, Cit-Val-Gly, Cit-Leu-Phe-Gly, Gly-Leu-Phe-Gly, Leu-Ala-Leu-Ala.

For Y = C(=0) or (C C6)alkyl-C(=0) and more particularly C(=0) or CH2-C(=0), RCG1-L may be one of the following (V86-101 ):

(V98)

in which:

■ (AA)'W represents a sequence of w amino acids AA connected together via peptide bonds as described above;

■ w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3;

■ i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50);

■ Mi , M2, M3 and M4 are chosen, independently of each other, from CR24 and N;

■ Rig, R2o, R21 , R22, R24 and R25 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group, more particularly a hydrogen atom or a methyl group.

The linker L may also be chosen from the illustrated compounds.

In accordance with the invention, the compounds of general formula (I), (II) and (III) prepared by the following processes.

Process for preparing the cryptophycin compounds

General route A for the preparation of the compounds of formula (I) in the case where

Fragments A, B, C and D allow the preparation of cryptophycin compounds Pi to P3 with the aid of the steps detailed below:

Step (i): peptidic coupling between fragments AD1 and BC in the presence of coupling reagents such as, for example, HOAt and HATU;

Step (ii): deprotection in acidic conditions using for example TFA and macrocyclization in the presence of coupling reagents such as, for example, HOAt and HATU;

Step (iii): oxidation of the olefine to form the epoxide using, for example, m-CPBA;

Step (v): reduction of the azido group using, for example, TCEP.

Fragments A and B were prepared according to the synthesis described below. Fragments C protected as methyl esters are commercially available for R4=H, R5=Me (R), R2=R3=H (CAS number [92535-26-7]); R4=H, R5=Me (S), R2=R3=H (CAS number [1 18138-56-0]); R4=R5=Me,

R2=R3=H (CAS number [25307-82-8]); R2=H, R3=Me (R), R4=H, R5=Me (R) (CAS number [86544-92-5]); R2=H, R3=Me (S), R4=H, R5=Me (R) (CAS number [86544-93-6]); R2=H, R3=Me (R), R4=R5=Me (CAS number [1315052-25-5]); R2=H, R3=Me (S), R4=R5=Me (CAS number [1315050-96-4]); R2=H, R3=iPr (R), R4=R5=Me (CAS number [1314999-06-8]); R2=H, R3=iPr (S), R4=R5=Me (CAS number [1315054-33-1]); R2=R3=R4=R5=Me (CAS number [90886-53-6]); R4 and R5 form together with the carbon atom to which they are attached a cyclopropyl group (CAS number [914226-26-9]). They may also be prepared as described in the examples. Non-commercially available fragments C were prepared as described in the examples. All fragments D are commercially available: L-Fmoc-Ala-OH (CAS number [35661-39-3]); L-Fmoc-Val-OH (CAS number [68858-20-8]); L-Fmoc-ferf-Leu-OH (CAS number [132684-60-7]); L-Fmoc-Leu-OH (CAS number [35661-60-0]); L-Fmoc-NMe-Leu-OH (CAS number [103478-62-2]); L-Fmoc-3-dimethylamino-Ala-OH (CAS number [587880-86-2]); L-Fmoc-(Oallyl)Asp-OH (CAS number [146982-24-3]); L-Fmoc-4-methyl-Leu-OH (CAS number [139551-74-9]). Building blocks AD1 and BC were prepared according to the synthesis described below.

General route B for the preparation of the compounds of formula (I) in the case where

W=CH;>OH or CHpNs or CHpNH?

Scheme 2

Fragments A, B, C and D allow the preparation of cryptophycin compounds P2 to P4 with the aid of the steps detailed below:

Step (i): peptidic coupling between fragments AD1 and BC in the presence of coupling reagents such as, for example, HOAt and HATU;

Step (ii): deprotection in acidic conditions using for example TFA, macrocyclization in the presence of coupling reagents such as, for example, HOAt and HATU and acetate hydrolysis at pH 6-7 using for example NaOH in a mixture of water and AcOEt;

Step (iii): benzylic alcohol protection as a silyl ether using for example chlorotriisopropylsilane in the presence of a base such as, for example, imidazole; oxidation of the olefine to form the epoxide using, for example, m-CPBA; deprotection of the silyl ether using, for example, a TBAF solution.

Step (iv): azidation in the presence of DPPA and a base such as, for example, DBU;

Step (v): reduction of the azido group using, for example, TCEP.

Fragments A and B were prepared according to the synthesis described below. Fragments C were either commercially available as reported in the previous section or prepared as described

in the examples. All fragments D are commercially available as reported in the previous section. Building blocks AD2 and BC were prepared according to the synthesis described below.

General route C for the preparation of the compounds of formula (I) in the case where W=

Scheme 3

Sakurai alcohol and fragments B, C and D allow the preparation of cryptophycin compounds P2 to P4 with the aid of the steps detailed below:

Step (i): peptidic coupling between fragments AD3 and alternative BC in the presence of coupling reagents such as, for example, HOAt and HATU;

Step (ii): macrocyclization by ring closing metathesis in the presence of a catalyst such as, for example, Grubbs I catalyst;

Step (iii): deprotection of the p-methoxybenzyl ether in acidic conditions such as, for example, 10% TFA;

Step (iv): oxidation of the alcohol using an oxidizing agent such as, for example, TEMPO in the presence of sodium hypochlorite;

Step (v): introduction of the epoxide by asymmetric Corey-Chaykovsky reaction using appropriately substituted isothiocineole-derived chiral sulfonium in the presence of a base such as, for example, phosphazene base P2-Et;

Step (vi): reduction of the azido group using, for example, TCEP.

Sakurai alcohol and fragment B were prepared according to the synthesis described below. Fragments C were either commercially available as reported in the previous section or prepared as described in the examples. All fragments D are commercially available as reported in the previous section. Building blocks AD3 and alternative BC were prepared according to the synthesis described below.

Preparation of the compounds of formula (I) in the case where W=CH2SH

P4 allows the preparation of other cryptophycin compounds P5 and Ρβ with the aid of the steps detailed below:

Step (i): introduction of the chloro group using methanesulfonyl chloride in the presence of a base such as, for example, DIEA;

Step (ii): functionalization using tetrabutylammonium trimethylsilylthiolate prepared in situ from TBAF and hexamethyldisilathiane according to Hu J., et al., J. Org. Chem. 1999, 64, 4959-496: in the course of this reaction, the intermediate dimer depicted in Scheme 3 is usually formed;

Step (iii): reduction of the dimer using a phosphine such as, for example, TCEP.

Schemes 1 , 2, 3 and 4 describe the cases n=1 but may also apply for the preparation of compounds of formula (I) in the case where n>1 starting from P2, P3 or P4 with the aid of the

Scheme 5

Step (i): opening of the epoxide ring in acidic medium so as to obtain the diol function; concentrated perchloric acid may be used, for example;

Step (ii): oxidative cleavage of the diol using, for example, sodium periodate;

Step (iii): Wittig reaction using a suitable phosphonium halide, for example a bromide, and a strong base, for instance BuLi;

Step (iv): oxidation of the olefine to form the epoxide using, for example, m-CPBA;

Step (v): deprotection of the silyl ether using, for example, a TBAF solution.

Preparation of the compounds of formula (I) in the case where W=C02H

Scheme 6

P4 allows the preparation of other cryptophycin compounds P7 and Pe with the aid of the steps detailed below:

Step (i): oxidation using the Dess-Martin reagent;

Step (ii): oxidation of Pinnick type in the presence of 2 methyl-2-butene (Pinnick H.W., Tetrahedron 1981 , 37, 2091-2096).

Scheme 6 describes the case n=0 but may also apply for the preparation of compounds of formula (I) in the case where W=(CH2)nC02H starting from an analog of P4 bearing a (CH2)n+ OH group. Schemes 1 , 2, 3, 4, 5 and 6 are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Process for preparing the cryptophycin payloads

The compounds of formula (II) might be prepared according to Scheme 7 starting with a

Scheme 7

W represents

• (Ci-C6)alkyl-NH(Rn), more particularly (CH2)nNHRn;

• (C C6)alkyl-OH, more particularly (CH2)nOH;

• (C C6)alkyl-SH, more particularly (CH2)nSH;

• C02H;

• (Ci-C6)alkyl-C02H, more particularly (CH2)nC02H; or

• (Ci-C6)alkyl-N3.

in which Rn represents a hydrogen atom or a group (Ci-Ce)alkyl, more particularly a methyl group.

The linker precursor LP has the function of introducing a precursor of the linker L into the cryptophycin compound after reaction between the group W and a chemical function present on LP.

In Scheme 7, several steps and/or reactions may be necessary to prepare the cryptophycin payload (II) starting from the cryptophycin compound (I). For example, in the case where ZaRa = -O-NHS, a linker L for which ZaRa = -O-allyl may be introduced using the corresponding linker precursor, followed by deprotecting the ester function and introducing -O-NHS. Deprotection may be performed by treatment with a palladium catalyst, for example Pd(PPh3)4 in the presence of a scavenger amine, for example morpholine; the activation may be performed with DSC in the presence of a base such as, for example, DIEA or with NHS in the presence of a coupling agent such as, for example, DCC. This conversion of a group ZaRa into another group ZaRa (e.g. -O-allyl - -O-NHS) may be applied to obtain other groups ZaRa, especially those described previously.

Scheme 7' similarly illustrates the preparation of a cryptophycin compound comprising a linker bearing respectively, a maleimido, a haloacetamido, an amino or an azido group (L* represents a fragment of a linker such that L= -L*-maleimido or L= -L*-haloacetamido or L= -L*-NH2- or L= -L*-N

Scheme 7'

These compounds are obtained by reaction between a cryptophycin compound comprising a linker L' comprising an amino group and a modifying agent for introducing, respectively, a maleimido, a haloacetamido, an amino or an azido group.

The compounds of formula (II) might alternatively be prepared according to Scheme 8 starting with the same cryptophycin compound of formula (I) and another linker precursor (LP'):

LP"

(II) with L=L'-L'

Scheme 8

In Scheme 8, several steps and/or reactions may be necessary to arrive at the cryptophycin payload (II) starting from the cryptophycin compound (I). For example, in the case where ZaRa = -O-NHS, a protected linker precursor L' may be introduced, followed by deprotection and coupling to a linker precursor L" to introduce the carboxylic acid function that may be directly activated to ZaRa = -O-NHS or first deprotected and subsequently activated (L' and L" represent fragments of linker such that L=L'-L"). Deprotection may be performed by treatment with a base, for example, piperidine; coupling to a linker L" may proceed through opening of a cyclic anhydride, for example, glutaric anhydride; the activation may be performed with DSC in the presence of a base such as, for example, DIEA or with NHS in the presence of a coupling agent such as, for example, DCC.

An example of reaction between the group W and a chemical function present on LP is an amidation either between a linker precursor LP bearing a carboxylic acid function and W=(CH2)nNH2 or between a linker precursor LP bearing an amine function and W=C02H or (CH2)nC02H: this reaction may be performed in the presence of a coupling agent such as, for example, EDCI or HOBt. It is also possible to react a linker precursor LP bearing an amine function and W'=(CH2)nO-C(=0)-0-(4-nitrophenyl) obtained from W=(CH2)nOH and p-nitrophenyl chloroformate (activation of the alcohol in the form of carbonate) according to the scheme below (Ri7 = H or (CrC6)alkyl):

-NHR-I7 + crypto-(CH2)nO-C(=0)-0-(4-nitrophenyl) crypto-(CH2)nO-C(=0)-NR17-The same kind of reaction can also be performed between a linker precursor LP bearing an alcohol function activated as a carbonate and W=(CH2)nNH2. Another example of reaction is an esterification between a linker precursor bearing an alcohol function and W=C02H or (CH2)nC02H: this reaction may be performed in the presence of a coupling agent such as, for example, MNBA. Another example of reaction is a copper-catalyzed azide-alkyne cycloaddition between a linker precursor bearing an alkyne function and W=(CH2)N3: this reaction may be performed in the presence of copper sulfate and sodium ascorbate.

Preparation of the compounds of formula (II) in the case where W=(CH2)nNH2 and L=(IV1)

Scheme 9

Step (i): peptidic coupling in the presence of coupling reagents such as, for example, EDC and HOBt, and a base such as, for example, DIEA;

Step (ii): deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis-(triphenylphosphine)palladium;

Step (iii): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

p3

Scheme 10

Step (i): peptidic coupling in the presence of coupling reagents such as, for example, EDC and HOBt and a base such as, for example, DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine;

Step (iii): coupling to glutaric anhydride;

Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Schemes 9 and 10 describe the case L(IV1) with n=1 , Rn=H and ALK=(CH2)3 but they may also apply to other linkers L with RCG1 =C(=0)ONHS, namely the case L(IV1) with n≠1 and/or Rn≠H and/or ALK≠(CH2)3 and the cases L(IV2) and L(IV3). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=(CH2)nNH2 with n=1 ,

Scheme 11

Step (i): peptidic coupling in the presence of coupling reagents such as, for example, EDC and HOBt and a base such as, for example, DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine.

Scheme 11 describes the cases L(IV4) with n=1 , Rn=H and ALK=(CH2)5, L(IV8) with n=1 and Rii=H, L(IV12) with n=1 , Rn=H and ALK=(CH2)4, L(IV14) with n=1 , Rn=H and ALK=(CH2)3 and L(IV16) with n=1 , Rn=H, ALK=(CH2)3 and ALK'=(CH2)2 but it may also apply to other linkers L(IV4) to L(IV17) with RCG1 =maleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=(CH2)nX with X=Q or S

Scheme 12

Step (i): activation of the benzylic alcohol or benzylic thiol as a p-nitrophenyl-(thio)carbonate by treatment with p-nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA; Step (ii): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA;

Step (iii): deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium;

Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Preparation of the compounds of formula (II) in the case where W=(CH2)nX with X=Q or S

Scheme 13

Step (i): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine;

Step (iii): coupling to glutaric anhydride;

Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Schemes 12 and 13 describe the case L(IV18) with n=1 , R17 and Ri8=H and ALK=(CH2)3 but

they may also apply to other linkers L with RCG1 =C(=0)ONHS, namely the case L(IV18) with n≠1 and/or R17, Ri8≠H and/or ALK≠(CH2)3 and the cases L(IV19) and L(IV20). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=(CH2)nX with X=Q or S and RCG1 =maleimido, haloacetamido, NH2, N3 or cyclooctyne namely linkers L(IV21) to

Scheme 14

Step (i): formation of the (thio)carbamate, the reaction is performed in the presence of a base such as, for example, DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine.

Scheme 14 describes the case L(IV21) with n=1 , R 7 and Ris=H and ALK=(CH2)5, the case L(IV25) with n=1 and Ri7 and Ri8=H, the case L(IV29) with n=1 , R17 and R 8=H and ALK=(CH2)4, the case L(IV31 ) with n=1 , R17 and Ri8=H and ALK=(CH2)3 and the case L(IV33) with n=1 , R17, Ri8 and R22= H, ALK=(CH2)3 and ALK'=(CH2)2 but it may also apply to other linkers L(IV21 to 34) with RCG1 =maleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=(CH2)nNH2 and L=(IV35)

Step (i): formation of the carbamate, the reaction is performed in the presence of a base such as, for example, DIEA;

Step (ii): deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis-(triphenylphosphine)palladium;

Step (iii): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Preparation of the compounds of formula (II) in the case where W=(CH2)nNH2 and L=(IV35)

Scheme 16

Step (i): formation of the carbamate, the reaction is performed in the presence of a base such as, for example, DIEA in the presence of a suitable p-nitrophenylcarbonate reagent;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine;

Step (iii): coupling to glutaric anhydride;

Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Schemes 15 and 16 describe the case L(IV35) with n=1 ,
a para-benzylic alcohol and ALK=(CH2)3 but they may also apply to other linkers L with

RCG1 =C(=0)ONHS, namely the case L(IV35) with n≠1 and/or Rn, R14, R15, Rie≠H and/or Ji, J2, J3, J4≠CH and/or an orffto-benzylic alcohol and/or ALK≠(CH2)3 and the cases L(IV36) and L(IV37). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=(CH2)nNH2 with n=1 ,
cyclooctyne namely linkers L(IV38) to L(IV151)

Scheme 17

Step (i): formation of the carbamate, the reaction is performed in the presence of a base such as, for example, DIEA in the presence of a suitable p-nitrophenylcarbonate reagent;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine.

Scheme 17 describes the cases L(IV38) with n=1 ,
a para-benzylic alcohol and ALK=(CH2)5, L(IV42) with n=1 , R11=R14=Ri5=Ri6=H and
and a para-benzylic alcohol, L(IV46) with n=1 ,
a para-benzylic alcohol and ALK=(CH2)4, L(IV48) with n=1 , R11=R14=Ri5=Ri6=H,
a para-benzylic alcohol and ALK=(CH2)3 and L(IV50) with n=1 , R11=R14=R15=R16=R22=H,
a para-benzylic alcohol, ALK=(CH2)3 and ALK'=(CH2)2 but it may also apply to other linkers L(IV38) to L(IV51) with RCG1 =maleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is given with a para-benzylic alcohol but it may also apply to an orf /o-benzylic alcohol. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=(CH2)nX with X=Q or S
Scheme 7

Scheme 18

Step (i): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA;

Step (ii): deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium;

Step (iii): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Preparation of the compounds of formula (II) in the case where W=(CH2)nX with X=Q or S
Scheme 8

Scheme 19

Step (i): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine;

Step (iii): coupling to glutaric anhydride;

Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Schemes 18 and 19 describe the case L(IV52) with n=1 ,

a para-benzylic alcohol and ALK=(CH2)3 but they may also apply to other linkers L with RCG1 =C(=0)ONHS, namely the case L(IV52) with n≠1 and/or R14, Ri5, Rie, R17, Ri8≠H and/or J-i, J2, J3, J4≠CH and/or an orffto-benzylic alcohol and/or ALK≠(CH2)3 and the cases L(IV53) and L(IV54). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=(CH2)nX with X=Q or S and RCG1 =maleimido, haloacetamido, NH2, N3 or cyclooctyne namely linkers L(IV55) to

Scheme 20

Step (i): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine.

Scheme 20 describes the case L(IV55) with n=1 ,
a para-benzylic alcohol, ALK=(CH2)2 and ALK'=(CH2)5, the case L(IV59) with n=1 ,
a para-benzylic alcohol and ALK=(CH2)2, the case L(IV63) with n=1 ,
a para-benzylic alcohol,

ALK=(CH2)2 and ALK'=(CH2)4, the case L(IV65) with n=1 ,
J1 =J2=J3=J4=CH, a para-benzylic alcohol, ALK=(CH2)2 and ALK'=(CH2)3 and the case L(IV67) with n=1 , R14=R15=R16=Ri7=Ri8=R22=H , Ji=J2=J3=J4=CH , a para-benzylic alcohol, ALK=(CH2)2, ALK'=(CH2)5 and ALK"=(CH2)2 but it may also apply to other linkers L(IV55 to 68) with RCG1 =maleimido, iodoacetamido, N H2, N3 or cyclooctyne. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=C(=Q)0 and L=(IV69)

Scheme 21

Step (i): esterification in the presence of a coupling reagent such as, for example, MNBA and a base such as, for example, DMAP and DIEA;

Step (ii): deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis-(triphenylphosphine)palladium;

Step (iii): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Preparation of the compounds of formula (II) in the case where W=C(=Q)0 and L=(IV69)

Scheme 22

Step (i): esterification in the presence of a coupling reagent such as, for example, MNBA and a base such as, for example, DMAP and DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine;

Step (iii): coupling to glutaric anhydride;

Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Schemes 21 and 22 describe the case L(IV69) with
a para-benzylic alcohol and ALK=(CH2)3 but they may also apply to other linkers L with RCG1 =C(=0)ONHS, namely the case L(IV69) with R14, Ri5, Rie≠H and/or J2, J3, J4≠CH and/or an orffto-benzylic alcohol and/or ALK≠(CH2)3 and the cases L(IV70) and L(IV71). They are given for a linker in the para position, but may identically apply for the ortho or meta positions.

Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially DrD 9.

Preparation of the compounds of formula (II) in the case where W=C(=Q)0 with
cyclooctyne namely linkers L(IV72) to L(IV85)

Scheme 23

Step (i): esterification in the presence of a coupling reagent such as, for example, MNBA and a base such as, for example, DMAP and DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example,

piperidine.

Scheme 23 describes the cases L(IV72) with n=1 , para-benzylic alcohol and ALK=(CH2)5, L(IV76) with n=1 ,
and a para-benzylic alcohol, L(IV80) with n=1 ,
a para-benzylic alcohol and ALK=(CH2)4, L(IV82) with n=1 , R14=Ri5=Ri6=H,
a para-benzylic alcohol and ALK=(CH2)3 and L(IV84) with n=1 , R14=Ri5=Ri6=R22=H,
a para-benzylic alcohol, ALK=(CH2)3 and ALK'=(CH2)2 but it may also apply to other linkers L(IV72) to L(IV85) with RCG1 =maleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is given with a para-benzylic alcohol but it may also apply to an orffto-benzylic alcohol. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D D19.

Preparation of the compounds of formula (II) in the case where W=C(=Q)0 and L=(V86)

Scheme 24

Step (i): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA;

Step (ii): peptidic coupling in the presence of a base such as, for example, DIEA;

Step (iii): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

Scheme 24 describes the case L(V86) with
and ALK=(CH2)7 but they may also apply to other linkers L with RCG1 =C(=0)ONHS, namely the case L(V86) with R25≠H and/or ALK≠(CH2)7 and the cases L(V87) and L(V88). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.

Preparation of the compounds of formula (II) in the case where W=C(=Q)0 with Ran=H and

R =maleimido, haloacetamido, NH2, N3 or cyclooctyne namely linkers L(V89) to L(V101)

Scheme 25

Step (i): peptidic coupling in the presence of a base such as, for example, DIEA;

Step (ii): reduction of the azido by treatment with a reducing agent such as, for example, TCEP. Scheme 25 describes the cases L(V89) with R25=H and ALK=(CH2)5, L(V93) with R25=H and ALK=(CH2)3, L(V96) with R25=H and ALK=(CH2)4, L(V98) with R25=H and ALK=(CH2)3 and L(V100) with R22=R25=H, ALK=(CH2)3 and ALK'=(CH2)2 but it may also apply to other linkers L(V89) to L(V101) with RCG1 =maleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially DrD 9.

Process for preparing the cryptophycin conjugates

The compounds of formula (III) might be prepared according to Scheme 26 starting with a

CLAIMS

1. Cryptophycin compound of formula (I):

wherein:

■ Ri represents a (CrC6)alkyl group;

■ R2 and R3 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group; or alternatively R2 and R3 form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl or a (C3-C6)heterocycloalkyl group;

■ R4 and R5 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group or a (Ci-C6)alkyl-NH(R12) group or a (CrC6)alkyl-OH group or a (CrC6)alkyl-SH group or a (C C6)alkyl-C02H group;

or alternatively R4 and R5 form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl or a (C3-C6)heterocycloalkyl group;

■ R6 represents a hydrogen atom or a (CrC6)alkyl group;

■ R7 and R8 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group or a (Ci-C6)alkyl-C02H group or a (Ci-C6)alkyl-N(Ci-C6)alkyl2 group;

or alternatively R7 and Re form together with the carbon atom to which they are attached a (C3- C6)cycloalkyl group or a (C3-C6)heterocycloalkyl group;

■ Rg represents one or more substituents of the phenyl nucleus chosen, independently of each other, from: a hydrogen atom, -OH, (Ci-C4)alkoxy, a halogen atom, -NH2, -NH(Ci-C6)alkyl or - N(Ci-C6)alkyl2 or -NH(C C6)cycloalkyl or (C3-C6)heterocycloalkyl;

■ R10 represents at least one substituent of the phenyl nucleus chosen from a hydrogen atom and (CrC4)alkyl;

■ W represents

• (Ci-C6)alkyl-NH(Rn),

• (CrC6)alkyl-OH,

• (CrC6)alkyl-SH,

· C02H or C(=0)NH2;

• (CrC6)alkyl-C02H or (C C6)alkyl-C(=0)NH2; or

• (CrC6)alkyl-N3.

■ R11 and R12 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group.

2. Cryptophycin compound of formula (I) according to claim 1 , having the following structure:

3. Cryptophycin compound of formula (I) according to any one of claim 1 or 2, characterized in that Ri represents a methyl group.

4. Cryptophycin compound of formula (I) according to any one of claims 1 to 3, characterized in that each of R2 and R3 represents a hydrogen atom.

5. Cryptophycin compound of formula (I) according any one of claims 1 to 3, characterized in that one of R2 and R3 represents a methyl group and the other one represents a hydrogen atom.

6. Cryptophycin compound of formula (I) according to any one of claims 1 to 3, characterized in that R2 and R3 form together with the carbon atom to which they are attached a cyclopropyl group.

7. Cryptophycin compound of formula (I) according to any one of claims 1 to 6, characterized in that each of R4 and R5 represents a methyl group.

8. Cryptophycin compound of formula (I) according to any one of claims 1 to 7, characterized in that R6 represents a hydrogen atom.

9. Cryptophycin compound of formula (I) according to any one of claims 1 to 8, characterized in that R7 and Re represent independently of each other a hydrogen atom or a (C C6)alkyl group.

10. Cryptophycin compound of formula (I) according to any one of claims 1 to 9, characterized in that R9 represents two substituents selected from a methoxy group and a chlorine atom.

1 1 . Cryptophycin compound of formula (I) according to any one of claims 1 to 10, characterized in that R10 represents a hydrogen atom.

12. Cryptophycin compound of formula (I) according to any one of claims 1 to 1 1 , characterized in that:

■ Ri represents a (CrC6)alkyl;

■ each of R2 and R3 represents a hydrogen atom;

■ R6 represents a hydrogen atom;

■ Rg represents two substituents selected from a methoxy group and a chlorine atom; and R10 represents a hydrogen atom.

13. Cryptophycin compound of formula (I) according to any one of claims 1 to 1 1 , characterized in that:

■ Ri represents a (C1-C6)alkyl;

■ R2 and R3 represents a (C1-C6)alkyl, and the other one represents a hydrogen atom;

■ R6 represents a hydrogen atom;

■ Rg represents two substituents selected from a methoxy group and a chlorine atom; and

■ R10 represents a hydrogen atom.

14. Cryptophycin compound of formula (I) according to any one of claims 1 to 13, characterized in that W is selected from (Ci-C6)alkyl-NHRn , (C C6)alkyl-OH, (C C6)alkyl-SH and (C C6)alkyl-C02H.

15. Cryptophycin compound of formula (I) according to claim 14, characterized in that W represents a CH2-NH2 or a CH2-OH group.

16. Cryptophycin compounds of formula (I) according to claim 1 which are selected from the following list:

17. Cryptophycin payload of formula (II):

wherein

Ri , R2, R3, R4, R5, R6, R7, Re, R9 and R10 are as defined in claim 1 to 13, and

■ Y is selected from (Ci-C6)alkyl-N Rn , (C C6)alkyl-0, (C C6)alkyl-S, C(=0)0, C(=0)NH, (C C6)alkyl-C(=0)0, (C C6)alkyl-C(=0)NH or (C C6)alkyl-triazole group;

■ R11 represents a hydrogen atom or a (CrC6)alkyl group;

■ L represents a linker;

■ RCG1 represents a reactive chemical group present at the end of the linker.

18. Cryptophycin payl llowing structure:

19. Cryptophycin payload of formula (II) according to any one of claim 17 or 18, characterized in that Y represents (Ci-C6)alkyl-NRn.

20. Cryptophycin payload of formula (II) according to claim 19, characterized in that Y represents CH2-NH.

21 . Cryptophycin payload of formula (II) according to any one of claims 17 to 20, characterized that L is of formula (IV):

in which:

■ L1 represents

■ a single bond or a N R16(hetero)aryl-CRi5Ri4-0-C(=0) group if Y = (CrC6)alkyl- N(Rn );

■ a N R18-(C2-C6)alkyl-NR17-C(=0) group or a N R16(hetero)aryl-CRi5Ri4-0-C(=0)- N Ri8-(C2-C6)alkyl-N R17-C(=0) group if Y = (C C6)alkyl- O or (C C6)alkyl- S;

■ a NR16(hetero)aryl-CRi5Ri4- group if Y = C(=0)0, C(=0)NH, (C C6)alkyl-C(=0)0 or (CrC6)alkyl-C(=0)NH;

■ Rii , Ri4, i5, Ri6, Ri7 and Ri8 represent, independently of each other, a hydrogen atom or a (CrC6)alkyl group;

■ (AA)W represents a sequence of w amino acids AA connected together via peptide bonds;

■ w represents an integer ranging from 1 to 12;

■ L2 represents a single bond or a (CrC6)alkyl group or a (Ci-C6)alkyl-(OCH2CH2)i group or a (Ci-C6)alkyl-(OCH2CH2)rO(Ci-C6)alkyl group or a (CH2CH20)i(Ci-C6)alkyl group or a CH(S03H)-(CrC6)alkyl group or a (Ci-C6)alkyl-CH(S03H) group or a (C C6)alkyl-cyclohexyl group or a NR19-(CrC6)alkyl group or a NR2o-(CH2CH20)i(Ci-C6)alkyl group or a NR21-aryl group or a NR21-heteroaryl group or a (Ci-C6)alkyl-NR22C(=0)-(Ci-C6)alkyl group or a(C C6)alkyl-NR22C(=0)-(Ci-C6)alkyl-(OCH2CH2)i group;

■ Rig, R2o, R21 and R22 represent, independently of each other, a hydrogen atom or a (Cr C6)alkyl group;

■ i represents an integer between 1 and 50 ;

AA denotes a natural or unnatural amino acid, of configuration D or L, chosen from: alanine (Ala), β-alanine, γ-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), α,α-dimethyl-γ-aminobutyric acid, β,β-dimethyl-γ-aminobutyric acid, glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), lysine (Lys), ε-acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val).

22. Cryptophycin payload of formula (II) according to claim 21 , characterized in that the sequence (AA)W has the formula:

in which R23 represents the side chain of AA.

23. Cryptophycin payload of formula (II) according to any one of claim 21 or 22, characterized in that the sequence AA represents alanine (Ala), citrulline (Cit), glutamine (Gin), glycine (Gly), ε-acetyl-lysine (AcLys), valine (Val).

24. Cryptophycin payload of formula (II) according to any one of claims 17 to 23, characterized in that RCG1 is chosen from:

- -C(=0)-ZaRa group for which

■ Za represents a single bond, O or NH, and

Ra represents H or a (CrC6)alkyl, (C3-C7)cycloalkyl, (C5-Ci0)aryl, Cio)heteroaryl or (C3-C7)heterocycloalkyl group or a succinimidyl group;

lowing reactive groups: the maleimido group; the haloacetamido

roup with R13 representing a hydrogen atom or a (CrC6)alkyl group; -CI; -N3;

group or a 0-(CrC6)alkyl hydroxylamine group.

25. Cryptophycin payload of formula (II) according to any one of claims 17 to 23, characterized in that L2 represents a (C1-C6)alkyl group or a (C1-C6)alkyl-(OCH2CH2)i group or a CH(S03H)-(C1-C6)alkyl group

26. Cryptophycin payloads of formula (II) according to claim 17, which are selected from the following list:

27. Conjugate of fo

wherein

■ Ri, R2, R3, R4, R5, R6, R7, Re, R9 and R10 are as defined in claim 1 to 13;

■ Y and L are as defined in claims 17 to 23;

■ G represents the product of reaction between RCG1 , a reactive group present at the end of the linker and RCG2, an orthogonal reactive group present on Ab;

■ Ab represents an antibody.

28. Conjugate of for ure:

29. Conjugate of formula (III) according to any one of claim 27 or 28, characterized in that RCG2 is selected from:

• ε-amino groups of lysines borne by the side chains of the lysine residues that are present at the surface of an antibody;

• oamino groups of N-terminal amino acids of antibody heavy and light chains;

• saccharide groups of the hinge region;

• thiols of cysteines generated by reducing intra-chain disulfide bonds or the thiols of engineered cysteines;

• amide groups borne by the side chains of some glutamine residues that are present at the surface of an antibody;

• aldehyde groups introduced using formylglycine generating enzyme.

30. Conjugate of formula (III) according to any one of claim 27 or 28, characterized in that:

• when RCG1 represents a N-hydroxysuccinimidyl ester, RCG2 represents a -NH2 group;

• when RCG1 represents a maleimido or haloacetamido function or a -CI group, RCG2 represents a -SH group;

• when RCG1 represents a -N3 group, RCG2 represents a -C≡CH group or an activated C≡C;

• when RCG1 represents a -OH or -NH2 group, RCG2 represents a carboxylic acid or amide function;

• when RCG1 represents a -SH group, RCG2 represents a maleimido or haloacetamido function;

· when RCG1 represents a -C≡CH function or an activated C≡C, RCG2 represents a -N3 group;

• when RCG1 represents a O-alkyI hydroxylamine function or a Pictet-Spengler reaction substrate, RCG2 represents an aldehyde or ketone function.

31. Conjugate of formula (III) according to any one of claims 27 to 30, characterized in that G is selected from:

32. Process for preparing the cryptophycin compound of formula (I) comprising the step:

wherein:

- Step (i) is a peptidic coupling between fragments AD3 and alternative BC in the presence of coupling reagents;

- Step (ii) is a macrocyclization by ring closing metathesis in the presence of a catalyst;

- Step (iii) is a deprotection of the p-methoxybenzyl ether in acidic conditions;

- Step (iv) is an oxidation of the alcohol using an oxidizing agent;

- Step (v) is an introduction of the epoxide by asymmetric Corey-Chaykovsky reaction using appropriately substituted isothiocineole-derived chiral sulfonium in the presence of a base;

- Step (vi) is a reduction of the azido group.

33. Process for preparing a conjugate of formula (III) comprising the steps of:

(i) placing in contact and leaving to react:

- an optionally buffered aqueous solution of an antibody, optionally modified by means of a modifying agent,

and

- a solution of a cryptophycin payload of formula (II) as defined in one of claims 17 to 26, the chemical group RCG1 of the cryptophycin payloadof formula (II) being reactive towards the chemical groups RCG2 present on the antibody especially towards the amino groups present on antibodies, the said chemical groups RCG2 having been introduced, where appropriate, by the modifying agent,

so as to attach the cryptophycin payload of formula (II) to the antibody by formation of a covalent bond;

(ii) and then optionally to separate the conjugate formed in step (i) from the cryptophycin payload of formula (II) and/or from the unreacted antibody and/or from any aggregates that may have formed.

34. Medicament, characterized in that it comprises a conjugate of formula (III) according to any one of claims 27 to 31.

35. Pharmaceutical composition, characterized in that it comprises a conjugate of formula (III) according to any one of claims 27 to 31 , and also at least one pharmaceutically acceptable excipient.

36. Use of the cryptophycin compound of formula (I) according to any one of claims 1 to 16 as an anticancer agent.

37. Use of the conjugate of formula (III) according to any one of claims 27 to 31 as an anticancer agent.

38. Cryptophycin compound of formula (I) according to any one of claims 1 to 16 for use in the treatment of cancer.

39. Conjugate of formula (III) according to any one of claims 27 to 31 for use in the treatment of cancer.