BR112018008490B1 - CRYPTOPHYCIN COMPOUNDS AND CONJUGATES, PROCESSES FOR THE PREPARATION THEREOF, MEDICINE AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

CRYPTOPHYCIN COMPOUNDS AND CONJUGATES, PROCESSES FOR THE PREPARATION THEREOF, MEDICINE AND PHARMACEUTICAL COMPOSITION. The present invention relates to cryptophycin compounds of formula (I): The invention also relates to cryptophycin payloads, cryptophycin conjugates, compositions containing them and their therapeutic use, especially as anticancer agents. The invention also relates to the process for preparing these conjugates.

Description

[0001] The present invention relates to new cryptophycin compounds, new cryptophycin payloads, new cryptophycin conjugates, compositions containing them and their therapeutic use, especially as anticancer agents. The invention also relates to the process for preparing these conjugates.

[0002] Cryptophycins are secondary metabolites that belong 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 the cyanobacterium N o sto c sp (ATCC 53789; see Eißler S., et al., Synthesis 2006, 22, 3747-3789) . Cryptophycins C-1 and C-52, which are characterized by epoxide function, were found to have antitumor activity in vitro and in vivo in the early 1990s. Their chlorohydrins, C-8 and C-55, were markedly more active, but could not be formulated as stable solutions (Bionpally RR, et al., Cancer Chemother Pharmacol 2003, 52, 2533). Its high activity was correlated by its putative ability to generate the corresponding epoxides in cells. With no method to adequately stabilize chlorohydrins at this time, cryptophycin C-52 (LY355703) entered clinical trials, produced marginal antitumor activity in two phase II cancer trials with 30 to 40% stable disease, and was, therefore discontinued (Edelman MJ, et al., Lung Cancer 2003, 39, 197-99 and Sessa C., et al., Eur J Cancer 2002, 38, 2388-96).

[0003] Considering its high potency and common mechanism of action with maytansinoids and auristatins, the two clinically validated cytotoxic molecules for antibody-drug conjugates (CAF), this series was considered as a potential tubulin ligand for CAF. Therefore, conjugates in the cryptophycin series were developed by starting from the derivation at the para-benzylic position of the macrocycle (AlAwar R.S., et al., J Med Chem 2003, 46, 2985-3007).

[0004] WO2011/001052 describes cryptophycin antibody-drug conjugates to which the cytotoxic moiety is linked to the antibody through the para-benzylic position using various species of linkers. They may be cleavable, disulfide or protease sensitive, or non-cleavable.

[0005] Another optimization of cryptophycin antibody-drug conjugates described in WO2011/001052 leads to potent cryptophycin conjugates that exhibited promising antitumor activity, but were found to be 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 stability properties.

[0006] The purpose of this invention is to propose new cryptophycin macrocycles, which once conjugated to an antibody are stable in mouse plasma, and new cryptophycin conjugates composed of those stable macrocycles.

Summary of the Invention

[0007] In this regard, the invention relates to the cryptophycin compounds of formula (I): where: Ri represents a (C1-C6) alkyl group; R2 and R3 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group; or alternatively R2 and R3 form together with the carbon atom to which they are attached a (C3-C6) cycloalkyl or (C3-C6) heterocycloalkyl group; R4 and R5 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group or a (C1-C6) alkyl-NH(R12) group or a (C1-C6) alkyl-OH group or a (C1-C6) alkyl-SH group or a (C1-C6) alkyl-CO2H group; or alternatively R4 and R5 form together with the carbon atom to which they are attached a (C3-C6) cycloalkyl or (C3-C6) heterocycloalkyl group; R6 represents a hydrogen atom or a (C1-C6) alkyl group; R7 and R8 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group or a (C1-C6) alkyl-CO2H group or a (C1-C6) alkyl-N(C1-C6) group alkyl2; or alternatively R7 and R8 form together with the carbon atom to which they are attached a (C3-C6)cycloalkyl group or a (C3-C6)heterocycloalkyl group; R9 represents one or more substituents of the phenyl nucleus chosen, independently of each other, from: a hydrogen atom, -OH, (C1-C4)alkoxy, a halogen atom, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl2, -NH(C1-C6)cycloalkyl or (C3-C6)heterocycloalkyl; Rio represents at least one phenyl nucleus substituent chosen from a hydrogen atom and a (C1-C4)alkyl group; W represents (C1-C6)alkyl-NH(Rii), more particularly (CH2)nNHRii; (C1-C6)alkyl-OH, more particularly (CH2)nOH; (C1-C6)alkyl-SH, more particularly (CH2)nSH; CO2H or C(=O)NH2; (C1-C6)alkyl-CO2H or (C1-C6)alkyl-C(=O)NH2; or (C1-C6)alkyl-N3. W being positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus; R11 and R12 represent, independently of each other, a hydrogen or (C1-C6)alkyl atom, more particularly a methyl group; n represents an integer between 1 and 6.

[0008] The invention also relates to cryptocurrency payloads of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in formula (I); Y represents (C1-C6)alkyl-NR11 or (C1-C6)alkyl-O or (C1-C6)alkyl-S; or alternatively Y represents C(=O)O, C(=O)NH, (C1-C6)alkyl-C(=O)O or (C1-C6)alkyl-C(=O)NH; or alternatively Y represents (C1-C6)alkyl-triazole- as Y being positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus; R11 represents a hydrogen atom or a (C1-C6) alkyl group; L represents a ligand; RCG1 represents a reactive chemical group present at the end of the linker, RCG1 being reactive towards a reactive chemical group present in an antibody.

[0009] The invention also refers to conjugates of formula (III): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in formula (I); Y and L are as defined in formula (II); G represents the reaction product between RCG1, a reactive chemical group present at the end of the ligand, and RCG2, an orthogonal reactive chemical group present on the antibody (Ab); Ab represents an antibody.

[00010] Each substituent R1 to R11 can also adopt one of the spatial configurations (for example, R or S or alternatively Z or E) as described in the examples.

[00011] Compounds of formula (I), (II) and (III) may contain one or more asymmetric carbon atoms. They can therefore exist in the form of enantiomers or diastereomers. These enantiomers and diastereoisomers, and also mixtures thereof, including racemic mixtures, form part of the invention.

[00012] The compounds of formula (I), including those illustrated, may exist in the form of acid or base addition salts, especially of pharmaceutically acceptable acids.

Definitions

[00013] In the context of the present invention, certain terms have the following definitions: • Alkenyl group: a hydrocarbon group obtained by removing a hydrogen atom from an alkene. The alkenyl group can be linear or branched. Examples that may be mentioned include ethenyl (-CH=CH2, also called vinyl) and propenyl (-CH2- CH=CH2, also called allyl). • Alkoxy group: an -O-alkyl group, where 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, tert-butyl, pentyl, neopentyl, 2,2-dimethylpropyl and hexyl groups; • Alkylene group: a saturated divalent group with empirical formula -CnH2n-, obtained by removing two hydrogen atoms from an alkane. The alkylene group can 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 A ; Preferably, the alkylene group is of the formula -(CH2)n-, n representing an integer; in value ranges, limits are included (for example, a range of type "n ranging from 1 to 6" or "between 1 and 6" includes the limits of 1 and 6); • Antibody: an antibody with affinity for the biological target, more particularly a monoclonal antibody. The function of the antibody is to direct the biologically active compound as a cytotoxic compound to 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" can 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-size antibody"). "). The terms "conventional (or full-size) antibody" refer to both an antibody comprising the signal peptide (or propeptide, if any), and the mature form obtained in the secretion and proteolytic processing of the chain(s). There are two types of light chains, lambda (l) and kappa (k). There are five main heavy chain classes (or isotypes) that 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 antigen specificity. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, association, secretion, trans-placental mobility, complement binding, and Fc receptor (FcR) binding. . The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of a light chain and a heavy chain. Antibody specificity 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 non-hypervariable or structural regions (FR) influence the global domain structure and, consequently, the combining site. Complementarity-Determining Regions or CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the natural Fv region of an 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 group of each heavy and light chain V region.

[00014] As used herein, the term "antibody" means both conventional (natural-size) antibodies and fragments thereof, as well as fragments and single-domain antibodies thereof, in particular, variable heavy chain of single-domain antibodies. Antibody fragments (conventional) typically comprise a portion of an intact antibody, in particular the antigen-binding region or variable region of the intact antibody, and maintain 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.

[00015] The function of the antibody is to direct the biologically active compound as a cytotoxic compound to the biological target. • Aryl group: a cyclic aromatic group containing between 5 and 10 carbon atoms. Examples of aryl groups include phenyl, tolyl, xylyl, naphthyl; • Biological target: an antigen (or group of antigens) preferably located on the surface of cancer cells or stromal cells associated with this tumor; these antigens may be, for example, a growth receptor, an oncogene product or a mutated "tumor suppressor" gene product, an angiogenesis-related molecule or an adhesion molecule; • conjugate: an antibody-drug conjugate or ADC, that is, an antibody to which at least one molecule of a cytotoxic compound is covalently linked via a linker; • cycloalkyl group: a cyclic alkyl group comprising between 3 and 6 carbon atoms committed to 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 linked 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 and 10 carbon atoms and between 1 and 5 heteroatoms such as nitrogen, oxygen or sulfur committed to 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 and 8 carbon atoms and between 1 and 3 heteroatoms, such as nitrogen, oxygen and sulfur committed to 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 link 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 a ligand is covalently linked; • reactive chemical group: group of atoms that can promote or undergo a chemical reaction.

abbreviations

[00016] CAF: antibody-drug conjugate; AcOH: acetic acid; AIBN: azobisisobutyronitrile; ALQ: (C1-C12)alkylene group, more particularly (C1-C6)alkylene, more particularly of the form - (CH2)n-, n being an integer from 1 to 12 and preferably from 1 to 6; aq.: aqueous; Air: argon; AUC: area under the curve; BEMP: 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine; BF3: boron trifluoride; Boc2O: di-tert-butyl-dicarbonate; BuLi: butyl lithium; CAN: ceric ammonium nitrate; Cbz: carboxybenzyl; CHCl3: chloroform; CH3CN: acetonitrile; CH3I: methyl iodide; CO2: carbon dioxide; CL: clearance; m-CPBA: m-chloroperbenzoic acid; CR: complete response; crypto means the formula unit , crypto especially denoting one of the cryptophycin compounds D1D19 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-diazabicyclo [5.4.0]undec-7-ene; DCC: N,N'-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's Medium; DMEM/F12: Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12; DMF: dimethylformamide; DMSO: dimethyl sulfoxide; DPBS: Dulbecco's phosphate-buffered saline; DPPA: diphenylphosphorazide (PhO)2P(=O)N3; DSC: N,N'-disuccinimidyl carbonate; EDA: ethylenediamine; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; EDTA: ethylenediaminetetraacetic acid; EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; ELSD: evaporative light scattering detector; eq.: equivalent; ES: electrovaporization; EtOAc: ethyl acetate; Et2O: diethyl ether; EtOH: ethanol; E.g.: example; FCS: fetal calf serum; Fmoc: 9-fluorenylmethoxycarbonyl; FmocOSu: N-(9-fluorenylmethoxycarbonyloxy) succinimide; GI: electroconductive group; Grubbs I: benzylidene-bis(tricyclohexylphosphine)dichlororuthenium; Grubbs II: (1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(phenylmethylene) (tricyclohexylphosphine)ruthenium; h: time; H2O: water; Hal: halogen; HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HCl: 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 chromatography; EMAR: high resolution mass spectrometry; IC50: average inhibitory concentration; ie: id est meaning that is; IEC: ion exchange chromatography; iPrOH: 2-propanol; iPr2O: diisopropyl ether; iv: intravenous; K2CO3: potassium carbonate; LDA: lithium diisopropylamide; LiOH: lithium hydroxide; marg.: marginally; MeOH: methanol; MeTHF: 2-methyl-tetrahydrofuran; MgSO4: magnesium sulfate; min: minute; MNBA: 2-methyl-6-nitrobenzoic anhydride; MTBE: methyl tert-butyl ether; DMT: maximum tolerated dose; NaH: sodium hydride; NaHSO4: sodium bisulfate; Na2S2O3: sodium thiosulfate; NaCl: sodium chloride; NaHCO3: sodium hydrogen carbonate; NaHSO3: sodium disulfide; NaHSO4: sodium hydrogen sulfate; NaOH: sodium hydroxide; NH4Cl: ammonium chloride; NHS: N-hydroxysuccinimide; NMP: 1-methyl-2-pyrrolidone; NMR: nuclear magnetic resonance; P2-Et: 1-ethyl-2,2,4,4,4-pentacyls (dimethylamino)- 2À5,4À5-catenadi(phosphazene); PBS: phosphate buffered saline; PEG: polyethylene glycol; PK: pharmacokinetics; PMB: para-methoxybenzyl; PNGase F: Peptide-N-Glycosidase F; PPh3: triphenylphosphine; ppm: parts per million; PR: partial response; QS: quantum satis meaning the quantity that is needed; Q-Tof: quadrupole trajectory time; quantity: quantitative production; RCG: reactive chemical group; RT: room temperature; sat.: saturated; sc: subcutaneously; SCID: severe combined immunodeficiency; SEC: size exclusion chromatography; TBAF: tetrabutylammonium fluoride; tBuOK: potassium tert-butoxide; TCEP: tris(2-carboxyethyl)phosphine hydrochloride; TEA: triethylamine; TIME: (2,2,6,6-tetramethylpiperidin-1-yl)oxyl TFA: trifluoroacetic acid; TFS: tumor-free survivor; THF: tetrahydrofuran; THP: tetrahydropyran; TLC: thin layer chromatography; t1/2: half life; tR: retention time; p-TsOH: para-toluenesulfonic acid; UPLC: Ultra Performance Liquid Chromatography; UV: ultraviolet; 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 11: 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 xenograft -231 in SCID mice Figure 13: In vivo PK profile of ADC1 following a single iv administration in SCID mice Figure 14: In vivo PK profile of ADC1 following a single iv administration in non-rodent species Figure 15: In vivo PK profile of Ex.3 after a single iv administration in SCID mice Figure 16: In vivo PK profile of Ex.7 after a single iv administration in SCID mice Figure 17: EMAR spectrum of deglycolyzed ADC1 at 96 hours after single iv administration in mice SCID Figure 18: EMAR spectrum of ADC2 light chain at 96 hours after single iv administration in mice SCID Figure 19: EMAR spectrum of deglycolyzed ADC1 at 6 days after single iv administration in non-rodent species Figure 20: EMAR spectrum of Ex.3 at 96 hours after single iv administration in SCID mice Figure 21: EMAR spectrum of Ex.7 at 96 hours after single iv administration in SCID mice Figure 22: EMAR spectrum of Ex.10 at 96 hours after simple iv administration in SCID mice Figure 23: EMAR spectrum of Ex.14 at 96 hours after single iv administration in SCID mice Figure 24: EMAR spectrum of Ex.20 at 96 hours after simple iv administration in SCID mice Figure 25: EMAR spectrum of Ex.23 at 96 hours after single iv administration in SCID mice

Detailed Description of the Invention

[00017] The invention relates to cryptophycin compounds of formula (I): where: Ri represents a (C1-C6) alkyl group; R2 and R3 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group; or alternatively R2 and R3 form together with the carbon atom to which they are attached a (C3-C6) cycloalkyl or (C3-C6) heterocycloalkyl group; R4 and R5 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group or a (C1-C6) alkyl-NH(R12) group or a (C1-C6) alkyl-OH group or a (C1-C6) alkyl-SH group or a (C1-C6) alkyl-CO2H group; or alternatively R4 and R5 form together with the carbon atom to which they are attached a (C3-C6) cycloalkyl or (C3-C6) heterocycloalkyl group; R6 represents a hydrogen atom or a (C1-C6) alkyl group; R7 and R8 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group or a (C1-C6) alkyl-CO2H group or a (C1-C6) alkyl-N(C1-C6) group alkyl2; or alternatively R7 and R8 form together with the carbon atom to which they are attached a (C3-C6)cycloalkyl group or a (C3-C6)heterocycloalkyl group; R9 represents one or more substituents of the phenyl nucleus chosen, independently of each other, from: a hydrogen atom, -OH, (C1-C4) alkoxy, a halogen atom, -NH2 group, - NH(C1-C6) alkyl or -N(C1-C6)alkyl2 or -NH(C1-C6)cycloalkyl or (C3-C6)heterocycloalkyl; R10 represents at least one substituent of the phenyl nucleus chosen from a hydrogen atom and a (C1-C4)alkyl group; W represents • (C1-C6)alkyl-NH(Rii), more particularly (CH2)nNHRii; • (C1-C6)alkyl-OH, more particularly (CH2)nOH; (C1-C6)alkyl-SH, more particularly (CH2)nSH; • CO2H or C(=O)NH2; • (C1-C6)alkyl-CO2H or (C1-C6)alkyl-C(=O)NH2; or • (C1-C6)alkyl-N3.

[00018] W is positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus; • R11 and R12 represent, independently of each other, a hydrogen atom or a (C1-C6)alkyl group, more particularly a methyl group; • n represents an integer between 1 and 6.

[00019] The cryptophycin compound can be one of the following D1-D19: where: • W and Ri2 are as defined in formula (I); or the cryptophycin compound may be an equivalent unit described in one of the examples.

[00020] Among the compounds of formula (I) which are the subject matter of the invention, a first group of compounds is composed of compounds for which R1 represents a (C1-C6)alkyl group, in particular a methyl group.

[00021] Among the compounds of formula (I) that are the subject matter of the invention, a second group of compounds is composed of compounds for which each of R2 and R3 represents a hydrogen atom.

[00022] Among the compounds of formula (I) which are the subject matter of the invention, a third group of compounds is composed of compounds for which one of R2 and R3 represents a (C1-C6)alkyl group, in particular, methyl , and the other represents a hydrogen atom.

[00023] Among the compounds of formula (I) which are the subject matter of the invention, a fourth group of compounds is composed of the compounds to which R2 and R3 form together with the carbon atom to which they are attached a group (C3 -C6)cycloalkyl, in particular a cyclopropyl group.

[00024] Among the compounds of formula (I) which are the subject matter of the invention, a fifth group of compounds is composed of compounds for which each of R4 and R5 represents a (C1-C6)alkyl group, in particular a methyl group.

[00025] Among the compounds of formula (I) that are the subject matter of the invention, a sixth group of compounds is composed of compounds for which R6 represents a hydrogen atom.

[00026] Among the compounds of formula (I) which are the subject matter of the invention, a seventh group of compounds is composed of compounds for which R7 and R8 represent independently of each other a hydrogen atom or a group (C1-C6 ) alkyl.

[00027] Among the compounds of formula (I) which are the subject matter of the invention, an eighth group of compounds is composed of compounds for which R9 represents two substituents selected from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents at positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-Cl and 4-methoxy.

[00028] Among the compounds of formula (I) that are the subject matter of the invention, a ninth group of compounds is composed of compounds for which R10 represents a hydrogen atom.

[00029] Among the compounds of formula (I) that are the subject matter of the invention, a tenth group of compounds is composed of compounds for which W is positioned in the para position of the phenyl nucleus.

[00030] Among the compounds of formula (I) which are the subject matter of the invention, an eleventh group of compounds is composed of the compounds for which W is selected from (C1-C6)alkyl-NHR11, in particular (C1- C3)alkyl-NHR11, particularly a CH2-NH2 group, (C1-C6)alkyl-OH, in particular (C1-C3)alkyl-OH, particularly a CH2-OH group, (C1-C6)alkyl-SH, in in particular (C1-C3)alkyl-SH and (C1-C6)alkyl-CO2H, in particular (C1-C3)alkyl-CO2H.

[00031] Among the compounds of formula (I) which are the subject matter of the invention, a twelfth group of compounds is composed of compounds for which R1 represents a (C1-C6)alkyl group, in particular a methyl group, each one 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 at positions 3 and 4 on the phenyl nucleus , preferably, it is 3-Cl and 4-methoxy and R10 represents a hydrogen atom.

[00032] Among the compounds of formula (I) which are the subject matter of the invention, a thirteenth group of compounds is composed of compounds for which R1 represents a (C1-C6)alkyl group, in particular a methyl group, R2 and R3 represents a (C1-C6)alkyl group, in particular a methyl group, and the other represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, further particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-Cl and 4-methoxy and R10 represents a hydrogen atom.

[00033] Alternatively, W is selected from (CH2)nNHR11, (CH2)nSH from (CH2)nOH, where n represents an integer between 1 and 6.

[00034] Among the compounds of formula (I) that are the subject matter of the invention, a fourteenth group of compounds is composed of compounds of the following structure (beta epoxide configuration):

[00035] All of these subgroups considered alone or in combination are part of the invention.

[00036] Among the compounds of formula (I) which are the subject matter of the invention, mention may be made in particular of the following compounds:

[00037] The invention also relates to cryptocurrency payloads of formula (II): where: • Ri, R2, R3, R4, R5, R6, R7, R7, R9 and R10 are as defined in formula (I); Y represents (C1-C6)alkyl-NR11 or (C1-C6)alkyl-O or (C1-C6)alkyl-S; or alternatively Y represents C(=O)O, C(=O)NH, (C1-C6)alkyl-C(=O)O or (C1-C6)alkyl-C(=O)NH; or alternatively Y represents (C1-C6)alkyl-triazole- as IY is positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus; Rii represents a hydrogen atom or a (C1-C6) alkyl group; L represents a ligand; RCGi represents a reactive chemical group present at the end of the linker, RCGi being reactive to a reactive chemical group present in an antibody.

[00038] Among the compounds of formula (II) which are the subject matter of the invention, a first group of compounds is composed of compounds for which Ri represents a (C1-C6)alkyl group, in particular a methyl group.

[00039] Among the compounds of formula (II) that are the subject matter of the invention, a second group of compounds is composed of compounds for which each of R2 and R3 represents a hydrogen atom.

[00040] Among the compounds of formula (II) which are the subject matter of the invention, a third group of compounds is composed of compounds for which one of R2 and R3 represents a (C1-C6) alkyl group, in particular a group methyl and the other represents a hydrogen atom.

[00041] Among the compounds of formula (II) which are the subject matter of the invention, a fourth group of compounds is composed of compounds to which R2 and R3 form together with the carbon atom to which they are attached a group (C3 -C6)cycloalkyl, in particular a cyclopropyl group.

[00042] Among the compounds of formula (II) which are the subject matter of the invention, a fifth group of compounds is composed of compounds for which each of R4 and R5 represents a (C1-C6) alkyl group, in particular a methyl group.

[00043] Among the compounds of formula (II) that are the subject matter of the invention, a sixth group of compounds is composed of compounds for which R6 represents a hydrogen atom.

[00044] Among the compounds of formula (II) which are the subject matter of the invention, a seventh group of compounds is composed of compounds for which R7 and R8 represent independently of each other a hydrogen atom or a group (C1-C6 ) alkyl.

[00045] Among the compounds of formula (II) that are the subject matter of the invention, an eighth group of compounds is composed of compounds for which R9 represents two substituents selected from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents at positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-Cl and 4-methoxy.

[00046] Among the compounds of formula (II) that are the subject matter of the invention, a ninth group of compounds is composed of compounds for which R10 represents a hydrogen atom.

[00047] Among the compounds of formula (II) that are the subject matter of the invention, a tenth group of compounds is composed of compounds for which Y is positioned in the para position of the phenyl nucleus.

[00048] Among the compounds of formula (II) which are the subject matter of the invention, an eleventh of compounds are composed of compounds for which Y represents (C1-C6)alkyl-NR11 in particular (C1-C3)alkyl- NR11 more particularly CH2-NH.

[00049] Among the compounds of formula (I) which are the subject matter of the invention, a twelfth group of compounds is composed of compounds for which R1 represents a (C1-C6)alkyl group, in particular a methyl group, each one 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 at positions 3 and 4 on the phenyl nucleus , preferably, it is 3-Cl and 4-methoxy and R10 represents a hydrogen atom.

[00050] Among the compounds of formula (I) which are the subject matter of the invention, a thirteenth group of compounds is composed of compounds for which R1 represents a (C1-C6)alkyl group, in particular a methyl group, R2 and R3 represents a (C1-C6)alkyl group, in particular a methyl group, and the other represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, further particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-Cl and 4-methoxy and R10 represents a hydrogen atom.

[00051] Among the compounds of formula (II) that are the subject matter of the invention, a fourteenth group of compounds is composed of compounds of the following structure (beta epoxide configuration):

[00052] All of these subgroups considered alone or in combination are part of the invention.

[00053] Among the compounds of formula (II) which are the subject matter of the invention, mention may be made in particular of the following compounds:

[00054] The invention also refers to conjugates of formula (III): R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in formula (I); Y and L are as defined in formula (II); G represents the reaction product between RCGi, a reactive chemical group present at the end of the ligand, and RCG2, an orthogonal reactive chemical group present in the antibody (Ab); Ab represents an antibody.

[00055] Among the compounds of formula (III) which are the subject matter of the invention, a first group of compounds is composed of compounds for which R1 represents a (C1-C6)alkyl group, in particular a methyl group.

[00056] Among the compounds of formula (III) that are the subject matter of the invention, a second group of compounds is composed of compounds for which each of R2 and R3 represents a hydrogen atom.

[00057] Among the compounds of formula (III) which are the subject matter of the invention, a third group of compounds is composed of compounds for which one of R2 and R3 represents a (C1-C6) alkyl group, in particular a group methyl and the other represents a hydrogen atom.

[00058] Among the compounds of formula (III) which are the subject matter of the invention, a fourth group of compounds is composed of compounds to which R2 and R3 form together with the carbon atom to which they are attached a group (C3 -C6)cycloalkyl, in particular a cyclopropyl group.

[00059] Among the compounds of formula (III) which are the subject matter of the invention, a fifth group of compounds is composed of compounds for which each of R4 and R5 represents a (C1-C6) alkyl group, in particular a methyl group.

[00060] Among the compounds of formula (III) that are the subject matter of the invention, a sixth group of compounds is composed of compounds for which R6 represents a hydrogen atom.

[00061] Among the compounds of formula (III) which are the subject matter of the invention, a seventh group of compounds is composed of compounds for which R7 and R8 represent independently of each other a hydrogen atom or a group (C1-C6 ) alkyl.

[00062] Among the compounds of formula (III) that are the subject matter of the invention, an eighth group of compounds is composed of compounds for which R9 represents two substituents selected from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents at positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-Cl and 4-methoxy.

[00063] Among the compounds of formula (III) that are the subject matter of the invention, a ninth group of compounds is composed of compounds for which R10 represents a hydrogen atom.

[00064] Among the compounds of formula (II) that are the subject matter of the invention, a tenth group of compounds is composed of compounds for which Y is positioned in the para position of the phenyl nucleus.

[00065] Among the compounds of formula (III) which are the subject matter of the invention, an eleventh of compounds are composed of compounds for which Y represents (C1-C6)alkyl-NR11 in particular (C1-C3)alkyl- NR11 more particularly CH2-NH.

[00066] Among the compounds of formula (I) that are the subject matter of the invention, a twelfth group of compounds is composed of compounds of the following structure (beta epoxide configuration):

[00067] All of these subgroups considered alone or in combination are part of the invention.

[00068] Among the compounds of formula (III) which are the subject matter of the invention, mention may be made in particular of the following compounds:

[00069] The link between the cryptophycin payload of formula (II) and the antibody, in order to obtain the conjugate of formula (III), is produced through a reactive chemical group RCG1 present in the payload that is reactive to an RCG2 reactive group present in the antibody. The reaction between RCG1 and RCG2 guarantees the binding of the compound of formula (II) to the antibody by forming a covalent bond. In the conjugate of formula (III), parts of RCG1 and RCG2 may remain forming the bond between the ligand and the antibody.

[00070] Examples of RCG1 that may be mentioned include: (i) The reactive -C(=O)-ZaRa group for which Za represents a single bond, O or NH, more particularly O, and Ra represents a hydrogen atom or a (C1-C6)alkyl, (C3-C7)cycloalkyl, alkenyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl group. The aryl group can be replaced by 1 to 5 chosen halogen groups, 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 (C1-C6) alkyl group, more particularly Me; -Cl; -N3; -OH; -SH; -NH2; -C?CH or an activated C?C, such as a cyclooctin moiety such as ; an Oalkyl hydroxylamine or a Pictet-Spengler reaction substrate such as N described in Agarwal P., et al., Bioconjugate Chem 2013, 24, 846-851.

[00071] More particularly, -ZaRa can represent -OH, -OCH3, - OCH2CH=CH2, (O-NHS) or where cation represents for example sodium, potassium or cesium or or the group wherein GI represents at least one electroinductive group such as -NO2 or -Hal, especially -F. They can be, for example, the following groups: Another type of group -C(=O)ZaRa is the following: More particularly, RCG1 may be chosen from one of those described in the examples.

[00072] Examples of RCG2 that may be mentioned include (Garnett M.C., et al., Advanced Drug Delivery Reviews 2001, 53, 171-216): (i) E-amino groups of lysines supported by the side chains of lysine residues that an antibody is present on the surface; (ii) α-amino groups of N-terminal amino acids of antibody heavy and light chains; (iii) The saccharide groups of the articulation region; (iv) Cysteine thiols generated by the reduction of intrachain disulfide bonds and engineered cysteine thiols; (v) Amide groups supported by the side chains of some glutamine residues that are present on the surface of an antibody; (vi) Aldehyde groups introduced using formylglycine generating enzyme.

[00073] More recently, other conjugation methods have been considered, for example, 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 chemistry (Axup J.Y., et al., PNAS 2012, 109, 40, 16101-16106) or conjugation on antibody glycans (Zhou Q., et al., Bioconjugate Chem. 2014, 25, 510-520). Another method 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 coupling technologies can also be applied to the cryptokine payloads described in the present invention.

[00074] It is also possible to chemically modify the antibody in order to introduce the new RCG2 reactive chemical groups. Thus, it is well known to those skilled in the art how to modify an antibody with the aid of a modifying agent by introducing, for example, activated disulfide, thiol groups, maleimide or haloacetamido (see especially, WO2005/077090 page 14 and WO2011/001052 ). The modification makes it possible to improve the conjugation reaction and use a wider variety of RCG1 groups.

[00075] More particularly, in the case where RCG1 is of type (ii) above, it is possible to chemically modify the antibody using a suitable modifying agent or introduce one or more unnatural amino acids in order to introduce the appropriate RCG2 functions. For example: when RCG1 represents an N-hydroxysuccinimidyl ester, RCG2 represents an -NH2 group; when RCG1 represents a maleimido or haloacetamido function or a -Cl group, RCG2 can be a -SH group; when RCG1 represents a -N3 group, RCG2 can be a -C=CH group or an activated C=C such as a cyclooctin moiety; when RCG1 represents a -OH or -NH2 group, RCG2 can be a carboxylic acid or amide function; when RCG1 represents a -SH group, RCG2 can be a maleimido or haloacetamido function; when RCG1 represents a -C=CH function or a C=C, activated RCG2 can be a -N3 group; When RCG1 represents an -O-alkyl hydroxyamine function or a Pictet-Spengler reaction substrate, RCG2 can be an aldehyde or ketone function.

[00076] Examples of G that may be mentioned include:

[00077] The invention relates to the cryptophyin payloads of formula (II) and the conjugates of formula (III): where the ligand L is of formula (IV): where: L1 represents (i) a single bond or an NR16(hetero)aryl-CR15R14-OC(=O) group, if Y = (C1-C6)alkyl-N(R11); (ii) an NR18-(C2-C6)alkyl-NR17-C(=O) group or an NR16(hetero)aryl-CR15R14-OC(=O)NR18-(C2-C6)alkyl-NR17-C( =O), if Y = (C1-C6)alkyl- O- or (C1-C6)alkyl- S; (iii) an NR16(hetero)aryl-CR15R14 group, if Y = C(=O)O, C(=O)NH, (C1-C6)alkyl-C(=O)O or (C1-C6)alkyl -C(=O)NH; R11, R14, R15, R16, R17 and R18 represent, independently of each other, H or a (C1-C6) alkyl group; (AA)w represents a sequence of w AA amino acids 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 (C1-C6)alkyl group or a (C1-C6)alkyl-(OCH2CH2)i group or a (C1-C6)alkyl-(OCH2CH2)iO(C1-C6)alkyl group or a (CH2CH2O)i(C1-C6)alkyl group or a CH(SO3H)-(C1-C6)alkyl group or a (C1-C6)alkyl-CH(SO3H) group or a (C1-C6)alkyl-cyclo group -hexyl or an NR19-(C1-C6)alkyl group or an NR20-(CH2CH2O)i(C1-C6)alkyl group or an NR21-aryl group or an NR21-heteroaryl group or a (C1-C6)alkyl group NR22C(=O)-(C1-C6)alkyl or a (C1-C6)alkyl-NR22C(=O)-(C1-C6)alkyl-(OCH2CH2)i group. More particularly L2 represents a (C1-C6) alkyl group or a (C1-C6)alkyl-(OCH2CH2)i or CH(SO3H)-(C1-C6)alkyl group; Ri9, R20, R21 and R22 represent, independently of each other, H or a (C1-C6) alkyl group; i represents an integer between 1 and 50 and preferably between 1 and 10 (i can consider all values between 1 and 50).

[00078] AA means a natural or unnatural amino acid, of D or L configuration, more particularly chosen from: alanine (Ala), ß-alanine, Y-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino acid -amino-2-phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), a,a-dimethyl-Y-aminobutyric acid, ß,ß-dimethyl acid -Y—aminobutyric, glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), e-acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tir), valine (Val). More particularly, AA is chosen from alanine (Ala), citrulline (Cit), glutamine (Gln), glycine (Gly), e-acetyl-lysine (AcLys), valine (Val).

[00079] The sequence (AA)w has the formula: wherein R23 represents the side chain of one of the amino acids described above. Example 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, Ile-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.

[00080] For Y = (C1-C6)alkyl-N(R11) and more particularly CH2NH, RCG1-L can be one of the following (IV1-17): wherein: (AA)w represents a sequence of w AA amino acids 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 can consider all values between 1 and 50); M1, M2, M3 and M4 are chosen independently of each other from CR24 and N;

[00081] R19, 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.

[00082] For Y = (C1-C6)alkyl-O or (C1-C6)alkyl-S and more particularly CH2O or CH2S, RCG1-L can be one of the following (IV18-34): wherein: (AA)w represents a sequence of w AA amino acids 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 can consider all values between 1 and 50); M1, M2, M3 and M4 are chosen independently of each other from CR24 and N; R17, R18, R19, 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.

[00083] For Y = (C1-C6)alkyl-N(R11) and more particularly CH2NH, RCG1-L can also be one of the following (IV35-51): AA)w represents a sequence of w AA amino acids 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 can consider all values between 1 and 50); J1, J2, J3 and J4 are chosen independently of each other from CR24 and N; M1, M2, M3 and M4 are chosen independently of each other from CR24 and N; R14, R15, R16, R19, 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.

[00084] For Y = (C1-C6)alkyl-O or (C1-C6)alkyl-S and more particularly CH2O or CH2S, RCG1-L can also be one of the following (IV52-68): (AA)w represents a sequence of w AA amino acids 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 can consider all values between 1 and 50); J1, J2, J3 and J4 are chosen independently of each other from CR24 and N; M1, M2, M3 and M4 are chosen independently of each other from CR24 and N; R14, R15, R16, R17, R18, R19, 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 .

[00085] For Y = C(=O)O, C(=O)NH, (C1-C6)alkyl-C(=O)O or (C1-C6) alkyl-C(=O)NH and more particularly C(=O)O or CH2-C(=O)O, RCG1-L can be one of the following (IV69-85): wherein: (AA)w represents a sequence of w AA amino acids 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 can consider all values between 1 and 50); J1, J2, J3 and J4 are chosen independently of each other from CR24 and N; M1, M2, M3 and M4 are chosen independently of each other from CR24 and N; R14, R15, R16, R19, 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.

[00086] For Y = C(=O) or (C1-C6)alkyl-C(=O), the invention also relates to cryptophycin payloads of formula (II) and conjugates of formula (III): on what: where the ligand L is of formula (V): where: (AA)'w represents a sequence of w AA amino acids connected together through 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 (C1 C6) alkyl group; L3 represents a (C1-C6)alkyl group or a (CH2CH2O)i-(C1-C6)alkyl group or a (C1-C6)alkyl-(OCH2CH2)i group or a CH(SO3H)-(C1-C6) group )alkyl or or a (C1-C6)alkyl-(OCH2 CH2)iO(C1-C6)alkyl group or a NR19-(C1-C6)alkyl group or an NR20-(CH2CH2O)iCH2CH2 group or a (C1- C6)alkyl-cyclohexyl-C(=O)NR19-(C1-C6)alkyl or a (C1-C6)alkyl-cyclohexyl-C(=O)NR20-(CH2CH2O)i-CH2CH2 group or a NR21-aryl-C(=O)NR19-(C1-C6)alkyl group or an NR21-heteroaryl-C(=O)NR19-(C1-C6)alkyl group or a (C1-C6)alkyl-NR22C( =O)(CH2CH2O)i-(C1-C6)alkyl; R19, R20, R21, R22 and R25 represent a hydrogen atom or a (C1-C6) alkyl group; RCG1 represents a reactive chemical group that is reactive to a reactive chemical group present in the antibody, as defined above; R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined above.

[00087] AA means a natural or unnatural amino acid, of D or L configuration, more particularly chosen from: alanine (Ala), ß-alanine, Y-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino acid - amino-2-phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), a,a-dimethyl-Y-aminobutyric acid, e,e-dimethyl acid -Y-aminobutyric acid, glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), e-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 (Gln), glycine (Gly), e-acetyl-lysine (AcLys), valine (Val).

[00088] The sequence (AA)'w has the formula: - . wherein R26 represents the side chain of one of the amino acids described above. Example 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-Ile, 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.

[00089] For Y = C(=O) or (C1-C6)alkyl-C(=O) and more particularly C(=O) or CH2-C(=O), RCG1-L may be one of the following ( V86-101): wherein: (AA)'w represents a sequence of w AA amino acids 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 can consider all values between 1 and 50); M1, M2, M3 and M4 are chosen independently of each other from CR24 and N; R19, R20, R21, R22, R24 and R25 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group, more particularly a hydrogen atom or a methyl group.

[00090] Ligand L can also be chosen from the illustrated compounds.

[00091] According to the invention, compounds of general formula (I), (II) and (III) can be prepared by the following processes. Process for the preparation of cryptophycin compounds General routine A for the preparation of compounds of formula (I), in the case where W=CH2N3 or CH2NH2

Scheme 1

[00092] Fragments A, B, C and D allow the preparation of cryptophycin compounds P1 to P3 with the aid of the steps detailed below: Step (i): peptide coupling between fragments AD1 and BC in the presence of coupling reagent such such as, for example, HOAt and HATU; Step (ii): deprotection under acidic conditions using, for example, TFA and macrocyclization in the presence of coupling reagent such as, for example, HOAt and HATU; Step (iii): oxidation of the olefin to form the epoxide using, for example, m-CPBA; Step (v): reduction of the azido group using, for example, TCEP.

[00093] Fragments A and B were prepared according to the synthesis described below. Protected C fragments such 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 [118138-56-0]); R4=R5=Me, R2=R3=H (CAS number [25307-828]); 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 together with the carbon atom to which they are attached form a cyclopropyl group (CAS number [914226-26-9]). They can also be prepared as described in the examples. Non-commercially available fragments C were prepared as described in the examples. All D fragments are commercially available: L-Fmoc-Ala-OH (CAS number [35661-39-3]); L-Fmoc-Val-OH (CAS number [68858-20-8]); L-Fmoc-tert-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 [13955174-9]). The AD1 and BC building blocks were prepared according to the synthesis described below. General routine B for the preparation of compounds of formula (I), in the case where W=CH2OH or CH2N3 or CH2NH2 Scheme 2

[00094] 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): peptide coupling between fragments AD1 and BC in the presence of coupling reagent such such as, for example, HOAt and HATU; Step (ii): deprotection under acidic conditions using for example TFA, macrocyclization in the presence of coupling reagent such as, for example, HOAt and HATU and hydrolysis of acetate at pH 6 to 7 using, for example, NaOH in a mixture of water and AcOEt; Step (iii): protecting benzyl alcohol as a silyl ether using, for example, chlorotriisopropylsilane in the presence of a base such as, for example, imidazole; oxidation of the olefin to form the epoxide using, for example, m-CPBA; deprotection of 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.

[00095] Fragments A and B were prepared according to the synthesis described below. The C fragments were commercially available as reported in the previous section or prepared as described in the examples. All D fragments are commercially available as reported in the previous section. AD2 and BC building blocks were prepared according to the synthesis described below. General routine C for the preparation of compounds of formula (I), in the case where W= CH2OH or CH2N3 or CH2NH2

Scheme 3

[00096] 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): peptide coupling between fragments AD3 and alternative BC in the presence of reagent coupling such as, for example, HOAt and HATU; Step (ii): macrocyclization ring-closing metathesis in the presence of a catalyst such as, for example, Grubbs I catalyst; Step (iii): deprotection of the p-methoxybenzyl ether under 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 chiral sulfonium derived from appropriately substituted isothiocineol 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.

[00097] Sakurai alcohol and fragment B were prepared according to the synthesis described below. C fragments were commercially available as reported in the previous section or prepared as described in the examples. All D fragments are commercially available as reported in the previous section. Alternative AD3 and BC building blocks were prepared according to the synthesis described below. Preparation of compounds of formula (I), in the case where W=CH2SH Scheme 4

[00098] P4 allows the preparation of other cryptophycin compounds P5 and P6 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 hexamethyldisilatian according to Hu J., et al., J. Org. Chem. 1999, 64, 4959-496: in the course of this reaction, the intermediate dimer described in Scheme 3 is generally formed; Step (iii): reduction of the dimer using a phosphine such as, for example, TCEP.

[00099] Schemes 1, 2, 3 and 4 describe the cases n=1, but they can also be applied in relation to the preparation of compounds of formula (I) in the case where n>1 starting from P2, P3 or P4 with the assistance of the steps detailed below and described in Scheme 2 of WO2011/001052: Scheme 5 Step (i): opening of the epoxide ring in an acidic medium, in order to obtain the diol function; concentrated perchloric acid can 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, e.g. a bromide, and a strong base, e.g. BuLi; Step (iv): oxidation of the olefin to form the epoxide using, for example, m-CPBA; Step (v): deprotection of the silyl ether using, for example, a TBAF solution. Preparation of compounds of formula (I), in the case where W=CO2H Scheme 6

[000100] P4 allows the preparation of other cryptophycin compounds P7 and P8 with the aid of the steps detailed below: Step (i): oxidation using the Dess-Martin reagent; Step (ii): Pinnick-type oxidation in the presence of 2 methyl-2-butene (Pinnick H.W., Tetrahedron 1981, 37, 2091-2096).

[000101] Scheme 6 describes the case n=0 but can also be applied to the preparation of compounds of formula (I) in the case where W=(CH2)nCO2H starting from an analogue of P4 carrying a group (CH2)n+ 1OH. Schemes 1, 2, 3, 4, 5 and 6 are given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, they are provided for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19. Process for preparing the Cryptoworks payloads

[000102] Compounds of formula (II) can be prepared according to Scheme 7, starting with a cryptophycin compound of formula (I) and a ligand precursor (LP): Scheme 7

[000103] W represents • (Ci-C6)alkyl-NH(Rii), more particularly (CH2)nNHRii; • (C1-C6)alkyl-OH, more particularly (CH2)nOH; • (C1-C6)alkyl-SH, more particularly (CH2)nSH; • CO2H; • (C1-C6)alkyl-CO2H, more particularly (CH2)nCO2H; or • (C1-C6)alkyl-N3. wherein R11 represents a hydrogen atom or a (C1-C6) alkyl group, more particularly a methyl group.

[000104] The precursor of the LP ligand has the function of introducing a precursor of the L ligand into the cryptophycin compound after a reaction between the W group and a chemical function present in the LP.

[000105] 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 ligand L for which ZaRa = -O-allyl can be introduced using the corresponding ligand precursor, followed by deprotection of the ester function and introducing -O-NHS. Deprotection can be carried out by treatment with a palladium catalyst, e.g. Pd(PPh3)4 in the presence of an amine scavenger, e.g. morpholine; activation can be carried out 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 convention of a ZaRa group into another ZaRa group (e.g. -O-allyl -> -O-NHS) can be applied to obtain other ZaRa groups, especially those described previously.

[000106] Scheme 7' similarly illustrates the preparation of a cryptophycin compound comprising a linker carrying, respectively, a maleimido, haloacetamido, amino or azido group (L* represents a fragment of a linker so that L= -L*- maleimido or L= -L*-haloacetamido or L= -L*-NH2- or L= -L*-N3 ).

[000107] These compounds are obtained by reaction between a cryptophycin compound comprising a ligand L' comprising an amino group and a modifying agent for introducing, respectively, a maleimido, haloacetamido, amino or azido group.

[000108] Compounds of formula (II) can alternatively be prepared according to Scheme 8 starting with the same cryptophycin compound of formula (I) and another ligand precursor (LP'): Scheme 8

[000109] In Scheme 8, several steps and/or reactions may be necessary to reach the cryptophycin payload (II) starting from the cryptophycin compound (I). For example, in the case where ZaRa = -O-NHS, an L'-protected ligand precursor can be introduced, followed by deprotection and coupling to an L'' ligand precursor to introduce the carboxylic acid function that can be directly activated to ZaRa = -O-NHS or first unprotected and subsequently activated (L' and L'' represent ligand fragments, so that L=L'-L''). Deprotection can be carried out by treatment with a base, for example piperidine; coupling to a ligand L'' can proceed through the opening of a cyclic anhydride, for example, glutaric anhydride; activation can be carried out 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.

[000110] An example of a reaction between the W group and a chemical function present in the LP is an amidation or between an LP ligand precursor carrying a carboxylic acid function and W=(CH2)nNH2 or between an LP ligand precursor carrying a carboxylic acid function amine and W=CO2H or (CH2)nCO2H: this reaction can be carried out in the presence of a coupling agent such as, for example, EDCI or HOBt. It is also possible to react an LP ligand precursor carrying an amine function and W'=(CH2)nO-C(=O)-O-(4-nitrophenyl) obtained from W=(CH2)nOH and p-nitrophenyl chloroformate ( activation of alcohol in the form of carbonate) according to the scheme below (R17 = H or (C1-C6)alkyl): -NHRi7 + crypto-(CH2)nO-C(=O)-O-(4-nitrophenyl) ^ crypto-(CH2)nO- C(=O)-NR17-

[000111] The same kind of reaction can also be carried out between an LP ligand precursor carrying an activated alcohol function as a carbonate and W=(CH2)nNH2. Another example of a reaction is an esterification between a ligand precursor carrying an alcohol function and W=CO2H or (CH2)nCO2H: this reaction can be carried out in the presence of a coupling agent such as, for example, MNBA. Another example reaction is a copper-catalyzed azide-alkyne cycloaddition between a ligand precursor carrying an alkyne function and W=(CH2)N3: this reaction can be carried out in the presence of copper sulfate and sodium ascorbate. Preparation of compounds of formula (II) in the case where W=(CH2)nNH2 and L=(IV1) with n=1, Ri5=H and ALQ=(CH2)3 based on Scheme 7 Scheme 9 Step (i): peptide coupling in the presence of coupling reagent 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, tetracis-(triphenylphosphine)palladium; Step (iii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA. Preparation of compounds of formula (II) in the case where W=(CH2)nNH2 and L=(IV1) with n=1, Rii=H and ALQ=(CH2)3 based on Scheme 8 Scheme 10 Step (i): peptide coupling in the presence of coupling reagent 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 an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

[000112] Schemes 9 and 10 describe the case L(IV1) with n=1, R11=H and ALQ=(CH2)3, however they can also apply to other ligands L with RCG1=C(=O) ONHS, namely the L(IV1) case with n#1 and/or Rn#H and/or ALK#(CH2)3 and the L(IV2) and L(IV3) cases. They are given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, they are provided for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=(CH2)nNH2 with n=1, Rii=H and RCG1=maleimide, haloacetamido, NH2, N3 or cyclooctin, namely, ligands L(IV4) to L (IV17) Scheme 11 Step (i): peptide coupling in the presence of coupling reagent 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.

[000113] Scheme 11 describes the cases L(IV4) with n=1, R11=H and ALQ=(CH2)5, L(IV8) with n=1 and R11=H, L(IV12) with n=1 , R11=H and ALQ=(CH2)4, L(IV14) with n=1, R11=H and ALQ=(CH2)3 and L(IV16) with n=1, R11=H, ALQ=(CH2) 3 and ALQ'=(CH2)2, however it can also apply to the other ligands L(IV4) to L(IV17) with RCG1=maleimide, iodoacetamido, NH2, N3 or cyclooctin. It is described for a reactive iodoacetamido group, but can equally apply to a reactive bromoacetamido group. It is given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, it is provided for a cryptophycin compound, but it may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=(CH2)nX with X=O or S and L=(IV18) with n=1, R17 and Ri8=H and ALQ=(CH2)3 based on the Scheme 7 Scheme 12 Step (i): activation of benzyl alcohol or benzyl 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 (thio)carbamate by reaction 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, tetracis(triphenylphosphine)palladium; Step (iv): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA. Preparation of compounds of formula (II) in the case where W=(CH2)nX with X=O or S and L=(IV18) with n=1, R17 and Ri8=H and ALQ=(CH2)3 based on the Scheme 8 Scheme 13 Step (i): formation of (thio)carbamate by reaction 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 an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

[000114] Schemes 12 and 13 describe the case L(IV18) with n=1, R17 and R18=H and ALQ=(CH2)3 but they can also apply to the other ligands L with RCG1=C(=O )ONHS, namely the case L(IV18) with p#1 and/or Ri7, Ri8#H and/or ALK#(CH2)3 and the cases L(IV19) and L(IV20). They are given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, they are provided for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially Di-Di9. Preparation of compounds of formula (II) in the case where W=(CH2)nX with Scheme 14 Step (i): formation of (thio)carbamate, the reaction is carried out 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.

[000115] Scheme 14 describes the case L(IV21) with n=1, R17 and R18=H and ALQ=(CH2)5, the case L(IV25) with n=1 and R17 and R18=H, the case L(IV29) with n=1, R17 and R18=H and ALQ=(CH2)4, the case L(IV31) with n=1, R17 and R18=H and ALQ=(CH2)3 and the case L( IV33) with n=1, R17, R18 and R22= H, ALQ=(CH2)3 and ALQ'=(CH2)2 however it can also apply to the other ligands L(IV21 to 34) with RCG1=maleimide, iodoacetamide, NH2, N3 or cyclooctin. It is described for a reactive iodoacetamido group, but can equally apply to a reactive bromoacetamido group. It is given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, it is provided for a cryptophycin compound, but it may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=(CH2)nNH2 and L=(IV35) with n=1, Rii=Ri4=Ri5=Ri6=H, JI=J2=J3=J4=CH and ALQ= (CH2)3 based on Scheme 7 Scheme 15 Step (i): formation of the carbamate, the reaction is carried out 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, tetracis-(triphenylphosphine)palladium; Step (iii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA. Preparation of compounds of formula (II) in the case where W=(CH2)nNH2 and L=(IV35) with n=1, R11=R14=R15=R16=H, J1=J2=J3=J4=CH and ALQ= (CH2)3 based on Scheme 8 Scheme 16 Step (i): formation of the carbamate, the reaction is carried out 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 an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

[000116] Schemes 15 and 16 describe the case L(IV35) with n=1, R11=R14=R15=R16=H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2 )3 however they can also apply to other ligands L with RCG1=C(=O)ONHS, namely the case L(IV35) with n#1 and/or R11, R14, R15, RI6#H and/ or J1, J2, J3, J4#CH and/or an ortho-benzyl alcohol and/or ALK#(CH2)3 and the cases L(IV36) and L(IV37). They are given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, they are provided for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=(CH2)nNH2 with n=1, Rii=Ri4=Ri5=Ri6=H, JI=J2=J3=J4=CH and RCG1=maleimido, haloacetamido, NH2, N3 or cyclooctin, namely linkers L(IV38) to L(IV151) Scheme 17 Step (i): formation of the carbamate, the reaction is carried out 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.

[000117] Scheme 17 describes the cases L(IV38) with n=1, R11=R14=R15=R16=H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2)5 , L(IV42) with n=1, R11=R14=R15=R16=H and J1=J2=J3=J4=CH and a para-benzyl alcohol, L(IV46) with n=1, R11=R14=R15 =R16=H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2)4, L(IV48) with n=1, R11=R14=R15=R16=H, J1=J2 =J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2)3 and L(IV50) with n=1, R11=R14=R15=R16=R22=H, J1=J2=J3=J4= CH , a para-benzyl alcohol, ALQ=(CH2)3 and ALQ'=(CH2)2 however it can also apply to the other ligands L(IV38) AL(IV51) with RCG1=maleimido, iodoacetamido, NH2, N3 or cyclooctin. It is supplied with a para-benzyl alcohol, but it can also be used with an ortho-benzyl alcohol. It is described for a reactive iodoacetamido group, but can equally apply to a reactive bromoacetamido group. It is given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, it is provided for a cryptophycin compound, but it may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=(CH2)nX with X=O or S and L=(IV52) with n=1, R14=R15=R16=R17=R18=H, J1=J2= J3=J4=CH and ALQ=(CH2)3 based on Scheme 7 Scheme 18 Step (i): formation of (thio)carbamate by reaction 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, tetracis(triphenylphosphine)palladium; Step (iii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA. Preparation of compounds of formula (II) in the case where W=(CH2)nX with X=O or S and L=(IV52) with n=1, Ri4=Ri5=Ri6=Ri7=Ri8=H, JI=J2= J3=J4=CH and ALQ=(CH2)3 based on Scheme 8 Scheme 19 Step (i): formation of (thio)carbamate by reaction 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 an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

[000118] Schemes 18 and 19 describe the case L(IV52) with n=1, R14=R15=R16=R17=R18=H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ= (CH2)3 however they can also apply to other ligands L with RCG1=C(=O)ONHS, namely the case L(IV52) with n#1 and/or R14, R15, R16, R17, RI8 #H and/or J1, J2, J3, J4#CH and/or an ortho-benzyl alcohol and/or ALQ#(CH2)3 and the cases L(IV53) and L(IV54). They are given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, they are provided for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=(CH2)nX with Scheme 20 Step (i): formation of (thio)carbamate by reaction 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.

[000119] Scheme 20 describes the case L(IV55) with n=1, R14=R15= R16=R17=R18=H, J1=J2=J3=J4=CH, a para-benzyl alcohol, ALQ= (CH2 )2 and ALQ'=(CH2)5, the case L(IV59) with n=1, R14=R15=R16=R17=R18= H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2)2, the case L(IV63) with n=1, R14=R15=R16=R17=R18=H, J1=J2=J3=J4=CH, a para-benzyl alcohol, ALQ=(CH2 )2 and ALQ'=(CH2)4, the case L(IV65) with n=1, R14=R15=R16=R17=R18=H, J1=J2=J3=J4=CH, a para-benzyl alcohol, ALQ=(CH2)2 and ALQ'=(CH2)3 and the case L(IV67) with n=1, R14=R15=R16= R17=R18=R22=H, J1=J2=J3=J4=CH, a para-benzyl alcohol, ALQ= (CH2)2, ALQ'=(CH2)5 and ALQ''=(CH2)2, however it can also apply to the other ligands L(IV55 to 68) with RCG1=maleimido , iodoacetamide, NH2, N3 or cyclooctin. It is described for a reactive iodoacetamido group, but can equally apply to a reactive bromoacetamido group. It is given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, it is provided for a cryptophycin compound, but it may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=C(=O)O and L=(IV69) with Ri4=Ri5=Ri6=H, JI=J2=J3=J4=CH and ALQ=(CH2)3 based on Scheme 7 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, tetracis-(triphenylphosphine)palladium; Step (iii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA. Preparation of compounds of formula (II) in the case where W=C(=O)O and L=(IV69) with RI4=RI5=RI6=H, JI=J2=J3=J4=CH and ALQ=(CH2)3 based on Scheme 8 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 an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

[000120] Schemes 21 and 22 describe the case L(IV69) with R14= R15=R16=H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2)3 but they can also apply to the other ligands L with RCG1=C(=O)ONHS, namely the case L(IV69) with R14, R15, Rw#H and/or J1, J2, J3, J4#CH and/or a ortho-benzyl alcohol and/or ALQ#(CH2)3 and the cases L(IV70) and L(IV71). They are given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, they are provided for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=C(=O)O with RI4=RI5=RI6=H, JI=J2=J3=J4=CH and RCG1=maleimide, haloacetamido, NH2, N3 or cyclo- octin, 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.

[000121] Scheme 23 describes the cases L(IV72) with n=1, R14=R15=R16=H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2)5, L (IV76) with n=1, R14=R15=R16=H and J1=J2=J3=J4=CH and a para-benzyl alcohol, L(IV80) with n=1, R14=R15=R16=H, J1 =J2=J3=J4= CH, a para-benzyl alcohol and ALQ=(CH2)4, L(IV82) with n=1, R14= R15=R16=H, J1=J2=J3=J4=CH, a para-benzyl alcohol and ALQ=(CH2)3 and L(IV84) with n=1, R14=R15=R16=R22=H, J1=J2=J3=J4=CH, a para-benzyl alcohol, ALQ=( CH2)3 and ALQ'=(CH2)2, however it can also apply to the other ligands L(IV72) to L(IV85) with RCG1=maleimido, iodoacetamido, NH2, N3 or cyclooctin. It is supplied with a para-benzyl alcohol but it can also be applied to an ortho-benzyl alcohol. It is described for a reactive iodoacetamido group, but can equally apply to a reactive bromoacetamido group. It is given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, it is provided for a cryptophycin compound, but it may also apply to the preparation of other compounds of formula (I), especially D1-D19. Preparation of compounds of formula (II) in the case where W=C(=O)O and L=(V86) with R25=H and ALQ=(CH2)7 Scheme 24 Step (i): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA; Step (ii): peptide coupling in the presence of a base such as, for example, DIEA; Step (iii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.

[000122] Scheme 24 describes the case L(V86) with R25=H and ALQ=(CH2)7, however they can also apply to the other ligands L with RCG1=C(=O)ONHS, namely the case L(V86) with R25#H and/or ALQ#(CH2)7 and cases L(V87) and L(V88). They are given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, they are provided for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1D19. Preparation of compounds of formula (II) in the case where W=C(=O)O with R30=H and RCG1=maleimide, haloacetamido, NH2, N3 or cyclooctin, namely, ligands L(V89) to L(V101 ) Scheme 25 Step (i): peptide coupling in the presence of a base such as, for example, DIEA; Step (ii): reduction of the azide by treatment with a reducing agent such as, for example, TCEP.

[000123] Scheme 25 describes the cases L(V89) with R25=H and ALQ=(CH2)5, L(V93) with R25=H and ALQ=(CH2)3, L(V96) with R25=H and ALQ=(CH2)4, L(V98) with R25=H and ALQ=(CH2)3 and L(V100) with R22=R25=H, ALQ=(CH2)3 and ALQ'=(CH2)2, however it can also apply to the other ligands L(V89) to L(V101) with RCG1=maleimide, iodoacetamido, NH2, N3 or cyclooctin. It is described for a reactive iodoacetamido group, but can equally apply to a reactive bromoacetamido group. It is given for a ligand in the para position, but can equally apply to the ortho or meta positions. Similarly, it is provided for a cryptophycin compound, but it may also apply to the preparation of other compounds of formula (I), especially D1-D19. Process for the preparation of cryptophycin conjugates

[000124] Compounds of formula (III) can be prepared according to Scheme 26 starting with a cryptophycin payload of formula (II) and an antibody (Ab): Scheme 26 wherein: Y represents (C1-C6)alkyl-NR11 or (C1-C6)alkyl-O or (C1-C6)alkyl-S; or alternatively Y represents C(=O)O or O(C1-C6)alkyl-C(=O)O; or alternatively Y represents (C1-C6)alkyl-triazole- as . Y is positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus; R11 represents a hydrogen atom or a (C1 C6) alkyl group; L represents a ligand positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus, as defined above; RCG1 represents a reactive chemical group that is reactive to a reactive chemical group present in the antibody, as defined above; Ab represents an antibody. Process for the preparation of building blocks for the synthesis of cryptophycin compounds of formula (I) General synthesis of fragment A Scheme 27

[000125] Fragment A can be prepared in 12 steps starting from appropriate hydroxide esters with the aid of the steps detailed below: Step (i): protection of the alcohol as a p-methoxybenzyl ether using activated p-methoxybenzyl alcohol as a trichloroacetimidate in presence of a catalytic amount of acid such as, for example, p-TsOH; Step (ii): reducing the ester using a reducing agent such as, for example, lithium borohydride; Step (iii): oxidation of the alcohol using an oxidizing agent such as, for example, TEMPO in the presence of sodium hypochlorite; Step (iv): diastereoselective allylation using allyltributyltin in the presence of tin tetrachloride; Step (v): cross-metathesis using tert-butylacrylate in the presence of a catalyst such as, for example, Grubbs II catalyst; Step (vi): deprotection of the p-methoxybenzyl ether using CAN and subsequent treatment with ethanedithiol in the presence of a catalytic amount of acid such as, for example, p-TsOH; Step (vii): protection of alcohols such as silyl ethers using chlorotriethylsilane in the presence of a base such as, for example, imidazole; Step (viii): Swern oxidation using DMSO and oxalyl chloride in the presence of a base such as, for example, DIEA; Step (ix): Wittig reaction using a suitable phosphonium halide, for example a bromide, and a strong base such as, for example, BuLi; Step (x): deprotection of the silyl ether using, for example, a TBAF solution. Step (xi): oxidizing the benzyl alcohol using an oxidizing agent such as, for example, manganese oxide; Step (xii): isomerization of the double bond using AIBN in the presence of benzenethiol.

[000126] Hydroxy esters are commercially available for R1=Me (CAS number [72657-23-9]), Et (CAS number [72604-810]) and iPr (CAS number [72604-82-1] ). Preparation of phosphonium halides is described in WO2011/001052. General synthesis of fragment B Scheme 28

[000127] Fragment B can be prepared in 2 steps starting from appropriate amino acids with the aid of the steps detailed below: Step (i): protection of the amine by treatment with Boc2O in the presence of a base such as, for example, TEA; Step (ii): saponification of the metal ester using a base such as, for example, LiOH.

[000128] Protected amino acids such as methyl esters are commercially available for R9=3-Cl, 4-OMe (CAS number [704870-54-2]); 3-Cl, 4-OEt (CAS number [1256963-00-9]); 3-Cl, 4-OPr (CAS number [1259709-59-9]); 3-Cl, 4-OMe, 5-F (CAS number [1213670-98-4]); 3-Cl, 4-OMe, 5-Cl (CAS number [1259701-422]); 3-Cl, 4-OMe, 5-OMe (CAS number [1212820-34-2]); 3-Cl, 4-OH, 5-OMe (CAS number [1213426-93-7]); 3-Cl, 4-NH2 (CAS number [1213672-64-0]); 2-Cl, 3-Cl, 4-NH2 (CAS number [1213607-49-8]); 3Cl, 4-NH2, 5-F (CAS number [1213400-66-8]); 3-Cl, 4-NH2, 5-Cl (CAS number [1213480-30-8]). General synthesis of AD1 building block Scheme 29

[000129] Fragments A and D allow the preparation of building block AD1 with the aid of the steps detailed below: Step (i): esterification of fragment D with fragment A in the presence of a coupling reagent such as, for example, MNBA is a base such as, for example, DMAP and DIEA; Step (ii): reducing the aldehyde to an alcohol using a reducing agent such as, for example, sodium trimethoxyborohydride; Step (iii): activation of the alcohol as a mesylate by treatment with methanesulfonyl chloride in the presence of a base such as, for example, TEA; replacement of the mesylate by an azido group by treatment with sodium azide; Step (iv): deprotection of the amine by treatment with a base such as, for example, piperidine. General synthesis of AD2 building block Scheme 30

[000130] Fragments A and D allow the preparation of building block AD2 with the aid of the steps detailed below: Step (i): esterification of fragment D with fragment A in the presence of a coupling reagent such as, for example, MNBA is a base such as, for example, DMAP and DIEA; Step (ii): reducing the aldehyde to an alcohol using a reducing agent such as, for example, sodium trimethoxyborohydride; Step (iii): deprotection of the amine by treatment with a base such as, for example, piperidine. General synthesis of AD3 building block Scheme 31 Step (i): esterification of fragment D with Sakurai Alcohol 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 amine by treatment with a base such as, for example, piperidine. General synthesis of BC building block Scheme 32

[000131] Fragments B and C allow the preparation of BC building block with the aid of the steps detailed below: Step (i): peptide coupling of fragment B with fragment C in the presence of coupling reagent such as, for example, HOBt and EDC and a base such as, for example, DIEA; Step (ii): saponification of the methyl ester using a base such as, for example, LiOH. General synthesis of an alternative BC building block Scheme 33

[000132] Fragments B and C allow the preparation of an alternative building block BC with the aid of the steps detailed below: Step (i): peptide coupling of fragment B with fragment C in the presence of coupling reagent such as, e.g. , HOBt and EDC and a base such as, for example, DIEA; Step (ii): deprotection of the Boc group under acidic conditions using, for example, TFA; Step (iii): formation of acrylamide by treatment with acryloyl chloride chloride in the presence of a base such as, for example, DIEA; s saponification of the methyl ester using a base such as, for example, tBuOK. Preparation of LP ligand precursors

[000133] LP may be one of the following; LP1 to LP101 are described using L amino acids, but can also apply to D amino acids. They are provided for w=2, but can similarly apply to w>2 by repeating the peptide coupling step as many times as necessary.

[000134] Lpi prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; The reaction is carried out at room temperature in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000135] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diacids such as allyl esters are commercially available for n=2 (monoallyl succinate) or can be prepared by transesterification of methyl or tert-butyl monoesters, which are commercially available for n=2 to 6.

[000136] LP2 prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000137] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected PEG diacids such as allyl esters can be prepared according to the scheme below: when ALQ=CH2CH2 Step (i): elongation of the PEG chain; the reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating an unsaturated protected acid with the alkoxide generated by the action of sodium in a catalytic amount; Step (ii): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid. In the latter case, trifluoroacetate of the alcohol function present in the structure can be formed. This trifluoroacetate is cleaved during the following step (iii); Step (iii): protection of the carboxylic acid as a methyl ester; the reaction is carried out in a polar aprotic solvent such as MeOH, by treatment with trimethylsilyldiazomethane; Step (iv): saponification of the methyl ester; the reaction is carried out in a mixture of polar solvents such as a THF/H2O mixture in the presence of LiOH; Step (v): protection of the carboxylic acid as an allyl ester; the reaction is carried out in a polar aprotic solvent such as DCM in the presence of allyl alcohol, a coupling agent such as, for example, EDC and a base such as, for example, DMAP; Step (vi): elongation of the PEG chain; The reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating a halogenated ester with the monoprotected PEG diol alkoxide as a THP ether. The preparation of this type of monoprotected PEG diol is well described in the literature: see, for example, Richard A., et al., Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou EG, et al., Tetrahedron 2003, 59, 9083-9090.

[000138] Starting PEG diols are commercially available for i=3 to 12.

[000139] LP3 prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar solvent such as a mixture of DMF/H2O by treatment with N,N'-disuccinimidyl carbonate in the presence of a base such as, for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; The reaction is carried out at room temperature in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000140] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected sulfo diacids such as allyl ester can be prepared according to the scheme below: Step (i): protection of the carboxylic acid as an allyl ester; the reaction is carried out in a polar aprotic solvent such as DCM in the presence of allyl alcohol, a coupling agent such as, for example, EDC and a base such as, for example, DMAP; Step (ii): a-sulfonation of the carboxylic acid; the reaction is carried out at 75°C in a polar aprotic solvent such as DCE by treatment with chlorosulfonic acid in the presence of a base such as, for example, DIEA. Step (iii): formation of the carboxylic acid moiety; the reaction is carried out by treatment with sodium hydroxide; Step (iv): replacement of the bromide by a thioacetyl; the reaction is carried out at -20°C in a polar aprotic solvent such as THF using thioacetic acid in the presence of a base such as, for example, DIEA; Step (v): formation of the sulfonic acid moiety; the reaction is carried out at room temperature by treatment with hydrogen peroxide and acetic acid.

[000141] Cyano carboxylic acids are commercially available for n=1 to 12.

[000142] prepared according to the scheme below: of peptide between Fmoc-L- the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000143] NHS esters of Fmoc-L-amino acids are commercially available; Maleimide carboxylic acids are commercially available for n=1 to 12.

[000144] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000145] NHS esters of Fmoc-L-amino acids are commercially available; Maleimido acids are commercially available for ALQ=ALQ'=CH2CH2 and i=1 to 7 and can otherwise be prepared according to the scheme below: Step (i): elongation of the PEG chain; the reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating an unsaturated protected acid with the alkoxide generated by the action of sodium in a catalytic amount; Step (ii): Mitsunobu reaction in maleimide; the reaction is carried out in a polar aprotic solvent such as THF by treatment of maleimide by PEG hydroxide acid in the presence of PPh3 and DIAD; Step (iii): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid; Step (iv): elongation of the PEG chain; The reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating a halogenated ester with the monoprotected PEG diol alkoxide such as THP ether. The preparation of this type of monoprotected PEG diol is well described in the literature: see, for example, Richard A., et al., Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou EG, et al., Tetrahedron 2003, 59, 9083-9090; Step (v): selective deprotection of THP ether; the reaction is carried out in a polar protic solvent such as MeOH using a catalytic amount of acetyl chloride; Step (vi): activation of alcohol as a mesylate; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with methanesulfonyl chloride in the presence of a base such as, for example, TEA; Step (vii): nucleophilic substitution; the reaction is carried out in a polar aprotic solvent such as DMF in the presence of a base such as, for example, sodium hydride; Step (viii): protection of the alcohol as a benzyl ether; the reaction is carried out in a polar aprotic solvent such as THF by treatment with benzyl bromide in the presence of a base such as, for example, sodium hydride; Step (ix): deprotection of the alcohol using a hydrochloric acid solution (e.g. dioxane solution); Step (x): deprotection of the alcohol by hydrogenolysis in the presence of a catalyst such as, for example, palladium on carbon.

[000146] Starting PEG diols are commercially available for i=3 to 12. Starting ALQ diols are commercially available for n=1 to 12.

[000147] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar solvent such as the DMF/H2O mixture by treatment with N,N'-disuccinimidyl carbonate in the presence of a base such as, for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000148] NHS esters of Fmoc-L-amino acids are commercially available; Maleimide cyclohexanecarboxylic acid is commercially available for ALQ=CH2 and can otherwise be prepared according to the scheme below: Step (i): hydrogenation; the reaction is carried out at 60°C in acetic acid in the presence of a catalyst such as platinum oxide under hydrogen pressure, for example, 4.21 kg/cm2 (60 psi); Step (ii): protection of the amine; the reaction is carried out in a polar protic solvent such as a dioxane/H2O mixture by treatment with benzylchloroformate in the presence of a base such as, for example, NaOH; Step (iii): protection of the carboxylic acid; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with tert-butanol in the presence of a coupling reagent such as, for example, EDC and a base such as, for example, DMAP; Step (iv): deprotection of the amine; the reaction is carried out by hydrogenolysis in a polar protic solvent such as MeOH in the presence of a catalyst such as, for example, palladium; Step (v): introduction of the maleimide moiety; the reaction is carried out in a polar aprotic solvent such as DMF by treatment with maleic anhydride in the presence of NHS and a coupling reagent such as, for example, EDC; Step (vi): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid; Step (vii): palladium-catalyzed coupling; The reaction is carried out in a polar aprotic solvent such as THF by treatment with the bromo-ALQ-cyano derivative in the presence of lithium chloride, zinc and trimethylchlorosilane and a catalyst such as, for example, tetracys (triphenylphosphine) palladium.

[000149] Suitable bromo-ALQ-cyano derivatives are commercially available for n=1 to 12.

[000150] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): a-sulfonation of the carboxylic acid; the reaction is carried out at 75°C in a polar aprotic solvent such as DCE by treatment with chlorosulfonic acid in the presence of a base such as, for example, DIEA; Step (iv): activation of the carboxylic acid as an NHS ester; the reaction is carried out at room temperature in a polar solvent such as a mixture of DMF/H2O by treatment with N,N'-disuccinimidyl carbonate in the presence of a base such as, for example, DIEA; Step (v): peptide coupling between the dipeptide and the NHS ester; The reaction is carried out at room temperature in a polar aprotic solvent such as a mixture of DCM/CH3CN. NHS esters of Fmoc-L-amino acids are commercially available; maleimide acids are commercially available for n=1 to 12.

[000151] prepared according to the scheme below: polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000152] NHS esters of Fmoc-L-amino acids are commercially available; N-succinimidyl bromo- and iodo-acetates are commercially available.

[000153] prepared according to the scheme below: Step (i): amino acid-ONHS and L-amino acid coupling; polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as an NHS ester; The coupling of N-succinimidyl bromo- and iodo-acetate with the amino carboxylic acid is carried out at room temperature in a polar aprotic solvent such as a mixture of DCM/DMF followed by the addition of N,N'-disuccinimidyl carbonate and a base, such as, for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000154] NHS esters of Fmoc-L-amino acids are commercially available; N-succinimidyl bromine and iodo-acetates are commercially available, the amino carboxylic acids also for n=1 to 12 and the N-methylated amino carboxylic acids for n=1 to 7.

[000155] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as an NHS ester; The coupling of N-succinimidyl bromine and iodine acetate with the amino carboxylic acid is carried out in a polar aprotic solvent such as a DCM/DMF mixture followed by the addition of N,N'-disuccinimidyl carbonate and a base such as , for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000156] NHS esters of Fmoc-L-amino acids are commercially available; N-succinimidyl bromine and iodoacetate are commercially available; in the case where ALQ=CH2CH2 and R20=H, amino PEG carboxylic acids are commercially available for i=1 to 6 and otherwise can be prepared from tert-butyl acrylate and the corresponding amino-PEG-alcohol; in the case where ALQ#CH2CH2, they can be prepared according to the schemes below: Step (i): protection of the amine; the reaction is carried out in a polar aprotic solvent such as DCM by treating the amine with di-tert-butyl dicarbonate in the presence of a base such as, for example, TEA; Step (ii): protection of the carboxylic acid; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with tert-butanol in the presence of a coupling reagent such as, for example, EDC and a base such as, for example, DMAP; Step (iii): alkylation of the nitrogen atom; the reaction is carried out in an anhydrous polar aprotic solvent such as THF by treatment with a base such as sodium hydride in the presence of a reagent carrying a leaving group such as a halide;

[000157] Step (iv): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid; Step (v): elongation of the PEG chain; the reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating a halogenated ester with the alkoxide of a benzophenone-imine-PEG-alcohol Generated through the action of sodium hydride or potassium naphthalenide as described in WO2007 /127440; Step (vi): saponification of the ester; The reaction is carried out by reacting the ester with LiOH in the presence of H2O.

[000158] Amino-PEG-alcohols are commercially available, for example, for i=3, 4, 7, 8 or can be prepared from PEG diols, which are commercially available for i=3 to 12, according to the procedure described in US7230101. Protection of the amine function with benzophenone can be accomplished by azeotropic dehydration in the presence of a Lewis acid such as BF3 etherate.

[000159] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid- NHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the Fmoc amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as an NHS ester; The coupling of N-succinimidyl bromine and iodine acetate with the amino carboxylic acid is carried out at room temperature in a polar aprotic solvent such as a DCM/DMF mixture followed by the addition of N,N'-disuccinimidyl carbonate and a base such as, for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; The reaction is carried out at room temperature in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000160] NHS esters of Fmoc-L-amino acids are commercially available; N-succinimidyl bromine and iodoacetate are commercially available; amino-(hetero)aryl-carboxylic acids are commercially available, such as, for example, 4-amino-2-benzoic acid, 6-amino-3-pyridine carboxylic acid, 5-amino-2-pyrazine carboxylic acid, 2-amino-5-pyrimidine carboxylic acid or 6-amino-1,2,4,5-tetrazine carboxylic acid.

[000161] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-Lamino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000162] NHS esters of Fmoc-L-amino acids are commercially available; amino carboxylic acids are commercially available for n=1 to 11.

[000163] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-Lamino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000164] NHS Fmoc-L-amino acid ester are commercially available; in the case where ALQ=CH2CH2, Fmoc-protected amino PEG carboxylic acids are commercially available for i=1 to 6 and otherwise can be prepared from tert-butyl acrylate and the corresponding amino-PEG-alcohol; in the case where ALQ#CH2CH2, they can be prepared according to the schemes described for the LP10 ligand precursor. Protection of the amine function with an Fmoc group can be accomplished by treatment with FmocOSu (CAS number [82911-69-1]) in the presence of a base such as, for example, DIEA.

[000165] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): introduction of the azido group; the reaction is carried out in a polar solvent such as an acetone/H2O mixture by treatment with sodium azide; Step (iv): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (v): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000166] NHS esters of Fmoc-L-amino acids are commercially available; Bromine carboxylic acids are commercially available for n=1 to 11.

[000167] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN.

[000168] NHS esters of Fmoc-L-amino acids are commercially available; PEG azido acids can be prepared according to the schemes below: Step (i): elongation of the PEG chain; the reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating an unsaturated protected acid with the alkoxide generated by the action of sodium in a catalytic amount; Step (ii): activation of the alcohol function; the reaction is carried out in a polar solvent such as DCM using methanesulfonyl chloride in the presence of a base such as, for example, TEA; Step (iii): replacement of the mesylate by an azido group; the reaction is carried out in a polar solvent such as an acetone/H2O mixture by treatment with sodium azide; Step (iv): deprotection using a hydrochloric acid solution (e.g. dioxane solution); Step (v): elongation of the PEG chain; The reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating a halogenated ester with the monoprotected PEG diol alkoxide such as a THP ether, alkoxide generated, for example, with sodium hydride. The preparation of this type of monoprotected PEG diol is well described in the literature: see, for example, Richard A. et al. Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou EG, et al. Tetrahedron 2003, 59, 9083-9090; Step (vi): saponification of the methyl ester; The reaction is carried out at room temperature in a mixture of polar solvents such as a THF/H2O mixture in the presence of LiOH.

[000169] Starting PEG diols are commercially available for i=3 to 12; Bromine methyl esters are commercially available for n=1 to 12.

[000170] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-Lamino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): cyclooctin coupling; the reaction is carried out in a polar solvent such as DCM; Step (iv): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (v): peptide coupling between the dipeptide and the NHS ester; The reaction is carried out in a polar aprotic solvent such as DCM.

[000171] NHS esters of Fmoc-L-amino acids are commercially available; cyclooctins are commercially available for n’=1, 2, 3 and 5; monoactivated diacids such as NHS ester are commercially available for n=1 to 3 and for n=4 to 10, they can be prepared by activating commercially available diacids with NHS in the presence of a coupling agent such as, for example, DCC and a base such as, for example, DMAP. according to the scheme below:

[000172] prepared according to the scheme below: Step (i): peptide coupling between Fmoc-L-amino acid-ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): cyclooctin coupling; the reaction is carried out in a polar solvent such as DCM; Step (iv): deprotection using a hydrochloric acid solution (e.g. dioxane solution); Step (v): activation of cyclooctin-PEG carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (vi): peptide coupling between the dipeptide and the NHS ester; The reaction is carried out in a polar aprotic solvent such as DCM.

[000173] NHS esters of Fmoc-L-amino acids are commercially available; cyclooctins are commercially available for n'=1, 2, 3 and 5; mono-activated PEG diacids as NHS ester are prepared according to the scheme below: Step (i): elongation of the PEG chain; the reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating an unsaturated protected acid with the alkoxide generated by the action of sodium in a catalytic amount; Step (ii): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid; Step (iii): monoactivation of an acid such as an NHS ester; the reaction is carried out in a polar solvent such as THF using NHS in the presence of a coupling agent such as, for example, DCC and a base such as, for example, DMAP; Step (iv): elongation of the PEG chain; The reaction is carried out in an anhydrous polar aprotic solvent such as THF or DMF by treating a halogenated ester with the monoprotected PEG diol alkoxide such as a THP ether. The preparation of this type of monoprotected PEG diol is well described in the literature: see, for example, Richard A., et al., Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou EG, et al., Tetrahedron 2003, 59, 9083-9090; Step (v): protection of the carboxylic acid as a methyl ester; the reaction is carried out in a polar aprotic solvent such as MeOH, by treatment with trimethylsilyldiazomethane; Step (vi): saponification of the methyl ester; the reaction is carried out in a mixture of polar solvents such as a THF/H2O mixture in the presence of LiOH; Step (vii): activation of the acid as an NHS ester; the reaction is carried out in a polar solvent such as THF using NHS in the presence of a coupling agent such as, for example, DCC and a base such as, for example, DMAP.

[000174] Starting PEG diols are commercially available for i=3 to 12.

[000175] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000176] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other. Monoprotected diacids such as allyl esters are commercially available for n=2 (monoallyl succinate) or can be prepared by transesterification of methyl or tert-butyl monoesters, which are commercially available for n'=2 to 6.

[000177] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000178] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; monoprotected PEG diacids as allyl esters are prepared according to the schemes described for the LP2 ligand precursor.

[000179] prepared according to the scheme below:according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000180] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Allyl ester monoprotected sulfo diacids are prepared according to the schemes described for the LP3 ligand precursor.

[000181] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD. Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000182] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Maleimide carboxylic acids are commercially available for n=1 to 12.

[000183] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/Me0H in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000184] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; PEG maleimido acids are commercially available for ALQ=ALQ'=CH2CH2 and i=1 to 7 and are otherwise prepared according to the schemes described for the LP5 ligand precursor.

[000185] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000186] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Maleimide cyclohexanecarboxylic acid is commercially available for ALQ=CH2 and can otherwise be prepared according to the schemes described for the LP6 ligand precursor.

[000187] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): a-sulfonation of the carboxylic acid; the reaction is carried out at 75°C in a polar aprotic solvent such as DCE by treatment with chlorosulfonic acid in the presence of a base such as, for example, DIEA; Step (iv): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (v): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (vi): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000188] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; maleimide acids are commercially available for n=1 to 12.

[000189] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (iv): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000190] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and Ri7 and Ri8 = H or Me independently of each other; N-succinimidyl bromine and iodo-acetates are commercially available.

[000191] prepared according to the scheme prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as an NHS ester; The coupling of N-succinimidyl bromine and iodine acetate with the amino carboxylic acid is carried out in a polar aprotic solvent such as a DCM/DMF mixture followed by the addition of N,N'-disuccinimidyl carbonate and a base such as , for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000192] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; N-succinimidyl bromine and iodo-acetates are commercially available; amino carboxylic acids are commercially available for n=1 to 12 and N-methylated amino carboxylic acids for n=1 to 7.

[000193] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as NHS ester; The coupling of N-succinimidyl bromine and iodine acetate with the amino carboxylic acid is carried out in a polar aprotic solvent such as a DCM/DMF mixture followed by the addition of N,N'-disuccinimidyl carbonate and a base such as , for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000194] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; N-succinimidyl bromine and iodoacetate are commercially available; in the case where ALQ=CH2CH2 and R20=H, amino PEG carboxylic acids are commercially available for e i=1 to 6 and otherwise can be prepared from tert-butyl acrylate and the corresponding amino-PEG-alcohol; in the case where ALQ#CH2CH2, they can be prepared according to the schemes described for the LP10 ligand precursor.

[000195] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as an NHS ester; The coupling of N-succinimidyl bromine and iodine acetate with the amino carboxylic acid is carried out in a polar aprotic solvent such as a DCM/DMF mixture followed by the addition of N,N'-disuccinimidyl carbonate and a base such as , for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000196] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; N-succinimidyl bromine and iodoacetate are commercially available; amino-(hetero)aryl-carboxylic acids are commercially available, such as, for example, 4-amino-2-benzoic acid, 6-amino-3-pyridine carboxylic acid, 5-amino-2-pyrazine carboxylic acid, 2-amino-5-pyrimidine carboxylic acid or 6-amino-1,2,4,5-tetrazine carboxylic acid.

[000197] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000198] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Amino carboxylic acids are commercially available for n=1 to 12.

[000199] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000200] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Fmoc-protected amino PEG carboxylic acids are commercially available for e i=1 to 6 and otherwise can be prepared from tert-butyl acrylate and the corresponding amino-PEG alcohol; in the case where ALQ#CH2CH2, they can be prepared according to the schemes for the LP10 ligand precursor. Protection of the amine function with an Fmoc group can be accomplished by treatment with FmocOSu (CAS number [82911-69-1]) in the presence of a base such as, for example,

[000201] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000202] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Azido carboxylic acids can be prepared according to the schemes described for the LP14 ligand precursor.

[000203] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, e.g. example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (v): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000204] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; PEG azido carboxylic acids can be prepared according to the schemes described for the LP15 ligand precursor.

[000205] prepared according to the scheme below: _dioxane) or trifluoroacetic acid.

[000206] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Activated cyclooctin ligands such as NHS esters can be prepared according to the schemes described for the LP16 ligand precursor.

[000207] prepared according to the scheme below: Step (i): peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, ASD; Step (ii): deprotection of the amine; the reaction is carried out in a polar solvent such as a mixture of DCM/MeOH in the presence of a base such as, for example, piperidine; Step (iii): peptide coupling between the dipeptide and the NHS ester; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (iv): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000208] NHS esters of Fmoc-L-amino acids are commercially available; monoprotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; Activated cyclooctin ligands such as NHS esters can be prepared according to the schemes described for the LP17 ligand precursor.

[000209] prepared according to the scheme below: Step (i): peptide coupling between the LP1 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000210] Many aniline derivatives are commercially available such as, for example, 4-(hydroxymethyl)-aniline (CAS number [623-04-1]), 4-(1-hydroxyethyl)-aniline (racemic (CAS number CAS [14572-89-5]) or enantiopure (R) (CAS number [210754-259]) or (S) (CAS number [500229-84-5])), 4-amino-a,a- dimethyl-benzene-methanol (CAS number [23243-04-1]), 4-amino-a-methoxy-a-methyl-benzenemethanol (CAS number [1379318-81-6]), 4-amino-a- methyl-a-trifluoromethyl-benzenemethanol (CAS number [851652-56-7]), 2-amino-benzenemethanol (CAS number [534490-1]), 2-amino-a-methyl-benzenemethanol (racemic (CAS number CAS [10517-50-7]) or enantiopure (R) (CAS number [3205-21-8]) or (S) (CAS number [3205-21-8])), 6-amino-3- pyridinemethanol (CAS number [113293-71-3]), 6-amino-a-methyl-3-pyridinemethanol (CAS number [1335054-83-5]), 6-amino-a-ethyl-3-pyridinemethanol ( CAS number [1355225-85-2]), 6-amino-a,a-dimethyl-3-pyridinemethanol (CAS number [843646-03-8]), 5-amino-3-pyridinemethanol (CAS number [ 873651-92-4]), 2-amino-3-pyridinemethanol (CAS number [23612-57-9]), 2-amino-a-methyl-3-pyridinemethanol (racemic (CAS number [869567-91- 9]) or enantiopure (R) (CAS number [936718-01-3]) or (S) (CAS number [936718-00-2])), 2-amino-a-ethyl-3-pyridinamethanol ( CAS number [914223-90-8]), 2-amino-a,a-dimethyl-3-pyridinamethanol (CAS number [213666-96-7]), 3-amino-4-pyridinamethanol (CAS number [ 152398-05-5]), 3-amino-a-methyl-4-pyridinamethanol (CAS number [1242470-88-7]), 3-amino-a,a-methyl-4-pyridinamethanol (CAS number [ 13357-81-8]), 4-amino-3-pyridinamethanol (CAS number [138116-34-4]), 4-amino-a-methyl-3-pyridinamethanol (CAS number [741223-49-4] ), 4-amino-a,a-methyl-3-pyridinamethanol (CAS number [1339013-26-1]), 3-amino-2-pyridinamethanol (CAS number [52378-63-9]), 3- amino-a-methyl-2-pyridinamethanol (CAS number [954240-54-1]), 3-amino-a,a-methyl-2-pyridinamethanol (CAS number [899438-57-4]).

[000211] prepared according to the scheme below: Step (i): peptide coupling between the LP2 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000212] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000213] prepared according to the scheme below: Step (i): peptide coupling between the LP3 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000214] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000215] prepared according to the scheme below: Step (i): peptide coupling between the Lp4 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000216] Many aniline derivatives are commercially available such as, for example, those listed for Lp35.

[000217] prepared according to the scheme below: J Step (i): peptide coupling between the LP5 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000218] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000219] prepared according to the scheme below: Step (i): peptide coupling between the LP6 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000220] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000221] prepared according to the scheme below: Step (i): peptide coupling between the LP7 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.

[000222] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000223] prepared according to the scheme below: Step (i): peptide coupling between the LP8 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000224] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000225] prepared according to the scheme below: Step (i): peptide coupling between the Lp9 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000226] Many aniline derivatives are commercially available such as, for example, those listed for Lp35.

[000227] prepared according to the scheme below: Step (i): peptide coupling between the LP10 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a mixture of DCM/Me0H in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000228] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000229] prepared according to the scheme below: Step (i): peptide coupling between the LP11 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a mixture of DCM/Me0H in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000230] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000231] prepared according to the scheme below: Step (i): peptide coupling between the LP12 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.

[000232] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000233] prepared according to the scheme below: Step (i): peptide coupling between the LP13 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.

[000234] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000235] prepared according to the scheme below: Step (i): peptide coupling between the LP14 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000236] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000237] prepared according to the scheme below: Step (i): peptide coupling between the LP15 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000238] prepared according to the scheme below: Step (i): peptide coupling between the LP16 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000239] Many aniline derivatives are commercially available such as, for example, those listed for LP35. prepared according to the scheme below: Step (i): peptide coupling between the LP17 ligand precursor and an aniline derivative; the reaction is carried out in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of benzyl alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenylchloroformate in the presence of a base such as, for example, DIEA.

[000240] Many aniline derivatives are commercially available such as, for example, those listed for LP35.

[000241] prepared according to the scheme below: Step (i): formation of the carbamate between the LP35 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000242] prepared according to the scheme below: Step (i): formation of the carbamate between the LP36 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000243] prepared according to the scheme below: Step (i): formation of the carbamate between the LP37 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000244] LP55 prepared according to the scheme below: polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000245] LP56 prepared according to the scheme below: Step (i): formation of the carbamate between the LP38 ligand precursor and a monoprotected diamine; the reaction is carried out in a Step (i): formation of the carbamate between the LP39 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000246] prepared according to the scheme below: Step (i): formation of the carbamate between the LP40 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000247] prepared according to the scheme below: Step (i): formation of the carbamate between the LP41 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000248] prepared according to the scheme below: Step (i): formation of the carbamate between the LP42 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000249] prepared according to the scheme below: Step (i): formation of the carbamate between the LP43 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000250] prepared according to the scheme below: Step (i): formation of the carbamate between the LP44 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000251] prepared according to the scheme below: Step (i): formation of the carbamate between the LP45 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000252] prepared according to the scheme below: Step (i): formation of the carbamate between the LP46 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000253] prepared according to the scheme below: Step (i): formation of the carbamate between the LP47 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000254] prepared according to the scheme below: Step (i): formation of the carbamate between the LP48 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000255] Prepared according to the scheme below: Step (i): formation of the carbamate between the LP49 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000256] LP67 prepared according to the scheme below: Step (i): formation of the carbamate between the LP50 ligand precursor and a monoprotected diamine; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000257] LP68 prepared according to the scheme below: Step (i): formation of carbamate; the reaction is carried out in a polar solvent such as CH3CN in the presence of a base such as, for example, DIEA; deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid.

[000258] prepared according to the 1st step described for the LP35 ligand precursor.

[000259] prepared according to the 1st step described for the LP36 ligand precursor.

[000260] prepared according to the 1st step described for the LP37 ligand precursor.

[000261] prepared according to the 1st step described for the LP38 ligand precursor.

[000262] prepared according to the 1st step described for the LP39 ligand precursor.

[000263] prepared according to the 1st step described for the LP40 ligand precursor.

[000264] prepared according to the 1st step described for the LP41 ligand precursor.

[000265] prepared according to the 1st step described for the LP42 ligand precursor.

[000266] prepared according to the 1st step described for the LP43 ligand precursor.

[000267] prepared according to the 1st step described for the LP44 ligand precursor.

[000268] prepared according to the 1st step described for the LP45 ligand precursor.

[000269] prepared according to the 1st step described for the LP46 ligand precursor.

[000270] prepared according to the 1st step described for the LP47 ligand precursor.

[000271] prepared according to the 1st step described for the LP48 ligand precursor.

[000272] prepared according to the 1st step described for the LP49 ligand precursor.

[000273] prepared according to the 1st step described for the LP50 ligand precursor.

[000274] prepared according to the 1st step described for the LP51 ligand precursor.

[000275] prepared according to the scheme below: Step (i): coupling of the acid to Wang resin by esterification; the reaction is carried out in a polar solvent such as DCM in the presence of a coupling reagent such as, for example, DIC and a base such as, for example, DMAP; Step (ii): deprotection of the Fmoc group; the reaction is carried out in a polar solvent such as DMF by treatment with a base such as, for example, piperidine; Step (iii): peptide coupling; the reaction is carried out in a polar solvent such as DMF in the presence of coupling reagent such as, for example, HOBt and HATU and a base such as, for example, DIEA; Step (iv): resin cleavage; the reaction is carried out in a polar solvent such as a mixture of DCM/H2O in the presence of an acid such as, for example, TFA.

[000276] Fmoc-protected alkyl amino acids are commercially available for n=1 to 9; Fmoc L-amino acids are commercially available.

[000277] prepared according to the scheme below: Step (i): coupling of the acid to Wang resin by esterification; the reaction is carried out in a polar solvent such as DCM in the presence of a coupling reagent such as, for example, DIC and a base such as, for example, DMAP; Step (ii): deprotection of the Fmoc group; the reaction is carried out in a polar solvent such as DMF by treatment with a base such as, for example, piperidine; Step (iii): peptide coupling; the reaction is carried out in a polar solvent such as DMF in the presence of coupling reagent such as, for example, HOBt and HATU and a base such as, for example, DIEA; Step (iv): resin cleavage; the reaction is carried out in a polar solvent such as a mixture of DCM/H2O in the presence of an acid such as, for example, TFA.

[000278] Fmoc-protected amino PEG carboxylic acids are commercially available for i=1 to 6 and can otherwise be prepared from tert-butyl acrylate and the corresponding amino-PEG-alcohol; in the case where ALQ?CH2CH2, they can be prepared according to the schemes described for the LP10 ligand precursor. Protection of the amine function with an Fmoc group can be accomplished by treatment with FmocOSu (CAS number [82911-69-1]) in the presence of a base such as, for example, DIEA; Fmoc Lamino acids are commercially available.

[000279] prepared according to the scheme below: Step (i): ONHS and L-amino acid coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with sulfo amino acids; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000280] L-amino acids are commercially available; commercially available Bocson-protected NHS esters of L-amino acids; Sulfo amino acids are commercially available for n=1 and 2 or otherwise, they can be prepared according to the scheme below:

[000281] Step (i): replacing the bromide with a thioacetyl; the reaction is carried out in a polar aprotic solvent such as, for example, THF using thioacetic acid in the presence of a base such as, for example, DIEA; Step (ii): formation of the sulfonic acid moiety; the reaction is carried out by treatment with hydrogen peroxide and acetic acid; Step (iii): replacement of the bromide by an azide; the reaction is carried out in a polar solvent such as, for example, DMA by treatment with sodium azide; Step (iv): methyl ester deprotection; the reaction is carried out under acidic conditions such as, for example, a mixture of HCl and AcOH; Step (v): azide reduction; The reaction is carried out by hydrogenolysis in a polar solvent such as H2O in the presence of a catalyst such as palladium on carbon.

[000282] Dibromo derivatives are commercially available for n=3, 4 and 9.

[000283] LP89 prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with maleimide amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000284] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; Maleimide amines are commercially available for n=2 to 6.

[000285] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with PEG maleimide amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000286] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; PEG maleimide amines are commercially available for ALQ=ALQ'=CH2CH2 and i=0 and 1 or otherwise can be prepared according to the scheme below: Step (i: Mitsunobu reaction in maleimide; the reaction is carried out in a polar aprotic solvent such as, for example, THF by treatment of malemide by PEG hydroxide acid in the presence of PPh3 and DIAD; Step (ii): deprotection using a solution of hydrochloric acid (e.g. solution in dioxane) or trifluoroacetic acid; Step (iii): activation of the alcohol as a tosylate; the reaction is carried out in a polar aprotic solvent such as, for example, DCM by treatment with chloride tosyl in the presence of a base such as, for example, TEA; Step (iv): substitution of the tosyl group; the reaction is carried out in a polar aprotic solvent such as, for example, THF, in the presence of a base such as, e.g. example, sodium hydride. Step (v): deprotection of the benzyl group; the reaction is carried out by hydrogenolysis in a polar solvent such as, for example, MeOH, in the presence of a catalytic amount of catalyst, such as, for example, palladium on carbon; Step (vi): activation of the alcohol as a mesylate; the reaction is carried out in a polar aprotic solvent such as, for example, DCM by treatment with methanesulfonyl chloride in the presence of a base such as, for example, TEA; Step (vii): nucleophilic substitution; the reaction is carried out in a polar aprotic solvent such as, for example, DMF in the presence of a base such as sodium hydride;

[000287] Boc-protected PEG amino alcohols are commercially available for i=2 to 4 or can be prepared by treating commercially available PEG amino alcohols by Boc2O for i=5 to 8; starting ALQ amino alcohols are commercially available for n=1 and 3 to 10; monoprotected starting PEG diols with a benzyl group are commercially available for i=1 to 9; starting ALQ’ diols are commercially available for n=1 to 12.

[000288] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with maleimide amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000289] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; Maleimide amines can be prepared according to the scheme below: Step (i): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar solvent such as the DMF/H2O mixture by treatment with N,N'-disuccinimidyl carbonate in the presence of a base such as, for example, DIEA; Step (ii): peptide coupling; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000290] Maleimide cyclohexanecarboxylic acid is commercially available for ALQ=CH2 or otherwise, it can be prepared according to the schemes described for LP6; monoprotected diamines with a Boc group are commercially available for n=1 to 10.

[000291] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acidONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with PEG maleimide amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000292] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; PEG maleimide amines can be prepared according to the scheme below: Step (i): activation of the carboxylic acid as an NHS ester; the reaction is carried out in a polar solvent such as a mixture of DMF/H2O by treatment with N,N'-disuccinimidyl carbonate in the presence of a base such as, for example, DIEA; Step (ii): peptide coupling; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000293] Maleimide cyclohexanecarboxylic acid is commercially available for ALQ=CH2 or can otherwise be prepared according to the schemes described for LP6; monoprotected PEG diamines with a Boc group are commercially available for i=1 to 9.

[000294] OR 29 HN O NH 2 R 30 NR 25 O NR 19 I or Br ALK prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with haloacetamido PEG amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000295] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; Haloacetamido amines are commercially available for n=2 to 4 and 8 or can be prepared according to the scheme below: Step (ii): peptide coupling; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000296] N-succinimidyl bromine and iodo-acetates are commercially available; monoprotected diamines with a Boc group are commercially available for n=1 to 10.

[000297] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with haloacetamido PEG amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000298] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; Haloacetamido amines are commercially available for n=2 to 4 and 8 or can be prepared according to the scheme below: Step (ii): peptide coupling; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000299] N-succinimidyl bromine and iodo-acetates are commercially available; monoprotected PEG diamines with a Boc group are commercially available for i=1 to 9.

[000300] prepared according to the scheme below: Step (i): ONHS and L-amino acid peptide coupling; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with haloacetamido amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000301] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; Haloacetamido amines can be prepared according to the scheme below: Step (ii): peptide coupling; the reaction is carried out in a polar aprotic solvent such as a mixture of DCM/CH3CN; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000302] NHS esters of haloacetamides can be prepared as described for the LP11 ligand precursor; monoprotected diamines with a Boc group are commercially available for n=1 to 10.

[000303] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with azido amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000304] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; Azido amines are commercially available for n=2 to 8 and 10.

[000305] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with azido PEG amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000306] L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; for ALQ=CH2CH2, azido PEG amines are commercially available for i=1 to 8 and 10, for ALQ#CH2CH2, azido PEG amines can be prepared according to the scheme below: Step (i): activation of alcohol as a mesylate; the reaction is carried out in a polar aprotic solvent such as, for example, DCM by treatment with methanesulfonyl chloride in the presence of a base such as, for example, TEA; Step (ii): replacement of the mesylate by an azido group; the reaction is carried out in a polar solvent such as an acetone/H2O mixture by treatment with sodium azide; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000307] Starting PEG amino alcohols prepared as described for the LP90 ligand precursor.

[000308] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with cyclooctin amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000309] Cycloctin amines are commercially available for n=1, 2, 3 and 5.

[000310] prepared according to the scheme below: Step (i): peptide coupling between Boc-L-amino acid- ONHS and L-amino acid; the reaction is carried out in a polar solvent such as a mixture of DME/THF/H2O in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptide coupling with cyclooctin PEG amines; the reaction is carried out in a polar solvent such as CH3CN in the presence of coupling reagent such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a hydrochloric acid solution (e.g. solution in dioxane).

[000311] Cycloctin PEG amines can be prepared according to the scheme below: Step (i): activation of alcohol as a mesylate; the reaction is carried out in a polar aprotic solvent such as, for example, DCM by treatment with methanesulfonyl chloride in the presence of a base such as, for example, TEA; Step (ii): nucleophilic substitution; the reaction is carried out in a polar aprotic solvent such as, for example, DMF in the presence of a base such as sodium hydride; Step (iii): deprotection using a hydrochloric acid solution (e.g. dioxane solution) or trifluoroacetic acid; Step (iv): protection of the amine with a Boc group; the reaction is carried out in a polar solvent such as, for example, DCM, by treatment with Boc2O in the presence of a base such as, for example, TEA; Step (v): peptide coupling; the reaction is carried out in a polar solvent such as, for example, DCM, in the presence of coupling reagent such as, for example, HOBt and EDC.

[000312] Starting ALQ amino alcohols are commercially available for n=1 to 10; cyclooctins are commercially available for n=1, 2, 3 and 5. Table captions l: or - Alq. 1 Examples are provided for a cryptophycin compound, but may apply to any cryptophycin compound of formula (I), especially D1-D19. 2 In Table I, L is given here in the para position, however it is possible to obtain compounds in a similar way with L in an ortho or meta position.

Process for preparing conjugates of formula (III)

[000313] The conjugates of the present invention can be obtained through the process that comprises the steps consisting of: (i) contacting and allowing to react an optionally buffered aqueous solution of an antibody, optionally first modified by means of an modification, and a solution of the cryptophycin payload of formula (II), the RCG1 chemical group of the compound of formula (II) being reactive to the RCG2 chemical groups present in the antibody, especially to the amino groups present in the antibodies, said RCG2 chemical groups were introduced, where appropriate, by the modifying agent, in order to link the compound of formula (II) to the compound by forming a covalent bond; (ii) and then optionally separating the conjugate formed in step (i) from the cryptophycin payload and/or the unreacted antibody and/or any aggregates formed.

[000314] According to one variant and more particularly, in step (ii) the conjugate from step (i) is separated from the unreacted cryptophycin payload and any aggregates formed, and any unreacted antibody is left in solution.

[000315] The function of contacting is to react the chemical groups RCG1 and RCG2 in order to guarantee the connection of the cryptophycin payload to the compound by forming a covalent bond; Preferably, when RCG1 represents -C(=O)-ZaRa, the reaction preferably occurs at the amino functions of the antibody, especially the e-amino groups supported by the side chains of antibody lysine (Lys) residues and the a-amino groups of amino acids N-terminal of heavy and light chains. A conjugate of the following formula is obtained in this case: mAb- [NH-C(=O)-L*-Cripto]d with L* = fragment of an L ligand comprising RCG1=-C(=O)- ZaRa and so that L= - L*C(=O)- ZaRa and d representing the drug-to-antibody ratio or DAR; when RCG1 represents -Cl or a maleimido or haloacetamido group, the antibody may comprise chemical thiol groups; when RCG1 represents an azido group, the antibody may comprise a -C=CH moiety or an activated triple bond, such as a cyclooctin; when RCG1 represents -NH2, the reaction can occur in the amide function of the antibody using enzymatic catalysis, especially the amide groups supported by the side chains of glutamine (Gln) residues of an antibody. A conjugate of the following formula is obtained in this case: mAb- [C(=O)-NH-L*-Cripto]d with L* = fragment of a ligand L comprising RCG1=-NH2 and so that L= -L* NH2 and d representing drug-to-antibody ratio or DAR; when RCG1 represents -C=CH or an activated C=C such as a cyclooctin moiety, the antibody may comprise azido groups.

[000316] The term "aggregates" means associations that can form between two or more antibodies, the antibodies possibly having been modified by conjugation. Aggregates are responsible for forming the influence of a wide variety of parameters, such as a high concentration of antibody in the solution, the pH of the solution, high shear forces, the number of grafted drugs and their hydrophobic nature, temperature (see references cited in the introduction of J. Membrane Sci. 2008, 318, 311-316), their influence, however, has not been clearly elucidated. In the case of proteins or antibodies, reference may be made to AAPS Journal, "Protein Aggregation and Bioprocessing" 2006, 8(3), E572-E579. The aggregate content can be determined by means of known techniques, such as SEC (see in this regard, Analytical Biochemistry 1993, 212 (2), 469-480).

[000317] The aqueous solution of the antibody can be buffered with buffers such as, for example, potassium phosphate or HEPES or a mixture of buffers such as buffer A described later. The buffer depends on the nature of the antibody. The cryptophycin payload is dissolved in a polar organic solvent such as, for example, DMSO or DMA.

[000318] The reaction occurs at a temperature generally between 20°C and 40°C. The reaction time can vary between 1 and 24 hours. The reaction between the antibody and the cryptophycin payload can be monitored by SEC with a refractometric and/or ultraviolet detector and/or EMAR in order to determine its degree of progress. If the degree of substitution is insufficient, the reaction can be continued for longer and/or the cryptophycin compound can be added. Reference can be made to the example section for further details considering particular conditions. Particular embodiments are described in Examples 3, 7, 10, 14, 20 and 23.

[000319] A person skilled in the art has at his disposal several chromatographic techniques for the separation of step (ii): the conjugate can be purified, for example, by steric exclusion chromatography (SEC), by adsorption chromatography (e.g. ion exchange, IEC), by hydrophobic interaction chromatography (HIC), by affinity chromatography, by chromatography on mixed supports such as ceramic hydroxyapatite, or by HPLC. Purification by dialysis or diafiltration can also be used.

[000320] After step (i) or (ii), the conjugate solution may go through an ultrafiltration and/or diafiltration step (iii). After these steps, the conjugate in aqueous solution is thus obtained.

Antibody

[000321] The antibody may be a monoclonal antibody selected from the group consisting of a murine, chimeric, humanized and human antibody.

[000322] In one embodiment, the antibody is a monospecific antibody, that is, an antibody that specifically binds to a single target, and may be a multispecific antibody.

[000323] In one embodiment, the antibody is an IgG antibody, for example, an IgG1, IgG2, IgG3 or IgG4 antibody.

[000324] The antibody according to the invention specifically binds to a target, thereby directing the biologically active compound as a cytotoxic compound to said target, as used herein, "specifically binds" or "specifically binds to" or " binds to" or the like, means that an antibody or antigen-binding fragment likewise forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (KD) of at least about 1x10-8 M or less (e.g., a lower KD means tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been characterized, for example, by their specific binding to the target and/or target antigen using surface plasmon resonance, e.g., BIACORE™.

[000325] The target typically corresponds to a protein expressed on the cell surface, for example, a protein expressed on the surface of tumor cells.

[000326] In one embodiment, the target is the EphA2 receptor. The EphA2 receptor is an Ephrin receptor, and is also referred to as the "EPH A2 receptor" or "Epithelial Cell Receptor Tyrosine Protein Kinase" (see, for example, OMIM Entry *176946, available at omim.org/entry /176946, version updated on July 21, 2016). The antibody that specifically binds to the EphA2 receptor may, for example, correspond to one of the antibodies described in WO2008/010101 or WO2011/039724.

[000327] In another embodiment, the target is CD19. CD19 is a cell surface molecule specifically expressed by B lymphocytes and follicular dendritic cells of the hematopoietic system, and is also referred to as "B lymphocyte antigen CD19" (see, for example, OMIM Entry *107265, available on the omim worldwide website. org/entry/107265, last version updated on May 11, 2015). The antibody that specifically binds to CD19 may, for example, correspond to Coltuximab, that is, the antibody part (portion) of Coltuximab Ravtansine.

[000328] The antibody can optionally be modified with a modifying agent in order to promote binding of the cryptophycin payload as previously described. The antibody may especially be monoclonal, polyclonal or multispecific. It may also be an antibody fragment. It may also be a murine, human, humanized or chimeric antibody. The antibody used in the examples of the present invention is hu2H11_R3574, an antagonist antibody against the EphA2 receptor. The sequence of hu2H11_R3574 is described in WO2011/039724 (SEQ ID NO: 18 for the heavy chain and SEQ ID NO: 16 for the light chain).

Conjugate

[000329] A conjugate generally comprises about 1 to 10 cryptophycin compounds covalently linked to the antibody (i.e., the degree of graft or drug-to-antibody ratio" or "DAR"). This number varies as a function of the nature of the antibody and the cryptophycin compound, and also the operating conditions used in the conjugation process (e.g., the number of cryptophycin compound equivalents with respect to the antibody, a reaction time, the nature of the solvent and of any cosolvents) Placing the antibody and the cryptophycin compound in contact produces a mixture comprising several conjugates that are individually distinguished from each other by different DARs, optionally aggregated. The DAR that is determined in the final solution thus corresponds to an average DAR. The DAR can be calculated from the deconvolution of the SEC-EMAR spectrum of the conjugate. The DAR (EMAR) is preferably greater than 0.5, more particularly between 1 and 10 and even more particularly between 2 and 7.

[000330] The conjugate can be used as an anti-cancer agent. Due to the presence of the antibody, the conjugate is highly selective towards tumor cells and not healthy cells. This makes it possible to direct the cryptophycin compound into an environment similar to it or directly into it; (In this regard, see the following publications describing the use of monoclonal antibody conjugates in cancer treatment: "Antibody-drug conjugates for cancer therapy" Carter P.J., et al., Cancer J. 2008, 14, 154-169; "Targeted cancer therapy: conferring specificity to cytotoxic drugs" Chari R., Acc. Chem. Res. 2008, 41, 98-107). It is possible to treat solid or liquid cancers. The conjugate can be used alone or in combination with at least one other anticancer agent.

[000331] The conjugate is formulated in the form of a buffered aqueous solution at a concentration of between 1 and 10 mg/mL. This solution can be injected as an infusion on its own or can be rediluted to form an infusion solution.

Examples

[000332] The following examples describe the preparation of certain compounds according to the invention. These examples are not limiting, and merely illustrate the present invention.

Analytical methods used High Pressure Liquid Chromatography - Mass Spectrometry (LCMS) Method A1

[000333] Spectra were obtained on a Waters UPLC-SQD system in a positive and/or negative electrospray mode (ES+/-) using ELSD and UV detection (210-400 nm). The chromatographic conditions were as follows: column: ACQUITY BEH C18 - 1.7 µm - 2.1 x 50 mm; solvents: A: H2O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 50°C; flow rate: 0.8 mL/min; gradient (2.5 min): from 5 to 100% B in 1.8 min; 2.4 min: 100% B; 2.45 min: from 100 to 5% B in 0.05 min.

Method A2

[000334] Spectra were obtained on a Waters XeVo-QTof system in positive electrospray mode (ES+) using ELSD and UV detection (210-400 nm). The chromatographic conditions were as follows: column: ACQUITY BEH C18 - 1.7 µm - 2.1 x 100 mm; solvents: A: H2O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 70°C; flow rate: 0.55 mL/min; gradient (11 min): from 5 to 97% B in 8.3 min; 8.6 min: 97% B; 9 min: from 97 to 5% B in 0.7 min and 5% B for 2 min. Method A3

[000335] The spectra were obtained on a Waters XeVo-QTof system in positive electrospray mode (ES+). The chromatographic conditions were as follows: column: ACQUITY BEH C18 - 1.7 µm - 2.1 x 100 mm; solvents: A: H2O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 45°C; flow rate: 0.6 mL/min; gradient (5.3 min): 5% B for 0.3 min; from 5 to 100% B in 3.7 min; 4.6 min: 100% B; 5.3 min 5% B.

A4 Method

[000336] Spectra were obtained on a Waters UPLC-SQD system in a positive and/or negative electrospray mode (ES+/-) using ELSD and UV detection (210-400 nm). The chromatographic conditions were as follows: column: ACQUITY BEH C18 - 1.7 µm - 2.1 x 50 mm; solvents: A: H2O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 45°C; flow rate: 0.8 mL/min; gradient (10 min): from 5 to 100% B in 8.6 min; 9.6 min: 100% B; 9.8 min: 5% B.

Method A5

[000337] Spectra were obtained on a Waters UPLC-SQD system in a positive and/or negative electrospray mode (ES+/-) using ELSD and UV detection (210-400 nm). The chromatographic conditions were as follows: column: ACQUITY CSH C18 - 1.7 µm - 2.1 x 50 mm; solvents: A: H2O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 40°C; flow rate: 0.85 mL/min; gradient (2.5 min): from 5 to 100% B in 1.8 min; 2.4 min: 100% B; 2.45 min: from 100 to 5% B in 0.05 min.

1H Nuclear Magnetic Resonance (NMR)

[000338] 1H NMR spectra were acquired on a Bruker Avance estrometer, model DRX-300, DRX-400 or DRX-500. Chemical shifts (d) are provided in ppm.

Size Exclusion Chromatography - High Resolution Mass Spectrometry (SEC-EMAR)

[000339] Chromatographic analysis was performed on an Agilent HP1100 machine and a Waters BEH SEC 200 1.7 µm (2.1 x 150 mm) column at 30°C with a flow rate of 0.5 mL/min and a isocratic elution of (A) 25 mM ammonium formate + 1% formic acid/(B) CH3CN + 0.1% formic acid 70/30 for 15 minutes. Mass spectrometry was performed on a Waters QTOF-II machine with electrospray ionization in positive mode (ES+). Mass spectra were deconvolved with Waters MaxEnt1 software.

Analytical Size Exclusion Chromatography (SEC)

[000340] Analysis was performed on a Waters Alliance HPLC system or a Hitachi Lachrom system equipped with a photodiode array detector and a Tosoh Bioscience TSKgel G3000 SWXL 5 µm (7.8 x 300 mm) column with a flow rate of 0. 5 mL/min and a 30 minute isocratic elution with a pH 7 buffer containing 0.2 M KCl, 0.052 M KH2PO4, 0.107 M K2HPO4 and 20% by volume isopropanol. n • Buffer A (pH 6.5): NaCl (50 mM), potassium phosphate (50 mM), EDTA (2 mM) • Buffer B (pH 6.5): NaCl (140 mM), potassium phosphate and sodium (9.6 mM) • PBS (pH 7.4): KH2PO4 (1.06 mM), NaCl (155.17 mM), Na2HPO4-7H2O (2.97 mM) • DPBS: KCl (2.67 mM ), KH2PO4(1.47 mM), NaCl (136.9 mM), Na2HPO4 (8.10 mM) adjusted to pH 6.5 with 5N HCl (1 mL per 1000 mL buffer)

General method for preparing antibody-drug conjugate (CAF)

[000341] An antibody solution in an aqueous buffer composed of a 96:4 mixture of buffer A and 1 N HEPES was treated with an excess of a solution in approximately 10 mM cryptophycin payload in DMA, so that the final antibody concentration is 3 mg/mL and the percentage of DMA in the aqueous buffer is 20%. After shaking for 2 hours, the mixture was analyzed by SEC-EMAR to determine the DAR in the monomeric antibody population. If the DAR was found to be insufficient, the mixture would be treated with another excess (1 to 5 equivalents) of cryptophycin solution in DMA for an additional 2 hours at room temperature under stirring. The mixture was purified by gel filtration using a 200 pg Superdex matrix (HiLoad 16/60 or 26/60 desalting column, GEHealthcare) pre-equilibrated in pH 6.5 aqueous buffer (buffer B or DPBS) containing 10 to 20% of NMP. The fractions containing the monomeric conjugate antibody were pooled and concentrated in Amicon Ultra-15 (10k or 50k Ultracel membrane, Millipore) to a concentration between 2 and 5 mg/mL. A buffer exchange or dilution into the appropriate buffer was then performed to formulate the conjugate in the buffer. In the case of a buffer exchange, it was performed by gel filtration using a pre-equilibrated SephadexTM G25 matrix (desalination columns NAP-5, NAP-10, NAP-25/PD-10 or Hiprep 26/10, GEHealthcare). with the final aqueous buffer whose composition and pH are adjusted for each conjugate. The conjugate was finally filtered through a Steriflip® filter unit (0.22 µm Durapore® PVDF membrane, Millipore). The final conjugate was tested by UV spectrometry or SEC-HPLC in order to measure the conjugate concentration, by SEC-HPLC in order to determine the monomeric purity and by SEC-EMAR in order to determine the DAR from the deconvolution of the spectrum. mass of the conjugate. Synthesis of fragment A: (2E,5S,6R,7E)-tert-butyl 8-(4-formylphenyl)-5-hydroxy-6-methylocta-2,7-dienoate Compound 1: (2E,5S,6R)-tert-butyl 5-hydroxy-7-((4-methoxybenzyl)oxy)-6-methylhept-2-enoate

[000342] Under argon, to a solution of (2R,3S)-1- [(4-methoxyphenyl)methoxy] -2-methyl-5-hexen-3-ol or Sakurai alcohol (CAS number [203926-55 -0], 100 g, 399.5 mmol) in DCM (350 mL) tert-butyl acrylate (354.7 mL, 2.435 mol) and Grubbs catalyst (4.849 g, 2.435 mol) were added. After stirring for 17 h at room temperature, the reaction mixture was filtered through 230 g of silica and eluted with a mixture of heptane/AcOEt (50/50, 6 x 300 mL). After concentration, the residue was purified by flash chromatography on 1.2 kg of silica gel (heptane/EtOAc gradient elution) to provide 140.8 g of a brown oil. The oil was dissolved in anhydrous DCM (550 mL), 10 g of quadraPureTM TU resin was added to remove excess catalyst. The mixture was stirred for 3 h at 35°C. The resin was filtered and washed with DCM, the filtrate was concentrated and dried in vacuo to provide 135.75 g of compound 1 as a brown oil (97%).

[000343] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.85 (d, J = 7.0 Hz, 3 H); 1.42 (s, 9 H); 1.72 (m, 1H); 2.18 (m, 1 H); 2.30 (m, 1H); 3.24 (dd, J = 7.0 and 9.3 Hz, 1 H); 3.46 (dd, J = 7.7 and 9.3 Hz, 1 H); 3.49 (m, 1H); 3.73 (s, 3H); 4.35 (s, 2H); 4.65 (d, J = 5.8 Hz, 1 H); 5.76 (td, J = 1.5 and 15.6 Hz, 1 H); 6.83 (td, J = 7.4 and 15.6 Hz, 1 H); 6.90 (d, J = 8.8 Hz, 2 H); 7.23 (d, J = 8.8 Hz, 2 H). Compound 2: (2E,5S, 6R)-tert-butyl 5,7-dihydroxy-6-methylhept-2-enoate

[000344] To a solution of compound 1 in CH3CN (910 mL), H2O (90 mL) was added and the mixture was cooled to 12°C before adding over 25 min a solution of CAN (196.7 g, 358. 9 mmol) in H2O (300 mL). Stirring was continued for 1 h at room temperature. Additional CAN (18.83 g, 34.36 mmol) dissolved in a mixture of CH3CN (70 mL) and H2O (30 mL) was added and stirred for 1 h at room temperature to complete the reaction. The mixture was quenched with NaCl until saturation of the aqueous layer, then MTBE (400 mL) was added. After decantation, the aqueous layer was extracted with MTBE (2 x 80 mL). The combined organic layers were washed with a 3:1 mixture of 5% NaHCO3/H2O (3 x 200 mL), saturated brine (2 x 130 mL), dried over MgSO4, filtered and concentrated. The aqueous layer was acidified with 2N HCl to pH 4, then saturated with NaCl and extracted with MTBE (2 x 150 mL). This second organic layer was washed with 5% NaHCO3 (60 mL), saturated brine (60 mL), dried over MgSO4, concentrated and combined with the first organic layer to provide an orange-brown oil.

[000345] The mixture was dissolved in MeTHF (300 mL), then 1,2-ethanedithiol (22 mL, 0.260 mol) and p-TsOH (1.651 g, 8.590 mmol) were added. After stirring at 60°C for 5 h, MTBE (200 mL) was added to the reaction mixture and it was washed with 5% NaHCO3 (2 x 120 mL), saturated brine (120 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 520 g of silica gel (DCM/MTBE gradient elution) to give 29.48 g of compound 2 as a yellow oil (75%). Compound 3: (2E,5S,6R)-tert-butyl 6-methyl-5,7-bis((triethylsilyl)oxy)hept-2-enoate

[000346] Under argon, to a solution of compound 2 (39.38 g, 0.171 mol) in anhydrous DCM (500 mL) imidazole (51.540 g, 0.749 mol) was added. The yellow mixture was cooled to 0°C, then triethylchlorosilane (63.99 mL, 0.374 mol) was added and the solution was allowed to warm to room temperature overnight. The reaction mixture was quenched with saturated NH4Cl (400 mL), MTBE (300 mL) and ice. The pH was adjusted to 4 with 2M NaHSO4, the layers were separated after vigorous shaking. The aqueous layer was extracted with MTBE (200 mL). The combined organic layers were washed with partially saturated NH4Cl (2 x 250 mL), pH 7 phosphate buffer (150 mL), saturated brine (150 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography at 490 g of silica gel (heptane/Et2O gradient elution) to give 74.52 g of compound 3 as a light yellow oil (95%). Compound 4: (2E,5S,6S)-tert-butyl 6-methyl-7-oxo-5-((triethylsilyl)oxy)hept-2-enoate

[000347] Under argon, to a solution of DMSO (102.400 mL, 1.427 mol) in DCM (300 mL) was added dropwise at -75°C oxalyl chloride (63 mL, 0.719 mol) in 1 h and stirring was continued for 15 min before adding, at -75°C, a solution of compound 3 in DCM (150 mL) while maintaining the temperature below -70°C. The reaction mixture was allowed to warm to -40°C and stirring continued for 2 h. The reaction mixture was cooled again to -75°C, then DIEA (425 mL, 2.432 mol) was added over 75 minutes, while maintaining the temperature below -65°C. The reaction mixture was allowed to warm to room temperature and then quenched with MTBE (500 mL), ice, saturated NH4Cl (200 mL) and 2M NaHSO4 to reach pH 4. The layers were separated after vigorous stirring. The aqueous layer was extracted with MTBE (200 mL). The combined organic layers were washed with NH4Cl (3 x 300 mL), pH 7 phosphate buffer (300 mL), saturated brine (300 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography at 1.25 kg of silica gel (heptane/Et2O gradient elution) to give 51.74 g of compound 4 as an orange oil (93%). Compound 5: (2E,5S,6R,7E)-tert-butyl 5-hydroxy-8-(4-(hydroxymethyl)phenyl)-6-methylocta-2,7-dienoate

[000348] Under argon, to a suspension of phosphonium bromide (synthesis described in WO2011/001052, 147 g, 0.237 mol) in anhydrous THF (1.5 L) was added at -50°C a 2.5 M solution of n-BuLi in hexane (90 mL, 0.225 mol). The temperature was allowed to warm to -40°C and stirring was continued for 15 min. After warming to room temperature, the red mixture was stirred for 1 h. The reaction medium was cooled to -70°C before adding a solution of compound 4 (51.63 g, 126.6 mmol) in THF (200 mL) while maintaining the temperature below -65°C. When the addition was complete, the reaction mixture was allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was filtered to remove the insoluble which was washed with MTBE (500 mL). The filtrate was partially concentrated at 42°C (1/3), then quenched with ice, NH4Cl (500 mL), MTBE (500 mL) and 2M NaHSO4 to reach pH 4. The layers were separated and the aqueous layer was extracted with MTBE (200 mL). The combined organic layers were washed with half saturated NH4Cl (250 mL), pH 7 phosphate buffer (250 mL), saturated brine (250 mL), dried over MgSO4, filtered and half concentrated in vacuo to 250 mbar. The suspension was diluted with 50:50 heptane/MTBE (600 mL) cooled with an ice bath, filtered and washed with 50:50 heptane/MTBE. The filtrate was concentrated in vacuo to provide 100.05 g as a brown oil.

[000349] The crude compound was dissolved in anhydrous THF (320 mL), then TBAF trihydrate (90 g, 282.40 mmol) was added and the reaction mixture was stirred for 3 h. After concentration in vacuum, the crude was diluted with 10:1 MTBE/THF (770 mL), washed with partially saturated NH4Cl (3 x 200 mL), 1M NaHSO4 (200 mL), phosphate buffer pH 7 (200 mL). mL), saturated brine (200 mL), dried over MgSO4, filtered and concentrated. The aqueous layer was extracted with MTBE. After decantation, the organic layer was washed with phosphate buffer pH 7 (30 mL), saturated brine (30 mL), dried over MgSO4 and filtered. After concentration, the combined organic layers were purified by flash chromatography on 1.25 kg of silica gel (heptane/Et2O gradient elution) to give 36.91 g of compound 5 as a reddish brown oil (88%). Fragment A: (2E,5S,6R,7E)-tert-butyl 8-(4-formylphenyl)-5-hydroxy-6-methylocta-2,7-dienoate

[000350] Under argon, to a solution of compound 5 (30.3 g, 91.7 mmol) in anhydrous DCM (600 mL) was added, at 10°C, manganese oxide (175 g, 3.013 mol) and to Reaction mixture was stirred for 4 h at 35°C. The oxidant was filtered through a pad of celite and washed with hot acetone. The filtrate was concentrated to provide 30.43 g of red-orange oil.

[000351] Under argon, the oil was diluted with benzene (800 mL) and refluxed in the presence of AIBN (880 mg, 4.58 mmol), and thiophenol (2.81 mL, 27.51 mmol) for 1 h. More AIBN (530 mg, 0.03 eq) and thiophenol (1.03 mL, 10.087 mmol) were added and reflux continued for another 1 h. More AIBN (530 mg, 0.03 eq) and thiophenol (1.03 mL, 10.087 mmol) were added and reflux continued for 3 h. More AIBN (353 mg, 1.834 mmol) and thiophenol (468 µL, 4.58 mmol) were added and reflux continued for 1 h. The E/Z conversion was 92/8, 352.6 mg of AIBN was added and reflux continued for 1 h. The reaction mixture was cooled to room temperature and concentrated in vacuum to provide 28.03 g of fragment A as an orange oil (92%) with an E/Z ratio greater than 98/2. Synthesis of fragment B: (R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxy-phenyl)propanoic acid

[000352] To a solution of methyl (R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphil)-propanoate (CAS number [162465-44-3], 30 g, 87.26 mmol) in THF (225 mL), H2O (30 mL) and LiOH monohydrate (4.4 g, 104.85 mmol) were added. The reaction medium was stirred for 2 hours at room temperature. After this time, the reaction medium was diluted with H2O (200 mL) then acidified to pH 2 with 5N HCl (20 mL) and extracted by EtOAc (2 x 250 mL). The combined organic phases were washed with H2O (500 mL), dried over MgSO4, filtered, concentrated in vacuo, diluted with Et2O and concentrated in vacuo to provide 26.9 g of fragment B as a white solid (94%).

[000353] 1H NMR (300 MHz, d in ppm, DMSO-d6): 1.21 to 1.37 (m, 9 H); 2.74 (dd, J = 11.0 and 14.3 Hz, 1 H); 2.96 (dd, J = 5.0 and 14.3 Hz, 1 H); 3.81 (s, 3H); 4.04 (m, 1H); 6.99 to 7.09 (m, 2H); 7.18 (dd, J = 2.3 and 8.7 Hz, 1 H); 7.29 (d, J = 2.3 Hz, 1 H); 12.60 (wide m, 1 H). Synthesis of fragment C1: methyl 3-amino-2,2-dimethylpropanoate hydrochloride Compound 6: methyl 3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropanoate

[000354] Under argon, to a solution of 3-([(tert-butoxy)carbonyl]amino)-2,2-dimethylpropanoic acid (CAS number [18018102-6], 250 mg, 1.09 mmol) in DCM (6 mL) and MeOH (2 mL) was added, dropwise at 0°C, (trimethylsilyl)diazomethane (819.86 µL, 1.64 mmol) until persistent yellow color. Then, AcOH was added until complete decolorization. The reaction mixture was concentrated in vacuo then diluted with H2O and extracted with DCM twice. The combined organic phases were washed with saturated brine, dried over MgSO4, filtered and concentrated in vacuo to provide 260 mg of compound 6 as a colorless oil (quant.). Fragment C1: methyl 3-amino-2,2-dimethylpropanoate hydrochloride

[000355] To a solution of compound 6 (260 mg, 1.12 mmol) in 1,4-dioxane (10 mL) was added 4N HCl in 1,4-dioxane (2.81 mL, 11.24 mmol) . The reaction mixture was stirred at room temperature overnight then concentrated in vacuo to provide 200 mg of C1 fragment (quant.). Synthesis of fragment C2: methyl 3-amino-2,2-dimethylbutanoate hydrochloride Compound 7: 3-((tert-butoxycarbonyl)amino)butanoic acid

[000356] To a solution of DL-3-aminobutyric acid (CAS number [541-48-02], 18.72 g, 176.09 mmol) in H2O (96 mL) was added di-tert-butyl dicarbonate (39.62 g, 176.09 mmol) and NaOH (8.03 g, 200.74 mmol) in H2O (76 mL), then tert-butyl alcohol (132 mL). The reaction mixture was stirred at room temperature overnight. After this time, the reaction medium was concentrated in vacuum, then diluted with EtOAc (200 mL) and acidified to pH 3 with diluted HCl. After settling, the aqueous phase was extracted with EtOAc (200 mL). The combined organic phases were dried over MgSO4, filtered and concentrated in vacuo to provide 34.5 g of compound 7 as a colorless oil (82%).

[000357] 1H NMR (300 MHz, d in ppm, CDCl3-d1): 1.25 (d, J = 7.0 Hz, 3 H); 1.45 (s, 9H); 2.55 (m, 2H); 4.04 (m, 1H); 4.93 (wide m, 1 H); 8.41 (wide m, 1 H). Compound 8: Methyl 3-((tert-butoxycarbonyl)amino)butanoate

[000358] Under argon, a solution of compound 7 (10 g, 49.20 mmol) in toluene (350 mL) and MeOH (100 mL) was stirred at +5°C. Between +5°C and +10°C, (trimethylsilyl)diazomethane (73.81 mL, 147.61 mmol) was added dropwise. The reaction mixture was stirred for 3 h then evaporated in vacuum to provide 10.8 g of compound 8 as a light yellow oil (quant.).

[000359] 1H NMR (300 MHz, d in ppm, CDCl3-d1): 1.20 (d, J = 7.0 Hz, 3 H); 1.45 (s, 9H); 2.50 (m, 2H); 3.69 (s, 3H); 4.03 (m, 1H); 4.90 (wide m, 1 H). Compound 9: Methyl 3-((tert-butoxycarbonyl)amino)-2-methylbutanoate

[000360] Under argon, to a solution of LDA (2M in THF, 25.32 mL, 50.63 mmol) in THF (48 mL) at -70°C was added dropwise a solution of compound 8 (5 g , 23.01 mmol) in THF (62 mL). The reaction mixture was stirred for 1 h at -75°C, then iodomethane (5.79 mL, 92.05 mmol) was added dropwise at -70°C. The reaction mixture was stirred for 3 h at -75°C, then at room temperature overnight. After this time, an aqueous solution of 20% NH4Cl (100 mL) and Et2O (125 mL) were added. After settling, the organic phase was washed with saturated NaHCO3 (80 mL), then saturated NaCl (80 mL). The combined aqueous phases were extracted with Et2O (125 mL). The combined organic phases were dried over MgSO4, filtered and concentrated in vacuo to provide 5.49 g of crude oil. Crystallization with pentane (15 mL) provided 2.65 g of compound 9 after vacuum drying. The pentane solution was concentrated in vacuo and purified by flash chromatography on 100 g of silica gel (heptane/Et2O gradient elution) to give 1.98 g of compound 9. The two batches were pooled (87%) and used in the state in which you are in the next stage. Compound 10: methyl 3-((tert-butoxycarbonyl)amino)-2-,2-dimethylbutanoate

[000361] Under argon, THF (46 mL) was cooled to -72°C, then LDA (2M in THF, 22 mL, 44.0 mmol) was added and dropwise to -72°C (+/- 2°C) a solution of compound 9 (4.60 g, 19.89 mmol) in THF (64 mL). The reaction mixture was stirred for 1h15 at -75°C, then iodomethane (5 mL, 79.55 mmol) was added dropwise at -72°C (+/- 2°C). The reaction medium was stirred 3 h at -75°C, then overnight at room temperature. After this time, an aqueous solution of 20% NH4Cl (50 mL) and Et2O (80 mL) were added. After settling, the organic phase was washed with sat. NaHCO3 (50 mL) and saturated brine (50 mL). The combined aqueous phases were extracted with Et2O (80 mL). The combined organic phases were dried over MgSO4, filtered and concentrated in vacuo to give 6.5 g of crude oil which was purified by flash chromatography on 200 g of silica gel (heptane/iPr2O gradient elution) to give 1.9 g of compound 10 as a colorless oil (39%).

[000362] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.92 (d, J = 7.1 Hz, 3 H); 1.02 (s, 3H); 1.04 (s, 3H); 1.48 (s, 9 H); 3.58 (s, 3H); 3.88 (m, 1 H); 6.62 (wide d, J = 10.5 Hz, 1 H). LCMS (A1): ES m/z = 146, m/z = 246 [M+H]+; tR = 1.2 min. Fragment C2: methyl 3-amino-2,2-dimethylbutanoate hydrochloride

[000363] In a round bottom flask, under magnetic stirring, compound 10 (0.3 g, 1.22 mmol) was introduced, followed by 1,4-dioxane (5 mL) and 4N HCl in 1,4- dioxane (5 mL). The reaction mixture was stirred at room temperature overnight then concentrated in vacuo. The crude solid obtained was precipitated in iPr2O (15 mL), filtered and dried to provide 210 mg of C2 fragment as a white solid (95%).

[000364] LCMS (A1): ES m/z = 146 [M+H]+; tR = 0.8 min. Synthesis of fragment C3: ethyl 2-(1-aminocyclopropyl)-2,2-dimethylpropanoate

[000365] Under argon, to a solution of ethyl 2-cyano-2-methylpropionate (CAS number [1572-98-1], 2 g, 13.88 mmol) in Et2O (48 mL) was added titanium isopropoxide (IV) (4.63 g, 15.97 mmol). The reaction mixture was stirred for 10 minutes at room temperature, then cooled to -5°C. A solution of 3M ethylmagnesium bromide in Et2O (9.72 mL, 29.16 mmol) was added dropwise at -5°C-0°C over 25 min, the reaction mixture was then stirred for 40 minutes without the cooling bath. At this time, a TLC showed that the reaction was completed. The reaction medium was cooled to 0°C and boron trifluoride diethyl etherate (3M in Et2O, 3.6 mL, 29.16 mmol) was added dropwise at 0°C. The reaction mixture was stirred for 30 minutes without a cooling bath. After this time, 1N HCl was added at 0°C to pH 1-2 (8 mL), then 2N NaOH to pH 8 (28 mL), the reaction mixture was extracted with EtOAc (3 x 150 mL ). The combined organic phases were dried over MgSO4, filtered and concentrated in vacuo to give 2.4 g of a yellow crude oil which was purified by flash chromatography on 70 g of silica gel (DCM/MeOH gradient elution) to give 915 mg of fragment C3 as a light yellow oil (39%).

[000366] 1H NMR (400 MHz, d in ppm, CDCl3-d1): 0.52 (m, 2 H); 0.70 (m, 2H); 1.11 (s, 6H); 1.27 (t, J = 7.2 Hz, 3 H); 1.64 (broad s, 2H); 4.18 (q, J = 7.2 Hz, 2 H). Synthesis of fragment C4: methyl 3-amino-2-(hydroxymethyl)-2-methylpropanoate hydrochloride Compound 11: 3-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid

[000367] To a solution of DL-3-aminoisobutyric acid (CAS number [10569-72-9], 5 g, 47.52 mmol) in 2N NaOH (24.7 mL) was added dropwise one solution of Boc2O (11.73 g, 53.22 mmol) in THF (75 mL) while maintaining the reaction medium temperature below 30 ° C with a cold water bath. The reaction medium was stirred at room temperature for 18 hours, then concentrated in vacuum, diluted in H2O (75 mL) and washed with MTBE (3 x 150 mL). The aqueous phase was acidified to pH 3 by adding 100 g/L citric acid (150 mL) and extracted with EtOAc (3 x 150 mL). The combined organic phases were washed with H2O (3 x 45 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 9.4 g of compound 11 as a colorless oil (97%).

[000368] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.01 (d, J = 7.1 Hz, 3H); 1.38 (s, 9H); 2.48 (m, 1H); 2.91 (dt, J = 6.1, 6.5 and 13.5 Hz, 1H); 3.15 (ddd, J = 6.1, 7.4 and 13.5 Hz, 1H); 6.80 (t, J = 6.1 Hz, 1H); 12.15 (broad s, 1H). Compound 12: methyl 3-((tert-butoxycarbonyl)amino)-2-methylpropanoate

[000369] To a solution of compound 11 (9.4 g, 46.25 mmol) in acetone (300 mL) were added K2CO3 (16.14 g, 115.63 mmol) and CH3I (13.26 g, 92. 5 mmol). A reaction medium, a yellow suspension, was stirred at room temperature for 20 hours, then filtered over Claricel. The mass thus obtained was washed with acetone, the filtrate concentrated in vacuo, diluted with DCM (100 mL), filtered over Clarcel, the mass thus obtained was washed with DCM and the filtrate concentrated in vacuo to give 9.4 g of compound 12 as a yellow liquid (93%).

[000370] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.01 (d, J = 7.1 Hz, 3H); 1.37 (s, 9H); 2.54 (m, 1H); 2.91 (dt, J = 6.2 and 13.5 Hz, 1H); 3.15 (ddd, J = 6.2, 6.9 and 13.5 Hz, 1H); 3.59 (s, 3H); 6.90 (t, J = 6.2 Hz, 1H). Compound 13: methyl 3-((tert-butoxycarbonyl)amino)-2-(((4-methoxybenzyl)oxy)methyl)-2-methylpropanoate

[000371] To a solution of DIEA (3.2 mL, 22.45 mmol) in THF (10 mL) cooled to -75°C was added dropwise a solution of 1.6 M n-BuLi in THF (14 mL , 22.4 mmol). The reaction medium was stirred at -75°C for 20 min; then a solution of compound 12 (2 g, 9.21 mmol) in THF (16 mL) was added dropwise at -75°C and the reaction mixture was stirred at -75°C for 10 min. A solution of 1-((chloromethoxy)methyl)-4-methoxybenzene (1.72 g, 9.21 mmol) in THF (16 mL) was then rapidly added to the reaction mixture and stirring carried out at -25°C for 4 hours. The reaction medium was diluted with DCM (100 mL) before adding a 100 g/L citric acid solution (50 mL) while maintaining the temperature below 5°C. The reaction medium was stirred at room temperature for 15 min. The organic phase was washed with a 100 g/L solution of citric acid (2 x 50 mL), H2O (3 x 50 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 4.08 g of a yellow oil -orange which was purified by flash chromatography on 150 g of silica gel (heptane/EtOAc gradient elution) to give 2.05 g of compound 13 as a colorless oil (60%).

[000372] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.03 (s, 3H); 1.37 (s, 9H); 3.14 (d, J = 6.8 Hz, 2H); 3.31 (d, J = 9.1 Hz, 1H); 3.48 (d, J = 9.1 Hz, 1H); 3.58 (s, 3H); 3.73 (s, 3H); 4.38 (s, 2H); 6.76 (t, J = 7.1 Hz, 1H); 6.90 (d, J = 8.9 Hz, 2H); 7.20 (m, 2H). Fragment C4: methyl 3-amino-2-(hydroxymethyl)-2-methylpropanoate hydrochloride

[000373] Compound 13 (1.5 g, 4.08 mmol) was treated with a solution of 4 M HCl in dioxane (24 mL) at room temperature for 1 h. The reaction medium was then concentrated in vacuum and co-evaporated in the presence of toluene to provide 794 mg of C4 fragment as a viscous oil (quant.). Synthesis of AD1: 5-(((S)-2-amino-4-methylpentanoyl)oxy)-8-(4-(azidomethyl)phenyl)-6-methylocta-2,7-dienoate (2E,5S ,6R,7E)-tert-butyl Compound 14: 5-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanoyl)oxy)-8-(4-formylphenyl)-6-methylocta (2E,5S,6R,7E)-tert-butyl -2,7-dienoate

[000374] Under argon, in a round-bottom flask under magnetic stirring, Fmoc-Leu-OH (4.9 g, 13.86 mmol) was introduced, followed by fragment A (3.5 g, 10.59 mmol) in DCM (100 mL) and DIEA (6.6 mL, 38.13 mmol). Then, MNBA (5 g, 14.52 mmol) and DMAP (620 mg, 5.07 mmol) were added and the reaction mixture was stirred overnight at room temperature. After this time, the reaction medium was washed with H2O (100 mL) and saturated brine (100 mL). The organic phase was dried over MgSO4, filtered and concentrated in vacuo to give 9.1 g of crude orange oil which was purified by flash chromatography on 400 g of silica gel (heptane/AcOEt gradient elution) to give 5 g of compound 14 as a light yellow oil (71%).

[000375] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.77 (d, J = 6.8 Hz, 6 H); 1.05 (d, J = 7.0 Hz, 3 H); 1.33 to 1.64 (m, 4H); 1.38 (s, 9 H); 2.39 to 2.63 (m partially masked, 2 H); 4.01 (m, 1 H); 4.13 to 4.31 (m, 3H); 4.95 (m, 1 H); 5.81 (d, J = 15.9 Hz, 1 H); 6.37 (dd, J = 8.8 and 16.2 Hz, 1 H); 6.52 (d, J = 16.2 Hz, 1 H); 6.70 (td, J = 7.3 and 15.9 Hz, 1 H); 7.29 (t, J = 7.9 Hz, 2 H); 7.40 (t, J = 7.9 Hz, 2 H); 7.56 (d, J = 8.4 Hz, 2 H); 7.67 (dd, J = 7.9 Hz, 2 H); 7.74 to 7.82 (m, 3H); 7.87 (d, J = 7.9 Hz, 2 H); 9.93 (s, 1 H). LCMS (A1): ES m/z = 666 [M+H]+, m/z = 689 [M+Na]+; tR = 1.92 min. Compound 15: 5-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanoyl)oxy)-8-(4-(hydroxymethyl)phenyl)- (2E,5S,6R,7E)-tert-butyl 6-methylocta-2,7-dienoate

[000376] Under argon, to a solution of compound 14 (5 g, 7.51 mmol) in MeTHF (60 mL), sodium trimethoxyborohydride (1.2 g, 8.91 mmol) was added portionwise. The reaction medium was stirred for 5 h at room temperature. After this time, saturated NH4Cl (100 mL) and acetone (20 mL) were added. The reaction medium was stirred for 1 h at room temperature. After settling, the organic phase was washed with H2O, then with saturated brine (50 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 5.3 g of crude yellow oil which was purified by flash chromatography in 300 g of silica gel (heptane/AcOEt gradient elution) to provide 3.15 g of compound 15 as a white semisolid (63%).

[000377] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.80 (m, 6 H); 1.02 (d, J = 7.0 Hz, 3 H); 1.35 to 1.66 (m, 4H); 1.38 (s, 9 H); 2.35 to 2.56 (m partially masked, 2 H); 4.02 (m, 1 H); 4.15 to 4.32 (m, 3H); 4.43 (d, J = 5.5 Hz, 2 H); 4.92 (m, 1H); 5.12 (t, J = 5.5 Hz, 1 H); 5.80 (d, J = 15.9 Hz, 1 H); 6.10 (dd, J = 8.8 and 16.2 Hz, 1 H); 6.39 (d, J = 16.2 Hz, 1 H); 6.69 (m, 1H); 7.20 (d, J = 8.4 Hz, 2 H); 7.28 to 7.33 (m, 4H); 7.41 (t, J = 7.9 Hz, 2 H); 7.69 (t, J = 7.9 Hz, 2 H); 7.80 (d, J = 8.2 Hz, 1 H); 7.88 (d, J = 7.9 Hz, 2 H). LCMS (A1): ES m/z = 668 [M+H]+; m/z = 690 [M+Na]+; m/z = 712 [M-H+HCO2H]-; tR = 1.84 min. Compound 16: 5-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanoyl)oxy)-8-(4-(azidomethyl)phenyl)- (2E,5S,6R,7E)-tert-butyl 6-methylocta-2,7-dienoate

[000378] Under argon, compound 15 (3.15 g, 4.72 mmol) and DCM (50 mL) were introduced into a round-bottom flask. At 0°C, TEA (987 µL, 7.08 mmol) was added, followed by methanesulfonyl chloride (438 µL, 5.66 mmol), the reaction medium was stirred for 1 hour at 0°C and 13 hours at room temperature. After this time, DCM (50 mL) and water (50 mL) were added. After settling, the organic phase was washed with saturated brine (3 x 25 mL), dried over MgSO4 and concentrated in vacuo. Under argon, the crude product thus obtained was dissolved in DMF (50 mL) and sodium azide (644 mg, 9.91 mmol) was added. The reaction medium was stirred overnight at room temperature. After this time, DMF was concentrated in vacuum and AcOEt was added. The obtained mixture was washed with 0.1N HCl (25 mL), saturated NaHCO3 (25 mL) and saturated brine (25 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 3.3 g of crude which was purified by flash chromatography on 200 g of silica gel (heptane/AcOEt gradient elution) to give 1.8 g of compound 16 as a colorless gum (55%) and 740 mg of compound AD1 (33%).

[000379] LCMS (A2): ES m/z = 715 [M+Na]+; tR = 8.95 min. Compound AD1: 5-(((S)-2-amino-4-methylpentanoyl)oxy)-8-(4-(azidomethyl)phenyl)-6-methylocta-2,7-dienoate (2E,5S,6R, 7E)-tert-butyl

[000380] Under argon, in a round bottom flask under magnetic stirring, compound 16 (1.8 g, 2.6 mmol) and DCM (50 mL) were introduced, followed by piperidine (1.6 mL, 16.2 mmol). The reaction medium was stirred overnight at room temperature. After this time, it was washed with 1N HCl then with saturated NaHCO3, saturated brine, dried over MgSO4, filtered and concentrated in vacuo to give 2 g of crude which was purified by flash chromatography on 130 g of silica gel (elution by heptane/EtOAc gradient) to provide 1.48 g of compound AD1 as a light yellow oil (quant.).

[000381] LCMS (A3): ES m/z = 471 [M+H]+; tR = 2.65 min. Synthesis of AD2: 5-(((S)-2-amino-4,4-dimethylpentanoyl)oxy)- 8-(4-(hydroxymethyl)phenyl)-6-methylocta-2,7-dienoate hydrochloride (2E ,5S,6R,7E)-tert-butyl Compound 17: 5-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4,4-dimethylpentanoyl)oxy)-8-(4-formylphenyl)-6 (2E,5S,6R,7E)-tert-butyl-methylocta-2,7- dienoate

[000382] Under argon, in a round bottom flask to a solution of compound A (1.633 g, 4.94 mmol) in DCM (60 mL) was added L-Fmoc-tert-Leu-OH (1.82 g, 4.94 mmol), DIEA (2.57 mL, 14.83 mmol), MNBA (1.70 g, 4.94 mmol) and DMAP (241.51 mg, 1.98 mmol). After stirring for 2 hours at room temperature, the reaction medium was diluted with H2O (50 mL) and extracted twice with DCM. The combined organic phases were washed with citric acid (2 x 50 mL), saturated NaHCO3 (50 mL) and saturated brine (50 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 3.24 g of crude compound 17 used. directly in the subsequent reduction (96%). Compound 18: 5-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4,4-dimethylpentanoyl)oxy)-8-(4-(hydroxymethyl)phenyl (2E,5S,6R,7E)-tert-butyl )-6-methylocta-2,7-dienoate

[000383] Under argon, in a round-bottom flask to a solution of compound 17 (3.24 g, 4.77 mmol) in MeTHF (60 mL) was added sodium trimethoxyborohydride (670.56 mg, 5.24 mmol ) at 0°C. After stirring for 1 hour at room temperature, more sodium trimethoxyborohydride (304 mg, 2.38 mmol) was added and stirred for 2 hours. Then, the reaction medium was cooled to 0°C, diluted with acetone (18 mL) and saturated NH4Cl (36 mL) and extracted with AcOEt. The combined organic phases were washed with saturated brine, dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 100 g of silica gel (Heptane/EtOAc gradient) to provide 2.61 g of compound 18 as a colorless amorphous solid. (80%).

[000384] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.80 (s, 9 H); 1.02 (d, J = 7.0 Hz, 3 H); 1.35 to 1.60 (m, 4 H); 1.38 (s, 9H); 2.37 to 2.58 (m partially masked, 2 H); 4.04 (m, 1H); 4.16 to 4.33 (m, 3H); 4.45 (d, J = 5.7 Hz, 2 H); 4.91 (m, 1H); 5.12 (t, J = 5.7 Hz, 1 H); 5.80 (d, J = 16.0 Hz, 1 H); 6.10 (dd, J = 8.8 and 16.2 Hz, 1 H); 6.39 (d, J = 16.2 Hz, 1 H); 6.69 (m, 1H); 7.21 (d, J = 8.4 Hz, 2 H); 7.25 to 7.33 (m, 4 H); 7.40 (t, J = 7.9 Hz, 2 H); 7.68 (t, J = 7.9 Hz, 2 H); 7.78 (d, J = 8.3 Hz, 1 H); 7.88 (d, J = 7.9 Hz, 2 H). LCMS (A1): ES m/z = 608; m/z = 682 [M+H]+; m/z = 726 [M-H+HCO2H]-; tR = 1.85 min. Compound AD2: 5-(((S)-2-amino-4,4-dimethylpentanoyl)oxy)-8-(4-(hydroxymethyl)phenyl)-6-methylocta-2,7-dienoate hydrochloride (2E, 5S, 6R,7E)-tert-butyl

[000385] Under argon, in a round bottom flask, to a solution of compound 18 (2.61 g, 3.83 mmol) in DCM (40 mL) was added piperidine (7.60 mL, 76.56 mmol) and the reaction medium was stirred for 1 hour at room temperature. It was then concentrated and extracted with AcOEt. The combined organic layers were washed with 1N HCl, H2O and saturated brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was diluted with iPr2O (50 mL) and stirred for 40 hours at room temperature. The crude product was filtered, washed with iPr2O and dried in vacuum 3 hours at 40°C to provide 1.706 g of compound AD2 as a colorless amorphous solid (90%).

[000386] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.88 (s, 9 H); 1.10 (d, J = 7.0 Hz, 3 H); 1.39 to 1.49 (m, 1 H); 1.42 (s, 9 H); 1.75 (dd, J = 6.1 and 15.1 Hz, 1 H); 2.47 to 2.64 (m partially masked, 2 H); 3.92 (t, J = 5.5 Hz, 1 H); 4.47 (d, J = 5.7 Hz, 2 H); 5.02 (m, 1H); 5.15 (t, J = 5.7 Hz, 1 H); 5.90 (d, J = 15.9 Hz, 1 H); 6.15 (dd, J = 8.3 and 16.1 Hz, 1 H); 6.44 (d, J = 16.1 Hz, 1 H); 6.77 (td, J = 7.3 and 15.9 Hz, 1 H); 7.26 (d, J = 8.4 Hz, 2 H); 7.34 (d, J = 8.4 Hz, 2 H); 8.10 (wide m, 3 H). LCMS (A1): ES m/z = 460 [M+H]+; tR = 0.95 min. Synthesis of AD3: (2R,3S)-1-((4-methoxybenzyl)oxy)-2-methyl-hex-5-en-3-yl (S)-2-amino-4,4-dimethylpentanoate Compound 19: (2R,3S)-1-((4-methoxybenzyl)oxy (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4,4-dimethylpentanoate -2-methyl-hex-5-en-3-yl

[000387] To a solution of Sakurai alcohol (1.02 g, 4.08 mmol) and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4 acid, 4-dimethylpentanoyl (1.5 g, 4.08 mmol) in THF (15 mL) was added 2,4,6-trichlorobenzoyl chloride (1.02 g, 4.08 mmol), dropwise TEA (1. 14 mL, 8.16 mmol) and DMAP (126.0 mg, 1.02 mmol). The reaction medium was stirred at room temperature for 5 hours, then cooled using an ice bath before adding 1N HCl (60 mL), while maintaining the temperature below 10°C. The resulting medium was stirred at room temperature for 15 minutes and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with sat. NaHCO3 (15 mL), saturated brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by three successive rapid chromatographies on silica gel (150 g, heptane/EtOAc gradient elution; 150 g and 20 g , DCM/MeOH gradient elution) to provide 1.78 g of compound 19 as a colorless oil (72%).

[000388] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (d, J = 7.1 Hz, 3H); 0.89 (s, 9H); 1.51 (dd, J = 2.8 and 14.3 Hz, 1H); 1.61 (dd, J = 9.0 and 14.3 Hz, 1H); 1.95 (m, 1H); 2.21 (m, 1H); 2.31 (m, 1H); 3.20 (dd, J = 6.5 and 9.6 Hz, 1H); 3.35 (m, 1H); 3.73 (s, 3H); 4.04 (m, 1H); 4.18 to 4.34 (m, 5H); 4.83 (m, 1H); 5.00 (dq, J = 2.1 and 10.3 Hz, 1H); 5.05 (dq, J = 2.1 and 7.3 Hz, 1H); 5.70 (m, 1H); 6.87 (d, J = 8.7 Hz, 2H); 7.17 (d, J = 8.7 Hz, 2H); 7.30 (m, 2H); 7.41 (t, J = 7.8 Hz, 2H); 7.70 (d, J = 7.8 Hz, 2H); 7.75 (d, J = 8.3 Hz, 1H); 7.90 (d, J = 7.8 Hz, 2H). LCMS (A5): ES m/z = 600 [M+H]+; m/z = 617 [M+H+NH3]+; m/z = 644 [M-H+HCO2H]-; tR = 1.86 min. Compound AD3: (2R,3S)-1-((4-methoxybenzyl)oxy)-2-methyl-hex-5-en-3-yl (S)-2-amino-4,4-dimethylpentanoate

[000389] To a solution of compound 19 (1.78 g, 2.98 mmol) in DCM (63 mL) was added dropwise piperidine (1.77 mL, 17.9 mmol). The reaction medium was stirred at room temperature for 4 h, diluted with DCM (150 mL), washed with 1 N HCl (2 x 20 mL), saturated NaHCO3 (20 mL), saturated brine (3 x 20 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 50 g of silica gel (heptane/EtOAc gradient elution) to give 644 mg of compound AD3 as a colorless oil (57%).

[000390] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.88 (s, 9H); 0.89 (d, J = 7.1 Hz, 3H); 1.21 (dd, J = 6, 9 and 13.9 Hz, 1H); 1.56 (dd, J = 5.0 and 13.9 Hz, 1H); 1.66 (s broad, 2H); 2.00 (m, 1H); 2.24 (m, 1H); 2.33 (m, 1H); 3.26 (m, 2H); 3.38 (dd, J = 5.5 and 9.4Hz, 1H); 3.74 (s, 3H); 4.36 (s, 2H); 4.84 (m, 1H); 5.00 to 5.10 (m, 2H); 5.72 (m, 1H); 6.89 (d, J = 8.8 Hz, 2H); 7.22 (d, J = 8.8 Hz, 2H). LCMS (A5): ES m/z = 378 [M+H]+; tR = 0.88 min. Synthesis of BC1: 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylpropanoic acid Compound 20: methyl 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylpropanoate

[000391] Under argon, the C1 fragment (981.45 mg, 4.00 mmol) and DCM (50 mL) were introduced into a round-bottom flask, followed by DIEA (1.84 mL, 10.92 mmol), fragment B (1.2 g, 3.64 mmol), HOBt (563.40 mg, 4.00 mmol) and EDC (1.45 mL, 8.01 mmol). The reaction medium was stirred overnight at room temperature. After this time, the reaction medium was diluted with H2O (30 mL) and extracted twice with DCM. The combined organic phases were washed with saturated brine, dried over MgSO4, filtered and concentrated in vacuo to provide 2.5 g of crude oil which was purified by flash chromatography on 110 g of silica gel (heptane/EtOAc gradient elution) to provide 1.06 g of compound 20 as a white meringue (66%).

[000392] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.06 (s, 3 H); 1.07 (s, 3H); 1.30 (s, 9 H); 2.65 (dd, J = 11.6 and 13.9 Hz, 1 H); 2.82 (dd, J = 4.0 and 13.9 Hz, 1 H); 3.18 (dd, J = 6.1 and 13.4 Hz, 1 H); 3.29 (partially masked m, 1 H); 3.60 (s, 3H); 3.81 (s, 3H); 4.11 (m, 1 H); 6.90 (d, J = 8.7 Hz, 1 H); 7.04 (d, J = 8.6 Hz, 1 H); 7.19 (dd, J = 2.3 and 8.6 Hz, 1 H); 7.33 (broad s, 1 H); 7.75 (large t, J = 6.1 Hz, 1 H). Compound BC1: 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylpropanoic acid

[000393] Under argon, compound 20 (1.06 g, 2.39 mmol) and THF (25 mL) were introduced into a round-bottom flask followed by LiOH (70.18 mg, 2.87 mmol) and H2O ( 1 mL). The reaction medium was stirred a few hours before adding 70 mg of LiOH and stirred overnight at room temperature. After this time, Amberlit resin was added until pH 4, filtered then washed with THF and concentrated in vacuo to provide 1 g of compound BC1 as a white solid (97%). Synthesis of BC2: 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylbutanoic acid Compound 21: methyl 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylbutanoate

[000394] Under argon, fragment B (1.02 g, 2.78 mmol) and DCM (25 mL) were introduced into a round-bottom flask, followed by EDC (592 mg, 3.03 mmol) and HOBt (478 mg, 3.03 mmol). The reaction medium was stirred for 15 minutes, then the C2 fragment (0.5 g, 2.75 mmol) and DIEA (1.7 mL, 9.73 mmol) were added. The reaction medium was stirred overnight at room temperature. After this time, the reaction medium was concentrated in vacuum, then diluted with AcOEt. The organic layer was washed with saturated brine, dried over MgSO4, filtered and concentrated in vacuo to give 1.45 g of crude oil which was purified by flash chromatography on 100 g of silica gel (heptane/AcOEt gradient elution) to give 845 mg of compound 21 as a white foam (67%).

[000395] 1H NMR (400 MHz, d in ppm, DMSO-d6): 50:50 diastereoisomeric mixture; 0.87 (d, J = 6.8 Hz, 1.5 H); 0.97 (d, J = 6.8 Hz, 1.5 H); 1.01 (s, 1.5 H); 1.03 (s, 1.5H); 1.07 (s, 3H); 1.31 (s, 9H); 2.58 to 2.87 (m, 2H); 3.59 (s, 1.5H); 3.60 (s, 1.5H); 3.81 (s, 3H); 3.99 to 4.20 (m, 2H); 6.90 (d, J = 9.0 Hz, 0.5 H); 6.97 (d, J = 9.0 Hz, 0.5 H); 7.05 (broad d, J = 8.6 Hz, 1 H); 7.19 (split dd, J = 2.4 and 8.6 Hz, 1 H); 7.32 (d, J = 2.4 Hz, 0.5 H); 7.34 (d, J = 2.4 Hz, 0.5 H); 7.54 (d, J = 10.1 Hz, 0.5 H); 7.62 (d, J = 10.1 Hz, 0.5 H). LCMS (A3): 50:50 diastereoisomeric mixture; ES m/z = 457 [M+H]+; m/z = 479 [M+Na]+; tR = 3.19-3.2 min.

[000396] Stereomers 1 and 2 of Compound 21: 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylbutanoate methyl - stereomers 1 and 2

[000397] Under argon, in a round bottom flask to a solution of fragment B (1.17 g, 3.55 mmol) in DCM (30 mL) EDC (741.75 mg, 3.87 mmol) was added, HOBt (592.57 mg, 3.87 mmol). After stirring for 15 minutes at room temperature, the C2 fragment (639 mg, 3.52 mmol) and DIEA (2.17 mL, 12.31 mmol) were added. The reaction medium was stirred for 4 hours, then concentrated and diluted with EtOAc (100 mL). The organic layers were washed with H2O (2 x 10 mL) and saturated brine (2 x 10 mL), dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography on 50 g of silica gel (heptane/EtOAc gradient elution) to provide 1.306 g of mixture of diastereoisomers (81%) which were separated by two successive flash chromatographies, the first on 100 g of silica gel (DCM/MeOH gradient elution) to provide 376 mg of stereomer 1 of compound 21 (23%), the Second in 70 g of silica gel (DCM/MeOH gradient elution) to provide 181 mg of stereomer 1 of compound 21 (11%), 279 mg of stereomer 2 of compound 21 (17%) and 476 mg of mixture of diastereoisomers.

Stereomer 1 of Compound 21

[000398] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (d, J = 6.8 Hz, 3 H); 1.07 (s, 6H); 1.31 (s, 9H); 2.57 (m, 1H); 2.80 (dd, J = 5.2 and 13.8 Hz, 1 H); 3.60 (s, 3H); 3.81 (s, 3H); 4.05 (m, 1H); 4.16 (m, 1H); 6.99 (d, J = 8.7 Hz, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.20 (dd, J = 2.0 and 8.6 Hz, 1 H); 7.34 (d, J=2.0 Hz, 1 H); 7.57 (d, J=10.1 Hz, 1 H). LCMS (A1): ES m/z = 381; m/z = 401; m/z = 455 [M-H]-; m/z = 457 [M+H]+; tR = 1.29 min.

Stereomer 2 of Compound 21

[000399] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.95 (d, J = 6.8 Hz, 3 H); 1.00 (s, 3H); 1.03 (s, 3H); 1.31 (s, 9H); 2.55 (m, 1H); 2.80 (dd, J = 5.0 and 14.3 Hz, 1 H); 3.59 (s, 3H); 3.81 (s, 3H); 4.09 to 4.22 (m, 2H); 6.92 (d, J = 8.8 Hz, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.20 (dd, J = 2.0 and 8.6 Hz, 1 H); 7.34 (d, J = 2.0 Hz, 1 H); 7.64 (d, J = 10.1 Hz, 1 H). LCMS (A1): ES m/z = 381; m/z = 401; m/z = 455 [M-H]-; m/z = 457 [M+H]+; tR = 1.29 min.

[000400] Compound BC2: 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylbutanoic acid

[000401] Compound 21 (0.845 g, 1.85 mmol) and MeOH (20 mL) were introduced into a round bottom flask followed by 1.8 mL of 10M NaOH. The solution was stirred and heated at 50°C overnight. The reaction medium was evaporated in vacuum then diluted with H2O (20 mL) and neutralized with 5N HCl. The solution was extracted twice with AcOEt. The combined organic layers were washed with saturated brine, dried over MgSO4, filtered and concentrated in vacuo to provide 800 mg of compound BC2 as a white foam (97%).

[000402] 1H NMR (400 MHz, d in ppm, DMSO-d6): diastereoisomeric mixture 50:50; 0.91 (d, J = 6.8 Hz, 1.5 H); 0.97 (d, J = 6.8 Hz, 1.5 H); 1.00 (s, 3H); 1.03 (s, 1.5H); 1.07 (s, 1.5 H); 1.31 (s, 9H); 2.57 (m, 1 H); 2.83 (m, 1H); 3.81 (s, 3H); 4.01 to 4.16 (m, 2H); 6.90 (d, J = 9.0 Hz, 0.5 H); 6.95 (d, J = 9.0 Hz, 0.5 H); 7.03 (d, J = 8.6 Hz, 1 H); 7.20 (broad, J = 8.6 Hz, 1 H); 7.31 (broad s, 1 H); 7.49 (d, J = 10.1 Hz, 0.5 H); 7.54 (d, J = 10.1 Hz, 0.5 H); 11.94 (wide m, 1 H). LCMS (A1): 50:50 diastereoisomeric mixture; ES m/z = 387; m/z = 443 [M+H]+; tR = 1.20-1.21 min.

[000403] Stereomer 1 of Compound BC2: stereomer 1 of 3- ((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylbutanoic acid

[000404] Stereomer 1 of compound 21 (0.325 g, 1.85 mmol) and MeOH (8 mL) were introduced into a round-bottom flask followed by 0.692 mL of 10M NaOH. The yellow solution was stirred and heated at 50°C overnight. The reaction medium was evaporated in vacuum, then diluted with H2O (20 mL) and extracted with AcOEt (3 x 5 mL). The aqueous layers were acidified with 5N HCl and extracted with AcOEt (3 x 30 mL). The combined organic phases were washed with saturated brine (5 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 303 mg of BC2 compound stereomer 1 as a white foam (96%) used directly in the subsequent reaction.

[000405] Stereomer 2 of Compound BC2: stereomer 2 of 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylbutanoic acid

[000406] Stereomer 2 of Compound 21 (1.094 g, 2.39 mmol), THF (5 mL) and H2O (5 mL) were introduced into a round bottom flask followed by LiOH (301 mg, 7.18 mmol). The solution was stirred for 44 hours at room temperature. The reaction was not completed, LiOH (301 mg) was added. The mixture was stirred for 48 h, then 301 mg of LiOH was added in THF (10 mL) and H2O (5 mL) and the reaction medium was stirred for 40 h at 60 ° C. The reaction medium was evaporated in vacuum. 1M citric acid was added until pH 2 and the mixture was extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with H2O, dried over MgSO4, filtered and concentrated in vacuo to provide 1.096 g of stereomer 2 of compound BC2 as a white amorphous solid (quant.) used directly in the subsequent reaction. Synthesis of BC3: ethyl 2-((R)-1-(2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)cyclopropyl)-2-methylpropanoic acid Compound 22: Ethyl 2-((R)-1-(2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)cyclopropyl)-2-methylpropanoate

[000407] Under argon, fragment C3 (1.2 g, 7.01 mmol) and THF (16.5 mL) were introduced into a round-bottom flask, followed by fragment B (2.54 g, 7.71 mmol). mmol), HOBt (1.77g, 8.76 mmol), EDC (1.23 g, 8.06 mmol) and DIEA (1.35 mL, 7.71 mmol). The reaction medium was stirred for 2 hours at room temperature. After this time, the reaction medium was diluted with H2O (25 mL) and extracted with AcOEt (250 mL). The organic layers were washed with H2O (2 x 25 mL), saturated brine (2 x 25 mL), dried over MgSO4, filtered, concentrated and purified by flash chromatography, the first on 200 g of silica gel (heptane gradient elution /EtOAc) to give 2.16 g of compound 22 as a colorless foam (64%) and 343 mg of mixture containing the expected compound which was also purified on 30 g of silica gel (heptane/EtOAc gradient elution) to give 160 mg of compound 22 as a colorless foam (4.7%).

[000408] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.62 (m, 1 H); 0.74 to 0.99 (m, 3H); 1.03 (s, 3H); 1.10 (s, 3H); 1.24 (t, J = 7.2 Hz, 3 H); 1.42 (s, 9H); 2.90 (m, 2H); 3.88 (s, 3H); 4.08 (m, 1H); 4.10 (q, J = 7.2 Hz, 2 H); 4.96 (m, 1H); 6.33 (broad s, 1 H); 6.85 (d, J = 8.5 Hz, 1 H); 7.03 (dd, J = 2.4 and 8.5 Hz, 1 H); 7.17 (d, J = 2.4 Hz, 1 H). Compound BC3: 2-((R)-1-(2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)cyclopropyl)-2-methylpropanoic acid

[000409] Compound 22 (2.09 g, 4.33 mmol), THF (10 mL) and H2O (8 mL) were introduced into a round bottom flask followed by LiOH (726.33 mg, 17.31 mmol ). The solution was stirred and heated to 65°C. After 16 h, the reaction was not completed, 726.33 mg of LiOH in 10 mL of H2O were added. The mixture was stirred for 48 h at 65°C. After cooling, the reaction medium was diluted with H2O (20 mL) and then extracted with AcOEt (3 x 40 mL). The organic layers were washed with H2O (2 x 10 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 1.29 g of ester/acid mixture. The aqueous layer was acidified with 5N HCl to pH 3, then extracted with EtOAc (3 x 50 mL). The organic layers were washed with H2O (2 x 10 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 787 mg of compound BC3 as a beige foam (40%).

[000410] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.50 to 0.90 (m, 4 H); 1.03 (s, 6H); 1.30 (s, 9 H); 2.60 (dd, J = 10.5 and 14.1 Hz, 1 H); 2.78 (dd, J = 5.0 and 14.1 Hz, 1 H); 3.81 (s, 3H); 3.97 (m, 1 H); 6.80 (d, J = 8.7 Hz, 1 H); 7.03 (d, J = 8.5 Hz, 1 H); 7.18 (dd, J = 2.0 and 8.5 Hz, 1 H); 7.31 (d, J = 2.0 Hz, 1 H); 7.86 (s, 1H); 12.11 (wide m, 1 H). Synthesis of BC4: (S)-3-((R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylbutanoic acid Compound 23: Methyl (S)-3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylbutanoate

[000411] To a solution of fragment B (2 g, 6.06 mmol) in DCM (60 mL) were added EDC (1.13 mL, 7.06 mmol) and HOBt (948 mg, 6.67 mmol). The reaction medium was stirred at room temperature for 15 minutes, then methyl (S)-3-amino-2,2-dimethylbutanoate (881 mg, 6.06 mmol) and DIEA (1.53 mL, 9 .10 mmol). The reaction medium was stirred at room temperature for 4 hours, concentrated in vacuo and diluted with EtOAc (100 mL) and H2O (20 mL). The aqueous phase was extracted with EtOAc (20 mL); the combined organic phases were washed with saturated brine (2 x 20 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 150 g of silica gel (heptane/EtOAc gradient elution) to give 2.21 g of compound 23 as a colorless lacquer (79%).

[000412] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.88 (d, J = 7.0 Hz, 3H); 1.08 (s, 6H); 1.32 (s, 9H); 2.67 (dd, J = 9.9 and 13.6 Hz, 1H); 2.82 (dd, J = 5.2 and 13.6 Hz, 1H); 3.62 (s, 3H); 3.83 (s, 3H); 4.06 (m, 1H); 4.77 (m, 1H) 6.97 (d, J = 8.6 Hz, 1H); 7.05 (d, J = 8.6 Hz, 1H); 7.20 (dd, J = 2.4 and 8.6 Hz, 1H); 7.33 (d, J = 2.4 Hz, 1H); 7.55 (d, J = 9.8 Hz, 1H). Compound 24: Methyl (S)-3-((R)-2-amino-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylbutanoate 2,2,2-trifluoroacetate

[000413] To a solution of compound 23 (2.2 g, 4.81 mmol) in DCM (25 mL) was added TFA (3.6 mL, 48.1 mmol). The reaction medium was stirred at room temperature overnight, concentrated in vacuum, and coevaporated in the presence of toluene to provide 2.0 g of compound 24 as a diastereoisomeric mixture (88%).

[000414] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.80 (d, J = 6.9 Hz, 3H); 1.04 (s, 3H); 1.10 (s, 3H); 2.95 (d, J = 7.0 Hz, 2H); 3.62 (s, 3H); 3.84 (s, 3H); 4.00 (m, 1H); 4.15 (m, 1H); 7.10 to 7.30 (m, 3H); 8.00 (d, J = 9.5 Hz, 1H); 8.22 (broad s, 3H). Compound 25: Methyl (S)-3-((R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylbutanoate

[000415] To a solution of compound 24 (2.0 g, 4.25 mmol) in DCM (20 mL) were added acryloyl chloride (536 µL, 6.37 mmol) and DIEA (2.5 mL, 12. 74 mmol). The reaction medium was stirred at room temperature for 2 hours, then diluted with H2O (20 mL). The aqueous phase was extracted with DCM (2 x 20 mL), the combined organic phases were washed with saturated brine (2 x 20 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 100 g of silica gel (heptane/EtOAc gradient elution) to provide 850 mg of compound 25 as an 85:15 diastereoisomeric mixture (68%).

[000416] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (d, J = 6.9 Hz, 3H); 1.03 (s, 3H); 1.04 (s, 3H); 2.72 (dd, J = 9.7 and 13.9 Hz, 1H); 2.86 (dd, J = 5.8 and 13.9 Hz, 1H); 3.58 (s, 3H); 3.80 (s, 3H); 4.15 (m, 1H); 4.56 (m, 1H); 5.56 (dd, J = 2.3 and 10.2 Hz, 1H); 6.03 (dd, J = 2.3 and 17.2 Hz, 1H); 6.28 (dd, J = 10.2 and 17.2 Hz, 1H); 7.02 (d, J = 8.5 Hz, 1H); 7.17 (dd, J = 2.2 and 8.5 Hz, 1H); 7.31 (d, J = 2.2 Hz, 1H); 7.72 (d, J = 9.8 Hz, 1H); 8.36 (d, J = 8.6 Hz, 1H). Compound BC4: (S)-3-((R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylbutanoic acid

[000417] To a solution of tBuOK (1.11 g, 9.86 mmol) in THF (4 mL) cooled to 0°C, H2O (47 µL) and compound 25 (450 mg, 1.10 mmol) were added. . The reaction medium was stirred at room temperature for 3 hours, then acidified with 1N HCl (5 mL). The aqueous phase was extracted with DCM (2 x 20 mL); the combined organic phases were washed with H2O (30 mL), saturated brine (20 mL), dried over MgSO4, filtered and concentrated in vacuo. The two diastereoisomers were separated by supercritical fluid chromatography on a Chiralpak AS 10 µm column (isocratic elution at 85/15 CO2/ [MeOH + 0.1% TEA] to give 385 mg of compound BC4 as an amorphous solid (89% ).

[000418] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.88 (d, J = 6.9 Hz, 3H); 0.99 (s, 3H); 1.00 (s, 3H); 2.71 (dd, J = 9.4 and 13.7 Hz, 1H); 2.88 (dd, J = 5.3 and 13.7 Hz, 1H); 3.80 (s, 3H); 4.10 (m, 1H); 4.54 (m, 1H); 5.55 (dd, J = 2.3 and 10.2 Hz, 1H); 6.01 (dd, J = 2.3 and 17.1 Hz, 1H); 6.28 (dd, J = 10.2 and 17.1 Hz, 1H); 7.02 (d, J = 8.7 Hz, 1H); 7.17 (dd, J = 2.4 and 8.7 Hz, 1H); 7.32 (d, J = 2.4 Hz, 1H); 7.80 (d, J = 9.8 Hz, 1H); 8.39 (d, J = 8.8 Hz, 1H); 12.00 (wide s, 1H). LCMS (A5): ES m/z = 395 [MH]-; m/z = 397 [M+H]+; tR = 0.92 min. Synthesis of BC5: (S)-3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4-methoxyphenyl)-propanamido)-2,2-dimethylbutanoic acid

[000419] Compound BC5 was prepared starting from methyl (3S)-3-amino-2,2-dimethylbutanoate (MFCD09256689) and following the general BC building block synthesis described in Scheme 32 and described for compounds BC1 and BC2 ,

[000420] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.89 (d, J = 7.0 Hz, 3H); 1.02 (s, 3H); 1.04 (s, 3H); 1.30 (s, 9H); 2.68 (dd, J = 13.6 and 10.2 Hz, 1H); 2.82 (dd, J = 5.2 and 13.6 Hz, 1H); 3.80 (s, 3H); 4.05 (m, 1H); 4.12 (m, 1H); 7.00 (d, J = 8.7 Hz, 1H); 7.05 (d, J = 8.7 Hz, 1H); 7.20 (dd, J = 2.2 and 8.7 Hz, 1H); 7.32 (d, J = 2.2 Hz, 1H); 7.52 (d, J = 9.9 Hz, 1H); 12.35 (wide s, 1H). LCMS (A1): ES m/z = 441 [MH]-; m/z = 443 [M+H]+; m/z = 883 [2M-H]-; tR = 1.16 min. Synthesis of BC6: 3-((R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanamido)-2-(hydroxymethyl)-2-methylpropanoic acid Methyl 3-((R)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-4- Compound 26: methoxyphenyl)-propanamido)-2-(hydroxymethyl)-2-methylpropanoate

[000421] To a solution of fragment C4 (1.059 g, 5.77 mmol) in THF (60 mL) were added DIEA (2.06 mL, 11.76 mmol), fragment B (1.90 g, 5.77 mmol), HOBt (935 mg, 6.92 mmol) and EDC (1.23 mL, 6.92 mmol). The reaction medium was stirred at room temperature for 48 hours, concentrated in vacuo, and purified by flash chromatography on 200 g of silica gel (heptane/EtOAc isocratic elution) to provide 1.05 g of compound 26 as a colorless oil (39 %).

[000422] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.98 (s, 3H); 1.30 (s, 9H); 2.63 (m, 1H); 2.83 (m, 1H); 3.18 to 3.48 (m, 4H); 3.60 (s, 3H); 3.80 (s, 3H); 4.10 (m, 1H); 4.81 (m, 1H); 6.98 (d, J = 8.9 Hz, 1H); 7.04 (d, J = 8.7 Hz, 1H); 7.20 (dd, J = 2.1 and 8.7 Hz, 1H); 7.35 (d, J = 2.1 Hz, 1H); 7.83 (m, 1H), LCMS (A5): ES m/z = 457 [M-H]-; m/z = 459 [M+H]+; m/z = 503 [M-H+HCO2H]-; tR = 1.1 min. Compound 27: methyl 3-((R)-2-amino-3-(3-chloro-4-methoxyphenyl)propanamido)-2-(hydroxymethyl)-2-methylpropanoate hydrochloride

[000423] Compound 26 (1.05 g, 2.29 mmol) was treated with 4M HCl in dioxane (16 mL, 64 mmol) for 1 hour at room temperature. The reaction medium was concentrated in vacuum and co-evaporated twice in the presence of toluene. The crude product was triturated with iPr2O (10 mL), filtered and washed twice with iPr2O (5 mL). The mass was then dissolved in DCM, filtered and concentrated in vacuo to provide 809 mg of compound 27 as a white foam (90%) which was used without further purification.

[000424] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.92 (s, 1.5H); 0.97 (s, 1.5H); 2.88 (m, 1H); 3.00 (m, 1H); 3.19 to 3.47 (m, 4H); 3.60 (s, 3H); 3.85 (s, 3H); 4.05 (m, 1H); 4.89 (m, 1H); 7.11 (d division, J = 8.6 Hz, 1H); 7.19 (split dd, J = 2.0 and 8.6 Hz, 1H); 7.38 (d division, J = 2.0 Hz, 1H); 8.15 (broad s, 3H); 8.39 (m, 1H). Compound 28: methyl 3-((R)-2-amino-3-(3-chloro-4-methoxyphenyl)propanamido)-2-methyl-2-(((triethylsilyl)oxy)methyl)propanoate

[000425] To a solution of compound 27 (809 mg, 2.05 mmol) in DCM (4 mL) cooled with an ice bath were added TEA (1.43 mL, 10.23 mmol) and chlorotriethylsilane (1.37 mL , 8.19 mmol) while maintaining the temperature below 4°C. Stirring at 4°C was carried out for 10 minutes, then the reaction medium was stirred at room temperature for 20 hours. Saturated brine (20 mL) and DCM were added to the medium which was stirred for 10 min. The organic phase was washed with saturated brine (3 x 10 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 70 g of silica gel (DCM/MeOH gradient elution) to provide 706 mg of compound 28 as a light yellow oil (73%).

[000426] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.52 (q, J = 8.0 Hz, 6H); 0.89 (t, J = 8.0 Hz, 9H); 0.98 (s, 3H); 1.70 (broad s, 2H); 2.54 (m, 1H); 2.82 (m, 1H); 3.18 to 3.45 (m, 3H); 3.58 (m, 2H); 3.60 (s, 3H); 3.80 (s, 3H); 7.03 (d, J = 8.7 Hz, 1H); 7.14 (dd, J = 2.3 and 8.7 Hz, 1H); 7.28 (split d, J = 2.3 Hz, 1H); 7.78 (m, 1H), LCMS (A5): ES m/z = 471 [M-H]-; m/z = 473 [M+H]+; m/z = 517 [M-H+HCO2H]-; tR = 0.97 min. Compound 29: 3-((R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanamido)-2-methyl-2-(((triethylsilyl)oxy)methyl)methylpropanoate

[000427] To a solution of compound 28 (704 mg, 1.49 mmol) in DCM (19 mL) cooled with an ice/acetone bath was added DIEA (780 µL, 4.46 mmol) and dropwise sodium chloride. acryloyl (181 µL, 2.23 mmol). The reaction medium was stirred at 0-5°C for 1 hour, then EtOAc was added (38 mL) and the medium was washed with 1N HCl (5 mL), saturated NaHCO3 (5 mL), saturated brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 30 g of silica gel (DCM/MeOH gradient elution) to give 742 mg of compound 29 as a colorless oil (94%).

[000428] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.52 (split q, J = 8.0 Hz, 6H); 0.89 (t division, J = 8.0 Hz, 9H); 0.99 (s, 1.5H); 1.01 (s, 1.5H); 2.69 (m, 1H); 2.87 (m, 1H); 3.20 (m, 1H); 3.28 (m, 1H); 3.50 (dd, J = 3.1 and 9.9 Hz, 1H); 3.58 (s, 3H); 3.68 (d, J = 9.9 Hz, 1H); 3.80 (s, 3H); 4.59 (m, 1H); 5.55 (d, J = 10.3 Hz, 1H); 6.00 (d, J = 17.3 Hz, 1H); 6.25 (dd division, J = 10.3 and 17.3 Hz, 1H); 7.02 (d, J = 8.5 Hz, 1H); 7.18 (dd division, J = 2.0 and 8.5 Hz, 1H); 7.31 (d division, J = 2.0 Hz, 1H); 7.96 (m, 1H); 8.39 (d, J = 8.9 Hz, 1H). LCMS (A5): ES m/z = 525 [M-H]-; m/z = 527 [M+H]+; m/z = 571 [M-H+HCO2H]-; tR = 1.54 min. Compound BC6: 3-((R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanamido)-2-(hydroxymethyl)-2-methylpropanoic acid

[000429] To a suspension of tBuOK (1.42 g, 12.65 mmol) in THF (7 mL) cooled with an ice/acetone bath, H2O (50 µL) was added, the medium was stirred for 10 minutes before addition of a solution of compound 29 (741 mg, 1.41 mmol) in THF (7 mL). Stirring was carried out at 0°C for 10 minutes, then at room temperature for 1 hour. The reaction medium was cooled with an ice bath before adding 1N HCl (16.9 mL). After 15 minutes of stirring, the reaction medium was extracted with DCM (3 x 25 mL). The combined organic phases were washed with saturated brine (2 x 15 mL), H2O (15 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 656 mg of compound BC6 as a yellow foam (quant.).

[000430] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.91 (s, 3H); 2.69 (m, 1H); 2.90 (dd, J = 4.8 and 13.6 Hz, 1H); 3.18 to 3.42 (m, 4H); 3.80 (s, 3H); 4.59 (m, 1H); 4.73 (broad, 1H); 5.55 (d, J = 2.3 and 10.3 Hz, 1H); 6.00 (dd, J = 2.3 and 17.2 Hz, 1H); 6.24 (dd, J = 10.3 and 17.2 Hz, 1H); 7.03 (d, J = 8.7 Hz, 1H); 7.19 (dd, J = 2.0 and 8.7 Hz, 1H); 7.35 (d, J = 2.0 Hz, 1H); 8.00 (m, 1H); 8.39 (d, J = 8.8 Hz, 1H); 12.30 (wide s, 1H). LCMS (A5): ES m/z = 397 [MH]-; m/z = 399 [M+H]+; tR = 0.77 min. Synthesis of Examples 1 to 3: benzylic aza-C52 amine, glutaryl-Val-Ala-aza-C52 benzylic amine NHS ester and corresponding ADC Compound 30: 6-(3-chloro-4-methoxybenzyl)-13-isobutyl-2,2,10, 10-tetramethyl-4,7,11-trioxo-3-oxa-5,8,12-triazatetradecan-14 -(6R,13S)-(1E,3R,4S,6E)-1-(4-(azidomethyl)phenyl)-8-(tert-butoxy)-3-methyl-8-oxo-octa-1-oate, 6-dien-4-ila

[000431] Under argon, in a round bottom flask, the compound BC1 (899.55 mg, 2.10 mmol) was introduced in DMF (25 mL), HATU (861 mg, 2.20 mmol) and HOAt (302 mg , 2.20 mmol). The mixture was stirred for 30 minutes at room temperature. After which, compound AD1 (940 mg, 2 mmol) and DIEA (1.05 mL, 5.99 mmol) were added. The reaction medium was stirred for 4 hours at room temperature. After this time, the reaction medium was diluted with H2O (50 mL) and extracted with AcOEt (2 x 40 mL). The organic layers were washed with saturated brine (25 ml), dried over MgSO4, filtered, concentrated and purified by flash chromatography on 80 g of silica gel (heptane/AcOEt gradient elution) to provide 1.011 g of compound 30 as a semisolid yellow (57%).

[000432] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.76 (d, J = 6.7 Hz, 3 H); 0.78 (d, J = 6.7 Hz, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.07 (s, 6H); 1.39 (s, 9 H); 1.37 to 1.63 (m, 3H); 1.41 (s, 9 H); 2.36 to 2.60 (m partially masked, 3 H); 2.64 (dd, J = 9.5 and 14.1 Hz, 1 H); 2.86 (dd, J = 5.6 and 14.1 Hz, 1 H); 3.18 (dd, J = 6.1 and 13.5 Hz, 1 H); 3.25 (dd, J = 6.9 and 13.5 Hz, 1 H); 3.79 (s, 3H); 4.09 (m, 1 H); 4.26 (m, 1 H); 4.40 (s, 2H); 4.92 (m, 1 H); 5.81 (d, J = 15.9 Hz, 1 H); 6.17 (dd, J = 8.4 and 16.1 Hz, 1 H); 6.44 (d, J = 15.9 Hz, 1 H); 6.70 (m, 1 H); 6.94 (d, J = 8.9 Hz, 1 H); 7.02 (d, J = 8.7 Hz, 1 H); 7.18 (dd, J = 2.3 and 8.9 Hz, 1 H); 7.31 (m, 3H); 7.42 (d, J = 8.5 Hz, 2 H); 7.59 (t, J = 6.3 Hz, 1 H); 7.77 (d, J = 7.9 Hz, 1 H), LCMS (A1): ES m/z = 879 [M-H]-; m/z = 881 [M+H]+; m/z = 925 [M- H+HCO2H]-; tR = 1.89 min. Compound 31: (3S,10R,16S,E)-16-((R,E)-4-(4-(azidomethyl)phenyl)but-3-en-2-yl)-10-(3-chloro- 4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1-oxa-4,8,11-triazacyclohexadec-13-ene- 2,5,9,12-tetraone

[000433] Compound 31 was prepared in two steps.

[000434] Step 1: compound 30 (1.011 g, 1.15 mmol) in DCM (10 mL) was introduced into a round-bottom flask. After cooling to 0°C, TFA (1.72 mL, 22.94 mmol) and 100 µL of H2O were added. The reaction medium was stirred for 72 hours at room temperature. Upon completion, toluene was added to the medium and concentrated in vacuo to provide 900 mg of amino/acid intermediate (quant.).

[000435] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.76 (d, J = 6.7 Hz, 3 H); 0.78 (d, J = 6.7 Hz, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.07 (s, 3H); 1.08 (s, 3H); 1.38 to 1.62 (m, 3H); 2.35 to 2.58 (m partially masked, 3 H); 2.79 (dd, J = 11.0 and 13.8 Hz, 1 H); 3.00 (dd, J = 4.1 and 13.8 Hz, 1 H); 3.16 (dd, J = 5.9 and 13.5 Hz, 1 H); 3.28 to 3.39 (masked m, 1 H); 3.81 (s, 3H); 4.03 (m, 1 H); 4.26 (m, 1H); 4.40 (s, 2H); 4.92 (m, 1H); 5.82 (d, J = 16.1 Hz, 1 H); 6.16 (dd, J = 8.8 and 16.4 Hz, 1 H); 6.42 (d, J = 16.4 Hz, 1 H); 6.73 (m, 1 H); 7.09 (d, J = 8.6 Hz, 1 H); 7.18 (dd, J = 2.0 and 8.6 Hz, 1 H); 7.32 (d, J = 8.6 Hz, 2 H); 7.37 (d, J = 2.0 Hz, 1 H); 7.41 (d, J = 8.6 Hz, 2 H); 7.84 (d, J = 7.9 Hz, 1 H); 8.01 (large s large, 3 H); 8.10 (t, J = 6.4 Hz, 1 H); 12.2 (wide s, 1 H). LCMS (A1): ES m/z = 723 [M-H]-; m/z = 725 [M+H]+; tR = 1.09 min.

[000436] Step 2: in a round bottom flask, to a solution of amino/acid intermediate (840 mg, 1.16 mmol) in 200 mL of CH3CN was added DIEA (1.95 mL, 11.58 mmol) , HOAt (159.23 mg, 1.16 mmol) and HATU (499.40 mg, 1.27 mmol). The reaction medium was stirred for 3 hours at room temperature. After concentration in vacuum, the crude was diluted with EtOAc (200 mL), neutralized with 0.5M citric acid (12 mL) and 1N HCl (6 mL). The organic layer was separated, washed with saturated NaHSO3, saturated NaHCO3, saturated brine, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 40 g of silica gel (DCM/MeOH gradient elution) to provide 558 mg of compound 31 as a white solid (68%).

[000437] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.60 (d, J = 6.7 Hz, 3 H); 0.61 (d, J = 6.7 Hz, 3 H); 0.97 (s, 3H); 1.07 (s, 3H); 1.09 (d, J = 7.0 Hz, 3 H); 1.15 (m, 1 H); 1.33 to 1.48 (m, 2H); 2.24 (m, 1 H); 2.53 to 2.70 (m, 2H); 2.88 (large d, J = 13.6 Hz, 1 H); 3.01 (dd, J = 3.5 and 14.5 Hz, 1 H); 3.23 to 3.32 (masked m, 1 H); 3.80 (s, 3H); 4.18 (m, 1H); 4.35 (m, 1 H); 4.40 (s, 2H); 4.95 (m, 1H); 5.86 (dd, J = 1.7 and 15.8 Hz, 1 H); 6.13 (dd, J = 8.8 and 16.1 Hz, 1 H); 6.40 (m, 1 H); 6.47 (d, J = 16.1 Hz, 1 H); 7.04 (d, J = 8.6 Hz, 1 H); 7.18 (dd, J = 2.0 and 8.6 Hz, 1 H); 7.29 (d, J = 2.0 Hz, 1 H); 7.32 (d, J = 8.6 Hz, 2 H); 7.41 (broad d, J = 8.6 Hz, 3 H); 7.89 (d, J = 8.9 Hz, 1 H); 8.1 (d, J = 8.2 Hz, 1 H), LCMS (A1): ES m/z = 705 [M-H]-; m/z = 707 [M+H]+; tR = 1.58 min. Compound 32: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(azidomethyl)phenyl)oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000438] In a round bottom flask, to a solution of compound 31 (410 mg, 0.579 mmol) in DCM (50 mL) was added, at 0°C, m-CPBA (259 mg, 1.16 mmol). After stirring for 16 h at room temperature, m-CPBA (130 mg) was added twice in 24 h. Once the reaction was complete, the crude mixture was stirred for 1 hour with saturated NaHCO3 (15 mL) and saturated Na2S2O3 (15 mL) then extracted with DCM (3 x 15 mL). The organic layers were washed with saturated brine (15 mL), dried over MgSO4, filtered and concentrated to provide 440 mg of a mixture of alpha and beta epoxides as a yellow semisolid. Alpha and beta epoxides were separated by chiral liquid chromatography which was performed on a 76 x 350 mm column packed with 1.1 kg of 10 µm Chiralpak AD (amylose tris-3,5-dimethylphenylcarbamate coated on a silica gel support, Chiral Technologies Europe) using isocratic elution with 80:20 heptane/EtOH. After concentration, 185 mg of compound 32 was obtained as a white solid (44%) and 118 mg of the alpha epoxide was obtained as a white solid (28%).

[000439] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.72 (d, J = 6.7 Hz, 3 H); 0.74 (d, J = 6.7 Hz, 3 H); 0.96 (s, 3H); 1.04 (d, J = 7.0 Hz, 3 H); 1.08 (s, 3H); 1.11 (m, 1 H); 1.40 to 1.55 (m, 2 H); 1.79 (m, 1 H); 2.26 (m, 1 H); 2.64 (m, 2H); 2.85 (wide d, J = 13.2 Hz, 1 H); 3.00 (m, 2H); 3.25 (dd, J = 10.2 and 13.2 Hz, 1 H); 3.80 (s, 3H); 3.90 (d, J = 2.0 Hz, 1 H); 4.17 (m, 1 H); 4.35 (m, 1 H); 4.46 (s, 2H); 5.12 (m, 1H); 5.80 (dd, J = 1.7 and 16.0 Hz, 1 H); 6.39 (ddd, J = 3.8, 11.6 and 16.0 Hz, 1 H); 7.04 (d, J = 8.6 Hz, 1 H); 7.18 (dd, J = 2.0 and 8.6 Hz, 1 H); 7.29 (d, J = 2.0 Hz, 1 H); 7.31 to 7.40 (m, 5 H); 7.94 (d, J = 9.1 Hz, 1 H); 8.40 (d, J = 8.1 Hz, 1 H), LCMS (A1): ES m/z = 723 [M+H]+; tR = 1.48 min.

Example 1: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl) oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000440] In a round bottom flask, to a solution of compound 32 (185 mg, 255.79 µmol) in DCM (6 mL), MeOH (6 mL) and H2O (0.8 mL), TCEP ( 81.47 mg, 281.37 µmol). The solution was stirred for 16 hours at room temperature. Once the reaction was complete, the crude mixture was diluted with saturated NaHCO3 (15 mL) and extracted with DCM (2 x 30 mL). The organic layers were washed with saturated brine, dried over MgSO4, filtered, concentrated and purified by two successive flash chromatographies on 15 g of silica gel (DCM/MeOH gradient elution) to give 90 mg of example 1 as a white solid. (51%).

[000441] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.72 (d, J = 6.6 Hz, 3 H); 0.75 (d, J = 6.6 Hz, 3 H); 0.97 (s, 3H); 1.03 (d, J = 7.1 Hz, 3 H); 1.07 (s, 3H); 1.14 (m, 1 H); 1.42 to 1.57 (m, 2H); 1.77 (m, 1H); 1.92 (wide m, 2 H); 2.26 (m, 1 H); 2.64 (m, 2H); 2.86 (wide d, J = 13.0 Hz, 1 H); 2.94 (dd, J = 2.0 and 8.0 Hz, 1 H); 3.00 (dd, J = 3.6 and 14.4 Hz, 1 H); 3.27 (dd, J = 10.2 and 13.0 Hz, 1 H); 3.70 (s, 2H); 3.80 (s, 3H); 3.84 (d, J = 2.0 Hz, 1 H); 4.18 (m, 1H); 4.34 (m, 1H); 5.11 (m, 1H); 5.79 (dd, J = 1.7 and 15.6 Hz, 1 H); 6.38 (ddd, J = 4.1, 11.6 and 15.6 Hz, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.16 (dd, J = 2.0 and 8.6 Hz, 1 H); 7.22 (d, J = 8.5 Hz, 2 H); 7.28 (d, J = 2.0 Hz, 1 H); 7.31 (d, J = 8.5 Hz, 2 H); 7.35 (d, J = 10.2 Hz, 1 H); 7.91 (d, J = 9.0 Hz, 1 H); 8.38 (d, J = 8.0 Hz, 1 H), LCMS (A1): ES m/z = 695 [M-H]-; m/z = 697 [M+H]+; tR = 0.83 min. Compound 33: (S)-2-amino-N-((S)-1-((4-((2R,3R)-3-((S)-1- ((3S,10R,16S,E) -10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en -16- yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[000442] Under argon, in a round bottom flask to a solution of example 1 (90 mg, 129.01 µmol) in DCM (20 mL), acid (S)-2-((S)-2- ((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-methylbutan-amido)propanoic or FmocValAla (CAS number [150114-97-9], 79.47 mg, 193.61 µmol) , EDC (34.27 µL, 193.61 µmol) and HOBt (20.93 mg, 154.9 µmol). The reaction medium was stirred overnight at room temperature. After this time, piperidine (129 µL, 1.29 mmol) was added and stirred for 2 h. The solvent was removed and the crude residue was purified by flash chromatography on 15 g of silica gel (DCM/MeOH gradient elution) to provide 50 mg of compound 33 (45%) as a white solid.

[000443] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.75 (m, 9 H); 0.88 (d, J = 7.1 Hz, 3 H); 0.96 (s, 3H); 1.03 (d, J = 7.1 Hz, 3 H); 1.08 (s, 3H); 1.18 (m, 1H); 1.23 (d, J = 7.0 Hz, 3 H); 1.42 to 1.57 (m, 2H); 1.70 (wide m, 2 H); 1.79 (m, 1 H); 1.92 (m, 1 H); 2.26 (m, 1H); 2.63 (m, 2H); 2.86 (wide d, J = 13.0 Hz, 1 H); 2.99 (m, 3H); 3.22 to 3.33 (m partially masked, 1 H); 3.80 (s, 3H); 3.86 (d, J = 2.0 Hz, 1 H); 4.18 (m, 1H); 4.28 (d, J = 6.3 Hz, 2 H); 4.33 (m, 2H); 5.11 (m, 1 H); 5.79 (dd, J = 1.7 and 15.6 Hz, 1 H); 6.39 (ddd, J = 4.1, 11.6 and 15.6 Hz, 1 H); 7.03 (d, J = 8.6 Hz, 1 H); 7.16 (dd, J = 2.0 and 8.6 Hz, 1 H); 7.24 (s, 4H); 7.28 (d, J = 2.0 Hz, 1 H); 7.37 (d, J = 10.2 Hz, 1 H); 7.92 (d, J = 9.1 Hz, 1 H); 8.07 (wide d, J = 8.3 Hz, 1 H); 8.38 (d, J = 8.1 Hz, 1 H); 8.42 (t, J = 6.3 Hz, 1 H). LCMS (A1): ES m/z = 434 [M+2H]2+; m/z = 865 [M-H]-; m/z = 867 [M+H]+; m/z = 911 [M-H+HCO2H]-; tR = 0.88 min. Compound 34: acid 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1- ((3S,10R,16S, E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13 -en-16- yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid

[000444] Under argon, in a round-bottom flask, to a solution of compound 33 (54 mg, 57.64 µmol) in DMF (5 mL) glutaric anhydride (8 mg, 69.17 µmol) was added. The reaction medium was stirred for 3.5 h at room temperature. After this time, the solvent was removed and the crude residue was purified by flash chromatography on 1.8 g of silica gel (DCM/MeOH gradient elution) to give 51 mg of compound 34 as a white solid (90%).

[000445] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.73 (d, J = 6.7 Hz, 3 H); 0.76 (d, J = 6.7 Hz, 3 H); 0.81 (d, J = 7.0 Hz, 3 H); 0.84 (d, J = 7.0 Hz, 3 H); 0.97 (s, 3H); 1.03 (d, J = 7.2 Hz, 3 H); 1.05 (s, 3H); 1.15 (m, 1 H); 1.22 (d, J = 7.2 Hz, 3 H); 1.43 to 1.52 (m, 2H); 1.70 (m, 2H); 1.77 (m, 1H); 1.97 (m, 1 H); 2.17 (m, 2H); 2.24 (m, 1 H); 2.55 to 2.68 (m, 2H); 2.85 (wide d, J = 12.7 Hz, 1 H); 2.96 (dd, J = 1.8 and 7.7 Hz, 1 H); 2.99 (dd, J = 3.0 and 14.6 Hz, 1 H); 3.23 to 3.40 (m partially masked, 1 H); 3.80 (s, 3H); 3.85 (d, J = 1.8 Hz, 1 H); 4.22 to 4.38 (m, 4H); 5.11 (m, 1 H); 5.79 (d, J = 14.9 Hz, 1 H); 6.37 (ddd, J = 4.1, 11.3 and 14.9 Hz, 1 H); 7.04 (d, J = 8.7 Hz, 1 H); 7.15 (dd, J = 2.0 and 8.7 Hz, 1 H); 7.23 (broad s, 4 H); 7.28 (d, J = 2.0 Hz, 1 H); 7.37 (wide d, J = 10.7 Hz, 1 H); 7.87 (d, J = 8.7 Hz, 1 H); 7.93 (d, J = 9.2 Hz, 1 H); 8.06 (large m, 1 H); 8.37 (wide m, 1 H); 8.41 (wide m, 1 H); 12.03 (wide m, 1 H). LCMS (A1): ES m/z = 979 [M-H]-; m/z = 981 [M+H]+; tR = 1.17 min.

Example 2: 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13- en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate of 2 ,5-dioxopyrrolidin-1-yl

[000446] Under argon, in a round bottom flask, to a solution of compound 34 (30 mg, 30.56 µmol) in THF (5 mL) DIEA (5.34 µL, 30.56 µmol) and DSC were added (16.31 mg, 61.13 µmol). The reaction medium was stirred for 2 h at room temperature. After this time, the solvent was removed and the crude residue was purified by flash chromatography on 1.3 g of silica gel (DCM/iPrOH gradient elution) to give 9 mg of example 2 as a white solid. A second batch containing the expected compound as well as an impurity was diluted with MeTHF, washed twice with H2O, saturated brine, dried over MgSO4, filtered and concentrated to provide 15 mg of example 2 as a white solid (73% overall yield). .

[000447] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.73 (d, J = 6.7 Hz, 3 H); 0.77 (d, J = 6.7 Hz, 3 H); 0.82 (d, J = 7.0 Hz, 3 H); 0.84 (d, J = 7.0 Hz, 3 H); 0.96 (s, 3H); 1.03 (d, J = 7.2 Hz, 3 H); 1.07 (s, 3H); 1.15 (m, 1 H); 1.23 (d, J = 7.2 Hz, 3 H); 1.43 to 1.56 (m, 2H); 1.78 (m, 1H); 1.81 (m, 1H); 1.97 (m, 1 H); 2.20 to 2.33 (m, 3H); 2.58 to 2.69 (m, 4H); 2.80 (s, 4H); 2.85 (wide d, J = 12.7 Hz, 1 H); 2.98 (dd, J = 2.1 and 7.9 Hz, 1 H); 3.00 (dd, J = 3.2 and 14.7 Hz, 1 H); 3.22 to 3.34 (m partially masked, 1 H); 3.80 (s, 3H); 3.85 (d, J = 2.1 Hz, 1 H); 4.15 (m, 2H); 4.22 to 4.38 (m, 4H); 5.11 (m, 1 H); 5.79 (d, J = 15.1 Hz, 1 H); 6.38 (ddd, J = 3.9, 11.2 and 15.1 Hz, 1 H); 7.04 (d, J = 8.7 Hz, 1 H); 7.15 (dd, J = 2.2 and 8.7 Hz, 1 H); 7.22 (broad s, 4 H); 7.29 (d, J = 2.2 Hz, 1 H); 7.36 (wide d, J = 10.3 Hz, 1 H); 7.89 (d, J = 8.7 Hz, 1 H); 7.92 (d, J = 8.9 Hz, 1 H); 8.04 (d, J = 7.4 Hz, 1 H); 8.32 (t, J = 6.1 Hz, 1 H); 8.39 (d, J = 8.0 Hz, 1 H), LCMS (A1): ES m/z = 540; m/z = 1076 [M-H]-; m/z = 1078 [M+H]+; m/z = 1122 [M-H+HCO2H]-; tR = 1.23 min. Example 3: mAb-Ex2

[000448] The general method described previously was used for the preparation of example 3. 60 mg of hu2H11_R35-74 was reacted with 161 µL of a 9.96 mM solution of example 2 in DMA (1.733 mg, 4 eq.) for 2 h. At that time, 121 µL of example solution 2 (3 eq.) was added and the medium was stirred for 2 hours. At that time, 121 µL of example solution 2 (3 eq.) was added and stirred for 2 hours. After purification in Superdex 200 pg in DPBS pH 6.5 + 20% NMP, concentration in Amicon Ultra-15, buffer exchange in PD-10 in buffer B pH 6.5 + 5% NMP and filtration in Steriflip, 39.9 mg of example 3 were obtained as a clear colorless solution at a concentration of 2.28 mg/mL with a DAR of 4.6 (EMAR), a monomer purity of 99% and an overall yield of 66%.

[000449] SEC-EMAR: spectrum for intact ADC in Figure 1; m/z = 150346 (D1); m/z = 151307 (D2); m/z = 152274 (D3); m/z = 153240 (D4); m/z = 154200 (D5); m/z = 155165 (D6); m/z = 156133 (D7); m/z = 157095 (D8). Synthesis of examples 4 to 7: (S)-3-neopentyl-aza-C52 benzylic amine, benzylic glutaryl-Val-Ala-(S)-3-neopentyl-aza-C52 amine NHS ester and corresponding ADC Compound 35: (6R,13S)-(1E,3R,4S,6E)-8-(tert-butoxy)-1-(4-(hydroxymethyl)phenyl)-3-methyl-8-oxo-octa-1, 6-dien-4-yl 6-(3-chloro-4-methoxybenzyl)-2,2,10,10-tetramethyl-13-neopentyl-4,7,11-trioxo-3-oxa- 5,8,12 -triazatetradecan-14-oate

[000450] Under argon, in a round bottom flask, to a solution of BC1 fragment (1.32 g, 3.08 mmol) in DCM (60 mL) DIEA (1.53 mL, 9.25 mmol) was added , HOAt (503.75 mg, 3.70 mmol) and HATU (1.41 g, 3.70 mmol). The yellow suspension was stirred for 30 minutes at RT, then the AD2 fragment (1.7 g, 3.08 mmol) was added. The reaction medium was stirred for 2 hours at RT. After this time, 1.53 mL of DIEA was added and stirred for 1 hour. The mixture was neutralized with 1M citric acid (50 mL) and extracted with AcOEt (2 x 80 mL). The organic layers were washed with 1M NaHSO4, H2O, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 100 g of silica gel (gradient elution heptane/EtOAc) to provide 1.475 g of compound 35 as a colorless solid (57%). Compound 36: (2E,5S,6R,7E)-5-(((S)-2-(3-((R)-2-amino-3-(3-chloro-4-methoxyphenyl)-propanamido) acid) -2,2-dimethylpropanamido)-4,4-dimethylpentanoyl)oxy)-8-(4-(hydroxymethyl)-phenyl)-6-methylocta-2,7-dienoic acid

[000451] In a round bottom flask, to a solution of compound 35 (1.475 g, 1.69 mmol) in DCM (20 mL), TFA (8 mL, 52.27 mmol) and H2O (2 mL) were added. . The reaction medium was stirred for 5 hours at RT. The solvent was removed. The residue was diluted with H2O (20 mL) and EtOAc (20 mL) and treated with 2M NaOH (2 mL) for 2 hours at RT. The organic layer was separated and the aqueous layer was extracted with AcOEt (2 x 10 mL), dried over MgSO4, filtered and concentrated to provide 1.24 g of a colorless solid. The solid was dissolved in EtOAc (10 mL) and H2O (10 mL) and treated with 2M NaOH (400 µL) for 2 hours at RT. The organic layer was separated, the aqueous layers were extracted with AcOEt (2 x 10 mL), dried over MgSO4, filtered, concentrated and purified by flash chromatography on 100 g of silica gel (heptane/AcOEt gradient elution) to give 990 mg of compound 36 as a white solid (82%).

[000452] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.80 (s, 9 H); 0.99 (s, 3H); 1.02 (d, J = 7.0 Hz, 3 H); 1.05 (s, 3H); 1.52 (dd, J = 2.0 and 14.8 Hz, 1 H); 1.69 (dd, J = 9.9 and 14.8 Hz, 1 H); 2.36 to 2.61 (m partially masked, 4 H); 2.84 (dd, J = 5.0 and 13.9 Hz, 1 H); 3.18 (d, J = 7.5 Hz, 2 H); 3.40 (dd, J = 5.1 and 8.1 Hz, 1 H); 3.80 (s, 3H); 4.29 (m, 1 H); 4.45 (s, 2H); 4.90 (m, 1H); 5.12 (wide m, 1 H); 5.81 (d, J = 15.5 Hz, 1 H); 6.10 (dd, J = 8.4 and 15.9 Hz, 1 H); 6.40 (d, J = 15.9 Hz, 1 H); 6.70 (td, J = 7.5 and 15.5 Hz, 1 H); 7.01 (d, J = 8.7 Hz, 1 H); 7.11 (dd, J = 2.4 and 8.7 Hz, 1 H); 7.25 (m, 3H); 7.32 (d, J = 8.2 Hz, 2 H); 7.72 (t, J = 6.5 Hz, 1 H); 7.79 (d, J = 7.8 Hz, 1 H). LCMS (A1): ES m/z = 712 [M-H]-; m/z = 714 [M+H]+; tR = 0.94 minute. Compound 37: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)- 16-((R,E)-4-(4-(hydroxymethyl)-phenyl)but-3-en -2-yl)-6,6-dimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000453] Under argon, in a round-bottom flask, compound 36 (990 mg, 1.39 mmol) and CH3CN (150 mL) were introduced, heated with a water bath at 50°C until complete solubilization and stirred for 10 minutes. After that, DIEA (687.20 µL, 4.16 mmol), HOAt (207.51 mg, 1.52 mmol) and HATU (579.7 mg, 1.52 mmol) were added and stirred for 30 minutes at RT. . The reaction medium was neutralized with 1M citric acid (30 mL). The solvent was removed and the aqueous layer was extracted with EtOAc (2 x 40 mL). The organic layers were washed with 1M NaHSO4, H2O, dried over MgSO4, filtered and concentrated. The crude solid was diluted with H2O (200 mL) and stirred for 1 hour. The solid was filtered, then diluted with AcOEt, dried over MgSO4, filtered and concentrated to provide a mixture of compound 37 and HATU. The solid was stirred with MeTHF (50 mL) and H2O (50 mL). The organic layer was separated, washed with H2O (4 x 20 mL), dried over MgSO4, filtered, concentrated and purified by flash chromatography on 10 g of silica gel (heptane/AcOEt gradient elution) to provide 680 mg of compound 37 as a colorless solid (70%).

[000454] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.68 (s, 9 H); 0.98 (s, 3H); 1.03 (s, 3H); 1.09 (d, J = 6.9 Hz, 3 H); 1.19 (wide d, J = 14.5 Hz, 1 H); 1.58 (dd, J = 10.3 and 14.5 Hz, 1 H); 2.23 (m, 1H); 2.52 to 2.70 (m, 3H); 2.86 (wide d, J = 12.7 Hz, 1 H); 3.00 (dd, J = 3.3 and 14.8 Hz, 1 H); 3.22 to 3.33 (m partially masked, 1 H); 3.80 (s, 3H); 4.18 (m, 1H); 4.42 (m, 1H); 4.46 (d, J = 6.0 Hz, 2 H); 4.91 (m, 1H); 5.13 (t, J = 6.0 Hz, 1 H); 5.85 (wide d, J = 15.0 Hz, 1 H); 6.05 (dd, J = 8.4 and 15.9 Hz, 1 H); 6.40 (m, 2H); 7.03 (d, J = 8.7 Hz, 1 H); 7.18 (dd, J = 2.2 and 8.7 Hz, 1 H); 7.25 (d, J = 8.5 Hz, 2 H); 7.29 (d, J = 2.2 Hz, 1 H), 7.31 (d, J = 8.5 Hz, 2 H); 7.42 (d, J = 10.6 Hz, 1 H); 7.94 (d, J = 9.1 Hz, 1 H); 8.41 (d, J = 8.2 Hz, 1 H). LCMS (A1): ES m/z = 694 [M-H]-; m/z = 696 [M+H]+; tR = 1.36 minutes. Compound 38: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6-dimethyl-3-neopentyl-16-((R,E)-4-(4-( ((triisopropylsilyl)oxy)methyl)phenyl)but-3-en-2-yl)-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000455] Under argon, at 0°C in a round bottom flask, to a solution of compound 37 (680 mg, 0.976 mmol) in CHCl3 (10 mL) was added 1H-imidazole (305.84 mg, 4.49 mmol) and chlorotriisopropylsilane (480.13 µl, 2.25 mmol). The reaction medium was stirred for 5 hours at room temperature, then diluted with saturated NH4Cl and MTBE (30 mL). The organic layer was washed with 1M NaHSO4, saturated NaHCO3, saturated brine, dried over MgSO4, filtered and concentrated to provide 970 mg of compound 38 as an orange amorphous solid (amount).

[000456] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.63 (s, 9 H); 0.87 to 1.17 (m, 31 H); 1.53 (dd, J = 10.9 and 14.3 Hz, 1 H); 2.22 (m, 1 H); 2.51 to 2.70 (m, 3H); 2.86 (wide d, J = 13.0 Hz, 1 H); 3.00 (dd, J = 3.2 and 14.9 Hz, 1 H); 3.24 to 3.35 (m partially masked, 1 H); 3.80 (s, 3H); 4.18 (m, 1H); 4.41 (m, 1H); 4.76 (s, 2H); 4.91 (m, 1H); 5.87 (wide d, J = 15.4 Hz, 1 H); 6.06 (dd, J = 8.9 and 15.9 Hz, 1 H); 6.40 (m, 2H); 7.03 (d, J = 8.5 Hz, 1 H); 7.18 (wide d, J = 8.5 Hz, 1 H); 7.25 (d, J = 8.3 Hz, 2 H); 7.28 (d, J = 8.3 Hz, 2 H); 7.30 (broad s, 1 H); 7.42 (d, J = 10.5 Hz, 1 H); 7.93 (d, J = 9.2 Hz, 1 H); 8.42 (d, J = 8.1 Hz, 1 H). LCMS (A1): ES m/z = 850 [M-H]-; m/z = 852 [M+H]+; m/z = 896 [M-H+HCO2H]-; tR = 2.15 minutes.

Example 4: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16-((S)-1-((2R, 3R)-3-(4-(hydroxy-methyl )phenyl)oxiran-2-yl)ethyl)-6,6-dimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000457] Example 4 was prepared in 2 steps.

[000458] Step 1: under argon, in a round bottom flask, to a solution of compound 38 (832 mg, 0.976 mmol) in DCM (10 mL) was added, in three times, m-CPBA (339.15 mg , 1.51 mmol). The reaction mixture was stirred for 50 hours at room temperature, then diluted with DCM (10 mL) and stirred for 15 minutes with saturated NaHCO3 (30 mL) and Na2S2O3 (30 mL). The organic layer was separated, washed with saturated brine, dried over MgSO4, filtered and concentrated to provide 1.1 g of mixture of alpha and beta epoxides as a colorless foam (amount).

[000459] Step 2: the mixture of alpha and beta epoxides was diluted in THF (30 mL) and 1M TBAF (952.32 µL) was added. After stirring for 2 hours, 952 µL of TBAF was added. After stirring for 1 hour, the mixture was diluted with H2O (50 mL) and extracted with EtOAc (3 x 50 mL). The organic layers were washed with saturated brine, dried over MgSO4, filtered and concentrated to provide 770 mg of the mixture of alpha and beta epoxides as a yellow solid (amount). Alpha and beta epoxides were separated by chiral liquid chromatography which was performed on a 76 x 350 mm column packed with 1.1 kg of 10 µm Chiralpak AD (mylose tris-3,5-dimethylphenylcarbamate coated on a silica gel support , Chiral Technologies Europe) using isocratic elution with 75:25 heptane/EtOH. After concentration, 190 mg of example 4 was obtained as a white solid (31%) and 125 mg of the alpha epoxide was obtained as a white solid (20%).

[000460] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.81 (s, 9 H); 0.98 (s, 3H); 1.03 (d, J = 7.0 Hz, 3 H); 1.05 (s, 3H); 1.26 (m, 1 H); 1.71 (dd, J = 10.4 and 14.5 Hz, 1 H); 1.82 (m, 1H); 2.26 (m, 1H); 2.52 (m, 2H); 2.84 (d, J = 12.8 Hz, 1 H); 2.92 (dd, J = 2.1 and 7.8 Hz, 1 H); 3.00 (dd, J = 3.1 and 14.5 Hz, 1 H); 3.24 to 3.36 (m partially masked, 1 H); 3.81 (s, 3H); 3.90 (d, J = 2.1 Hz, 1 H); 4.17 (ddd, J = 3.4, 8.0 and 11.7 Hz, 1 H); 4.42 (m, 1 H); 4.50 (d, J = 6.0 Hz, 2 H); 5.10 (m, 1 H); 5.20 (t, J = 6.0 Hz, 1 H); 5.79 (dd, J = 1.8 and 15.4 Hz, 1 H); 6.39 (ddd, J = 3.8, 11.5 and 15.4 Hz, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.16 (dd, J = 2.1 and 8.6 Hz, 1 H); 7.26 (d, J = 8.3 Hz, 2 H); 7.29 (d, J = 2.1 Hz, 1 H); 7.32 (d, J = 8.3 Hz, 2 H); 7.41 (d, J = 10.3 Hz, 1 H); 8.02 (d, J = 9.2 Hz, 1 H); 8.38 (d, J = 8.2 Hz, 1H). LCMS (A1): ES m/z = 710 [M-H]-; m/z = 712 [M+H]+; tR = 1.28 minutes. Compound 39: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(azidomethyl)phenyl)oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-6,6-dimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000461] Under argon, in a round bottom flask, to an example solution 4 (100 mg, 140 µmol) in THF (5 mL) were added DPPA (156.82 µl, 701.98 µmol) and DBU (110 .22 µl, 701.98 µmol). The solution was stirred for 6 hours at room temperature, then diluted with H2O and extracted with AcOEt (3 x 30 mL). The organic layer was separated, washed with saturated brine, dried over MgSO4, filtered, concentrated and purified on 15 g of silica gel (DCM/iPrOH gradient elution) to provide 100 mg of compound 39 as a white solid (40%) .

[000462] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.80 (s, 9 H); 0.97 (s, 3H); 1.03 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3H); 1.28 (d, J = 14.5 Hz, 1 H); 1.69 (dd, J = 10.7 and 14.5 Hz, 1 H); 1.84 (m, 1H); 2.28 (m, 1H); 2.62 (m, 2H); 2.84 (d, J = 12.8 Hz, 1 H); 2.93 to 3.03 (m, 2H); 3.22 to 3.34 (m partially masked, 1 H); 3.80 (s, 3H); 3.92 (broad s, 1 H); 4.17 (m, 1 H); 4.40 to 4.49 (m, 3H); 5.10 (m, 1H); 5.80 (d, J = 15.8 Hz, 1 H); 6.39 (ddd, J = 3.8, 11.7 and 15.8 Hz, 1 H); 7.03 (d, J = 8.7 Hz, 1 H); 7.16 (dd, J = 2.2 and 8.7 Hz, 1 H); 7.28 (d, J = 2.1 Hz, 1 H); 7.32 (d, J = 8.3 Hz, 2 H); 7.39 (d, J = 8.3 Hz, 2 H); 7.41 (m, 1H); 8.00 (d, J = 9.1 Hz, 1 H); 8.36 (d, J = 8.2 Hz, 1H). LCMS (A1): ES m/z = 735 [M-H]-; m/z = 737 [M+H]+; tR = 1.54 minutes.

Example 5: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl) oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-6,6-dimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000463] In a round bottom flask, to a solution of compound 39 (100 mg, 122.07 µmol) in DCM (2.5 mL) and MeOH (2.5 mL) a solution of TCEP was added dropwise (38.88 mg, 134.28 µmol) in H2O (500 µL). The reaction medium was stirred for 24 hours at room temperature. The reaction mixture was diluted with H2O and saturated NaHCO3, extracted with DCM (3 x 10 mL). The organic layer was separated, washed with saturated brine, dried over MgSO4, filtered and concentrated to provide 73 mg of example 5 as a white solid (84%) used without further purification in the next step.

[000464] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.82 (s, 9 H); 0.97 (s, 3H); 1.03 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3H); 1.29 (d, J = 14.4 Hz, 1 H); 1.70 (dd, J = 10.2 and 14.4 Hz, 1 H); 1.80 (m, 1H); 2.02 (wide m, 2 H); 2.25 (m, 1 H); 2.62 (m, 2H); 2.83 (d, J = 13.0 Hz, 1 H); 2.93 (dd, J = 2.2 and 7.8 Hz, 1 H); 2.99 (dd, J = 3.4 and 14.5 Hz, 1 H); 3.23 to 3.35 (m partially masked, 1 H); 3.71 (s, 2H); 3.80 (s, 3H); 3.88 (d, J = 2.2 Hz, 1 H); 4.17 (ddd, J = 3.5, 8.5 and 11.5 Hz, 1 H); 4.41 (m, 1H); 5.09 (m, 1H); 5.79 (d, J = 15.7 Hz, 1 H); 6.39 (ddd, J = 3.7, 11.4 and 15.7 Hz, 1 H); 7.05 (d, J = 8.7 Hz, 1 H); 7.16 (dd, J = 2.3 and 8.7 Hz, 1 H); 7.22 (d, J = 8.4 Hz, 2 H); 7.28 (d, J = 2.3 Hz, 1 H); 7.34 (d, J = 8.4 Hz, 2 H); 7.40 (d, J = 10.3, 1 H); 8.02 (d, J = 8.9 Hz, 1 H); 8.38 (d, J = 8.2 Hz, 1H). LCMS (A1): ES m/z = 709 [M-H]-; m/z = 711 [M+H]+; tR = 0.86 minute. Compound 40: ((S)-1 -(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R, 16S,E)-10 -(3-chloro-4-methoxybenzyl)-6,6-dimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16 (9H-fluoren-9-yl)-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate methyl

[000465] Under argon, in a round bottom flask, example 5 (73 mg, 82.10 µmol) and DMF (1 mL) were introduced, followed by FmocValAla (50.55 mg, 123.16 µmol), HOBt ( 17.75 mg, 131.37 µmol), DCM (10 mL) and EDC (14.53 µl, 82.10 µmol). The solution was stirred for 4 hours at room temperature, then diluted with H2O (10 mL) and extracted with DCM (3 x 20 mL). The organic layer was separated, washed with H2O, dried over MgSO4, filtered, concentrated and purified on 15 g of silica gel (DCM/iPrOH gradient elution) to provide 90 mg of compound 40 as a colorless solid (99%).

[000466] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.80 (s, 9 H); 0.84 (d, J = 7.1 Hz, 3 H); 0.86 (d, J = 7.1 Hz, 3 H); 0.93 (s, 3H); 1.02 (d, J = 7.1 Hz, 3 H); 1.04 (s, 3H); 1.24 (d, J = 7.3 Hz, 3 H); 1.29 (d, J = 14.5 Hz, 1 H); 1.70 (dd, J = 10.5 and 14.5 Hz, 1 H); 1.81 (m, 1H); 1.99 (m, 1 H); 2.25 (m, 1 H); 2.60 (m, 2H); 2.82 (d, J = 13.0 Hz, 1 H); 2.91 (dd, J = 1.9 and 7.6 Hz, 1 H); 2.99 (dd, J = 3.4 and 14.5 Hz, 1 H); 3.29 (m, 1H); 3.80 (s, 3H); 3.88 (d, J = 1.9 Hz, 1 H); 3.90 (m, 1H); 4.16 (ddd, J = 3.4, 8.0 and 11.8 Hz, 1 H); 4.20 to 4.35 (m, 6 H); 4.41 (m, 1 H); 5.09 (m, 1 H); 5.79 (d, J = 15.7 Hz, 1 H); 6.39 (ddd, J = 3.7, 11.6 and 15.7 Hz, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.15 (dd, J = 2.5 and 8.6 Hz, 1 H); 7.21 (d, J = 8.5 Hz, 2 H); 7.24 (d, J = 8.5 Hz, 2 H); 7.28 (d, J = 2.5 Hz, 1 H); 7.32 (t, J = 7.9 Hz, 2 H); 7.38 to 7.47 (m, 4 H); 7.73 (t, J = 7.9 Hz, 2 H); 7.89 (d, J = 7.9 Hz, 2 H); 8.03 (d, J = 9.1 Hz, 1 H); 8.05 (d, J = 7.9 Hz, 1 H); 8.39 (m, 2 H). LCMS (A1): ES m/z = 1103 [M+H]+; m/z = 1147 [M-H+HCO2H]-; tR = 1.71 minutes. Compound 41: (S)-2-amino-N-((S)-1-((4-((2R,3R)-3-((S)-1- ((3S,10R,16S,E) -10-(3-chloro-4-methoxybenzyl)-6,6-dimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en -16- yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[000467] Under argon, in a round bottom flask, compound 40 (104 mg, 76.65 µmol) in DCM (5 mL) was introduced followed by piperidine (138.87 µl, 1.40 mmol). The solution was stirred for 5 hours at room temperature, then concentrated and purified on 10 g of silica gel (DCM/MeOH/H2O gradient elution) to provide 50 mg of compound 41 as a colorless solid (60%).

[000468] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.76 (d, J = 7.0 Hz, 3 H); 0.81 (s, 9H); 0.88 (d, J = 7.0 Hz, 3 H); 0.97 (s, 3H); 1.02 (d, J = 7.1 Hz, 3 H); 1.04 (s, 3H); 1.22 (d, J = 7.3 Hz, 3 H); 1.29 (d, J = 14.6 Hz, 1 H); 1.66 (wide m, 2 H); 1.70 (dd, J = 10.6 and 14.6 Hz, 1 H); 1.81 (m, 1H); 1.92 (m, 1 H); 2.25 (m, 1 H); 2.60 (m, 2H); 2.83 (d, J = 13.0 Hz, 1 H); 2.93 (dd, J = 1.9 and 7.4 Hz, 1 H); 2.99 (m, 2H); 3.28 (dd, J = 10.4 and 13.0 Hz, 1 H); 3.80 (s, 3H); 3.89 (d, J = 1.9 Hz, 1 H); 4.16 (ddd, J = 3.2 and 8.2 and 11.8 Hz, 1 H); 4.29 (d, J = 6.0 Hz, 2 H); 4.34 (m, 1H); 4.41 (m, 1 H); 5.09 (m, 1 H); 5.79 (dd, J = 1.7 and 15.5 Hz, 1 H); 6.38 (ddd, J = 3.8, 11.3 and 15.5 Hz, 1 H); 7.04 (d, J = 8.7 Hz, 1 H); 7.16 (dd, J = 2.2 and 8.7 Hz, 1 H); 7.25 (m, 4H); 7.29 (d, J = 2.2 Hz, 1 H); 7.40 (d, J = 10.4 Hz, 1 H); 8.02 (d, J = 9.1 Hz, 1 H); 8.08 (d large, J = 7.7 Hz, 1 H); 8.39 (d, J = 8.2 Hz, 1 H); 8.45 (t, J = 6.0 Hz, 1 H). LCMS (A1): ES m/z = 441 2+ - + - [M+2H]; m/z = 879 [M-H]-; m/z = 881 [M+H] ; m/z = 925 [M-H+HCO2H]-; tR = 0.99 minute. Compound 42: acid 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1- ((3S,10R,16S, E)-10-(3-chloro-4-methoxy-benzyl)-6,6-dimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec -13-en-16- yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5- oxopentanoic

[000469] Under argon, in a round bottom flask, compound 41 (50 mg, 51.05 µmol) in DCM (10 mL) was introduced followed by glutaric anhydride (10.48 mg, 91.89 µmol). The reaction medium was stirred for 2 hours at room temperature, concentrated and purified on 10 g of silica gel (DCM/MeOH/H2O gradient elution) to provide 42 mg of compound 42 as a colorless solid (82%).

[000470] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.80 (s, 9 H); 0.82 (d, J = 7.0 Hz, 3 H); 0.85 (d, J = 7.0 Hz, 3 H); 0.97 (s, 3H); 1.02 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3H); 1.23 (d, J = 7.2 Hz, 3 H); 1.29 (d, J = 14.2 Hz, 1 H); 1.70 (m, 3H); 1.81 (m, 1H); 1.96 (m, 1H); 2.18 to 2.25 (m, 5 H); 2.61 (m, 2H); 2.83 (d, J = 13.0 Hz, 1 H); 2.93 (dd, J = 2.0 and 7.4 Hz, 1 H); 3.00 (dd, J = 3.1 and 14.5 Hz, 1 H); 3.29 (dd, J = 10.4 and 13.0 Hz, 1 H); 3.80 (s, 3H); 3.89 (d, J = 2.0 Hz, 1 H); 4.16 (m, 2H); 4.25 to 4.31 (m, 3H); 4.42 (m, 1H); 5.09 (m, 1 H); 5.79 (dd, J = 1.9 and 15.5 Hz, 1 H); 6.39 (ddd, J = 3.8, 11.6 et 15.5 Hz, 1 H); 7.05 (d, J = 8.7 Hz, 1 H); 7.15 (dd, J = 2.1 and 8.7 Hz, 1 H); 7.23 (m, 4H); 7.28 (d, J = 2.1 Hz, 1 H); 7.40 (d, J = 10.4 Hz, 1 H); 7.83 (d, J = 8.7 Hz, 1 H); 8.02 (m, 2H); 8.35 (t, J = 6.1 Hz, 1 H); 8.39 (d, J = 8.0 Hz, 1 H); 12.04 (wide m, 1 H). LCMS (A1): ES m/z = 498 [M+2H]2+; m/z = 993 [M-H]-; m/z = 995 [M+H]+; tR = 1.27 minutes.

Example 6: 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )-10- (3-chloro-4-methoxybenzyl)-6,6-dimethyl-3-neopentyl-2,5,9,12-tetraoxo-1- oxa-4,8,11 -triazacyclohexadec-13- en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate of 2 ,5-dioxopyrrolidin-1-yl

[000471] Under argon, in a round-bottom flask, compound 42 (23 mg, 23.10 µmol) in DCM (5 mL) was introduced, followed by DSC (8.29 mg, 32.34 µmol) and DIEA ( 5.63 µL, 32.34 µmol). The reaction medium was stirred for 2 hours at room temperature. After this time, 2 mg of DSC, 1 µL of DIEA and DCM (2 mL) were added and stirred for 1 hour at room temperature. The solvent was removed and the crude residue was purified by flash chromatography on 10 g of silica gel (DCM/iPrOH gradient elution) to provide 20 mg of example 6 as a colorless solid (79%).

[000472] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.80 (s, 9 H); 0.82 (d, J = 7.0 Hz, 3 H); 0.85 (d, J = 7.0 Hz, 3 H); 0.97 (s, 3H); 1.02 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3H); 1.23 (d, J = 7.2 Hz, 3 H); 1.30 (d, J = 14.2 Hz, 1 H); 1.70 (dd, J = 10.7 and 14.7 Hz, 1 H); 1.81 (m, 3H); 1.97 (m, 1 H); 2.21 to 2.32 (m, 3H); 2.61 (m, 2H); 2.68 (t, J = 7.8 Hz, 2 H); 2.80 (broad s, 4 H); 2.83 (d, J = 13.0 Hz, 1 H); 2.94 (dd, J = 2.0 and 7.6 Hz, 1 H); 3.00 (dd, J = 3.4 and 14.9 Hz, 1 H); 3.28 (dd, J = 10.5 and 13.0 Hz, 1 H); 3.80 (s, 3H); 3.88 (d, J = 2.0 Hz, 1 H); 4.16 (m, 2H); 4.23 to 4.31 (m, 3H); 4.42 (m, 1H); 5.10 (m, 1H); 5.79 (dd, J = 1.9 and 15.3 Hz, 1 H); 6.39 (ddd, J = 3.8, 11.4 and 15.3 Hz, 1 H); 7.05 (d, J = 8.8 Hz, 1 H); 7.16 (dd, J = 2.2 and 8.8 Hz, 1 H); 7.23 (m, 4H); 7.28 (d, J = 2.2 Hz, 1 H); 7.40 (d, J = 10.5 Hz, 1 H); 7.89 (d, J = 8.7 Hz, 1 H); 8.02 (d, J = 9.1 Hz, 1 H); 8.06 (d, J = 7.5 Hz, 1 H); 8.34 (t, J = 6.1 Hz, 1 H); 8.39 (d, J = 8.1 Hz, 1 H). LCMS (A1): ES m/z = 546.5 [M+2H]2+; m/z = 1090 [M-H]-; m/z = 1092 [M+H]+; m/z = 1136 [M-H+HCO2H]-; tR = 1.34 minutes.

Example 7: mAb-Ex6

[000473] The general method described previously was used for the preparation of example 7. 60 mg of hu2H11_R35-74 was reacted with 233 µL of a 10.6 mM solution of example 6 in DMA (5 equivalents) for 2 hours. After purification in SuperDex 200 pg in DPBS pH 6.5 + 20% NMP, concentration in Amicon Ultra-15, buffer exchange in NAP-25 in buffer B pH 6.5 + 5% NMP and filtration in Steriflip, 39 mg of example 7 were obtained as a clear, colorless solution at a concentration of 1.98 mg/mL with a DAR of 4.1 (HRMS), a monomer purity of 100% and an overall yield of 66%.

[000474] SEC-HRMS: spectrum for intact ADC in Figure 2; m/z = 149370 (naked mAb); m/z = 150357 (D1); m/z = 151330 (D2); m/z = 152307 (D3); m/z = 153285 (D4); m/z = 154262 (D5); m/z = 155238 (D6); m/z = 156222 (D7). Synthesis of examples 8 to 10: 7-Me-aza-C52 benzylic amine stereoisomer 1, glutaryl-Val-Ala-7-Me-aza-C52 benzylic amine stereomer NHS ester 1 and corresponding ADC Example Compound 43: 6-(3-chloro-4-methoxybenzyl)-13-isobutyl-2,2,9,10, 10-pentamethyl-4,7,11-trioxo-3-oxa-5,8,12- (6R,13S)-(1E,3R,4S,6E)-1-(4-(azidomethyl)phenyl)-8-(tert-butoxy)-3-methyl-8-oxo-octa triazatetradecan-14-oate -1,6-dien-4-yl

[000475] Under argon, in a round-bottom flask, BC2 fragment (1.10 g, 2.48 mmol) was introduced in DMF (5 mL), followed by HATU (950 mg, 2.5 mmol) and HOAt ( 340 mg, 2.5 mmol). The mixture was stirred for 30 minutes at room temperature, then the AD1 fragment (1.24 g, 2.18 mmol) and DIEA (1.2 mL, 6.87 mmol) were added. The yellow solution was stirred for 16 hours at room temperature, quenched with H2O and extracted with EtOAc (3 x 30 mL). The organic layers were washed with H2O, saturated brine, dried over MgSO4, filtered, concentrated and purified on 200 g of silica gel (heptane/AcOEt gradient elution) to give 1.55 g of compound 43 as a white meringue (77 %).

[000476] 1H NMR (400 MHz, d in ppm, DMSO-d6): 55/45 diastereoisomer mixture; 0.82 to 0.89 (m, 6H); 0.85 (d, J = 7.0 Hz, 1.65 H); 0.91 (d, J = 7.0 Hz, 1.35 H); 0.99 to 1.09 (m, 9 H); 1.30 (s, 9 H); 1.40 (s, 10 H); 1.50 to 1.70 (m, 2 H); 2.38 to 2.59 (m partially masked, 3 H); 2.68 (m, 1H); 2.84 (m, 1 H); 3.79 (s, 1.35H); 3.81 (s, 1.65H); 4.00 to 4.18 (m, 2H); 4.30 (m, 1H); 4.40 (broad s, 2H); 4.92 (m, 1H); 5.81 (d, J = 15.7 Hz, 1 H); 6.18 (dd, J = 8.3 and 16.1 Hz, 1 H); 6.45 (d, J = 16.1 Hz, 1 H); 6.71 (td, J = 7.3 and 15.7 Hz, 1 H); 6.94 (d, J = 8.1 Hz, 0.45 H); 6.96 (d, J = 8.1 Hz, 0.55 H); 7.04 (split d, J = 8.7 Hz, 1 H); 7.19 (wide d, J = 8.7 Hz, 1 H); 7.30 (d, J = 7.8 Hz, 2 H); 7.32 (broad s, 1 H); 7.41 (d, J = 8.7 Hz, 2 H); 7.52 (d, J = 10.1 Hz, 0.45 H); 7.59 (d, J = 10.1 Hz, 0.55 H); 7.74 (m, 1 H). LCMS (A1): 55/45 diastereoisomer mixture; ES m/z = 895 [M+H]+; m/z = 917 [M+Na]+; tR = 6.94-6.98 minutes. Compounds 44 & 45: (3S,10R,16S,E)-16-((R,E)-4-(4-(azido-methyl)phenyl)but-3-en-2-yl)-10-( 3-chloro-4-methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000477] Compounds 44 & 45 were obtained in two steps.

[000478] Step 1: at 0°C, in a round bottom flask, to a solution of compound 43 (1.50 g, 1.67 mmol) in 11 mL of DCM was added TFA (2.6 mL, 34 .65 mmol) and 100 µL of H2O. The mixture was stirred for 15 minutes at 0°C and at room temperature for 6.5 hours. The reaction medium was then evaporated in vacuum and co-evaporated in the presence of toluene to provide 1.7 g of deprotected intermediate as an orange solid.

[000479] 1H NMR (500 MHz, d in ppm, DMSO-d6): 55/45 diastereoisomer mixture; 0.70 to 0.80 (m, 7.65 H); 0.90 (s, 1.35H); 0.95 (m, 3H); 1.01 to 1.09 (m, 6H); 1.38 to 1.68 (m, 3H); 2.40 to 2.60 (m partially masked, 3 H); 2.80 to 3.02 (m, 2H); 3.81 (s, 1.35 H); 3.82 (s, 1.65 H); 3.96 to 4.08 (m, 1 H); 4.18 (m, 1H); 4.28 (m, 1H); 4.41 (s, 2H); 4.91 (m, 1H); 5.83 (d, J = 15.7 Hz, 0.45 H); 5.85 (d, J = 15.7 Hz, 0.55 H); 6.18 (m, 1 H); 6.43 (d, J = 16.1 Hz, 0.45 H); 6.46 (d, J = 16.1 Hz, 0.55 H); 6.74 (m, 1H); 7.08 to 7.20 (m, 2H); 7.30 (masked m, 0.45 H); 7.32 (d, J = 7.8 Hz, 2 H); 7.37 (d, J = 2.0 Hz, 0.55 H); 7.42 (m, 2H); 7.76 (d, J = 8.00 Hz, 0.45 H); 7.79 (d, J = 8.00 Hz, 0.55 H); 7.86 (d, J = 10.1 Hz, 0.55 H); 8.00 (d, J = 10.1 Hz, 0.45 H); 8.11 (wide m, 3 H); 12.22 (wide m, 1 H). LCMS (A1): 55/45 diastereoisomer mixture; ES m/z = 737 [M-H]-; m/z = 739 [M+H]+; tR = 1.07 to 1.09 minutes.

[000480] Step 2: in a round bottom flask, to a solution of deprotected intermediate (1.43 g, 1.68 mmol) in 25 mL of CH3CN were added DIEA (3 mL, 16.23 mmol), HOAt ( 250.91 mg, 1.84 mmol) and HATU (700.91 mg, 1.84 mmol). The mixture was stirred for 1 hour at room temperature. Then, the solvent was removed, the medium was diluted with EtOAc (200 mL), neutralized with 0.5 M citric acid and HCl. The organic layer was separated, washed with saturated NaHSO3, saturated NaHCO3, saturated brine, dried over MgSO4, filtered, concentrated and purified on 100 g of silica gel (DCM/MeOH gradient elution) to give 640 mg of compounds 44 & 45 as a yellow solid (53%).

[000481] C7 diastereoisomers were separated by chiral liquid chromatography which was carried out on a 76.5 x 350 mm column packed with 1.1 kg of Whelk 01 SS at 10 µm (4-(3,5-dinitrobenzamido)tetra- hydrophenanthrene, Regis Technologies) using isocratic elution with 50:50 heptane/EtOH. After concentration, 210 mg of compound 44 (stereoisomer 1) was obtained as a white solid (17%) and 236 mg of compound 45 (stereoisomer 2) was obtained as a white solid (19%).

Compound 44 (stereoisomer 1):

[000482] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.60 (d, J = 6.7 Hz, 3 H); 0.62 (d, J = 6.7 Hz, 3 H); 0.98 (s, 3H); 1.05 (d, J = 7.0 Hz, 3 H); 1.10 (d, J = 7.0 Hz, 3 H); 1.14 (s, 3H); 1.18 (m, 1H); 1.40 to 1.54 (m, 2H); 2.24 (m, 1H); 2.48 to 2.61 (m partially masked, 2 H); 2.65 (dd, J = 11.8 and 14.5 Hz, 1 H); 3.04 (dd, J = 3.2 and 14.5 Hz, 1 H); 3.55 (m, 1H); 3.81 (s, 3H); 4.19 (m, 2H); 4.40 (s, 2H); 4.96 (m, 1H); 5.91 (dd, J = 1.4 and 15.5 Hz, 1 H); 6.13 (dd, J = 8.9 and 16.1 Hz, 1 H); 6.40 (ddd, J = 3.9, 11.3 and 15.5 Hz, 1 H); 6.46 (d, J = 16.1 Hz, 1 H); 7.03 (d, J = 8.7 Hz, 1 H); 7.19 (dd, J = 2.1 and 8.7 Hz, 1 H); 7.30 (d, J = 2.1 Hz, 1 H); 7.32 (d, J = 8.4 Hz, 2 H); 7.41 (d, J = 8.4 Hz, 2 H); 7.87 (d, J = 8.9 Hz, 1 H); 8.36 (d, J = 9.9 Hz, 1 H); 8.40 (d, J = 8.0 Hz, 1 H). LCMS (A1): ES m/z = 719 [M-H]-; m/z = 721 [M+H]+; tR = 1.61 minutes

Compound 45 (stereoisomer 2):

[000483] 1H NMR (d in ppm, DMSO-d6): 0.59 (d, J = 6.7 Hz, 3 H); 0.67 (d, J = 6.7 Hz, 3 H); 0.88 (d, J = 6.7 Hz, 3 H); 1.03 (s, 3H); 1.10 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3H); 1.21 (m, 1 H); 1.49 to 1.60 (m, 2H); 2.23 (m, 1H); 2.46 to 2.61 (m partially masked, 2 H); 2.71 (dd, J = 11.3 and 14.5 Hz, 1 H); 2.98 (dd, J = 3.7 and 14.5 Hz, 1 H); 3.48 (m, 1H); 3.80 (s, 3H); 4.05 (m, 1 H); 4.11 (ddd, J = 3.7, 7.6 and 11.3 Hz, 1 H); 4.40 (s, 2H); 4.90 (m, 1 H); 5.93 (d, J = 15.7 Hz, 1 H); 6.14 (dd, J = 8.7 and 16.1 Hz, 1 H); 6.47 (d, J = 16.1 Hz, 1 H); 6.51 (ddd, J = 5.2, 10.3 and 15.5 Hz, 1 H); 7.02 (d, J = 8.7 Hz, 1 H); 7.21 (dd, J = 2.4 and 8.7 Hz, 1 H); 7.31 (d, J = 8.4 Hz, 2 H); 7.37 (d, J = 2.4 Hz, 2 H); 7.41 (d, J = 8.4 Hz, 2 H); 7.87 (d, J = 6.9 Hz, 1 H); 7.89 (d, J = 9.0 Hz, 1 H); 8.51 (d, J = 7.6 Hz, 1 H). LCMS (A1): ES m/z = 719 [M-H]-; m/z = 721 [M+H]+; tR = 1.61 minutes. Compound 46: (3S,10R,16S,E)-16-((S)-1-(3-(4-(azidomethyl)phenyl)oxiran-2-yl)ethyl)-10-(3-chloro-4 -methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000484] At 0°C, under argon, in a round bottom flask to a solution of compound 44 (154 mg, 213.51 µmol) in DCM (10 mL) was added m -CPBA (80 mg, 324.51 µmol). The reaction medium was stirred at room temperature for 5 days. The reaction mixture was diluted with DCM (15 mL) and stirred for 15 minutes with saturated NaHCO3 (6 mL) and Na2S2O3 (6 mL). The organic layer was separated, washed with saturated brine (2 x 3 mL), dried over MgSO4, filtered and concentrated to provide 150 mg of compound 46 as a white solid (amount).

[000485] 1H NMR (500 MHz, d in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.75 (d, J = 6.8 Hz, 1.8 H); 0.79 (d, J = 6.8 Hz, 3 H); 0.85 (d, J = 6.8 Hz, 1.2 H); 0.95 to 0.99 (m, 4.2 H); 1.02 to 1.09 (m, 4.8 H); 1.15 (s, 1.8H); 1.18 (s, 1.2H); 1.20 (m, 0.6H); 1.38 (m, 0.4H); 1.59 to 1.62 (m, 2H); 1.80 (m, 0.6H); 1.89 (m, 0.4H); 2.25 (m, 0.6H); 2.40 to 2.71 (m partially masked, 2.4 H); 2.96 to 3.08 (m, 2H); 3.54 (m, 1H); 3.80 (s, 3H); 3.82 (d, J = 1.9 Hz, 0.4 H); 3.92 (d, J = 1.9 Hz, 0.6 H); 4.14 to 4.29 (m, 2H); 4.40 to 4.50 (m, 2 H); 5.11 (m, 1 H); 5.85 (dd, J = 2.0 and 15.5 Hz, 0.6 H); 5.95 (dd, J = 2.0 and 15.5 Hz, 0.4 H); 6.38 (m, 1H); 7.04 (split d, J = 8.8 Hz, 1 H); 7.16 (dd, J = 2.2 and 8.8 Hz, 0.6 H); 7.19 (dd, J = 2.2 and 8.8 Hz, 0.4 H); 7.28 (d, J = 2.2 Hz, 0.6 H); 7.30 (d, J = 2.2 Hz, 0.4 H); 7.30 to 7.40 (m, 4 H); 7.89 (d, J = 8.1 Hz, 0.6 H); 7.92 (d, J = 8.1 Hz, 0.4 H); 8.29 (d, J = 10.0 Hz, 0.6 H); 8.32 (d, J = 8.1 Hz, 0.6 H); 8.35 (d, J = 10.0 Hz, 0.4 H); 8.40 (d, J = 8.1 Hz, 0.4 H). LCMS (A1): ES m/z = 735 [M-H]-; m/z = 737 [M+H]+; tR = 1.52 minutes.

Example 8: (3S,10R,16S,E)-16-((S)-1-(3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-(3-chloro-4 -methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-1-oxa-4,8,11 - triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000486] At 0°C, in a round-bottom flask, to a solution of compound 46 (60 mg, 81.38 µmol) in DCM (3 mL) and MeOH (3 mL) was added dropwise a solution of TCEP (38.9 mg, 134.3 µmol) in 1 mL of H2O. The reaction mixture was stirred at room temperature for 35 hours, then diluted with DCM (15 mL) and saturated NaHCO3. After stirring for 10 minutes, the organic layer was separated, washed with saturated brine, dried over MgSO4, filtered, concentrated in vacuo and purified on 5 g of silica gel (DCM/MeOH gradient elution) to give 23 mg of example 8 as a white solid (40%).

[000487] 1H NMR (500 MHz, d in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.77 (d, J = 7.0 Hz, 1.8 H); 0.81 (split d, J = 7.0 Hz, 3 H); 0.88 (d, J = 7.0 Hz, 1.4 H); 0.95 (d, J = 7.0 Hz, 1.2 H); 0.98 (s, 1.8H); 1.00 (s, 1.2H); 1.02 to 1.09 (m, 4.8 H); 1.17 (s, 1.8H); 1.19 (s, 1.2H); 1.22 (m, 0.6H); 1.40 (m, 0.4H); 1.51 to 1.64 (m, 2H); 1.78 (m, 0.6H); 1.89 (m, 0.4H); 2.28 (m, 0.6H); 2.40 to 2.71 (m partially masked, 2.4 H); 2.96 (dd, J = 2.2 and 7.5 Hz, 0.6 H); 2.99 (dd, J = 2.2 and 7.5 Hz, 0.4 H); 3.03 (m, 1 H); 3.55 (m, 1H); 3.70 (s, 1.2 H); 3.72 (s, 1.8H); 3.78 (d, J = 2.2 Hz, 0.4 H); 3.81 (s, 3H); 3.88 (d, J = 2.2 Hz, 0.6 H); 4.12 to 4.30 (m, 2H); 5.12 (m, 1H); 5.84 (dd, J = 1.8 and 15.6 Hz, 0.6 H); 5.96 (dd, J = 1.8 and 15.6 Hz, 0.4 H); 6.39 (m, 1H); 7.05 (d, J = 8.7 Hz, 1 H); 7.16 to 7.38 (m, 6H); 7.90 (d, J = 8.1 Hz, 0.6 H); 7.93 (d, J = 8.1 Hz, 0.4 H); 8.28 (d, J = 10.0 Hz, 0.6 H); 8.32 (d, J = 8.1 Hz, 0.6 H); 8.37 (d, J = 10.0 Hz, 0.4 H); 8.41 (d, J = 8.1 Hz, 0.4 H). LCMS (A1): ES m/z = 709 [M-H]-; m/z = 711 [M+H]+; tR = 0.86 minute. Compound 47: ((S)-1-(((S)-1-((4-(3-((S)-1-((3S,10R,16S,E)-10- (3-chloro- 4-methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl (9H-fluoren-9-yl)methyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[000488] Under argon, in a round bottom flask example 8 (110 mg, 154.65 µmol) and DMF (2 mL) were introduced, followed by FmocValAla (90 mg, 219 µmol), HOBt (30 mg, 222 µmol ), DCM (10 mL) and EDC (35 µl, 197.52 µmol). The solution was stirred at room temperature for 3 hours and 30 minutes, then quenched with H2O (10 mL) and extracted by DCM (3 x 10 mL). The organic layer was separated, washed with saturated NaHCO3, saturated brine, dried over MgSO4, filtered, concentrated in the presence of toluene and purified on 15 g of silica gel (DCM/MeOH gradient elution) to provide 49 mg of compound 47 as a white solid (46%).

[000489] 1H NMR (500 MHz, d in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.76 (d, J = 7.0 Hz, 1.8 H); 0.80 (m, 3H); 0.82 to 0.89 (m, 6.4 H); 0.95 (d, J = 7.0 Hz, 1.2 H); 0.98 (s, 1.8H); 1.00 (s, 1.2H); 1.02 to 1.09 (m, 4.8 H); 1.17 (s, 1.8H); 1.19 (s, 1.2H); 1.20 to 1.32 (m, 3.6 H); 1.40 (m, 0.4H); 1.51 to 1.63 (m, 2H); 1.78 (m, 0.6H); 1.85 (m, 0.4H); 2.00 (m, 1 H); 2.27 (m, 0.6H); 2.40 to 2.72 (m partially masked, 2.4 H); 2.96 (m, 1 H); 3.04 (m, 1H); 3.55 (m, 1 H); 3.77 (d, J = 2.2 Hz, 0.4 H); 3.81 (s, 3H); 3.88 (d, J = 2.2 Hz, 0.6 H); 3.90 (m, 1 H); 4.15 to 4.38 (m, 6H); 5.12 (m, 1H); 5.85 (dd, J = 1.8 and 15.6 Hz, 0.6 H); 5.95 (dd, J = 1.8 and 15.6 Hz, 0.4 H); 6.39 (m, 1 H); 7.05 (split d, J = 8.7 Hz, 1 H); 7.15 to 7.48 (m, 19 H); 7.73 (t, J = 8.1 Hz, 2 H); 7.90 (d, J = 8.1 Hz, 2.6 H); 7.92 (d, J = 8.1 Hz, 0.4 H); 8.05 (d, J = 8.1 Hz, 1 H); 8.25 to 8.45 (m, 3 H). LCMS (A1): ES m/z = 552 [M+2H]2+; m/z = 1103 [M+H]+; m/z = 1147 [M-H+HCO2H]-; tR = 1.55 to 1.57 minutes. Compound 48: (2S)-2-amino-N-((2S)-1-((4-((2R,3R)-3-((1S)-1- ((3S,10R,16S,E) -10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[000490] In a round bottom flask, piperidine (60 µL, 600.6 µmol) was added to a solution of compound 47 (50 mg, 45.30 µmol) in DCM (5 mL). The resulting mixture was stirred for 24 hours at room temperature, concentrated in vacuo and purified on 5 g of silica gel (DCM/MeOH gradient elution) to provide 70 mg of afa and beta epoxides.

[000491] 1H NMR (500 MHz, d in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.75 (d, J = 7.0 Hz, 1.8 H); 0.80 (m, 3H); 0.82 to 0.92 (m, 6.4 H); 0.96 (d, J = 7.0 Hz, 1.2 H); 0.98 (s, 1.8H); 1.00 (s, 1.2H); 1.02 to 1.09 (m, 4.8 H); 1.15 (s, 1.8 H); 1.19 (s, 1.2H); 1.20 to 1.42 (m, 4 H); 1.50 to 1.70 (masked m, 2 H); 1.78 (m, 0.6H); 1.85 (m, 0.4H); 2.00 (m, 1 H); 2.25 (m, 0.6H); 2.40 to 2.72 (m partially masked, 2.4 H); 2.92 to 3.08 (m, 3H); 3.54 (m, 1H); 3.79 (d, J = 2.1 Hz, 0.4 H); 3.81 (s, 3H); 3.88 (d, J = 2.1 Hz, 0.6 H); 4.13 to 4.41 (m, 6 H); 5.12 (m, 1H); 5.85 (d, J = 15.4 Hz, 0.6 H); 5.93 (d, J = 15.4 Hz, 0.4 H); 6.38 (m, 1H); 7.05 (d, J = 8.7 Hz, 1 H); 7.15 to 7.32 (m, 6 H); 7.90 (d, J = 8.3 Hz, 0.6 H); 7.93 (d, J = 8.3 Hz, 0.4 H); 8.10 to 8.55 (m, 7 H). LCMS (A1): ES m/z = 441 [M+2H]2+; m/z = 879 [M-H]-; m/z = 881 [M+H]+; m/z = 925 [M-H+HCO2H]-; tR = 0.92 minute.

[000492] Alpha and beta epoxides were separated by chiral liquid chromatography which was performed on a 76 x 350 mm column packed with 1.1 kg of 10 µm Chiralpak AD (amylose tris-3,5-dimethylphenylcarbamate coated on a support silica gel, Chiral Technologies Europe) using isocratic elution with 70:30 heptane/EtOH. After concentration, 28 mg of compound 48 was obtained as a white solid (70%) and 19 mg of the alpha epoxide was obtained as a white solid.

[000493] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.75 (d, J = 7.0 Hz, 6 H); 0.80 (d, J = 7.0 Hz, 3 H); 0.88 (d, J = 7.0 Hz, 3 H); 0.98 (s, 3H); 1.03 (d, J = 7.0 Hz, 6 H); 1.17 (s, 3H); 1.23 (d, J = 7.0 Hz, 3 H); 1.35 (m, 1 H); 1.50 to 1.68 (m, 2H); 1.78 (m, 1H); 1.91 (m, 1H); 2.26 (m, 1 H); 2.58 to 2.70 (m, 2H); 2.96 (dd, J = 1.9 and 7.7 Hz, 1 H); 2.99 (d, J = 4.8 Hz, 1 H); 3.02 (dd, J = 3.3 and 14.8 Hz, 1 H); 3.53 (m, 1 H); 3.80 (s, 3H); 3.89 (d, J = 1.9 Hz, 1 H); 4.10 to 4.23 (m, 3H); 4.28 (d, J = 6.1 Hz, 2 H); 4.35 (m, 1 H); 5.11 (m, 1 H); 5.83 (dd, J = 1.9 and 15.2 Hz, 1 H); 6.37 (ddd, J = 4.0, 11.2 and 15.2 Hz, 1 H); 7.03 (d, J = 8.8 Hz, 1 H); 7.18 (dd, J = 2.4 and 8.8 Hz, 1 H); 7.26 (s, 4H); 7.29 (d, J = 2.4 Hz, 1 H); 7.90 (d, J = 8.2 Hz, 1 H); 8.08 (wide d, J = 7.7 Hz, 1 H); 8.29 (d, J = 10.1 Hz, 1 H); 8.33 (d, J = 8.2 Hz, 1 H); 8.46 (t, J = 6.7 Hz, 1 H). LCMS (A1): ES m/z = 441 [M+2H]2+; m/z = 879 [M-H]-; m/z = 881 [M+H]+; m/z = 925 [M-H+HCO2H]-; tR = 0.92 minute. Compound 49: acid 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1- ((3S,10R,16S, E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec -13-en-16- yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5- oxopentanoic

[000494] Under argon, in a round-bottom flask, a solution of glutaric anhydride (3.78 mg, 32.44 µmol) in DCM (4 mL) was added to a solution of compound 48 (26 mg, 29.5 µmol) in DCM (9 mL). The resulting mixture was stirred for 2 hours at room temperature, concentrated in vacuo and purified on 2.5 g of silica gel (DCM/MeOH/H2O gradient elution) to provide 18 mg of compound 49 as a white solid (61% ).

[000495] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.78 (d, J = 7.0 Hz, 3 H); 0.80 (d, J = 7.0 Hz, 3 H); 0.82 (d, J = 7.0 Hz, 3 H); 0.84 (d, J = 7.0 Hz, 3 H); 0.97 (s, 3H); 1.03 (d, J = 7.0 Hz, 6 H); 1.15 (s, 3H); 1.23 (wide d, J = 7.0 Hz, 4 H); 1.49 to 1.61 (m, 2H); 1.70 (m, 2H); 1.78 (m, 1H); 1.97 (m, 1H); 2.20 (m, 4H); 2.25 (m, 1 H); 2.62 (m, 2H); 2.95 (m, 1 H); 3.02 (m, 1H); 3.53 (m, 1 H); 3.80 (s, 3H); 3.88 (s, 1 H); 4.10 to 4.22 (m, 3H); 4.24 to 4.31 (m, 3H); 5.11 (m, 1 H); 5.83 (d, J = 15.7 Hz, 1 H); 6.36 (m, 1H); 7.03 (d, J = 8.7 Hz, 1 H); 7.19 (wide d, J = 8.7 Hz, 1 H); 7.23 (m, 4H); 7.29 (broad s, 1 H); 7.87 (d, J = 8.9 Hz, 1 H); 7.90 (d, J = 8.1 Hz, 1 H); 8.06 (d large, J = 7.3 Hz, 1 H); 8.29 (d, J = 10.1 Hz, 1 H); 8.37 (m, 2H); 12.0 (wide m, 1 H). LCMS (A1): ES m/z = 993 [M-H]-; m/z = 995 [M+H]+; tR = 1.21 minutes.

Example 9: 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec- 13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate 2,5-dioxopyrrolidin-1-yl

[000496] Under argon, in a round bottom flask, to a solution of compound 49 (15 mg, 15.07 µmol) in DCM (5 mL) were added DSC (5.63 mg, 21.09 µmol) and DIEA (3.56 µL, 21.09 µmol). The resulting mixture was stirred for 1 hour at room temperature, concentrated in vacuo and purified on 2.5 g of silica gel (DCM/MeOH gradient elution) to give 7.7 mg of example 9 as a white solid (47% ).

[000497] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.78 (d, J = 6.7 Hz, 3 H); 0.80 (d, J = 6.7 Hz, 3 H); 0.82 (d, J = 7.0 Hz, 3 H); 0.85 (d, J = 7.0 Hz, 3 H); 0.99 (s, 3H); 1.04 (d, J = 7.2 Hz, 6 H); 1.18 (s, 3H); 1.26 (m, 4H); 1.57 (m, 2H); 1.79 (m, 1 H); 1.83 (m, 2H); 1.99 (m, 1 H); 2.22 to 2.33 (m, 3H); 2.60 to 2.71 (m, 4 H); 2.82 (s, 4H); 2.97 (dd, J = 2.1 and 7.7 Hz, 1 H); 3.04 (dd, J = 3.4 and 14.7 Hz, 1 H); 3.55 (m, 1 H); 3.81 (s, 3H); 3.89 (d, J = 2.1 Hz, 1 H); 4.19 (m, 3H); 4.26 to 4.34 (m, 3H); 5.12 (m, 1H); 5.86 (dd, J = 2.0 and 15.7 Hz, 1 H); 6.38 (ddd, J = 3.8, 11.2 and 15.7 Hz, 1 H); 7.05 (d, J = 8.7 Hz, 1 H); 7.19 (dd, J = 2.2 and 8.7 Hz, 1 H); 7.26 (m, 4H); 7.29 (d, J = 2.2 Hz, 1 H); 7.91 (m, 2H); 8.09 (m, 1H); 8.29 (d, J = 9.9 Hz, 1 H); 8.35 (m, 2 H). LCMS (A1): ES m/z = 1092 [M+H]+; m/z = 1136 [M-H+HCO2H]-; tR = 1.26 minutes.

Example 10: mAb-Ex9

[000498] The general method described previously was used for the preparation of example 10. 60 mg of hu2H11_R35-74 was reacted with 198 µL of a 10.78 mM solution of example 9 in DMA (5 equivalents) for 2 hours. At this time, 120 µL of example solution 9 (3 equivalents) were added and the medium was stirred for 2 hours. After purification in SuperDex 200 pg in DPBS pH 6.5 + 20% NMP, concentration in Amicon Ultra-15, buffer exchange in PD-10 in buffer B pH 6.5 + 5% NMP and filtration in Steriflip, 46 mg of example 10 were obtained as a clear, colorless solution at a concentration of 2.23 mg/mL with a DAR of 4.7 (HRMS), a monomer purity of 99.2% and an overall yield of 78%.

[000499] SEC-HRMS: spectrum for intact ADC in Figure 3; m/z = 150345 (D1); m/z = 151319 (D2); m/z = 152297 (D3); m/z = 153274 (D4); m/z = 154251 (D5); m/z = 155222 (D6); m/z = 156202 (D7); m/z = 157183 (D8). Synthesis of examples 11 to 14: benzyl amine of 3-(S)-neopentyl-7-Me-aza-C52 stereoisomer 1, glutaryl-Val-Ala-3-(S)-neopentyl-7-Me-NHS ester aza-C52 benzyl amine stereomer 1 and Compound 50: 6-(3-chloro-4-methoxybenzyl)-2,2,9,10,10-pentamethyl-13-neopentyl-4,7,11-trioxo-3-oxa-5,8,12 (6R,13S)-(1E,3R,4S,6E)-8-(tert-butoxy)-1-(4-(hydroxymethyl)phenyl)-3-methyl-8-oxo--triazatetradecan-14-oate octa-1,6-dien-4-yl

[000500] Under argon, in a round bottom flask, to a solution of BC2 fragment (742 mg, 1.68 mmol) in DMF (20 mL) were added HATU (716 mg, 1.83 mmol) and HOAt (251 mg, 1.83 mmol). The mixture was stirred for 30 minutes at room temperature. Then, a solution of AD1 fragment (730 mg, 1.59 mmol) in DMF (10 mL) and DIEA (981 µL, 5.56 mmol) was added. The reaction medium was stirred for 24 hours at room temperature. After this time, the reaction medium was diluted with ice (200 g) and extracted with AcOEt (4 x 200 mL). The organic layers were washed with H2O (80 mL), saturated brine (2 x 80 mL), dried over MgSO4, filtered, concentrated and purified by two successive rapid chromatographies, the first on 300 g of silica gel (elution by gradient of DCM/MeOH) and the second one in 70 g of silica gel (gradient elution of DCM/MeOH) to provide 428 mg of compound 50 as a colorless foam (30%). Compound 51: (2E,5S,6R,7E)-5-(((2S)-2-(3-((R)-2-amino-3-(3-chloro-4-methoxyphenyl)-propanamido) acid) -2,2-dimethylbutanamido)-4,4-dimethylpentanoyl)oxy)-8-(4-(hydroxymethyl)phenyl)-6-methylocta-2,7- dienoic

[000501] In a round bottom flask, compound 50 (428 mg, 483.87 µmol) and DCM (27 mL) were introduced. The solution was cooled to 0°C, then TFA (8 mL, 106.62 mmol) was added. The mixture was stirred for 1.5 hours at room temperature. The solvent was removed and co-evaporated under reduced pressure with toluene (3 x 100 mL). The crude oil was diluted with 1:1 EtOAc/H2O (75 mL) and neutralized with 2M NaOH (250 µL) until pH 6 to 7. The mixture was stirred for 6 hours at room temperature. The layers were separated. The aqueous layer was extracted with EtOAc (3 x 50 mL). The organic layers were washed with saturated brine (2 x 15 mL), dried over MgSO4, filtered and concentrated to provide 374 mg of compound 51 as a colorless foam (amount). Compound 52: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)- 16-((R,E)-4-(4-(hydroxymethyl)-phenyl)but-3-en -2-yl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000502] Under argon, in a round bottom flask, to a solution of compound 51 (352 mg, 483.31 µmol) in CH3CN (60 mL) were added HATU (208 mg, 531.64 mmol), HOAt (73 .09 mg, 531.64 µmol) and DIEA (244.52 µL, 1.45 mmol). The reaction medium was stirred for 45 minutes at room temperature. After this time, the reaction medium was neutralized with 0.5 N citric acid until pH 4, partially concentrated in vacuum and extracted with AcOEt (150 mL). The organic layer was washed with saturated NaHCO3 (10 mL), saturated brine (3 x 10 mL), dried over MgSO4, filtered, concentrated and purified by flash chromatography on 15 g of silica gel (DCM/MeOH gradient elution) to provide 188 mg of compound 52 as a colorless foam (54%). Compound 53: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-16-((R,E)-4-(4 -(((triisopropylsilyl)oxy)methyl)phenyl)but-3-en-2-yl)-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12 -tetraone

[000503] At 0°C, under argon, in a round-bottom flask, to a solution of compound 52 (188 mg, 264.68 µmol) in DCM (7 mL), 1H-imidazole (83.72 mg) was added , 1.2 mmol) and 1M chlorotriisopropylsilane (134.15 µl). The reaction medium was stirred for 5 hours at room temperature, then quenched with saturated NH4Cl and stirred for 15 minutes. The layers were separated. The aqueous layer was extracted with DCM (3 x 25 mL). The organic layers were washed with 1M NaHSO4 (10 mL), saturated NaHCO3 (10 mL), saturated brine, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 10 g of silica gel (DCM/MeOH gradient elution ) to provide 137 mg of compound 53 as a colorless foam (59%).

Example 11: (3S,10R,16S,E)-10-(^chloro-4-methoxybenzyl)-16-((S)-1- ((2R,3R)-3-(4-(hydroxymethyl)phenyl) oxiran-2-yl)ethyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000504] Example 11 was prepared in two steps.

[000505] Step 1: at 0°C under argon, in a round bottom flask, to a solution of compound 53 (137 mg, 158.78 µmol) in DCM (5.5 mL) was added a solution of m - CPBA (50.66 mg, 205.51 µmol) in DCM (2 mL) and the reaction medium was stirred for 2 hours. Then, 39 mg of m-CPBA was added twice after 2 hours of stirring. After 16 hours of stirring, the mixture was quenched with saturated NaHCO3 (30 mL) and Na2S2O3 (30 mL), stirred for 15 minutes and diluted with DCM (40 mL). The layers were separated. The aqueous layer was extracted with DCM (2 x 20 mL). The organic layers were washed with saturated brine (2 x 8 mL), dried over MgSO4, filtered and concentrated to provide 160 mg of alpha and beta epoxides as a colorless solid (amount).

[000506] 1H NMR (500 MHz, d in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.80 to 0.91 (m, 12 H); 0.97 to 1.09 (m, 24 H); 1.12 to 1.21 (m, 6 H); 1.32 (d, J = 14.7 Hz, 0.6 H); 1.40 (d, J = 14.7 Hz, 0.4 H); 1.70 to 1.87 (m, 1 H); 1.90 (dd, J = 10.1 and 14.7 Hz, 0.4 H); 1.97 (dd, J = 10.1 and 14.7 Hz, 0.6 H); 2.28 (m, 0.6H); 2.40 (m, 0.4H); 2.55 to 2.79 (m, 2H); 2.90 to 3.00 (m, 2H); 3.44 (m, 1 H); 3.79 (d, J = 2.1 Hz, 0.4 H); 3.80 (s, 3H); 3.90 (d, J = 2.1 Hz, 0.6 H); 4.03 to 4.22 (m, 2H); 4.78 (s, 0.8H); 4.80 (s, 1.2 H); 5.03 (m, 1H); 5.90 (dd, J = 1.5 and 15.5 Hz, 0.6 H); 5.99 (dd, J = 1.5 and 15.5 Hz, 0.4 H); 6.40 to 6.55 (m, 1 H); 7.02 to 7.07 (m, 1 H); 7.13 to 7.38 (m, 6H); 7.85 to 7.93 (m, 2H); 8.40 (d, J = 7.3 Hz, 0.6 H); 8.51 (d, J = 7.3 Hz, 0.4 H). LCMS (A1): ES m/z = 880 [M-H]-; m/z = 882 [M+H]+; m/z = 926 [M-H+HCO2H]-; tR = 2.05 to 2.06 minutes.

[000507] Step 2: at 0°C under argon, in a round bottom flask, to a solution of alpha and beta epoxides (177 mg, 200.53 µmol) in THF (7.5 mL) was added dropwise 1M TBAF (221 µL, 221 µmol). After stirring for 2 hours at room temperature, 50 µL of TBAF was added and stirred for 3.5 hours. The reaction medium was diluted with H2O (9 mL), stirred for 10 minutes and extracted with DCM (3 x 25 mL). The combined organic layers were washed with saturated brine (3 x 8 mL), dried over MgSO4, filtered and concentrated to provide 210 mg of alpha and beta epoxides as a colorless solid (amount).

[000508] 1H NMR (500 MHz, d in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.82 to 1.25 (m, 21 H); 1.32 (d, J = 14.5 Hz, 0.6 H); 1.42 (d, J = 14.5 Hz, 0.4 H); 1.80 (m, 1 H); 1.91 (dd, J = 10.0 and 14.5 Hz, 0.4 H); 1.98 (dd, J = 10.0 and 14.5 Hz, 0.6 H); 2.22 to 2.48 (m, 1 H); 2.55 to 2.78 (m, 2H); 2.89 to 3.03 (m, 2H); 3.44 (m, 1 H); 3.79 (d, J = 2.2 Hz, 0.4 H); 3.80 (s, 3H); 3.90 (d, J = 2.2 Hz, 0.6 H); 4.04 to 4.22 (m, 2H); 4.48 (d, J = 5.8 Hz, 0.8 H); 4.50 (d, J = 5.8 Hz, 1.2 H); 5.03 (m, 1H); 5.16 (t, J = 5.8 Hz, 0.4 H); 5.19 (t, J = 5.8 Hz, 0.6 H); 5.89 (dd, J = 1.9 and 15.6 Hz, 0.6 H); 5.99 (dd, J = 1.9 and 15.6 Hz, 0.4 H); 6.40 to 6.52 (m, 1 H); 7.02 (d, J = 8.7 Hz, 0.6 H); 7.04 (d, J = 8.7 Hz, 0.4 H); 7.16 to 7.37 (m, 6H); 7.83 to 7.94 (m, 2H); 8.39 (d, J = 7.4 Hz, 0.6 H); 8.50 (d, J = 7.4 Hz, 0.4 H). LCMS (A1): ES m/z = 724 [M-H]-; m/z = 726 [M+H]+; tR = 1.29 minutes.

[000509] Alpha and beta epoxides were separated by chiral liquid chromatography which was performed on a 76 x 350 mm column packed with 1.1 kg of 10 µm Chiralpak AD (amylose tris-3,5-dimethylphenylcarbamate coated on a support silica gel, Chiral Technologies Europe) using isocratic elution with 75:25 heptane/EtOH. After concentration, 66 mg of example 11 was obtained as a white solid (45%) and 45 mg of the alpha epoxide was obtained as a white solid (31%).

[000510] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.82 to 0.89 (m, 12 H); 0.99 (s, 3H); 1.03 (d, J = 6.9 Hz, 3 H); 1.19 (s, 3H); 1.34 (d, J = 14.6 Hz, 1 H); 1.80 (m, 1 H); 1.98 (dd, J = 10.1 and 14.6 Hz, 1 H); 2.26 (m, 1H); 2.61 (m, 1H); 2.69 (dd, J = 11.1 and 14.6 Hz, 1 H); 2.91 (dd, J = 2.1 and 7.8 Hz, 1 H); 2.94 (dd, J = 3.7 and 14.6 Hz, 1 H); 3.42 (m, 1H); 3.80 (s, 3H); 3.91 (d, J = 2.1 Hz, 1 H); 4.03 to 4.14 (m, 2H); 4.50 (d, J = 5.8 Hz, 2 H); 5.03 (m, 1H); 5.19 (t, J = 5.8 Hz, 1 H); 5.89 (dd, J = 2.1 and 15.5 Hz, 1 H); 6.43 (ddd, J = 4.7, 10.9 and 15.5 Hz, 1 H); 7.03 (d, J = 8.7 Hz, 1 H); 7.20 (dd, J = 2.3 and 8.7 Hz, 1 H); 7.23 (d, J = 8.4 Hz, 2 H); 7.30 (d, J = 2.3 Hz, 1 H); 7.33 (d, J = 8.4 Hz, 2 H); 7.86 (d, J = 8.4 Hz, 1 H); 7.92 (d, J = 7.1 Hz, 1 H); 8.39 (d, J = 9.1 Hz, 1 H). LCMS (A1): ES m/z = 724 [M-H]-; m/z = 726 [M+H]+; tR = 1.29 minutes. Compound 54: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(azidomethyl)phenyl)oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000511] Under argon, in a round bottom flask, to an example solution 11 (66 mg, 90.87 µmol) in THF (5 mL) was added, at 0°C, DPPA (100.23 µl, 454 .36 µmol) and DBU (69.27 µl, 454.36 µmol). The solution was stirred for 5 hours at room temperature, then diluted with H2O and extracted with EtOAc (2 x 15 mL). The organic layer was separated, washed with saturated brine (2 x 5 mL), dried over MgSO4, filtered, concentrated and purified on 15 g of silica gel (DCM/iPrOH gradient elution) to provide 64 mg of compound 54 as an colorless solid (94%).

Example 12: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl) oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000512] In a round bottom flask, to a solution of compound 54 (64 mg, 85.18 µmol) in DCM (3 mL), MeOH (3 mL) and H2O (400 µL) was added TCEP (26.86 mg, 93.70 µmol). The reaction medium was stirred for 24 hours at room temperature. The reaction mixture was diluted with DCM (15 mL) and saturated NaHCO3, stirred for 10 minutes and extracted with DCM (3 x 15 mL). The combined organic layers were washed with saturated brine, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 4.5 g of amino-propyl modified silica gel (DCM/MeOH gradient elution) to give 42 mg of example 12 as a white solid (68%). Compound 55: (9H-fluoren-9-yl)methyl ((2S)-1-(((2S)-1-((4- ((2R,3R)-3-((1S)-1-(( 3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8 ,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl )carbamate

[000513] Under argon, in a round bottom flask, example 12 (42 mg, 57.91 µmol) and DMF (1 mL) were introduced, followed by FmocValAla (34.44 mg, 86.86 µmol), HOBt ( 13.20 mg, 93.81 µmol), DCM (10 mL) and EDC (10.36 µl, 57.91 µmol). The reaction medium was stirred for 3 hours at room temperature and then diluted with H2O (15 mL), stirred for 10 minutes at room temperature and extracted with DCM (3 x 20 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to provide 83 mg of compound 55 as a white solid (amount). Compound 56: (2S)-2-amino-N-((2S)-1-((4-((2R,3R)-3-((1S)-1- ((3S,10R,16S,E) -10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[000514] In a round bottom flask, piperidine (57.8 µL, 579.10 µmol) was added to a solution of compound 55 (64.73 mg, 57.91 µmol) in DCM (10 mL). After stirring for 5 hours, 57.8 µL of piperidine was added and the medium was stirred overnight at room temperature. The reaction medium was concentrated in vacuo and purified on 15 g of silica gel (DCM/MeOH/H2O gradient) to provide 30 mg of compound 56 as a colorless solid (58%).

[000515] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.86 (d, J = 7.0 Hz, 3 H); 0.82 to 0.90 (m, 15 H); 1.00 (s, 3H); 1.03 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3H); 1.24 (d, J = 7.0 Hz, 3 H); 1.34 (d, J = 14.6 Hz, 1 H); 1.66 (wide m, 2 H); 1.80 (m, 1 H); 1.91 (m, 1H); 1.98 (dd, J = 10.2 and 14.6 Hz, 1 H); 2.27 (m, 1 H); 2.60 (m, 1 H); 2.69 (dd, J = 11.1 and 14.6 Hz, 1 H); 2.91 (dd, J = 2.0 and 7.7 Hz, 1 H); 2.94 (dd, J = 3.7 and 14.6 Hz, 1 H); 2.99 (d, J = 4.9 Hz, 1 H); 3.43 (m, 1 H); 3.80 (s, 3H); 3.91 (d, J = 2.0 Hz, 1 H); 4.03 to 4.12 (m, 2H); 4.29 (d, J = 6.2 Hz, 2 H); 4.34 (m, 1H); 5.03 (m, 1H); 5.89 (dd, J = 1.8 and 15.6 Hz, 1 H); 6.43 (ddd, J = 4.8, 10.9 and 15.6 Hz, 1 H); 7.02 (d, J = 8.6 Hz, 1 H); 7.20 (dd, J = 2.2 and 8.6 Hz, 1 H); 7.22 (d, J = 8.4 Hz, 2 H); 7.26 (d, J = 8.4 Hz, 2 H); 7.32 (d, J = 2.2 Hz, 1 H); 7.86 (d, J = 8.4 Hz, 1 H); 7.92 (d, J = 7.2 Hz, 1 H); 8.07 (wide d, J = 7.9 Hz, 1 H); 8.40 (d, J = 7.3 Hz, 1 H); 8.45 (t, J = 6.2 Hz, 1 H). LCMS (A1): ES m/z = 448 [M+2H]2+; m/z = 893 [M-H]-; m/z = 895 [M+H]+; m/z = 939 [M-H+HCO2H]-; tR = 1.29 minutes. Compound 57: acid 5-(((2S)-1-(((2S)-1-((4-((2R,3R)-3-((1S)- 1-((3S,10R,16S, E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec -13-en-16- yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5- oxopentanoic

[000516] Under argon, in a round bottom flask, a solution of glutaric anhydride (4.29 mg, 36.85 µmol) in DCM (2 mL) was added to a solution of compound 56 (30 mg, 33.5 µmol) in DCM (6 mL). The reaction medium was stirred for 2 hours at room temperature, partially concentrated in vacuum and purified on 2.5 g of silica gel (DCM/MeOH/H2O gradient elution) to provide 34 mg of compound 57 as a colorless lake ( amount).

[000517] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.80 to 0.89 (m, 18 H); 0.99 (s, 3H); 1.03 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3H); 1.24 (d, J = 7.0 Hz, 3 H); 1.34 (d, J = 14.6 Hz, 1 H); 1.70 (m, 2H); 1.80 (m, 1 H); 1.97 (m, 2H); 2.19 (m, 4H); 2.26 (m, 1H); 2.60 (m, 1H); 2.70 (dd, J = 11.2 and 14.6 Hz, 1 H); 2.90 (dd, J = 2.2 and 7.8 Hz, 1 H); 2.95 (dd, J = 3.7 and 14.6 Hz, 1 H); 3.43 (m, 1H); 3.80 (s, 3H); 3.91 (d, J = 2.2 Hz, 1 H); 4.03 to 4.18 (m, 3H); 4.22 to 4.33 (m, 3H); 5.03 (m, 1H); 5.90 (d, J = 15.5 Hz, 1 H); 6.43 (ddd, J = 4.9, 11.0 and 15.5 Hz, 1 H); 7.02 (d, J = 8.6 Hz, 1 H); 7.19 to 7.28 (m, 5 H); 7.32 (d, J = 2.2 Hz, 1 H); 7.85 (wide d, J = 8.9 Hz, 2 H); 7.91 (d, J = 7.2 Hz, 1 H); 8.06 (wide d, J = 7.5 Hz, 1 H); 8.36 (wide t, J = 6.6 Hz, 1 H); 8.42 (d, J = 7.8 Hz, 1 H); 12.10 (wide m, 1 H). LCMS (A1): ES m/z = 1007 [M-H]-; m/z = 1009 [M+H]+; tR = 1.21 minutes.

Example 13: 2,5-dioxopyrrolidin-1-yl 5-(((2S)-1-(((2S)-1-((4-((2R,3R)-3- ((1S)-1- ((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4 ,8,11 -triazacyclohexadec-13-en-16- yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2 -yl)amino)-5-oxopentanoate

[000518] Under argon, in a round bottom flask, to a solution of compound 57 (33 mg, 32.69 µmol) in DCM (8 mL) were added DSC (11.72 mg, 45.76 µmol) and DIEA (7.7 µL, 45.76 µmol). The reaction medium was stirred for 1 hour at room temperature, concentrated in vacuo and purified by two successive flash chromatographies on 2.5 g of silica gel (DCM/iPrOH gradient elution) to give 17.9 mg of example 13 as a colorless solid (49%).

[000519] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.80 to 0.90 (m, 18 H); 1.00 (s, 3H); 1.04 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3H); 1.24 (d, J = 7.0 Hz, 3 H); 1.34 (d, J = 14.6 Hz, 1 H); 1.80 (m, 1 H); 1.83 (m, 2H); 1.98 (m, 2H); 2.26 (m, 1H); 2.29 (m, 2H); 2.60 (m, 1H); 2.68 (t, J = 7.5 Hz, 2 H); 2.70 (dd, J = 11.2 and 14.6 Hz, 1 H); 2.80 (s, 4H); 2.91 (dd, J = 2.2 and 7.6 Hz, 1 H); 2.95 (dd, J = 3.7 and 14.6 Hz, 1 H); 3.43 (m, 1H); 3.80 (s, 3H); 3.90 (d, J = 2.2 Hz, 1 H); 4.06 to 4.15 (m, 2H); 4.18 (dd, J = 6.9 and 8.7 Hz, 1 H); 4.21 to 4.33 (m, 3H); 5.03 (m, 1H); 5.89 (d, J = 15.5 Hz, 1 H); 6.43 (ddd, J=4.9, 10.9 and 15.5 Hz, 1 H); 7.02 (d, J=8.7 Hz, 1 H); 7.20 (dd, J = 2.3 and 8.7 Hz, 1 H); 7.23 (d, J = 8.4 Hz, 2 H); 7.25 (d, J = 8.4 Hz, 2 H); 7.31 (d, J = 2.3 Hz, 1 H); 7.84 (d, J = 8.6 Hz, 1 H); 7.89 (m, 2H); 8.04 (d, J = 7.5 Hz, 1 H); 8.33 (t, J = 6.5 Hz, 1 H); 8.39 (d, J = 7.6 Hz, 1 H). LCMS (A1): ES m/z = 796; m/z = 1104 [M-H]-; m/z = 1106 [M+H]+; m/z = 1150 [M-H+HCO2H]-; tR = 1.26 minutes.

Example 14: mAb-Ex13

[000520] The general method described previously was used for the preparation of example 14. 60 mg of hu2H11_R35-74 was reacted with 200 µL of a 10.05 mM solution of example 13 in DMA (5 equivalents) for 2 hours. At this time, 180 µL of example solution 13 (4.5 equivalents) was added and the medium was stirred for 2 hours. After purification in SuperDex 200 pg in buffer B pH 6.5 + 10% NMP, concentration in Amicon Ultra-15, buffer exchange in PD-10 in buffer B pH 6.5 + 5% NMP and filtration in Steriflip, 46 mg of example 14 was obtained as a clear, colorless solution at a concentration of 2 mg/mL with a DAR of 3.5 (HRMS), a monomeric purity of 99.7% and an overall yield of 77%.

[000521] SEC-HRMS: spectrum for intact ADC in Figure 4; m/z = 149336 (naked mAb); m/z = 150328 (D1); m/z = 151319 (D2); m/z = 152311 (D3); m/z = 153302 (D4); m/z = 154295 (D5); m/z = 155290 (D6); m/z = 156282 (D7). Example 15: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16-((S)-1-(3- (4-(hydroxy-methyl)phenyl)oxiran-2 -yl)ethyl)-3,6,6-trimethyl-1-oxa-4,8,11 - triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000522] Example 15 was prepared following the general routine B represented in Scheme 2 and represented by examples 4 and 11.

[000523] 1H NMR (500 MHz, d in ppm, DMSO-d6): 65/35 diastereoisomer mixture; 0.93 to 1.26 (m, 12 H); 1.75 (m, 0.65 H); 1.83 (m, 0.35 H); 2.22 (m, 0.65H); 2.41 (m, 0.35 H); 2.55 to 2.70 (m, 2H); 2.81 (d, J = 13.7 Hz, 0.65 H); 2.88 (d, J = 13.7 Hz, 0.35 H); 2.93 (dd, J = 2.1 and 8.2 Hz, 0.65 H); 3.00 (m, 1.35 H); 3.22 to 3.35 (m partially masked, 1 H); 3.77 (d, J = 2.1 Hz, 0.35 H); 3.81 (s, 3H); 3.88 (d, J = 2.1 Hz, 0.65 H); 4.25 to 4.39 (m, 1.65 H); 4.34 (m, 0.35 H); 4.48 (d, J = 6.0 Hz, 0.7 H); 4.50 (d, J = 6.0 Hz, 1.3 H); 5.08 (m, 1 H); 5.17 (t, J = 6.0 Hz, 0.35 H); 5.20 (t, J = 6.0 Hz, 0.65 H); 5.78 (dd, J = 1.9 and 15.5 Hz, 0.65 H); 5.89 (dd, J = 1.9 and 15.5 Hz, 0.35 H); 6.40 (m, 1 H); 7.04 (d, J = 8.7 Hz, 1 H); 7.16 (dd, J = 2.2 and 8.7 Hz, 0.65 H); 7.19 (dd, J = 2.2 and 8.7 Hz, 0.35 H); 7.20 to 7.34 (m, 5 H); 7.58 (wide d, J = 10.2 Hz, 0.65 H); 7.64 (wide d, J = 10.2 Hz, 0.35 H); 8.00 (d, J = 8.4 Hz, 0.65 H); 8.07 (d, J = 8.4 Hz, 0.35 H); 8.35 (d, J = 8.3 Hz, 0.65 H); 8.44 (d, J = 8.3 Hz, 0.35 H). LCMS (A1): ES m/z = 654 [MH]-; m/z = 656 [M+H]+; tR = 1.06 minutes. Example 16: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16-((S)-1-(3- (4-(hydroxy-methyl)phenyl)oxiran-2 -yl)ethyl)-6,6-dimethyl-3-isopropyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000524] Example 16 was prepared following the general routine B represented in Scheme 2 and represented by examples 4 and 11.

[000525] 1H NMR (500 MHz, d in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.72 (d, J = 7.0 Hz, 1.8 H); 0.82 (d, J = 7.0 Hz, 1.2 H); 0.84 (d, J = 7.0 Hz, 1.8 H); 0.87 (d, J = 7.0 Hz, 1.2 H); 0.98 (s, 1.8H); 0.99 (d, J = 7.0 Hz, 1.8 H); 1.01 (s, 1.2H); 1.05 (d, J = 7.0 Hz, 1.2 H); 1.09 (s, 1.8 H); 1.11 (s, 1.2H); 1.82 (m, 1H); 1.91 (m, 0.6H); 2.01 (m, 0.4H); 2.30 (m, 1 H); 2.52 to 2.72 (m, 2H); 2.88 to 3.04 (m, 3H); 3.25 to 3.35 (masked m, 1 H); 3.79 (d, J = 2.2 Hz, 0.4 H); 3.80 (s, 3H); 3.88 (d, J = 2.2 Hz, 0.6 H); 4.09 to 4.21 (m, 2H); 4.48 (d, J = 5.8 Hz, 0.8 H); 4.50 (d, J = 5.8 Hz, 1.2 H); 5.14 to 5.29 (m, 2H); 5.79 (dd, J = 1.7 and 15.4 Hz, 0.6 H); 5.90 (dd, J = 1.7 and 15.4 Hz, 0.4 H); 6.40 to 6.52 (m, 1 H); 7.05 (d, J = 8.7 Hz, 1 H); 7.10 (dd, J = 2.0 and 10.2 Hz, 1 H); 7.17 (m, 1H); 7.21 to 7.35 (m, 5 H); 7.70 (d, J = 9.3 Hz, 0.6 H); 7.80 (d, J = 9.3 Hz, 0.4 H); 8.39 (d, J = 8.0 Hz, 0.6 H); 8.44 (d, J = 8.0 Hz, 0.4 H). LCMS (A1): ES m/z = 682 [MH]-; m/z = 684 [M+H]+; tR = 1.16 minutes. Example 17: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16-((S)-1-(3- (4-(hydroxy-methyl)phenyl)oxiran-2 -yl)ethyl)-3-tert-butyl-6,6-dimethyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000526] Example 17 was prepared following the general routine B represented in Scheme 2 and represented by examples 4 and 11.

[000527] 1H NMR (400 MHz, d in ppm, DMSO-d6): 50/50 diastereoisomeric mixture; 0.90 (s, 4.5H); 0.91 (s, 4.5H); 1.00 (d, J = 7.0 Hz, 1.5 H); 1.03 (s, 1.5H); 1.05 (s, 1.5 H); 1.06 (d, J = 7.0 Hz, 1.5 H); 1.10 (s, 1.5H); 1.12 (s, 1.5H); 1.85 (m, 1H); 2.32 (m, 1H); 2.55 to 2.72 (m, 2H); 2.90 to 3.03 (m, 3H); 3.25 to 3.35 (masked m, 1 H); 3.81 (s, 3H); 3.90 (d, J = 2.2 Hz, 1 H); 4.15 (m, 1 H); 4.36 (d, J = 10.0 Hz, 0.5 H); 4.43 (d, J = 10.0 Hz, 0.5 H); 4.49 (d, J = 5.9 Hz, 1 H); 4.51 (d, J = 5.9 Hz, 1 H); 5.15 (t, J = 5.9 Hz, 0.5 H); 5.18 (t, J = 5.9 Hz, 0.5 H); 5.29 (m, 1H); 5.79 (dd, J = 2.0 and 15.4 Hz, 0.5 H); 5.90 (d, J = 15.4 Hz, 0.5 H); 6.39 (m, 1H); 6.91 (dd, J = 2.5 and 10.4 Hz, 0.5 H); 6.98 (dd, J = 2.5 and 10.4 Hz, 0.5 H); 7.05 (split d, J = 8.7 Hz, 1 H); 7.15 (split dd, J = 2.4 and 8.7 Hz, 1 H); 7.20 to 7.39 (m, 6H); 8.32 (d, J = 7.9 Hz, 0.5 H); 8.40 (d, J = 7.9 Hz, 0.5 H). LCMS (A4): ES m/z = 696 [MH]-; m/z = 698 [M+H]+; tR = 4.13 to 4.16 minutes. Example 18 (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-( 3-chloro-4-methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000528] Example 18 was prepared following the general routine A represented in Scheme 1 and described for Example 8.

[000529] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.78 (d, J = 7.0 Hz, 3 H); 0.84 (d, J = 7.0 Hz, 3 H); 0.87 (d, J = 7.0 Hz, 3 H); 1.00 (s, 3H); 1.03 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3H); 1.30 (m, 1 H); 1.50 to 1.90 (m, 5 H); 2.28 (m, 1 H); 2.55 to 2.76 (m, 2H); 2.90 to 3.00 (m, 2 H); 3.48 (m, 1 H); 3.70 (s, 2H); 3.80 (s, 3H); 3.89 (s, 1 H); 4.02 to 4.15 (m, 2H); 5.06 (m, 1H); 5.88 (d, J = 15.5 Hz, 1 H); 6.47 (m, 1 H); 7.03 (d, J = 8.7 Hz, 1 H); 7.20 (m, 3H); 7.31 (m, 3H); 7.80 (d, J = 8.7 Hz, 1 H); 7.89 (d, J = 7.3 Hz, 1 H); 8.41 (d, J = 8.2 Hz, 1 H). Example 19: 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )- 10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6,7-trimethyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec- 13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate 2,5-dioxopyrrolidin-1-yl

[000530] Example 19 was prepared as represented in Scheme 3 and represented by examples 2, 6, 9 and 13.

[000531] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.79 (d, J = 7.0 Hz, 3 H); 0.81 to 0.89 (m, 18 H); 1.01 (s, 3H); 1.03 (d, J = 7.0 Hz, 3 H); 1.20 (s, 3H); 1.24 (d, J = 7.0 Hz, 3 H); 1.31 (m, 1 H); 1.60 to 1.74 (m, 2H); 1.78 to 1.86 (m, 3H); 1.96 (m, 1H); 2.21 to 2.31 (m, 3H); 2.55 to 2.72 (m, 4H); 2.80 (s, 4H); 2.96 (m, 2H); 3.47 (m, 1H); 3.80 (s, 3H); 3.89 (d, J = 2.2 Hz, 1 H); 4.03 to 4.14 (m, 2H); 4.17 (dd, J = 6.8 and 8.6 Hz, 1 H); 4.21 to 4.33 (m, 3H); 5.06 (m, 1H); 5.89 (d, J = 15.5 Hz, 1 H); 6.47 (ddd, J = 5.2, 10.5 and 15.5 Hz, 1 H); 7.02 (d, J = 8.7 Hz, 1 H); 7.20 (dd, J = 2.3 and 8.7 Hz, 1 H); 7.23 (m, 4H); 7.32 (d, J = 2.3 Hz, 1 H); 7.80 (d, J = 8.3 Hz, 1 H); 7.89 (m, 2H); 8.04 (d, J = 7.6 Hz, 1 H); 8.32 (t, J = 6.3 Hz, 1 H); 8.42 (d, J = 7.6 Hz, 1 H). LCMS (A1): ES m/z = 1092 [M+H]+; m/z = 1136 [M-H+HCO2H]-; tR = 1.23 minutes. Example 20: mAb-Ex19

[000532] Example 20 was prepared in a medium similar to examples 3, 7, 10 and 14. 45 mg of example 20 was obtained as a clear, colorless solution at a concentration of 2.55 mg/mL with a DAR of 4 .4 (HRMS), a monomeric purity of 99.1% and an overall production of 78%.

[000533] SEC-HRMS: spectrum for intact ADC in Figure 5; m/z = 150368 (D1); m/z = 151350 (D2); m/z = 152327 (D3); m/z = 153304 (D4); m/z = 154281 (D5); m/z = 155255 (D6); m/z = 156237(D7); m/z = 157217 (D8). Example 21: (3S,10R,16S,E)-16-((S)-1-(3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-(3-chloro-4 -methoxybenzyl)-7-cylopropyl-3-isobutyl-6,6-dimethyl-1-oxa-4,8,11-triazacylohexadec-13-ene-2,5,9,12-tetraone

[000534] Example 21 was prepared following the general routine A represented in Scheme 1 and represented by examples 1 and 8. Example 22: 5-(((S)-1-(((S)-1-(( ^((2R,3R)-^((S)-1-((3S,10R,16S,E)- 10-(3-chloro-4-methoxybenzyl)-7-cylopropyl-3-isobutyl-6,6 -dimethyl-2,5,9,12- tetraoxo-1-oxa-4,8,11 -triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1- oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)- 2,5-dioxopyrrolidin-1-yl 5-oxopentanoate

[000535] Example 22 was prepared as represented in Scheme 3 and represented by examples 2, 6, 9 and 13.

[000536] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.34 (m, 1 H); 0.66 (m, 1 H); 0.74 (d, J = 7.0 Hz, 3 H); 0.76 (d, J = 7.0 Hz, 3 H); 0.78 (s, 3H); 0.80 (m, 1 H); 0.82 (d, J = 7.0 Hz, 3 H); 0.85 (d, J = 7.0 Hz, 3 H); 1.04 (d, J = 7.0 Hz, 3 H); 1.08 (s, 3H); 1.11 (m, 1H); 1.20 (m, 1H); 1.23 (d, J = 7.0 Hz, 3 H); 1.43 to 1.54 (m, 2H); 1.78 (m, 1H); 1.82 (m, 2H); 1.98 (m, 1H); 2.22 (m, 1H); 2.28 (m, 2H); 2.65 (m, 3H); 2.79 (m, 1H); 2.81 (s, 4H); 2.97 (dd, J = 2.2 and 7.9 Hz, 1 H); 3.80 (s, 3H); 3.89 (d, J = 2.2 Hz, 1 H); 4.02 (m, 1 H); 4.17 (dd, J = 6.7 and 8.7 Hz, 1 H); 4.27 (d, J = 6.3 Hz, 2 H); 4.30 (m, 1 H); 4.37 (m, 1H); 5.10 (m, 1 H); 5.79 (dd, J = 1.8 and 15.5 Hz, 1 H); 6.39 (ddd, J = 4.0, 11.6 and 15.5 Hz, 1 H); 7.02 (d, J = 8.7 Hz, 1 H); 7.11 (dd, J = 2.3 and 8.7 Hz, 1 H); 7.20 (d, J = 2.3 Hz, 1 H); 7.22 (m, 4H); 7.46 (s, 1 H); 7.77 (d, J = 9.0 Hz, 1 H); 7.89 (d, J = 8.6 Hz, 1 H); 8.05 (d, J = 7.5 Hz, 1 H); 8.29 (d, J = 7.6 Hz, 1 H); 8.32 (t, J = 6.3 Hz, 1 H). Example 23: mAb-Ex22

[000537] Example 23 was prepared in a medium similar to examples 3, 7, 10 and 14. 45 mg of example 23 was obtained as a clear, colorless solution at a concentration of 2.14 mg/mL with a DAR of 3 .6 (HRMS), a monomeric purity of 100% and an overall production of 75%.

[000538] SEC-HRMS: spectrum for intact ADC in Figure 6; m/z = 150341 (D1); m/z = 151329 (D2); m/z = 152317 (D3); m/z = 153308 (D4); m/z = 154296 (D5); m/z = 155287 (D6); m/z = 156279 (D7); m/z = 157267 (D8). Example 24: (3S,7S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(azidomethyl) phenyl)oxiran-2-yl)-ethyl) -10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12 -tetraone

[000539] Example 24 was prepared following the general routine C represented in Scheme 3 and described for Example 32.

[000540] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.83 (s, 9H); 0.88 (d, J = 6.8 Hz, 3H); 1.00 (s, 3H); 1.03 (d, J = 7.1 Hz, 3H); 1.19 (s, 3H); 1.33 (d, J = 14.5 Hz, 1H); 1.84 (m, 1H); 1.97 (dd, J = 9.8 and 14.5 Hz, 1H); 2.27 (dd, J = 11.1 and 14.5 Hz, 1H); 2.61 (m, 1H); 2.69 (dd, J = 11.1 and 14.2 Hz, 1H); 2.92 (dd, J = 2.1 and 7.7 Hz, 1H); 2.96 (dd, J = 3.6 and 14.2 Hz, 1H); 3.43 (m, 1H); 3.80 (s, 3H); 3.96 (d, J = 2.1 Hz, 1H); 4.08 (ddd, J = 3.6, 7.1 and 11.1 Hz, 1H); 4.12 (m, 1H); 4.46 (s, 2H); 5.05 (ddd, J = 1.3, 3.9 and 11.3 Hz, 1H); 5.90 (dd, J = 1.3 and 15.3 Hz, 1H); 6.45 (ddd, J = 4.7, 10.7 and 15.3 Hz, 1H); 7.02 (d, J = 8.7 Hz, 1H); 7.20 (dd, J = 2.4 and 8.7 Hz, 1H); 7.32 (m, 3H); 7.40 (d, J = 8.4 Hz, 2H); 7.88 (d, J = 8.4 Hz, 1H); 7.92 (d, J = 7.1 Hz, 1H); 8.40 (d, J = 7.4 Hz, 1H). LCMS (A5): ES m/z = 749 [MH]-; m/z = 751 [M+H]+; m/z = 795 [M-H+HCO2H]-; tR = 1.52 minutes. Example 25: (3S,7S, 10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(amino-methyl)phenyl)oxiran-2-yl)ethyl )-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9, 12-tetraone

[000541] Example 25 was prepared as described for examples 1, 5, 8 and 11.

[000542] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.86 (s, 9H); 0.88 (d, J = 6.9 Hz, 3H); 0.98 (s, 3H); 1.02 (d, J = 7.1 Hz, 3H); 1.18 (s, 3H); 1.34 (d, J = 14.5 Hz, 1H); 1.80 (m, 1H); 1.98 (dd, J = 9.8 and 14.5 Hz, 1H); 2.27 (dd, J = 11.1 and 14.5 Hz, 1H); 2.40 (wide m, 2H); 2.62 (m, 1H); 2.68 (dd, J = 11.1 and 14.2 Hz, 1H); 2.90 (dd, J = 2.1 and 7.7 Hz, 1H); 2.96 (dd, J = 3.6 and 14.2 Hz, 1H); 3.43 (m, 1H); 3.76 (s, 2H); 3.80 (s, 3H); 3.91 (d, J = 2.1 Hz, 1H); 4.02 to 4.15 (m, 2H); 5.04 (ddd, J = 1.3, 3.9 and 11.3 Hz, 1H); 5.88 (dd, J = 1.3 and 15.4 Hz, 1H); 6.45 (ddd, J = 4.7, 10.7 and 15.3 Hz, 1H); 7.03 (d, J = 8.5 Hz, 1H); 7.20 (dd, J = 2.2 and 8.5 Hz, 1H); 7.23 (d, J = 8.3 Hz, 2H); 7.32 (d, J = 2.2 Hz, 2H); 7.36 (d, J = 8.3 Hz, 2H); 7.86 (d, J = 8.3 Hz, 1H); 7.93 (d, J = 6.9 Hz, 1H); 8.40 (d, J = 7.1 Hz, 1H). LCMS (A5): ES m/z = 723 [MH]-; m/z = 725 [M+H]+; m/z = 769 [M-H+HCO2H]-; tR = 0.87 minute. Synthesis of examples 26 to 28: 3-(S)-neopentyl-7-(S)-Me-aza-C52 benzyl alcohol, glutaryl-Val-Ala-EDA-3-(S)- benzyl alcohol NHS ester neopentyl-7-(S)-Me-aza-C52 and corresponding ADC Compound 58: (S)-2-((S)-3-((R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylbutanamido)-4, (2R,3S)-1-((4-methoxybenzyl)oxy)-2-methylex-5-en-3-yl 4-dimethylpentanoate

[000543] To a solution of compound BC4 (365 mg, 919.7 µmol) in DMF (12 mL) were added HATU (402 mg, 1.06 mmol) and HOAt (144 mg, 1.06 mmol), the medium The reaction mixture was stirred at room temperature for 30 minutes, then a solution of compound AD3 (365 mg, 965.7 µmol) in DMF (5 mL) and DIEA (562 µL, 3.22 mmol) were added. The reaction medium was stirred at room temperature for 4 hours, then diluted with H2O (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with H2O (15 mL), saturated brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by two successive flash chromatographies on 30 g of silica gel (DCM gradient elution /MeOH and heptane/EtOAc) to provide 564 mg of compound 58 as a colorless foam (81%).

[000544] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.83 (d, J = 6.9 Hz, 3H); 0.85 (s, 9H); 0.87 (d, J = 6.9 Hz, 3H); 1.00 (s, 3H); 1.03 (s, 3H); 1.55 (dd, J = 2.4 and 14.5 Hz, 1H); 1.78 (dd, J = 9.5 and 14.5 Hz, 1H); 1.96 (m, 1H); 2.20 (m, 1H); 2.30 (m, 1H); 2.72 (dd, J = 9.6 and 13.9 Hz, 1H); 2.87 (dd, J = 5.3 and 13.9 Hz, 1H); 3.20 (dd, J = 6.5 and 9.5 Hz, 1H); 3.37 (dd, J = 5.4 and 9.5 Hz, 1H); 3.76 (s, 3H); 3.80 (s, 3H); 4.19 (m, 1H); 4.28 (m, 1H); 4.34 (s, 2H); 4.55 (m, 1H); 4.82 (m, 1H); 5.00 (d, J = 10.2 Hz, 1H); 5.07 (d, J = 17.4 Hz, 1H); 5.55 (dd, J = 2.3 and 10.2 Hz, 1H); 5.71 (m, 1H); 6.01 (dd, J = 2.3 and 17.1 Hz, 1H); 6.28 (dd, J = 10.2 and 17.1 Hz, 1H); 6.90 (d, J = 8.7 Hz, 2H); 7.02 (d, J = 8.6 Hz, 1H); 7.17 (dd, J = 2.3 and 8.6 Hz, 1H); 7.22 (d, J = 8.7 Hz, 2H); 7.32 (d, J = 2.3 Hz, 1H); 7.65 (d, J = 9.5 Hz, 1H); 7.70 (d, J = 7.9 Hz, 1H); 8.35 (d, J = 8.4 Hz, 1H). LCMS (A5): ES m/z = 754 [M-H]-; m/z = 756 [M+H]+; m/z = 800 [M-H+HCO2H]-; tR = 1.62 minutes. Compound 59: (3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16-((R)-1-((4-methoxy-benzyl)oxy)propan-2- il)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000545] To a solution of compound 58 (560 mg, 740.4 µmol) in DCM (56 mL) was added under Grubbs Ar catalyst I (31.0 mg, 37.02 µmol). The reaction medium was stirred at room temperature for 1 hour and 30 minutes before adding 31.0 mg of catalyst. Stirring was carried out at room temperature for 1 hour and 30 minutes before adding 31.0 mg of catalyst. Stirring was carried out at room temperature for 1 hour and 30 minutes before adding 31.0 mg of catalyst and the reaction medium was stirred at room temperature for 1 hour and 30 minutes. It was concentrated in vacuo and purified by flash chromatography on 25 g of silica gel (DCM/MeOH gradient elution) to give 330 mg of compound 59 (61%) and 220 mg which were also purified by flash chromatography on 15 g of silica gel (DCM/MeOH gradient elution) to provide 204 mg of compound 59 (37%) as a mixture with tricyclohexylphosphine oxide.

[000546] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (s, 9H); 0.88 (d, J = 6.9 Hz, 3H); 0.91 (d, J = 7.1 Hz, 3H); 1.04 (s, 3H); 1.20 (s, 3H); 1.29 (d, J = 14.5 Hz, 1H); 1.95 (dd, J = 9.8 and 14.5 Hz, 1H); 1.99 (m, 1H); 2.26 (m, 1H); 2.43 (m, 1H); 2.72 (dd, J = 10.1 and 14.3 Hz, 1H); 2.97 (dd, J = 3.6 and 14.3 Hz, 1H); 3.27 (dd, J = 6.1 and 9.3 Hz, 1H); 3.40 (dd, J = 5.8 and 9.3 Hz, 1H); 3.46 (m, 1H); 3.72 (s, 3H); 3.80 (s, 3H); 4.10 (m, 1H); 4.13 (m, 1H); 4.38 (s, 2H); 4.97 (m, 1H); 5.93 (d, J = 15.4 Hz, 1H); 6.43 (ddd, J = 4.8, 10.7 and 15.4 Hz, 1H); 6.90 (d, J = 8.7 Hz, 2H); 7.02 (d, J = 8.6 Hz, 1H); 7.17 (d, J = 8.6 Hz, 3H); 7.36 (d, J = 2.3 Hz, 1H); 7.88 (m, 2H); 8.48 (d, J = 7.5 Hz, 1H). LCMS (A5): ES m/z = 726 [M-H]-; m/z = 728 [M+H]+; m/z = 772 [M-H+HCO2H]-; tR = 1.54 minutes. Compound 60: (3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16-((R)-1-hydroxypropan-2-yl)-6,6,7-trimethyl -3-neopentyl-1- oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000547] Compound 59 (532 mg, 730.4 µmol) was treated with a solution of TFA (3.93 mL) in DCM (36 mL) at room temperature for 30 minutes. The reaction medium was poured into 9% aqueous NaHCO3 (130 mL) under magnetic stirring. Stirring was carried out for 30 minutes, then the aqueous phase was extracted with DCM (2 x 70 ml). The combined organic phases were washed with H2O (3 x 20 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 30 g of silica gel (DCM/MeOH gradient elution) to provide 299 mg of compound 60 as a white foam (67%).

[000548] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.88 (d, J = 6.9 Hz, 3H); 0.89 (s, 9H); 0.90 (d, J = 7.0 Hz, 3H) 1.03 (s, 3H); 1.20 (s, 3H); 1.36 (dd, J = 1.8 and 14.5 Hz, 1H); 1.80 (m, 1H); 1.96 (dd, J = 9, 6 and 14.5 Hz, 1H); 2.25 (m, 1H); 2.43 (m, 1H); 2.72 (dd, J = 11.1 and 14.3 Hz, 1H); 2.97 (dd, J = 3.8 and 14.3 Hz, 1H); 3.25 (m, 1H); 3.45 (m, 2H); 3.80 (s, 3H); 4.10 (m, 1H); 4.16 (m, 1H); 4.57 (t, J = 5.3 Hz, 1H); 4.98 (m, 1H); 5.93 (dd, J = 1.3 and 15.3 Hz, 1H); 6.45 (ddd, J = 4.9, 10.7 and 15.3 Hz, 1H); 7.02 (d, J = 8.6 Hz, 1H); 7.22 (dd, J = 2.3 and 8.6 Hz, 1H); 7.36 (d, J = 2.3 Hz, 1H); 7.89 (m, 2H); 8.48 (d, J = 7.3 Hz, 1H). LCMS (A5): ES m/z = 606 [M-H]-; m/z = 608 [M+H]+; tR = 1.2 minutes. Compound 61: (S)-2-((3S,7S,10R,16S,E)-10-(3-chloro—4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5, 9,12-tetraoxo-1-oxa-4,8,11- triazacyclohexadec-13-en-16-yl)propanal

[000549] To a solution of compound 60 (297 mg, 488.4 µmol) in DCM (6 mL) cooled with an ice/acetone bath was added at 0°C a solution of KBr (58.11 mg, 488. 4 µmol) in H2O (1.18 mL), TEMPO (1.57 mg, 9.8 µmol) and, drop by drop, a 1.56M aqueous solution of sodium hypochlorite, pH 9.5 (467.6 µL, 732.52 µmol). Stirring was carried out at 0°C for 15 minutes, then saturated Na2S2O3 at 0°C (3.56 mL) was added, the ice bath was removed and the reaction medium was stirred for 10 minutes. It was then extracted with DCM (3 x 25 mL), the combined organic phases were washed with H2O (2 x 10 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 15 g of silica gel. (DCM/MeOH gradient elution) to provide 139 mg of compound 61 as a white lacquer (47%).

[000550] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (s, 9H); 0.90 (d, J = 7.0 Hz, 3H); 1.03 (s, 3H); 1.04 (d, J = 6.9 Hz, 3H); 1.20 (s, 3H); 1.26 (d, J = 15.3 Hz, 1H); 1.95 (m, 1H); 2.44 (m, 1H); 2.57 (m, 1H); 2.72 (dd, J = 11.2 and 14.4 Hz, 1H); 2.80 (m, 1H); 2.98 (dd, J = 3.1 and 14.4 Hz, 1H); 3.80 (s, 3H); 4.10 (m, 3H); 5.22 (m, 1H); 5.98 (d, J = 15.3 Hz, 1H); 6.47 (ddd, J = 4.9, 10.8 and 15.3 Hz, 1H); 7.04 (d, J = 8.7 Hz, 1H); 7.22 (dd, J = 2.3 and 8.7 Hz, 1H); 7.37 (s, 1H); 7.90 (m, 1H); 7.92 (d, J = 8.2 Hz, 1H); 8.53 (d, J = 7.6 Hz, 1H); 9.68 (s, 1H). LCMS (A5): ES m/z = 604 [MH]-; m/z = 606 [M+H]+; m/z = 650 [M-H+HCO2H]-; tR = 1.28 minutes. Compound 62: (4-(((triisopropylsilyl)oxy)methyl)phenyl)methanol

[000551] To a solution of 1,4-benzenedimethanol (2 g, 14.33 mmol) in THF (100 mL) imidazole (1.13 g, 16.48 mmol) was added. The reaction medium was stirred at room temperature for 15 minutes, then triisopropylsilyl chloride (3.14 mL, 14.33 mmol) was added and stirring was carried out at room temperature overnight. Et2O (50 mL) was added to the reaction mixture and the organic phase was washed twice with saturated brine (100 mL), filtered over MgSO4, concentrated in vacuo, dissolved in DCM, filtered, concentrated in vacuo and purified by flash chromatography at 200 g of silica gel (8:2 isocratic elution of heptane/EtOAc) to provide 1.9 g of Compound 62 as a colorless oil (45%).

[000552] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.04 (d, J = 6.7 Hz, 18H); 1.15 (m, 3H); 4.48 (d, J = 5.7 Hz, 2H); 4.78 (s, 2H); 5.13 (t, J = 5.7 Hz, 1H); 7.29 (s, 4H). Compound 63: ((4-(bromomethyl)benzyl)oxy)triisopropylsilane

[000553] To a suspension of N-bromosuccinimide (1.29 g, 7.16 mmol) in DCM (65 mL) cooled to 0°C was added dropwise dimethyl sulfide (953 µL, 12.9 mmol). Stirring was carried out at 0°C for 10 minutes, then the reaction medium was cooled to -20°C and stirred at -20°C for 10 minutes. A solution of Compound 62 (1.9 g, 6.45 mmol) in DCM (30 mL) cooled to -20°C was then added. Stirring was carried out at -20°C for 15 minutes, then at 0°C for 15 minutes and at room temperature overnight. The reaction medium was washed twice with saturated brine (100 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by two successive rapid chromatographies on 40 g of silica gel (heptane/EtOAc gradient elution). Fractions containing the expressed compound were combined, concentrated in vacuo and dissolved in heptane (8 mL). The suspension was filtered, the filtrate concentrated in vacuo, dissolved in Et2O (5 mL), cooled to -20°C, filtered, concentrated in vacuo to give 1.18 g of compound 63 as a pale yellow oil (51%).

[000554] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.04 (d, J = 6.8 Hz, 18H); 1.15 (m, 3H); 4.70 (s, 2H); 4.81 (s, 2H); 7.29 (d, J = 8.4 Hz, 2H); 7.41 (d, J = 8.4 Hz, 2H). LCMS (A5): ES m/z = 104; m/z = 356 [M]+; tR = 1.99 minutes. Compound 64: (1R,4S,5R,6S)-4,7,7-trimethyl-6-(4-(((triisopropylsilyl)oxy)methyl)benzyl)-6-thiabicyl [3,2,1] octan-6-ium trifluoromethanesulfonate

[000555] Ao (1R,4R,5R)-4,7,7-trimethyl-6-thiabicil [3,2,1]octane (CAS number [5718-75-2], 562.3 mg, 3, 3 mmol) a solution of compound 63 (1.18 g, 3.3 mmol) in DCM (3.4 mL) was added. A solution of lithium trifluoromethanesulfonate (2.63 g, 16.51 mmol) in H2O (3 mL) was then added dropwise and the reaction medium was stirred at room temperature overnight. H2O (15 mL) and DCM (15 mL) were then added to the reaction medium and stirring was carried out at room temperature for 10 minutes. The aqueous phase was extracted with DCM (3 x 20 mL), the combined organic phases were washed with saturated brine (3 x 8 mL), dried over MgSO4, filtered and concentrated in vacuo. The pale yellow oil was triturated in iPr2O (5 mL), the solid thus obtained was filtered, washed with iPr2O (2 x 5 mL) and dried under vacuum to provide 1.125 g of compound 64 as a white solid (57%).

[000556] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.04 (m, 21H); 1.15 (m, 3H); 1.45 (m, 1H); 1.60 (m, 2H); 1.68 (s, 3H); 1.71 (m, 1H); 1.74 (s, 3H); 1.98 (m, 1H); 2.40 (d, J = 14.6 Hz, 1H); 2.45 (m, 1H); 2.58 (wide d, J = 14.6 Hz, 1H); 3.85 (m, 1H); 4.56 (d, J = 12.6 Hz, 1H); 4.83 (s, 2H); 4.90 (d, J = 12.6 Hz, 1H); 7.44 (d, J = 8.4 Hz, 2H); 7.57 (d, J = 8.4 Hz, 2H). Compound 65: (3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-16-((S)-1-(( 2R,3R)-3-(4-(((triisopropylsilyl)oxy)methyl)phenyl)oxiran-2-yl)ethyl)-1-oxa-4,8,11-triazacyclohexadec-13-ene- 2,5,9,12-tetraone

[000557] To a solution of compounds 61 (138 mg, 227.7 µmol) and 64 (149.5 mg, 250.4 µmol) in DCM (4 mL) cooled to -70°C was added dropwise BEMP ( CAS number [98015-45-3], 90.2 µL, 296.0 µmol). The reaction mixture was stirred at -70°C for 2 hours, saturated brine (7 ml) was added to the reaction mixture, the bath removed and stirring carried out vigorously until RT. The aqueous phase was extracted with DCM (3 x 20 mL), the combined organic phases were dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 10 g of silica gel (DCM/MeOH gradient elution) to give 163 mg of Compound 65 as a colorless oil (30%) and 60 mg of compound 61 (43%).

[000558] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.84 (s, 9H); 0.88 (d, J = 6.9 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J = 7.1 Hz, 3H); 1.05 (d, J = 7.3 Hz, 18H); 1.15 (m, 3H); 1.19 (s, 3H); 1.32 (d, J = 14.8 Hz, 1H); 1.82 (m, 1H); 1.97 (dd, J = 9.9 and 14.8 Hz, 1H); 2.28 (dt, J = 11.1 and 14.9 Hz, 1H); 2.61 (m, 1H); 2.69 (dd, J = 11.2 and 14.3 Hz, 1H); 2.91 (dd, J = 2.2 and 7.6 Hz, 1H); 2.95 (dd, J = 3.8 and 14.3 Hz, 1H); 3.43 (m, 1H); 3.80 (s, 3H); 3.91 (d, J = 2.2 Hz, 1H); 4.08 (ddd, J = 3.8, 7.4 and 11.2 Hz, 1H); 4.12 (m, 1H); 4.81 (s, 2H); 5.04 (ddd, J = 1.5, 4.5 and 11.1 Hz, 1H); 5.89 (dd, J = 1.5 and 15.2 Hz, 1H); 6.44 (ddd, J = 4.5, 10.6 and 15.2 (d, J = 2.3 Hz, 1H); 7.36 (d, J = 8.4 Hz, 2H); 7, 88 (d, J = 8.4 Hz, 1H); 7.91 (d, J = 7.1 Hz, 1H); 8.39 (d, J = 7.4 Hz, 1H). LCMS (A4) : ES m/z = 120; m/z = 882 [M+H]+; tR = 7.65 minutes Example 26: (3S,7S,10R,16S,E)-10-(3-chloro-4 -methoxybenzyl)- 16-((S)-1-((2R,3R)-3-(4-(hydroxymethyl)phenyl)oxiran-2-yl)ethyl)-6,6,7-trimethyl- 3-neopentyl -1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000559] To a solution of compound 65 in THF cooled to 0°C, TBAF was added. The reaction medium was stirred at 0°C for 1 hour and 30 minutes, then H2O (5 mL) was added and stirring was carried out for 20 minutes. The reaction medium was extracted with DCM (3 x 20 mL), the combined organic phase was washed with saturated brine (3 x 5 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 15 g of silica gel (EtOAc/MeOH/H2O gradient elution) to provide 69 mg of Example 26 as a white lacquer (91%).

[000560] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (s, 9H); 0.89 (d, J = 6.9 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J = 7.1 Hz, 3H); 1.19 (s, 3H); 1.35 (d, J = 14.8 Hz, 1H); 1.80 (m, 1H); 1.99 (dd, J = 9.9 and 14.8 Hz, 1H); 2.27 (dt, J = 11.1 and 14.9 Hz, 1H); 2.61 (m, 1H); 2.69 (dd, J = 11.2 and 14.3 Hz, 1H); 2.90 (dd, J = 2.2 and 7.6 Hz, 1H); 2.95 (dd, J = 3.8 and 14.3 Hz, 1H); 3.43 (m, 1H); 3.80 (s, 3H); 3.91 (d, J = 2.2 Hz, 1H); 4.06 to 4.15 (m, 2H); 4.50 (d, J = 5.9 Hz, 2H); 5.03 (dd, J = 1.5, 3.7 and 11.5 Hz, 1H); 5.20 (t, J = 5.9 Hz, 1H); 5.89 (dd, J = 1.5 and 15.3 Hz, 1H); 6.43 (ddd, J = 4.6, 10.7 and 15.3 Hz, 1H); 7.02 (d, J = 8.6 Hz, 1H); 7.20 (dd, J = 2.2 and 8.6 Hz, 1H); 7.23 (d, J = 8.3 Hz, 2H); 7.32 (m, 3H); 7.87 (d, J = 8.3 Hz, 1H); 7.92 (d, J = 7.0 Hz, 1H); 8.40 (d, J = 7.4 Hz, 1H). LCMS (A5): ES m/z = 724 [MH]-; m/z = 726 [M+H]+; m/z = 770 [M-H+HCO2H]-; tR = 1.29 minutes. Compound 66: 4-((2R,3R)-3-((S)-1- ((3S,7S,10R,16S,E)-10-(3-chloro-4-) (4-nitrophenyl)carbonate methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran -2-yl)benzyl

[000561] To a solution of example 26 (68 mg, 93.6 µmol) in DCM (5 mL) was added bis (4-nitrophenyl)carbonate (118.7 mg, 374.5 µmol) and dropwise DIEA ( 49 µL, 281.0 µmol). The reaction medium was stirred at room temperature for 6 days, then saturated brine was added and extracted with DCM (3 x 10 mL). The combined organic phases were dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 5 g of silica gel (DCM/MeOH gradient elution) to give 79 mg of Compound 66 as a white lacquer (94%).

[000562] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (s, 9H); 0.89 (d, J = 6.9 Hz, 3H); 1.00 (s, 3H); 1.05 (d, J = 7.1 Hz, 3H); 1.19 (s, 3H); 1.34 (d, J = 14.6 Hz, 1H); 1.83 (m, 1H); 1.97 (dd, J = 9.9 and 14.6 Hz, 1H); 2.28 (dt, J = 11.1 and 14.9 Hz, 1H); 2.61 (m, 1H); 2.69 (dd, J = 11.2 and 14.3 Hz, 1H); 2.93 (m, 2H); 3.42 (m, 1H); 3.79 (s, 3H); 3.99 (d, J = 2.2 Hz, 1H); 4.09 (m, 2H); 5.05 (dd, J = 1.3, 4.0 and 11.1 Hz, 1H); 5.31 (s, 2H); 5.89 (dd, J = 1.3 and 15.3 Hz, 1H); 6.47 (ddd, J = 4.8, 10.8 and 15.3 Hz, 1H); 7.01 (d, J = 8.6 Hz, 1H); 7.21 (dd, J = 2.2 and 8.6 Hz, 1H); 7.31 (d, J = 2.2 Hz, 1H); 7.36 (d, J = 8.5 Hz, 2H); 7.50 (d, J = 8.5 Hz, 2H); 7.59 (d, J = 9.3 Hz, 2H); 7.87 (d, J = 8.3 Hz, 1H); 7.92 (d, J = 7.0 Hz, 1H); 8.32 (d, J = 9.3 Hz, 2H); 8.39 (d, J = 7.4 Hz, 1H). LCMS (A5): ES m/z = 889 [MH]-; m/z = 891 [M+H]+; m/z = 935 [M-H+HCO2H]-; tR = 1.55 minutes. Compound 67: ((S)-1-(((S)-1-((2-NBoc-aminoethyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan- hydrochloride (9H-fluoren-9-yl)methyl 2-yl)carbamate

[000563] To a suspension of Fmoc-Val-Ala-OH (3.5 g, 8.53 mmol) in DCM (100 mL) TEA charge (3.6 mL, 25.58 mL) was added to complete dissolved of starting material, N-Boc-EDA (1.62 mL, 10.23 mmol) and a 50% solution of propylphosphonic anhydride (6.51 g, 10.23 mmol) in DCM. The reaction medium was stirred at room temperature for 3 hours, then 1N NaOH (50 mL) was added and the suspension filtered, washed with H2O and DCM. The organic phase of the filtrate was washed with H2O. The mass and organic phase were combined, partially concentrated in vacuo, filtered, washed with DCM and dried to provide 3.952 g of Compound 67 as a white powder (84%).

[000564] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.84 (d, J = 7.0 Hz, 3H); 0.86 (d, J = 7.0 Hz, 3H); 1.19 (d, J = 7.1 Hz, 3H); 1.37 (s, 9H); 1.99 (m, 1H); 2.92 to 3.14 (m, 4H); 3.88 (dd, J = 6.9 and 9.1 Hz, 1H); 4.19 to 4.35 (m, 4H); 6.71 (m, 1H); 7.32 (m, 2H); 7.38 (m, 1H); 7.42 (m, 2H); 7.73 (m, 2H); 7.84 (t, J = 6.8 Hz, 1H); 7.89 (d, J = 7.8 Hz, 2H); 7.92 (d, J = 7.8 Hz, 1H). Compound 68: ((S)-1-(((S)-1-((2-aminoethyl)amino)- 1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2- hydrochloride (9H-fluoren-9-yl)methyl yl)carbamate

[000565] To the suspension of Compound 67 (100 mg, 159.5 µmol) in dioxane (3 mL) was added 4N HCl in dioxane (905 µL, 3.62 mmol). The reaction medium was stirred at room temperature for 20 hours, then concentrated in vacuum. The crude product was resuspended in iPr2O (3 mL), immersed in an ultrasonic bath and filtered. The mass thus obtained was washed with iPr2O (2 x 3 mL) and dried under vacuum to provide 78 mg of compound 68 as a white solid (88%).

[000566] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.85 (d, J = 6.9 Hz, 3H); 0.87 (d, J = 6.9 Hz, 3H); 1.23 (d, J = 7.1 Hz, 3H); 1.99 (m, 1H); 2.83 (m, 2H); 3.29 (m, 2H); 3.89 (dd, J = 6.9 and 8.9 Hz, 1H); 4.19 to 4.38 (m, 4H); 7.33 (m, 2H); 7.42 (m, 3H); 7.75 (m, 2H); 7.88 (broad s, 3H); 7.90 (d, J = 7.6 Hz, 2H); 8.08 (d, J = 7.3 Hz, 1H); 8.14 (d, J = 5.9 Hz, 1H). Compound 69: 4-((2R,3R)-3-((S)-1- (2-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)ethyl)carbamate ((3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa -4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl

[000567] To a suspension of compounds 66 (78 mg, 87.5 µmol) and 68 (51.4 mg, 105.0 µmol) in DCM was added DIEA (44.0 µL, 262.5 µmol). The reaction medium is stirred at room temperature overnight, then compound 68 (24 mg, 49 µoml) and DIEA (15 µL) were added and stirring was carried out at room temperature for 1 day. Piperidine (87.0 µL, 875.0 µmol) was added and stirring was carried out at room temperature overnight. The reaction medium was concentrated in vacuo and purified by two successive flash chromatographies on 5 g of silica gel (EtOAc/MeOH/H2O gradient elution) to give 57 mg of Compound 69 as a white lacquer (66%).

[000568] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.75 (d, J = 7.0 Hz, 3H); 0.85 (s, 9H); 0.86 (d, J = 7.0 Hz, 3H); 0.87 (d, J = 6.7 Hz, 3H); 0.99 (s, 3H); 1.04 (d, J = 7.0 Hz, 3H); 1.18 (m, 6H); 1.33 (d, J = 14.5 Hz, 1H); 1.75 (broad s, 2H); 1.81 (m, 1H); 1.92 (m, 1H); 1.98 (dd, J = 10.0 and 14.5 Hz, 1H); 2.28 (m, 1H); 2.61 (m, 1H); 2.69 (dd, J = 11.3 and 14.7 Hz, 1H); 2.90 (dd, J = 2.0 and 7.5 Hz, 1H); 2.96 (m, 1H); 2.98 (d, J = 4.7 Hz, 1H); 3.05 (m, 2H); 3.12 (m, 2H); 3.43 (m, 1H); 3.81 (s, 3H); 3.93 (d, J = 2.0 Hz, 1H); 4.09 (m, 2H); 4.26 (m, 1H); 5.01 (s, 2H); 5.04 (m, 1H); 5.89 (dd, J = 1.5 and 15.2 Hz, 1H); 6.45 (ddd, J = 4.5, 10.4 and 15.2 Hz, 1H); 7.02 (d, J = 8.8 Hz, 1H); 7.20 (dd, J = 2.4 and 8.8 Hz, 1H); 7.26 (t, J = 6.0 Hz, 1H); 7.29 (d, J = 8.4 Hz, 2H); 7.32 (d, J = 2.5 Hz, 1H); 7.37 (d, J = 8.4 Hz, 2H); 7.88 (d, J = 8.4 Hz, 1H); 7.93 (d, J = 7.1 Hz, 1H); 8.00 (t, J = 6.0 Hz, 1H); 8.03 (d, J = 8.1 Hz, 1H); 8.41 (d, J = 7.2 Hz, 1H). LCMS (A5): ES m/z = 492 [M+2H]2+; m/z = 980 [M-H]-; m/z = 982 [M+H]+; m/z = 1026 [M-H+HCO2H]-; tR = 0.91 minute. Compound 70: Acid (9S,12S)-1-(4-((2R,3R)-3-((S)-1-((3S,7S, 10R,16S,E)-10-(3-chloro -4-methoxy-benzyl)-6,6,7-trimethyl-3-neopentyl-2,5, 9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16- yl)ethyl)oxiran-2-yl)phenyl)-12-isopropyl-9-methyl-3,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetra-azaoctadecan-18-oic acid

[000569] To a solution of compound 69 (56.0 mg, 56.8 µL) in DCM (8 mL) was added a solution of glutaric anhydride (7.27 mg, 62.5 µmol) in DCM (4 mL) . The reaction medium was stirred at room temperature for 2 hours, concentrated in vacuo and purified by flash chromatography on 5 g of silica gel (DCM/MeOH/H2O gradient elution) to provide 35.5 mg of compound 70 as a lacquer. white (56%).

[000570] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.81 (d, J = 7.0 Hz, 3H); 0.83 (d, J = 7.0 Hz, 3H); 0.85 (s, 9H); 0.87 (d, J = 6.5 Hz, 3H); 0.99 (s, 3H); 1.04 (d, J = 7.1 Hz, 3H); 1.19 (m, 6H); 1.32 (d, J = 14.3 Hz, 1H); 1.70 (m, 2H); 1.81 (m, 1H); 1.92 (m, 1H); 1.98 (m, 2H); 2.15 (m, 2H); 2.19 (t, J = 7.5 Hz, 2H); 2.27 (m, 1H); 2.61 (m, 1H); 2.69 (dd, J = 11.1 and 14.1 Hz, 1H); 2.91 (d, J = 7.6 Hz, 1H); 2.95 (m, 1H); 3.04 (m, 2H); 3.10 (m, 2H); 3.41 (m, 1H); 3.80 (s, 3H); 3.93 (s, 1H); 4.03 to 4.16 (m, 3H); 4.19 (m, 1H); 5.01 (s, 2H); 5.04 (m, 1H); 5.89 (d, J = 15.6 Hz, 1H); 6.43 (ddd, J = 5.3, 11.0 and 15.6 Hz, 1H); 7.02 (d, J = 8.7 Hz, 1H); 7.20 (dd, J = 2.3 and 8.7 Hz, 1H); 7.28 (d, J = 8.4 Hz, 2H); 7.30 (m, 1H); 7.32 (d, J = 2.3 Hz, 1H); 7.36 (d, J = 8.4 Hz, 2H); 7.88 (m, 2H); 7.93 (d, J = 7.4 Hz, 1H); 7.98 (broad s, 1H); 8.06 (broad s, 1H); 8.42 (d, J = 7.4 Hz, 1H). LCMS (A5): ES m/z = 549 [M+2H]2+; m/z = 1094 [M-H]-; m/z = 1096 [M+H]+; tR = 1.21 minutes. Example 27: (9S,12S)-1-(4-((2R,3R)-3-((S)-1-((3S,7S,10R, 16S,E)-10-(3-chloro- 4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacylohexadec-13-en-16-yl)ethyl )oxiran-2-yl)phenyl)-12-isopropyl-9-methyl-3,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetra-azaoctadecan-18-oate of 2, 5-dioxopyrrolidin-1-yl

[000571] To a solution of compound 70 (14.6 mg, 13.3 µmol) in DCM (5 mL) under Ar were added DSC (4.3 mg, 16.0 µmol) and DIEA (2.8 µL, 16.0 µmol). The reaction medium was stirred at room temperature for 3 hours, then DSC (1.5 mg, 5.6 µmol) and DIEA (1 µL, 5.7 µmol) were added and stirring was carried out at room temperature for 1 hour. The reaction medium was concentrated in vacuo and purified by flash chromatography on 16.4 g of diol-modified silica gel (DCM/iPrOH gradient elution) to give 11.4 mg of example 27 as a white lacquer (72%) .

[000572] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.81 (d, J = 7.0 Hz, 3H); 0.83 (d, J = 7.0 Hz, 3H); 0.85 (s, 9H); 0.88 (d, J = 6.5 Hz, 3H); 0.99 (s, 3H); 1.04 (d, J = 7.1 Hz, 3H); 1.19 (m, 6H); 1.34 (d, J = 14.4 Hz, 1H); 1.83 (m, 3H); 1.98 (m, 2H); 2.26 (m, 1H); 2.29 (m, 2H); 2.60 (m, 1H); 2.68 (t, J = 7.6 Hz, 2H); 2.70 (dd, J = 11.2 and 14.3 Hz, 1H); 2.80 (s, 4H); 2.91 (dd, J = 2.3 and 7.6 Hz, 1H); 2.95 (dd, J = 3.7 and 14.3 Hz, 1H); 2.99 to 3.20 (m, 4H); 3.43 (m, 1H); 3.80 (s, 3H); 3.93 (d, J = 2.2 Hz, 1H); 4.05 to 4.25 (m, 4H); 5.01 (m, 2H); 5.03 (m, 1H); 5.89 (dd, J = 1.6 and 15.4 Hz, 1H); 6.44 (ddd, J = 4.6, 10.9 and 15.4 Hz, 1H); 7.02 (d, J = 8.5 Hz, 1H); 7.20 (m, 2H); 7.28 (d, J = 8.5 Hz, 2H); 7.31 (d, J = 2.3 Hz, 1H); 7.37 (d, J = 8.5 Hz, 2H); 7.86 (m, 3H); 7.91 (d, J = 7.1 Hz, 1H); 7.95 (d, J = 7.6 Hz, 1H); 8.39 (d, J = 7.3 Hz, 1H). LCMS (A5): ES m/z = 311; m/z = 1191 [M-H]-; m/z = 1193 [M+H]+; m/z = 1237 [M-H+HCO2H]-; tR = 1.26 minutes. Example 28: mAb-Ex27

[000573] Example 28 was prepared in a medium similar to examples 3, 7, 10 and 14. 1.56 mg of example 28 was obtained as a clear, colorless solution at a concentration of 0.78 mg/mL with a DAR of 4 (HRMS), a monomeric purity of 100% and an overall production of 13%.

[000574] SEC-HRMS: m/z = 149405 (naked mAb); m/z = 150486 (D1); m/z = 151568 (D2); m/z = 152645 (D3); m/z = 153725 (D4); m/z = 154802 (D5); m/z = 155882 (D6); m/z = 156961 (D7); m/z = 158039 (D8). Example Synthesis 29: Maleimido-mc-vc-PABA-3-(S)-neopentyl-7-(S)-Me-aza-C52 benzylic amine Compound 71: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-) (4-nitrophenyl)carbonate ila)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl

[000575] Compound 72 was prepared as described by Verma V.A., et al., in Bioorg Med Chem Lett 2015, 25, 864-868.

[000576] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.83 (d, J = 7.0 Hz, 3H); 0.87 (d, J = 7.0 Hz, 3H); 1.18 (m, 2H); 1.31 to 1.75 (m, 8H); 1.96 (m, 1H); 2.10 (m, 1H); 2.18 (m, 1H); 2.96 (m, 1H); 3.01 (m, 1H); 3.37 (t, J = 7.2 Hz, 2H); 4.19 (dd, J = 7.0 and 8.6 Hz, 1H); 4.38 (m, 1H); 5.24 (s, 2H); 5.42 (s, 2H); 5.98 (t, J = 6.0 Hz, 1H); 7.00 (s, 2H); 7.40 (d, J = 8.7 Hz, 2H); 7.57 (d, J = 9.2 Hz, 2H); 7.66 (d, J = 8.7 Hz, 2H); 7.81 (d, J = 8.6 Hz, 1H); 8.12 (d, J = 7.9 Hz, 1H); 8.31 (d, J = 9.2 Hz, 2H); 10.08 (s, 1H). LCMS (A5): ES m/z = 738 [M+H]+; m/z = 782 [M-H+HCO2H]-; tR = 1.1 minutes. Example 29: (4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6 ,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl )4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamido)-3-carbamate methyl-butanamido)-5-ureidopentanamido)benzyl

[000577] To Compound 71 (16.0 mg, 21.7 µmol) was added a solution of Example 25 (14.3 mg, 19.7 µmol) in DCM (2.5 mL) and DIEA (3.4 µL , 19.7 µmol). The reaction medium was stirred at room temperature overnight, then DMF (1 mL) was added and stirring was carried out at room temperature for 1 day. H2O (8 mL) was added to the reaction medium, stirring was carried out for 15 minutes, then the aqueous phase was extracted with DCM (3 x 10 mL). The combined organic phases were dried over MgSO4, filtered, concentrated in vacuo, co-evaporated twice with toluene and purified by flash chromatography on 5 g on silica gel (DCM/MeOH gradient elution) to give 15.8 mg of Example 29 as a white solid (60%).

[000578] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.81 (d, J = 7.0 Hz, 3H); 0.85 (d, J = 7.0 Hz, 3H); 0.86 (s, 9H); 0.89 (d, J = 6.8 Hz, 3H); 0.99 (s, 3H); 1.04 (d, J = 7.0 Hz, 3H); 1.18 (s, 3H); 1.19 (m, 2H); 1.31 to 1.75 (m, 9H); 1.80 (m, 1H); 1.96 (m, 2H) 2.10 (m, 1H); 2.18 (m, 1H); 2.27 (m, 1H); 2.61 (m, 1H); 2.69 (dd, J = 10.8 and 14.3 Hz, 1H); 2.90 (dd, J = 2.1 and 7.6 Hz, 1H); 2.96 (m, 2H); 3.01 (m, 1H); 3.37 (t, J = 7.2 Hz, 2H); 3.42 (m, 1H); 3.80 (s, 3H); 3.90 (d, J = 2.1 Hz, 1H); 4.08 (m, 1H); 4.11 (m, 1H); 4.20 (m, 3H); 4.39 (m, 1H); 4.97 (s, 2H); 5.04 (m, 1H); 5.40 (s, 2H); 5.89 (dd, J = 1.7 and 15.5 Hz, 1H); 5.97 (t, J = 6.2 Hz, 1H); 6.45 (ddd, J = 4.8, 10.7 and 15.5 Hz, 1H); 6.99 (s, 2H); 7.01 (d, J = 8.7 Hz, 1H); 7.20 (dd, J = 2.2 and 8.7 Hz, 1H); 7.21 to 7.30 (m, 6H); 7.31 (d, J = 2.2 Hz, 1H); 7.59 (d, J = 8.7 Hz, 2H); 7.78 (m, 2H); 7.85 (d, J = 8.5 Hz, 1H); 7.90 (d, J = 6.9 Hz, 1H); 8.07 (d, J = 7.8 Hz, 1H); 8.39 (d, J = 7.3 Hz, 1H); 9.97 (s, 1H). LCMS (A5): ES m/z = 662 [M+2H]2+; m/z = 1322 [MH]-; m/z = 1324 [M+H]+; m/z = 1368 [M-H+HCO2H]-; tR = 1.3 minutes. Synthesis of Examples 30 & 31: Triazolo-3-(S)-neopentyl-7-(S)-Me-aza-C52 non-cleavable NHS ester and corresponding ADC Compound 72: tert-butyl 3-(prop-2-in-1-yloxy)propanoate

[000579] To a solution of propargyl alcohol (1.362 mL, 23.4 mmol) in THF (23 mL) sodium (20.08 mg, 0.874 mmol) was added, the reaction medium was heated to 60°C until complete sodium solubilization, then cooled to room temperature before adding tert-butyl acrylate (2.286 mL, 15.6 mmol). The reaction medium was stirred at room temperature overnight, then H2O (25 mL) was added and the aqueous phase was extracted with EtOAc (3 x 25 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated in vacuo to provide 2 g of compound 72 as a colorless oil (70%). Compound 73: 3-(prop-2-yn-1-yloxy)propanoic acid

[000580] To a solution of compound 72 (2.0 g, 10.86 mmol) in DCM (50 mL) was added TFA (8.765 mL, 108.6 mmol). The reaction medium was stirred at room temperature overnight, concentrated in vacuum, co-evaporated with toluene and purified by flash chromatography on 80 g of silica gel (DCM/MeOH gradient elution) to provide 1.1 g of compound 73 as an oil (80%).

[000581] 1H NMR (400 MHz, d in ppm, DMSO-d6): 2.46 (t, J = 6.4 Hz, 2H); 3.43 (t, J = 2.4 Hz, 1H); 3.63 (t, J = 6.4 Hz, 2H); 4.11 (d,J = 2.4 Hz, 2H); 12.20 (broad, 1H). Compound 74: acid 3-((1-(4-((2R,3R)-3-((S)-1-((3S,7S,10R, 16S,E)-10-(3-chloro-4 -methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl) oxiran-2-yl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)propanoic acid

[000582] To a solution of Example 24 (50 mg, 66.6 µmol) in THF (0.5 mL) was added Compound 73 (17.05 mg, 113.1 µmol), aqueous copper (II) sulfate to 0.1M (266 µL, 26.6 µmol) and 0.2M aqueous sodium ascorbate (266 µL, 53.2 µmol). The reaction medium was stirred at room temperature for 1 day, then H2O (1 mL) was added and the aqueous phase was extracted with EtOAc (3 x 1 mL). The combined organic phases were washed with saturated brine, dried over MgSO4, filtered, concentrated in vacuo and purified by Sephadex LH20 filtration (DCM elution) to provide 40 mg of an oil which was also purified by flash chromatography on 5 g of silica gel (DCM/MeOH gradient elution) to provide 20 mg of compound 74 (35%).

[000583] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.82 (s, 9H); 0.88 (d, J = 6.7 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J = 7.1 Hz, 3H); 1.18 (s, 3H); 1.31 (d, J = 14.6 Hz, 1H); 1.80 (m, 1H); 1.97 (dd, J = 10.0 and 14.6 Hz, 1H); 2.25 (m, 1H); 2.42 (t, J = 6.6 Hz, 2H); 2.60 (m, 1H); 2.70 (dd, J = 10.9 and 14.4 Hz, 1H); 2.90 (dd, J = 2.2 and 7.6 Hz, 1H); 2.95 (dd, J = 3.6 and 14.4 Hz, 1H); 3.44 (m, 1H); 3.62 (t, J = 6.6 2H); 3.80 (s, 3H); 3.94 (d, J = 2.2 Hz, 1H); 4.08 (m, 2H); 4.49 (s, 2H); 5.03 (m, 1H); 5.59 (s, 2H); 5.89 (dd, J = 1.7 and 15.3 Hz, 1H); 6.43 (ddd, J = 4.8, 10.8 and 15.3 Hz, 1H); 7.02 (d, J = 8.7 Hz, 1H); 7.20 (dd, J = 2.4 and 8.7 Hz, 1H); 7.29 (d, J = 8.4 Hz, 2H); 7.32 (d, J = 2.4 Hz, 1H); 7.35 (d, J = 8.4 Hz, 2H); 7.85 (d, J = 8.4 Hz, 1H); 7.90 (d, J = 7.0 Hz, 1H); 8.13 (s, 1H); 8.46 (m, 1H); 12.20 (broad, 1H). LCMS (A5): ES m/z = 440 [M+2H]2+; m/z = 877 [M-H]-; m/z = 879 [M+H]+; tR = 1.24 minutes. Example 30: 3-((1-(4-((2R,3R)-3-((S)-1- ((3S,7S,10R,16S,E)-10-(3-chloro-4- methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triaza-cyclohexadec-13-en-16-yl)ethyl 2,5-dioxopyrrolidin-1-yl)oxiran-2-yl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)propanoate

[000584] To a solution of Compound 74 (20 mg, 22.7 µmol) in DCM (5 mL) were added DIEA (5.0 µL, 28.5 µmol) and DSC (6.34 mg, 23.8 µmol ). The reaction medium was stirred at room temperature for 3 hours, then DSC (3 mg, 11.3 µmol) and DIEA (5 µL, 28.5 µmol) were added and stirring was carried out at room temperature for 2 hours. The reaction medium was then concentrated in vacuo and purified by two successive flash chromatographies on 1.5 g of diol-modified silica gel (DCM/iPrOH gradient elution) to provide 11.5 mg of Example 30 (50 %).

[000585] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.83 (s, 9H); 0.87 (d, J = 6.6 Hz, 3H); 0.99 (s, 3H); 1.03 (d, J = 7.1 Hz, 3H); 1.04 (d, J = 6.6 Hz, 3H); 1.19 (s, 3H); 1.32 (d, J = 14.5 Hz, 1H); 1.81 (m, 1H); 1.96 (dd, J = 10.2 and 14.5 Hz, 1H); 2.25 (m, 1H); 2.60 (m, 1H); 2.70 (dd, J = 11.2 and 14.5 Hz, 1H); 2.80 (s, 4H); 2.90 (dd, J = 2.0 and 7.4 Hz, 1H); 2.94 (t, J = 6.1 Hz, 2H); 2.97 (dd, J = 3.6 and 14.1 Hz, 1H); 3.42 (m, 1H); 3.74 (t, J = 6.1 Hz, 2H); 3.80 (s, 3H); 3.92 (d, J = 2.0 Hz, 1H); 4.09 (m, 2H); 4.55 (s, 2H); 5.03 (m, 1H); 5.59 (s, 2H); 5.89 (dd, J = 1.6 and 15.2 Hz, 1H); 6.44 (ddd, J = 4.7, 10.6 and 15.2 Hz, 1H); 7.02 (d, J = 8.6 Hz, 1H); 7.20 (dd, J = 2.4 and 8.6 Hz, 1H); 7.29 (d, J = 8.3 Hz, 2H); 7.32 (m, 3H); 7.86 (d, J = 8.4 Hz, 1H); 7.90 (d, J = 6.9 Hz, 1H); 8.14 (s, 1H); 8.39 (d, J = 7.5 Hz, 1H). LCMS (A5): ES m/z = 120; m/z = 974 [M-H]-; m/z = 976 [M+H]+; m/z = 1020 [M-H+HCO2H]-; tR = 1.29 minutes. Example 31: mAb-Ex.30

[000586] Example 31 was prepared in a medium similar to examples 3, 7, 10 and 14. 5.4 mg of Example 31 was obtained as a clear and colorless solution at a concentration of 1.8 mg/mL with a DAR of 4 (HRMS), a monomeric purity of 98.1% and an overall production of 45%.

[000587] SEC-HRMS: m/z = 149399 (naked mAb); m/z = 150245 (D1); m/z = 151101 (D2); m/z = 151965 (D3); m/z = 152831 (D4); m/z = 153679 (D5); m/z = 154546 (D6); m/z = 155408 (D7); m/z = 156273 (D8); m/z = 157284(D9). Example Synthesis 32: 3-(S)-neopentyl-6-Me-6-CH2OH-aza-C52 benzylic amine 32 Compound 75: (2R,3S)-1-((4-methoxybenzyl)oxy)-2-methylex-5-en-3-yl (2S)-2-((4-((R)-2-acrylamido -3-(3-chloro-4-methoxyphenyl)propanamido)-3-(hydroxymethyl)-3-methyl-2-oxobutyl)amino)-4,4-dimethylpentanoate

[000588] To a solution of compound BC6 (385.6 mg, 773.5 µmol) in DMF (12 mL) were added HATU (348.7 mg, 889.5 µmol) and HOAt (122.3 mg, 889. 5 µmol). The reaction medium was stirred at room temperature for 30 minutes, then a solution of compound AD3 (292 mg, 773.5 µmol) in DMF (5 mL) and DIEA (475.2 µL, 2.71 mmol) were added. and stirring was carried out at room temperature for 4 hours. H2O (50 mL) was added and the aqueous phase was extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with H2O (15 mL), saturated brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 30 g of silica gel (DCM/MeOH gradient elution ) to provide 383 mg of Compound 75 as a colorless oil (65%).

[000589] 1H NMR (500 MHz, d in ppm, DMSO-d6): 55/45 diastereoisomer mixture; 0.85 to 0.88 (m, 12H); 0.91 (s, 1.65H); 0.92 (s, 1.35H); 1.56 (m, 1H); 1.60 (m, 1H); 1.98 (m, 1H); 2.21 (m, 1H); 2.30 (m, 1H); 2.71 (m, 1H); 2.92 (m, 1H); 3.19 to 3.45 (m, 6H); 3.73 (s, 3H); 3.80 (s, 3H); 4.29 (m, 1H); 4.33 (m, 2H); 4.58 (m, 1H); 4.82 (m, 1H); 4.95 to 5.09 (m, 3H); 5.55 (dd, J = 2.2 and 10.2 Hz, 1H); 5.71 (m, 1H); 6.01 (dd, J = 2.2 and 17.2 Hz, 1H); 6.23 (dd, J = 10.2 and 17.2 Hz, 1H); 6.89 (m, 2H); 7.01 (d, J = 8.6 Hz, 1H); 7.17 (dd, J = 2.2 and 8.6 Hz, 1H); 7.21 (m, 2H); 7.34 (d, J = 2.2 Hz, 1H); 7.82 (d, J = 8.7 Hz, 0.45H); 7.84 (d, J = 8.7 Hz, 0.55H); 7.98 (t, J = 6.5 Hz, 0.45H); 8.70 (t, J = 6.5 Hz, 0.55H); 8.39 (d, J = 8.6 Hz, 1H). LCMS (A5): ES m/z = 756 [M-H]-; m/z = 758 [M+H]+; m/z = 802 [M-H+HCO2H]-; tR = 1.54 to 1.56 minutes. Compound 76: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6-(hydroxymethyl)-16-((R)-1-((4-methoxybenzyl)oxy)propan- 2-yl)-6-methyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000590] To a solution of Compound 75 (380 mg, 501.1 µmol) in DCM (38 mL) under Ar was added Grubbs catalyst I (20.97 mg, 25.05 µmol). The reaction medium was stirred under Ar at room temperature for 4 hours, then Grubbs catalyst I (20.97 mg, 25.05 µmol) was added. Stirring was carried out at room temperature for 3 hours, then Grubbs catalyst I (20.97 mg, 25.05 µmol) was added and stirring was carried out at room temperature. After 24 hours of reaction, Grubbs I catalyst (20.97 mg, 25.05 µmol) was added and stirring was carried out for 4 hours. The reaction medium was concentrated in vacuo and purified by flash chromatography on 20 g of silica gel (heptane/EtOAc isocratic elution) to provide 170 mg of stereoisomer 1 of Compound 76 (56%) and 138 mg of stereoisomer 2 of compound 76 ( 37%). Stereoisomer 1 of Compound 76

[000591] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.83 (s, 9H); 0.91 (d, J = 7.1 Hz, 3H); 1.02 (s, 3H); 1.30 (dd, J = 2.4 and 14.6 Hz, 1H); 1.53 (dd, J = 9.9 and 14.6 Hz, 1H); 1.99 (m, 1H); 2.28 (m, 1H); 2.45 (m, 1H); 2.67 (m, 1H); 2.91 (dd, J = 3.0 and 13.7 Hz, 1H); 2.97 (dd, J = 4.0 and 14.5 Hz, 1H); 3.17 to 3.49 (m, 5H); 3.72 (s, 3H); 3.80 (s, 3H); 4.18 (m, 1H); 4.37 (m, 2H); 4.50 (td, J = 2.4 and 9.6 Hz, 1H); 5.00 (m, 3H); 5.82 (dd, J = 1.9 and 15.4 Hz, 1H); 6.39 (ddd, J = 4.2, 11.6 and 15.4 Hz, 1H); 6.90 (d, J = 8.7 Hz, 2H); 7.04 (d, J = 8.6 Hz, 1H); 7.12 (m, 1H); 7.15 (dd, J = 2.3 and 8.6 Hz, 1H); 7.21 (d, J = 8.7 Hz, 2H); 7.28 (d, J = 2.3 Hz, 1H); 8.79 (d, J = 9.6 Hz, 1H); 8.38 (d, J = 8.7 Hz, 1H). LCMS (A4): ES m/z = 728 [M-H]-; m/z = 730 [M+H]+; m/z = 774; tR = 5.15 minutes.

Compound 76 stereoisomer 2

[000592] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.83 (s, 9H); 0.88 (s, 3H); 0.91 (d, J = 7.1 Hz, 3H); 1.28 (dd, J = 2.4 and 14.6 Hz, 1H); 1.67 (dd, J = 9.9 and 14.6 Hz, 1H); 1.99 (m, 1H); 2.28 (m, 1H); 2.45 (m, 1H); 2.67 (m, 1H); 3.02 (dd, J = 3.3 and 14.2 Hz, 1H); 3.08 (dd, J = 1.9 and 13.5 Hz, 1H); 3.25 to 3.42 (m, 4H); 3.55 (dd, J = 5.1 and 11.0 Hz, 1H); 3.73 (s, 3H); 3.81 (s, 3H); 4.17 (m, 1H); 4.37 (m, 2H); 4.44 (td, J = 2.1 and 9.3 Hz, 1H); 4.80 (t, J = 5.1 Hz, 1H); 5.00 (m, 2H); 5.86 (dd, J = 1.6 and 15.1 Hz, 1H); 6.38 (ddd, J = 4.0, 11.4 and 15.3 Hz, 1H); 6.90 (d, J = 8.8 Hz, 2H); 7.04 (d, J = 8.7 Hz, 1H); 7.19 (dd, J = 2.2 and 8.7 Hz, 1H); 7.22 (d, J = 8.8 Hz, 2H); 7.30 (d, J = 2.2 Hz, 1H); 7.42 (d, J = 10.4 Hz, 1H); 7.92 (d, J = 8.9 Hz, 1H); 8.45 (d, J = 8.7 Hz, 1H). LCMS (A4): ES m/z = 728 [M-H]-; m/z = 730 [M+H]+; m/z = 774; tR = 4.79 minutes. Compound 77: (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)- 16-((R)-1-((4-methoxy-benzyl)oxy)propan-2-yl) -6-methyl-3-neopentyl-6- (((triethylsilyl)oxy)methyl)-1-oxa-4,8,11-triaza-cyclohexadec-13-ene- 2,5,9,12-tetraone

[000593] To a solution of stereoisomer 1 of compound 76 (170 mg, 232.8 µmol) in DCM (3 mL) cooled with an ice bath was added at 4°C chlorotriethylsilane (159.3 µL, 931.1 µmol) and , dropwise, TEA (131.1 µL, 931.1 µmol). The reaction medium was stirred at 4°C for 30 minutes, then at room temperature for 20 hours. The reaction medium was diluted with DCM (50 mL) and brine (10 mL) and stirring was carried out for 10 minutes. The organic phase was washed with saturated brine (2 x 10 mL), dried over MgSO4, filtered and concentrated in vacuo. The other diastereoisomer of Compound 76 (138 mg, 189.0 µmol) was similarly protected, both batches were mixed and purified by flash chromatography on 20 g of silica gel (heptane/EtOAc isocratic elution) to give 139 mg of Compound 77 stereoisomer 1 as a colorless foam (39%) and 107 mg of stereoisomer 2 of Compound 77 as a colorless foam (30%).

Compound 77 stereoisomer 1

[000594] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.62 (q, J = 8.1 Hz, 6H); 0.83 (s, 9H); 0.91 (d, J = 7.1 Hz, 3H); 0.96 (t, J = 8.1 Hz, 9H); 1.04 (s, 3H); 1.66 (dd, J = 3.4 and 14.4 Hz, 1H); 1.43 (dd, J = 9.3 and 14.4 Hz, 1H); 1.99 (m, 1H); 2.28 (m, 1H); 2.47 (m, 1H); 2.68 (m, 1H); 2.92 (dd, J = 3.0 and 13.5 Hz, 1H); 2.99 (dd, J = 4.0 and 14.8 Hz, 1H); 3.21 to 3.40 (m, 3H); 3.48 (m, 1H); 3.63 (d, J = 10.3 Hz, 1H); 3.73 (s, 3H); 3.81 (s, 3H); 4.17 (m, 1H); 4.36 (m, 2H); 4.51 (td, J = 2.9 and 9.4 Hz, 1H); 5.02 (m, 1H); 5.82 (dd, J = 1.8 and 15.4 Hz, 1H); 6.39 (ddd, J = 4.3, 11.6 and 15.4 Hz, 1H); 6.89 (d, J = 8.7 Hz, 2H); 7.02 (m, 1H); 7.04 (d, J = 8.7 Hz, 1H); 7.15 (dd, J = 2.3 and 8.7 Hz, 1H); 7.21 (d, J = 8.7 Hz, 2H); 7.29 (d, J = 2.3 Hz, 1H); 7.96 (d, J = 9.4 Hz, 1H); 8.31 (d, J = 8.7 Hz, 1H). LCMS (A4): ES m/z = 842 [M-H]-; m/z = 844 [M+H]+; m/z = 888 [M-H+HCO2H]-; tR = 7.08 minutes.

Compound 77 stereoisomer 2

[000595] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.52 (q, J = 8.1 Hz, 6H) 0.84 (s, 9H); 0.88 to 0.93 (m, 15H); 1.29 (dd, J = 2.6 and 14.5 Hz, 1H); 1.68 (dd, J = 10.1 and 14.5 Hz, 1H); 1.99 (m, 1H); 2.28 (m, 1H); 2.47 (m, 1H); 2.68 (m, 1H); 3.01 (dd, J = 3.4 and 14.5 Hz, 1H); 3.15 (d, J = 12.8 Hz, 1H); 3.33 (m, 1H); 3.48 (d, J = 10.4 Hz, 1H); 3.73 (s, 3H); 3.81 (s, 3H); 3.83 (d, J = 10.4 Hz, 1H); 4.17 (m, 1H); 4.36 (m, 2H); 4.45 (td, J = 2.4 and 9.2 Hz, 1H); 5.01 (m, 1H); 5.84 (dd, J = 1.8 and 15.3 Hz, 1H); 6.39 (ddd, J = 3.9, 11.4 and 15.3 Hz, 1H); 6.90 (d, J = 8.8 Hz, 2H); 7.05 (d, J = 8.6 Hz, 1H); 7.19 (dd, J = 2.2 and 8.6 Hz, 1H); 7.22 (d, J = 8.8 Hz, 2H); 7.30 (d, J = 2.2 Hz, 1H); 7.47 (d, J = 10.4 Hz, 1H); 8.71 (d, J = 9.2 Hz, 1H); 8.41 (d, J = 8.7 Hz, 1H). LCMS (A4): ES m/z = 842 [M-H]-; m/z = 844 [M+H]+; m/z = 888 [M-H+HCO2H]-; tR = 6.88 minutes. Compound 78: 3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16- ((R)-1-hydroxypropan-2-yl)-6-methyl-3-neopentyl-6- (((triethylsilyl)oxy)methyl)-1- oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone

[000596] To a solution of both diastereoisomers of compound 77 (245 mg, 290.1 µmol) in DCM (10 mL) and H2O (2.5 mL) cooled with an ice/acetone bath was added DDQ (339, 4 mg, 1.45 mmol) and 2,6-di-tert-butylpyridine (171.7 mg, 870.3 µmol). The reaction medium was stirred at 0°C for 2 hours, then it was diluted with aqueous NaHCO3 (10 mL) and DCM (10 mL) and stirring was carried out for 10 minutes. The aqueous phase was extracted with DCM (3 x 20 mL), the combined organic phases were washed with saturated brine (3 x 5 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 20 g of silica gel (DCM/MeOH gradient elution) to provide 155 mg of compound 78 as a white foam (74%).

[000597] 1H NMR (400 MHz, d in ppm, DMSO-d6): 0.49 to 0.65 (m, 6H); 0.80 to 1.07 (m, 24H); 1.35 to 1.71 (m, 2H); 1.83 (m, 1H); 2.28 (m, 1H); 2.47 (m, 1H; 2.68 (m, 1H); 2.90 to 3.50 (m, 5.96H); 3.65 (d, J = 10.6 Hz, 0.52H); 3.80 (s, 3H); 3.82 (d, J = 10.6 Hz, 0.52H); 4.18 (m, 1H); 4.29 to 4.70 (m, 2H); 5 .02 (m, 1H); 5.80 to 5.89 (m, 1H); 6.05 (m, 0.11H); 6.39 (m, 0.89H); 6.98 to 7.31 (m, 3.48H); 7.47 (d, J = 10.6 Hz, 0.52H); 7.87 (d, J = 9.4 Hz, 0.11H); 7.96 (d, J = 9.4 Hz, 0.52H); 8.70 (d, J = 9.4 Hz, 0.37H); 8.31 (d, J = 8.7 Hz, 0.63H); 8, 42 (d, J = 8.4 Hz, 0.37H). LCMS (A4): ES m/z = 722 [M-H]-; m/z = 724 [M+H]+; m/z = 768 [ M-H+HCO2H]- ; tR = 5.68 to 5.78 minutes. Compound 79: 2S)-2-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)- 6-methyl-3-neopentyl-2,5,9,12-tetraoxo-6- (((triethylsilyl)oxy)methyl)-1-oxa-4,8,11-triazacyclohexadec-13-en-16- il)propanal

[000598] To a solution of compound 78 (154 mg, 212.6 µmol) in DCM (1.5 mL) cooled with an ice/acetone bath was added at 0°C a solution of KBr (25.6 mg, 212.6 µmol) in H2O (612 µL), a solution of TEMPO at 5 mg/mL in DCM (137 µL, 4.25 µmol) and, drop by drop, an aqueous solution of sodium hypochlorite at 1.56M pH 9.5 (204 µL, 318.9 µmol). Stirring was carried out at 0°C for 15 minutes, then saturated Na2S2O3 at 0°C (1.9 mL) was added, the ice bath was removed and the reaction medium was stirred for 10 minutes. It was then extracted with DCM (3 x 20 mL), the combined organic phases were washed with H2O (2 x 10 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 15 g of silica gel (elution by DCM/MeOH gradient) to provide 36 mg of compound 61 (23%).

[000599] 1H NMR (400 MHz, d in ppm, DMSO-d6): 72/28 diastereoisomer mixture: 0.49 to 0.65 (m, 6H); 0.80 to 1.08 (m, 24H); 1.32 to 1.69 (m, 2H); 2.40 (m, 1H); 2.68 (m, 2H); 2.79 (m, 1H); 2.89 to 3.70 (m, 5H); 3.80 (s, 3H); 4.10 to 4.53 (m, 2H); 5.02 (m, 0.28H); 5.28 (m, 0.72H); 5.78 to 5.91 (m, 1H); 6.07 (m, 0.28H); 6.40 (m, 0.72H); 7.00 to 7.51 (m, 4H); 7.90 (d, J = 9.4 Hz, 0.28H); 7.98 (d, J = 9.4 Hz, 0.72H); 8.34 (m, 1H); 9.59 (d, J = 2.5 Hz, 0.28H); 9.63 (d, J = 1.8 Hz, 0.72H). LCMS (A5): m/z = 722 [M+H]+; tR = 1.71 minutes. Compound 80: 1-(azidomethyl)-4-(bromomethyl)benzene

[000600] To a solution of p-xylene dibromide (2.0 g, 7.2 mmol) in DMF (20 mL) was added over 20 minutes a solution of sodium azide (584.9 mg, 8.64 mmol ) in DMF (20 mL). The reaction medium was stirred at room temperature for 20 hours, then poured onto ice (100 g) and extracted with EtOAc (3 x 50 mL). The combined organic phase was washed with saturated brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo, coevaporated with toluene and purified by flash chromatography on 70 g of silica gel (Et2O elution) to give 773 mg of Compound 80 as a colorless oil (47%).

[000601] 1H NMR (400 MHz, d in ppm, DMSO-d6): 4.45 (s, 2H); 4.71 (s, 2H); 7.35 (d, J = 8.5 Hz, 2H); 7.47 (d, J = 8.5 Hz, 2H). Compound 81: (1R,4S,5R,6S)-6-(4-(azidomethyl)benzyl)-4,7,7-trimethyl-6-thiabicyl [3,2,1]octan-6-ium trifluoromethanesulfonate

[000602] Ao (1R,4R,5R)-4,7,7-trimethyl-6-thiabicil [3,2,1]octane (CAS number [5718-75-2], 580.1 mg, 3, 41 mmol) a solution of compound 80 (770 mg, 3.41 mmol) in DCM (2.2 mL) was added. A solution of lithium trifluoromethanesulfonate (2.71 g, 17.03 mmol) in 2 mL H2O) was then added dropwise and the reaction medium was stirred at room temperature for 2 days. H2O (15 mL) and DCM (15 mL) were then added to the reaction medium and stirring was carried out at room temperature for 10 minutes. The aqueous phase was extracted with DCM (3 x 15 mL), the combined organic phases were washed with saturated brine (3 x 8 mL), dried over MgSO4, filtered and concentrated in vacuo. The pale yellow oil was triturated in iPr2O (5 mL), the solid thus obtained was filtered, washed with iPr2O (2 x 5 mL) and dried under vacuum to provide 1.266 g of compound 81 as a white solid (79%).

[000603] 1H NMR (400 MHz, d in ppm, DMSO-d6): 1.08 (d, J = 7.2 Hz, 3H); 1.45 (m, 1H); 1.55 to 1.80 (m, 3H); 1.69 (s, 3H); 1.75 (s, 3H); 1.99 (m, 1H); 2.40 (d, J = 13.8 Hz, 1H); 2.45 (m, 1H); 2.59 (dm, J = 13.8 Hz, 1H); 3.88 (t, J = 4.6 Hz, 1H); 4.52 (s, 2H); 4.60 (d, J = 12.8 Hz, 1H); 4.90 (d, J = 12.8 Hz, 1H); 7.47 (d, J = 8.5 Hz, 2H); 7.61 (d, J = 8.5 Hz, 2H). Compound 82: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(azidomethyl)phenyl)oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-6-methyl-3-neopentyl-6-(((triethylsilyl)oxy)methyl)-1-oxa-4,8,11-triazacyclohexadec-13-ene-2, 5,9,12-tetraone

[000604] To a solution of compounds 79 (35 mg, 48.5 µmol) and 81 (24.8 mg, 53.3 µmol) in DCM (1.5 mL) cooled to -70°C was added dropwise BEMP (CAS number [98015-45-3], 18.6 µL, 63.0 µmol). The reaction mixture was stirred at -70°C for 2 hours and 30 minutes, saturated brine (1.5 ml) was added to the reaction mixture, the bath removed and stirring carried out vigorously until RT. The aqueous phase was extracted with DCM (3 x 10 mL), the combined organic phases were dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 5 g of silica gel (DCM/MeOH gradient elution) to give 10.2 mg of Compound 82 as a colorless lacquer (24%).

[000605] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.61 (q, J = 8.1 Hz, 6H); 0.80 (s, 9H); 0.98 (t, J = 8.1 Hz, 9H); 1.02 (s, 3H); 1.03 (d, J = 7.1 Hz, 3H); 1.34 (dd, J = 2.5 and 14.4 Hz, 1H); 1.44 (dd, J = 10.0 and 14.4 Hz, 1H); 1.85 (m, 1H); 2.27 (m, 1H); 2.62 (m, 2H); 2.88 (dd, J = 2.6 and 13.5 Hz, 1H); 2.97 (dd, J = 3.7 and 14.2 Hz, 1H); 3.00 (dd, J = 2.3 and 7.3 Hz, 1H); 3.35 (m, 1H); 3.48 (dd, J = 9.5 and 14.2 Hz, 1H); 3.61 (d, J = 10.3 Hz, 1H); 3.81 (s, 3H); 3.92 (d, J = 2.3 Hz, 1H); 4.15 (m, 1H); 4.46 (s, 2H); 4.48 (td, J = 2.5 and 9.7 Hz, 1H); 5.10 (m, 1H); 5.78 (dd, J = 2.2 and 15.3 Hz, 1H); 6.40 (ddd, J = 4.3, 11.6 and 15.3 Hz, 1H); 7.04 (m, 2H); 7.14 (dd, J = 2.3 and 8.6 Hz, 1H); 7.28 (d, J = 2.3 Hz, 1H); 7.33 (d, J = 8.4 Hz, 2H); 7.39 (d, J = 8.4 Hz, 2H); 7.96 (d, J = 9.7 Hz, 1H); 8.28 (d, J = 8.7 Hz, 1H). LCMS (A5): ES m/z = 120; m/z = 865 [M-H]-; m/z = 867 [M+H]+; m/z = 911 [M-H+HCO2H]-; tR = 1.87 minutes. Example 32: (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10- (3-chloro-4-methoxybenzyl)-6-methyl-3-neopentyl-6-(((triethylsilyl)oxy)methyl)-1-oxa-4,8,11-triazacyclohexadec-13-ene-2, 5,9,12-tetraone

[000606] To a solution of Compound 82 (9.4 mg, 10.84 µmol) in DCM (1.3 mL) and MeOH (0.7 mL) was added a solution of TCEP (3.45 mg, 11. 9 µmol) in H2O (0.7 mL). The reaction medium was stirred at room temperature overnight, then aqueous NaHCO3 (2.5 mL) was added and stirring was carried out for 10 minutes. The aqueous phase was extracted with DCM (3 x 10 mL), the combined organic phases were dried over MgSO4, filtered, concentrated in vacuo and purified by slash chromatography on 5 g of silica gel (DCM/MeOH/H2O gradient elution). to provide 4.55 mg of example 32 as a white lacquer (57%).

[000607] 1H NMR (500 MHz, d in ppm, DMSO-d6): 0.82 (s, 9H); 1.01 (s, 3H); 1.03 (d, J = 7.1 Hz, 3H); 1.33 (dd, J = 2.3 and 14.5 Hz, 1H); 1.56 (dd, J = 10.1 and 14.5 Hz, 1H); 1.82 (m, 1H); 2.27 (m, 1H); 2.64 (m, 2H); 2.88 (dd, J = 2.6 and 13.5 Hz, 1H); 2.92 (dd, J = 2.2 and 7.3 Hz, 1H); 2.96 (dd, J = 4.0 and 11.2 Hz, 1H); 3.19 (dd, J = 4.3 and 10.9 Hz, 1H); 3.41 (m, 2H); 3.74 (s, 2H); 3.81 (s, 3H); 3.88 (d, J = 2.2 Hz, 1H); 4.18 (m, 1H); 4.47 (td, J = 2.1 and 9.4 Hz, 1H); 4.91 (dd, J = 4.2 and 6.2 Hz, 1H); 5.08 (m, 1H); 5.76 (dd, J = 2.3 and 15.5 Hz, 1H); 6.40 (ddd, J = 3.7, 11.5 and 15.5 Hz, 1H); 7.05 (d, J = 8.8 Hz, 1H); 7.11 (m, 1H); 7.14 (dd, J = 2.4 and 8.8 Hz, 1H); 7.22 (d, J = 8.3 Hz, 2H); 7.25 (d, J = 2.4 Hz, 1H); 7.35 (d, J = 8.3 Hz, 2H); 8.78 (d, J = 9.4 Hz, 1H); 8.31 (d, J = 8.7 Hz, 1H). LCMS (A5): ES m/z = 725 [M-H]-; m/z = 727 [M+H]+; tR = 0.79 minute.

Pharmacological Results

[000608] The compounds of the invention were subjected to pharmacological tests to determine their antitumor effect. The ADC of formula (III) was also evaluated in terms of plasma stability.

[000609] For illustrative purposes, two conjugates, ADC1 and ADC2 described below, derived from WO2011/001052 were also tested.

Assessment of inhibition of proliferation of HCT116 and MDA-MB-231 cell lines by cryptophycin compounds of formula (I)

[000610] HCT116 and MDA-MB-231 cells in their exponential growth phase were trypsinized and resuspended in their respective culture medium (DMEM/F12 Gibco #21331, 10% FCS Gibco #10500-056, 2 nM Gibco glutamine #25030 for MDA-MB-231 cells; DMEM Gibco #11960, 10% FCS Gibco #10500-056, 2 mM glutamine Gibco #25030 for HCT116 cells). The suspension was seeded into Cytostar 96-well culture plates (GE Healthcare Europe, #RPNQ0163) in complete culture medium containing serum at a density of 5000 cells/well. After incubation for 4 hours, successive dilutions of the cryptophycin compound were added to the wells in concentrations decreasing from 10-7 to 10-12 M (in triplicate for each concentration). The cells were cultured at 37°C in an atmosphere containing 5% CO2 in the presence of the cryptophycin compound for 3 days. On the 4th day, 10 µl of a 14C-thymidine solution (0.1 µCi/well, Perkin Elmer #NEC56825000) was added to each well. 14C-thymidine incorporation was measured 96 hours after the start of the experiment with a microbeta radioactivity counter (Perkin Elmer). Data were expressed in the form of a percentage of survival by determining the relationship between the reduced count obtained with cells treated with the cryptophycin compound and the count obtained with cells from control wells (treated with the culture medium alone).

[000611] The inhibitory activity is provided by the concentration that inhibits 50% of the activity. 1 Cryptophycin compounds of formula (I) were tested as pure ß epoxides or as a mixture of a and ß epoxides as specified in the column entitled "structural comment". It is known from the literature that ß epoxides are generally 50 to 100 times more potent than a epoxide (see, for example, Al-Awar RS, et al., J Med Chem 2003, 46, 2985-3007).

[000612] The cryptophycin compounds of formula (I) were found to inhibit the proliferation of HCT116 and MDA-MB-231 cell lines with the IC50 ranging from 50 pM to 2.815 nM. Assessment of proliferation inhibition of the MDA-MB- cell line

231 by ADC of formula (III)

[000613] MDA-MB-231 cells in their exponential growth phase were trypsinized and resuspended in their culture medium (DMEM/F12 Gibco #21331, 10% FCS Gibco #10500-056, 2 nM glutamine Gibco #25030 ). The cell suspension was seeded into Cytostar 96-well culture plates (GE Healthcare Europe, #RPNQ0163) in serum-containing complete culture medium at a density of 5000 cells/well. After incubation for 4 hours, successive dilutions of the ADC are added to the wells at concentrations decreasing from 10-7 to 10-12 M (in triplicate for each concentration). Cells were cultured at 37°C in an atmosphere containing 5% CO2 in the presence of ADC for 3 days. On the 4th day, 10 µl of a 14C-thymidine solution (0.1 µCi/well, Perkin Elmer #NEC56825000) was added to each well. 14C-thymidine incorporation was measured 96 hours after the start of the experiment with a microbeta radioactivity counter (Perkin Elmer). Data were expressed in the form of a percentage of survival by determining the relationship between the reduced cell count obtained with cells treated with the ADC and the cell count obtained with control cells (treated with the culture medium alone). In certain experiments, naked antibody was added to the wells at a concentration of 1 µM at the beginning of the experiment and proliferation inhibition was measured as previously described.

[000614] Cryptophycin ADC of formula (III), as well as ADC1 and ADC2, were found to inhibit the proliferation of MDA-MB-231 cell line with IC50 ranging from 20 pM to 130 pM and selectivity ratio of ADC alone vs ADC + naked antibody between 185 and 500. Determination of the MTD of the ADC of formula (III) after single i.v. administration in SCID mice

[000615] DMT was determined as the maximum dose that does not induce 15% body weight loss over 3 consecutive days for an individual mouse or 20% body weight loss over 1 day or mortality. It was evaluated following a single intravenous (iv) bolus injection into 3 female SCID mice and over a period of 28 days post-treatment.

[000616] Cryptophycin ADC of formula (III) exhibited DMT in SCID mice ranging from 20 mg/kg to > 50 mg/kg.

Assessment of in vivo antitumor activity of ADC of formula (III) against MDA-MB-231 in SCID mice after single i.v. administration

[000617] In vivo antitumor activity was evaluated at 3 dose levels against measurable breast MDA-MB-231 xenografts implanted s.c. into female SCID mice. Control groups were left untreated. Conjugates were administered by a single i.v. bolus injection, the day of treatment was indicated on each graph by an arrow (?).

[000618] To evaluate the antitumor activity of the conjugates, the animals were weighed twice weekly and the tumors were measured twice weekly by caliper. Animal body weights included tumor weights. Tumor volume was calculated using the formula mass (mm3) = [length (mm) x width (mm)2]/2. The primary efficacy endpoints were ?T/?C, mean regression percentage, partial and complete regressions (PR and CR). Changes in tumor volume for each treated (T) and control (C) were calculated for each tumor by subtracting the tumor volume on the first treatment (preparation day) from the tumor volume on the specified observation day. The mean ?T was calculated for the treated group and the mean ?C was calculated for the control group. Then, the ?T/?C ratio was calculated and expressed as a percentage: ?T/?C = (delta T/delta C)x100.

[000619] The percentage of tumor regression was defined as the % decrease in tumor volume in the treated group on a specified observation day (t) compared to its volume on the first day of the first treatment (t0). At a specific time point and for each animal, % regression was calculated. The average % regression was then calculated for the group. Regression % (at) = ((Volume - Volumet)/Volume)x1QQ. Regressions were defined as partial (PR) when the tumor volume decreased to 5Q % of the tumor volume at the start of treatment and complete (CR) when the tumor volume could not be measured (Q mm3). Tumor-free survival (TFS) was defined as animals with undetectable tumors at study termination (>100 days after last treatment). Assessment of in vivo antitumor activity of Ex.3 against MDA-MB-231 in SCID mice after single iv administration

[000620] These results showed that all tested examples of the invention, as well as ADC2, exhibited antitumor activity at doses ranging from 0.625 mg/kg to 5 mg/kg.

Determination of PK parameters of Ex.3 and Ex.7 after single i.v. administration in SCID mice (5 mg/kg)

[000621] The in vivo pharmacokinetics of ADC and total antibody were evaluated in female SCID mice after administration of the conjugate by a simple iv bolus injection. Female SCID mice (5-6 weeks old, average weight 20 to 25 g) were housed in sterile, aseptic conditions in a laminar hood and were fed ad libitum. The ADC was administered (non-serial design, n=3/sampling time) as an iv bolus at one dose level (10 mL/kg). For each animal at each selected time point (i.e., 0.083, 0.25, 24, 72, 96, 168, 240, 336 h), 600 µL of blood was collected via cardiac puncture and blood samples were then centrifuged (15 minutes at 4°C and 3500 tr/min). Quantification of ADC and total antibody in plasma samples was performed using immunoassays. Plasma concentration versus time profiles and PK parameters of conjugate and total antibody in mice were characterized using noncompartmental analysis (Phoenix, WinNonLin version 6.3) and are described in Figures 15 and 16. The area under the concentration-time curve, AUC (µg.day/mL) was estimated using the trapezoidal rule. Concentration at=0 C0 (µg/mL), Clearance CL (L/(day.kg), and terminal elimination half-life t1/2 (day) were derived and calculated from the curve. a: Informational data only as AUCext >> 30% (i.e. 42%); b: informational data only as AUCext >> 30% (i.e. 50%).

EMAR evaluation of plasma stability of cryptophycin ADC of formula (III) after single i.v. administration in SCID mice at 5 mg/kg

[000622] Plasma samples from the PK study were also analyzed by LC-EMAR. Chromatographic analysis was performed on a Waters Acquity I Class column and a Water Mass Prep Micro Desalination 20 µm (2.1 x 5 mm) at 80°C with gradient elution of (A) water + formic acid at 0. 1%/(B) CH3CN + 0.1% formic acid described below.

[000623] Mass spectrometry was performed on a Waters QTOF Synapt G2-S machine with electrospray ionization in positive mode (ES+). Mass spectra deconvolved with Waters Biopharmalynx software. EMAR spectra were obtained for all tested examples and are described in Figures 20 to 25.

[000624] These results showed that all tested examples of the invention do not exhibit the metabolism observed with reference to ADC, ADC1 and ADC2.

[000625] It is therefore evident that the compounds of the invention have anticancer activity and that the conjugates of the invention exhibit improved stability in mouse plasma.

[000626] Consequently, in another of its aspects, the invention also relates to the use of cryptophycin compounds of formula (I) or conjugates of formula (III) as anti-cancer agents.

[000627] The present invention, according to another of its aspects, also provides medicaments comprising a conjugate of formula (III).

[000628] These medicines are used therapeutically, especially in the treatment of cancer.

[000629] According to another of its aspects, the present invention refers to pharmaceutical compositions comprising as an active ingredient a conjugate of formula (III) according to the invention. These pharmaceutical compositions comprise an effective dose of at least one conjugate of formula (III) according to the invention and also at least one pharmaceutically acceptable excipient.

[000630] Said excipients are selected, according to the desired pharmaceutical form and method of administration, from the usual excipients, which are known to a person skilled in the art.

[000631] The present invention, according to other aspects of it, also provides a method of treating the pathologies indicated above, which comprises administering to a patient an effective dose of a conjugate of formula (III) according to the invention.

Claims (31)

1. Cryptophycin compound, characterized by the fact that it has formula (I): where: Ri represents a (C1-C6) alkyl group; R2 and R3 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group; or alternatively R2 and R3 form together with the carbon atom to which they are attached a (C3-C6) cycloalkyl or (C3-C6) heterocycloalkyl group; R4 and R5 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group or a (C1C6) alkyl-NH(R12) group or a (C1-C6) alkyl-OH group or a group ( C1-C6) alkyl-SH or a (C1-C6) alkyl-CO2H group; or alternatively R4 and R5 form together with the carbon atom to which they are attached a (C3-C6) cycloalkyl or (C3-C6) heterocycloalkyl group; R6 represents a hydrogen atom or a (C1 C6) alkyl group; R7 and R8 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group or a (C1C6) alkyl-CO2H group or a (C1-C6) alkyl-N(C1-C6)alkyl2 group; or alternatively R7 and R8 form together with the carbon atom to which they are attached a (C3-C6)cycloalkyl group or a (C3-C6)heterocycloalkyl group; R9 represents one or more substituents of the phenyl nucleus chosen, independently of each other, from: a hydrogen atom, -OH, (C1-C4)alkoxy, a halogen atom, -NH2, -NH(C1-C6)alkyl or -N(C1-C6)alkyl2 or -NH(C1-C6)cycloalkyl or (C3-C6)heterocycloalkyl; R10 represents at least one phenyl nucleus substituent chosen from a hydrogen atom and (C1-C4)alkyl; W represents (C1-C6)alkyl-NH(Rii), (C1-C6)alkyl-OH, (C1-C6)alkyl-SH, CO2H or C(=O)NH2; (C1-C6)alkyl-CO2H or (C1-C6)alkyl-C(=O)NH2; or (C1-C6)alkyl-N3. R11 and R12 represent, independently of each other, a hydrogen atom or a (Ci-C6) alkyl group. 2. Cryptocin compound of formula (I) according to claim i, characterized by the fact that it has the following structure: 3. Cryptophycin compound of formula (I) according to claim i or 2, characterized by the fact that R1 represents a methyl group. 4. Cryptophycin compound of formula (I) according to any one of claims 1 to 3, characterized by the fact that: - each of R2 and R3 represents a hydrogen atom, or - one of R2 and R3 represents a group methyl and the other represents a hydrogen atom, or - R2 and R3 together with the carbon atom to which they are attached form a cyclopropyl group. 5. Cryptophycin compound of formula (I) according to any one of claims 1 to 4, characterized in that each of R4 and R5 represents a methyl group. 6. Cryptophycin compound of formula (I) according to any one of claims 1 to 5, characterized in that R6 represents a hydrogen atom. 7. Cryptophycin compound of formula (I) according to any one of claims 1 to 6, characterized in that R7 and R8 independently represent a hydrogen atom or a (C1-C6) alkyl group. 8. Cryptophycin compound of formula (I) according to any one of claims 1 to 7, characterized in that R9 represents two substituents selected from a methoxy group and a chlorine atom. 9. Cryptophycin compound of formula (I) according to any one of claims 1 to 8, characterized by the fact that R10 represents a hydrogen atom. 10. Cryptophycin compound of formula (I) according to any one of claims 1 to 9, characterized by the fact that: Ri represents a (Ci-C6)alkyl; 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; and Rio represents a hydrogen atom. 11. Cryptophycin compound of formula (I) according to any one of claims 1 to 9, characterized by the fact that: R1 represents a (C1-C6)alkyl; R2 and R3 represent a (C1-C6)alkyl, and the other represents a hydrogen atom; R6 represents a hydrogen atom; R9 represents two substituents selected from a methoxy group and a chlorine atom; and R10 represents a hydrogen atom. 12. Cryptophycin compound of formula (I) according to any one of claims 1 to 11, characterized in that W is selected from (C1-C6)alkyl-NHR11, (C1-C6)alkyl-OH, (C1 - C6)alkyl-SH and (C1-C6)alkyl-CO2H, preferably W represents a CH2-NH2 group or a CH2-OH group. 13. Cryptocin compounds of formula (I) according to claim 1, characterized in that they are selected from the following list: 14. Cryptoficin payload, characterized by the fact that it has formula (II): wherein R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in claims 1 to 11, and Y is selected from the group (C1-C6)alkyl-NRii, (C1-C6) alkyl-O, (C1-C6)alkyl-S, C(=O)O, C(=O)NH, (C1-C6)alkyl-C(=O)O, or (C1-C6)alkyl-C (=O)NH; R11 represents a hydrogen atom or a (C1 C6) alkyl group; L represents a ligand; RCG1 represents a reactive chemical group present at the end of the ligand, L is of formula (IV): where: L1 represents a single bond or an NR16(hetero)aryl-CR15R14-OC(=O) group, if Y = (C1-C6)alkyl-N(R11); an NR18-(C2-C6)alkyl-NR17-C(=O) group or an NR16(hetero)aryl-CR15R14-OC(=O)-NR18-(C2-C6)alkyl-NR17-C(=O) group ), if Y = (C1-C6)alkyl- O or (C1-C6)alkyl- S; an NR16(hetero)aryl-CR15R14- group, if Y = C(=O)O, C(=O)NH, (C1-C6)alkyl-C(=O)O or (C1-C6)alkyl-C (=O)NH; Rii, Ri4, Ri5, Ri6, Ri7 and Ri8 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group; (AA)w represents a sequence of w AA amino acids connected together via peptide bonds; w represents an integer ranging from 1 to 12; L2 represents a single bond or a (C1-C6)alkyl group or a (C1-C6)alkyl-(OCH2CH2)i group or a (C1-C6)alkyl-(OCH2CH2)iO(C1-C6)alkyl group or a (CH2CH2O)i(C1-C6)alkyl group or a CH(SO3H)-(C1-C6)alkyl group or a (C1-C6)alkyl-CH(SO3H) group or a (C1-C6)alkyl-cyclo group -hexyl or an NR19-(C1-C6)alkyl group or an NR20-(CH2CH2O)i(C1-C6)alkyl group or an NR21-aryl group or an NR21-heteroaryl group or a (C1-C6)alkyl group NR22C(=O)-(C1-C6)alkyl or a (C1-C6)alkyl-NR22C(=O)-(C1-C6)alkyl-(OCH2CH2)i group; Ri9, R20, R2i and R22 represent, independently of each other, a hydrogen atom or a (C1-C6) alkyl group; i represents an integer between 1 and 50; AA means a natural or unnatural amino acid, of D or L configuration, chosen from: alanine (Ala), ß-alanine, Y-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, e,e-dimethyl-Y-aminobutyric acid, glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), e-acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val). 15. Cryptocurrency payload of formula (II) according to claim 14, characterized by the fact that it has the following structure: 16. Cryptophycin payload of formula (II) according to claim 14 or 15, characterized in that Y represents (C1-C6) alkyl-NR11, preferably Y represents CH2-NH. 17. Cryptocurrency payload of formula (II) according to claim 14, characterized by the fact that the sequence (AA)w has the formula: where R23 represents the side chain of AA. 18. Cryptophycin payload of formula (II) according to claim 14 or 17, characterized in that the sequence AA represents alanine (Ala), citrulline (Cit), glutamine (Gln), glycine (Gly), and - acetyl-lysine (AcLys), valine (Val). 19. Cryptophycin payload of formula (II) according to any one of claims 14 to 18, characterized by the fact that RCG1 is chosen from: - group -C(=O)-ZaRa for which Za represents a single bond , O or NH, and Ra represents H or a (C1-C6)alkyl, (C3-C7)cycloalkyl, (C5-C10)aryl, (C5-C10)heteroaryl or (C3-C7)heterocycloalkyl group or a succinimidyl group ; - one of the following reactive groups: the maleimido group haloacetamido group with R13 representing a hydrogen atom or a (C1-C6) alkyl group; -Cl; -N3; -OH; -SH; -NH2; -C=CH, a group or an O-(C1-C6)alkylhydroxylamine group. 20. Cryptophycin payload of formula (II) according to any one of claims 14 to 18, characterized in that L2 represents a (C1-C6) alkyl group or a (C1-C6)alkyl-(OCH2CH2) group i or a CH(SO3H)-(C1-C6)alkyl group. 21. Cryptocurrency payloads of formula (II) according to claim 14, characterized in that they are selected from the following list: 22. Conjugated, characterized by the fact that it presents formula (III): wherein R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in claims 1 to 11; Y and L are as defined in claim 17 or 18; G represents the reaction product between RCGi, a reactive group present at the end of the ligand, and RCG2, an orthogonal reactive group present in Ab; Ab represents an antibody. 23. Conjugate of formula (III) according to claim 22, characterized by the fact that it has the following structure: 24. Conjugate of formula (III) according to any one of claims 22 or 23, characterized by the fact that RCG2 is selected from: • e-amino groups of lysines supported by the side chains of lysine residues that are present on the surface of an antibody; • α-amino groups of N-terminal amino acids of antibody heavy and light chains; • saccharide groups in the articulation region; • cysteine thiols generated by intrachain disulfide bonds or engineered cysteine thiols; • amide groups supported by the side chains of some glutamine residues that are present on the surface of an antibody; • aldehyde groups introduced using formylglycine generating enzyme. 25. Conjugate of formula (III) according to any one of claims 22 or 23, characterized by the fact that: • when RCG1 represents an N-hydroxysuccinimidyl ester, RCG2 represents an -NH2 group; • when RCG1 represents a maleimido or haloacetamido function or a -Cl 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 a C=C, activated RCG2 represents a -N3 group; • when RCG1 represents an O-alkyl hydroxylamine function or a Pictet-Spengler reaction substrate, RCG2 represents an aldehyde or ketone function. 26. Conjugate of formula (III) according to any one of claims 22 to 25, characterized by the fact that G is selected from: 27. Process for preparing the cryptophycin compound of formula (I), characterized by the fact that it comprises the step of: wherein: - step (i) is a peptide coupling between the alternative AD3 and BC fragments 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 p-methoxybenzyl ether under 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 isothiocineol-derived chiral sulfonium in the presence of a base; - step (vi) is a reduction of the azido group. 28. Process for preparing a conjugate of formula (III), characterized by the fact that it comprises the steps of: (i) contacting and letting it react: - an optionally buffered aqueous solution of an antibody, optionally modified by means of an modifying agent, and - a solution of a cryptophycin payload of formula (II) as defined in any one of claims 14 to 21, the RCG1 chemical group of the cryptophycin payload of formula (II) being reactive to the RCG2 chemical groups present in the antibody especially to the amino groups present in the antibodies, said RCG2 chemical groups having been introduced, where appropriate, by the modifying agent, in order to link the cryptophycin payload of formula (II) to the antibody by formation of a covalent bond ; (ii) and then optionally separating the conjugate formed in step (i) from the cryptophycin payload of formula (II) and/or the unreacted antibody and/or any aggregates that may be formed. 29. Medication, characterized by the fact that it comprises a conjugate of formula (III) as defined in any one of claims 22 to 26. 30. Pharmaceutical composition, characterized by the fact that it comprises a conjugate of formula (III) as defined in any one of claims 22 to 26, and also at least one pharmaceutically acceptable excipient. 31. Conjugate of formula (III) according to any one of claims 22 to 26, characterized in that it is for use in the treatment of cancer.