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NZ741734B2 - Novel cryptophycin compounds and conjugates, their preparation and their therapeutic use - Google Patents

Novel cryptophycin compounds and conjugates, their preparation and their therapeutic use Download PDF

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Publication number
NZ741734B2
NZ741734B2 NZ741734A NZ74173416A NZ741734B2 NZ 741734 B2 NZ741734 B2 NZ 741734B2 NZ 741734 A NZ741734 A NZ 741734A NZ 74173416 A NZ74173416 A NZ 74173416A NZ 741734 B2 NZ741734 B2 NZ 741734B2
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New Zealand
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group
alkyl
formula
reaction
ome
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NZ741734A
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NZ741734A (en
Inventor
Antony Bigot
Herve Bouchard
Marie Priscille Brun
Francois Clerc
Jidong Zhang
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Sanofi
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Priority claimed from PCT/EP2016/076603 external-priority patent/WO2017076998A1/en
Publication of NZ741734A publication Critical patent/NZ741734A/en
Publication of NZ741734B2 publication Critical patent/NZ741734B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Abstract

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

Description

NOVEL PHYCIN COMPOUNDS AND CONJUGATESI THEIR PREPARATION AND THEIR THERAPEUTIC USE The present invention relates to new cryptophycin compounds, to new cryptophycin ds, to new cryptophycin conjugates, to compositions containing them and to their therapeutic use, especially as anticancer agents. The invention also relates to the process for preparing these ates.
Cryptophycins are secondary metabolites ing 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 acterium Nostoc sp (ATCC 53789; see EifSler S., et al., Synthesis 2006, 22, 3747-3789). Cryptophycins C-1 and C-52, which are terized by an e function, were discovered to have in vitro and in vivo antitumor activity in the early 1990s. The chlorohydrins of these, C-8 and C-55, were markedly more active but could not be formulated as stable ons (Bionpally R.R., et al., Cancer Chemother Pharmacol 2003, 52, 25-33). Their higher activity was correlated by their putative ability to generate the corresponding epoxides in cells. With no method to adequately stabilize the chlorohydrins at the time, cryptophycin C-52 (LY355703) entered clinical trials, produced marginal antitumor activity in two phase II lung cancer trials with 30-40% stable disease and was thus tinued (Edelman M.J., et al., Lung Cancer 2003, 39, 197-99 and Sessa C., et al., Eur J Cancer 2002, 38, 6).
R: H, C-1 R= Me, C-52 Considering their high potency and common mechanism of action with maytansinoids and auristatins, the two cytotoxic molecules validated in the clinic for dy-drug conjugates (ADC), this series was considered as a potential tubulin binder for ADC. Therefore conjugates in the cryptophycin series were developped starting from derivatization at the para-benzylic position of the macrocycle (Al-Awar R.S., et al., J Med Chem 2003, 46, 2985-3007).
W02011/001052 describes cryptophycin antibody-drug conjugates for which the cytotoxic moiety is linked to the antibody through the para-benzylic position using various kinds of linkers. They might be cleavable, disulfide or protease-sensitive, or non-cleavable.
Further optimization of cryptophycin antibody-drug ates described in WO2011/001052 led to potent cryptophycin conjugates which displayed promising antitumor activity but were found unstable in the plasma of mice while being stable in the plasma of non-rodent species. Therefore, there was a need for cryptophycin conjugates exhibiting improved stability properties.
The sure proposes new cryptophycin ycles which once conjugated to an antibody are stable in mice plasma and new cryptophycin conjugates composed of those stable macrocycles.
Summary of the ion In this respect, the disclosure s to cryptophycin compounds of formula (I): o O 1 m O p o O O HN m O R R R 3 2 R 8 9 R N N O 7 R R R H 4 6 (I) wherein: ? R1 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 a (C3-C6)heterocycloalkyl group; ? R4 and R5 represent, independently of each other, a en 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 a (C3-C6)heterocycloalkyl group; ? R6 represents a en 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)alkyl2 group; or atively R7 and R8 form er with the carbon atom to which they are attached a (C3- C6)cycloalkyl group or a )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; ? R10 represents at least one substituent of thephenyl nucleus chosen from a hydrogen atom and a group (C1-C4)alkyl; ? W represents • (C1-C6)alkyl-NH(R11), more particularly (CH2)nNHR11; 20266725_1 (GHMatters) P44029NZ00 • )alkyl-OH, more particularly (CH2)nOH; • (C1-C6)alkyl-SH, more ularly (CH2)nSH; • CO2H or C(=O)NH2; • )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 andR12 represent, independently of each other, a hydrogen atom or (C1-C6)alkyl, more particularly a methyl group; ? n represents an integer between 1 and 6.
In one aspect, there is provided phycin compound of formula (I): o O 1 m O p o O O HN R m O R R 3 R R 2 8 9 R N N O 7 R R R H 4 6 (I) wherein: ? R1 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 group or a -(C3-C6)heterocycloalkyl group; ? R4 and R5 represent, independently of each other, a hydrogen atom, a -(C1-C6)alkyl group, a - (C1-C6)alkyl-NH(R12) group, a -(C1-C6)alkyl-OH group, a -(C1-C6)alkyl-SH group or a -(C1- C6)alkyl-CO2H group; or alternatively R4 and R5 form er with the carbon atom to which they are attached a -(C3- C6)cycloalkyl or a -(C3-C6)heterocycloalkyl group; ? R6 represents a hydrogen atom or a -(C1-C6)alkyl group; ? R7 and R8 represent, independently of each other, a hydrogen atom, a -(C1-C6)alkyl group, a - )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, a -(C1-C4)alkoxy group, a halogen atom, -NH2, a -NH(C1- C6)alkyl group, a -N(C1-C6)alkyl2 group, a -NH(C1-C6)cycloalkyl group or a (C3- C6)heterocycloalkyl group; ? R10 ents at least one substituent of the phenyl nucleus chosen from a hydrogen atom and 20266725_1 (GHMatters) P44029NZ00 a -(C1-C4)alkyl group; ? W represents • (C1-C6)alkyl-NH(R11), • (C1-C6)alkyl-OH, • (C1-C6)alkyl-SH, • CO2H or C(=O)NH2; • (C1-C6)alkyl-CO2H or )alkyl-C(=O)NH2; or • (C1-C6)alkyl-N3.
R11 and R12 ent, independently of each other, a hydrogen atom or a -(C1-C6)alkyl group.
The disclosure further relates to cryptophycin payloads of formula (II): o O 1 m O RCG -L-Y p o O O HN R m R R R 3 2 R 8 9 R N N O 7 R R R H 6 (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 )alkyl-C(=O)NH; (C -C )alkyl N or alternatively Y represents (C1-C6)alkyl-triazole- like 1 6 ; Y being positioned in an ortho (o), meta (m) or para (p) position of the phenyl s; ? R11 represents a hydrogen atom or a (C1-C6)alkyl group; ? L represents a linker; ? RCG1represents a reactive al group present at the end of the linker, RCG1 being reactive towards a reactive chemical group present on an antibody.
[PAGE 4a TO FOLLOW 20266725_1 (GHMatters) P44029NZ00 In another aspect, there is provided cryptophycin payload of formula (II): o O 1 m O RCG -L-Y p o O O HN R m R R R 3 2 R 8 9 R N N O 7 R R H R 6 (II) wherein R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in claim 1 to 13, and ? Y isselected from a –(C1-C6)alkyl-NR11- group, a -(C1-C6)alkyl-O- group, a -(C1-C6)alkyl-S- group , a -C(=O)O, C(=O)NH- group, a -(C1-C6)alkyl-C(=O)O- group, and a 6)alkyl- C(=O)NH- group; ? R11 represents a hydrogen atom or a -(C1-C6)alkyl group; ? L represents a linker; ? RCG1 ents a reactive chemical group present at the end of the linker, L being of a (IV): L2 (AA)w L1 in which: ? L1 represents ? a single bond or a -NR16(hetero)aryl-CR15R14-O-C(=O)- group if Y is -(C1-C6)alkyl- N(R11)-; ? a -NR18-(C2-C6)alkyl-NR17-C(=O)- group or a -NR16(hetero)aryl-CR15R14-O-C(=O)- NR18-(C2-C6)alkyl-NR17-C(=O)- group if Y is -(C1-C6)alkyl- O- or -(C1-C6)alkyl- S-; ? a -NR16(hetero)aryl-CR15R14- group if Y is -C(=O)O-, -C(=O)NH-, (-C1-C6)alkyl- C(=O)O- or -(C1-C6)alkyl-C(=O)NH-; ? R14, R15, R16, R17 and R18 ent, independently of each other, a hydrogen atom or a - (C1-C6)alkyl group; ? (AA)w represents a sequence of w amino acids AA connected together via peptide bonds; ? w ents an integer g from 1 to 12; ? L2 represents a single bond , a -(C1-C6)alkyl- group, a –(C1-C6)alkyl-(OCH2CH2)i- group, a - (C1-C6)alkyl-(OCH2CH2)i-O(C1-C6)alkyl- group, a -(CH2CH2O)i(C1-C6)alkyl- group, a - CH(SO3H)-(C1-C6)alkyl- group, a 6)alkyl-CH(SO3H)- group, a -(C1-C6)alkyl-cyclohexylgroup , a -NR19-(C1-C6)alkyl- group, a -NR20-(CH2CH2O)i(C1-C6)alkyl- group, a -NR21-arylgroup , a -NR21-heteroaryl- group, a -(C1-C6)alkyl-NR22C(=O)-(C1-C6)alkyl- group, or a -(C1- C6)alkyl-NR22C(=O)-(C1-C6)alkyl-(OCH2CH2)i- group; [PAGE 4b TO FOLLOW] 20266725_1 (GHMatters) P44029NZ00 ? R19,R20, R21 and R22 represent, independently of each other, a hydrogen atom or a -(C1- C6)alkyl group; ? i represents an r between 1 and 50 ; AA denotes a natural or unnatural amino acid, of configuration D or L, chosen from: alanine (Ala), ?-alanine, ?-aminobutyric acid, 2-aminocyclohexylacetic acid, 2-aminophenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), line (Cit), cysteine (Cys), ?,?-dimethyl- ?-aminobutyric acid, ?,?-dimethyl-?-aminobutyric acid, glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), cine (Ile), leucine (Leu), lysine (Lys), ?-acetyl-lysine (AcLys), nine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
The disclosure also relates to conjugates of formula (III): o O 1 Ab-G-L-Y p o O O HN m O R R R R 3 2 R 8 9 R N N O 7 R R R H 4 6 (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 ents the product of reaction between RCG1, a reactive chemical group t at the end of the linker and RCG2, an orthogonal reactive chemical group present on the antibody (Ab); ? Ab represents an antibody.
In another aspect, there is provided conjugate of formula (III): o O 1 Ab-G-L-Y p o O O HN O R R R 3 2 R 8 9 R N N O 7 R R R H 6 (III) wherein ? R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in claim 1 to 13; ? Y and L are as defined in claims 17 to 22; [PAGE 4c TO FOLLOW] 20266725_1 (GHMatters) P44029NZ00 ? Grepresents the t of reaction between RCG1, a reactive group present at the end of the linker and RCG2, an orthogonal reactive group present on Ab; ? Ab represents an antibody.
Each substituent R1 to R11 may also adopt one of the spatial configurations (e.g. R or S or alternatively Z or E) as described in the examples.
The compounds of formula (I), (II) and (III) may contain one or more asymetric carbon atoms.
They may therefore exist in the form of enantiomers or diastereoisomers. These enantiomers and diastereoisomers, and also mixtures thereof, including racemic mixtures, form part of the invention.
The compounds of formula (I), including those that are illustrated, may exist in the form of bases or of acid-addition salts, especially of ceutically acceptable acids.
Definitions In the context of the present invention, certain terms have the following definitions: • l group: a hydrocarbon group obtained by ng one hydrogen atom from an alkene. The l group may be linear or branched. Examples that may be mentionned include ethenyl (-CH=CH2, also named vinyl) and propenyl CH=CH2, also named allyl). • alkoxy group: a group -O-alkyl, in which the alkyl group is as defined below; • alkylgroup: a linear or branched saturated aliphatic hydrocarbon-based group obtained by removing a hydrogen atom from an alkane. es that may be mentioned include methyl, ethyl, , isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, 2,2-dimethylpropyl and hexyl groups; • alkylene group: a saturated nt group of empirical formula -CnH2n-, obtained by removing two hydrogen atoms from an alkane. The alkylene group may be linear or branched. Examples that may be ned include methylene (-CH2-), ethylene (-CH2CH2-), ene (- CH2CH2CH2-), butylene (-CH2CH2CH2CH2-) and hexylene H2CH2CH2CH2CH2-) groups or the following branched groups , , , ; preferably, the alkylene group is of the formula -(CH2)n-, n representing an integer; in the ranges of values, the limits are included (e.g. a range of the type "n ranging from 1 to 6" or "between 1 and 6" includes limits 1 and 6); • antibody: an dy with affinity for a biological target, more particularly a monoclonal [PAGE 5 TO FOLLOW] 20266725_1 (GHMatters) P44029NZ00 antibody. The function of the antibody is to direct the biologically active nd as a cytotoxic compound towards the biological target. The antibody may be monoclonal, polyclonal or multispecific; it may also be an dy fragment; it may also be a murine, chimeric, humanized or human antibody. An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by ide bonds and each heavy chain is linked to a light chain by a disulfide bond (also referred to as a "full-length antibody"). The terms "conventional (or full-length) antibody" refers both to an dy comprising the signal peptide (or pro-peptide, if any), and to the mature form obtained upon secretion and proteolytic processing of the chain(s). There are two types of light chain, lambda (l) and kappa (k). There are five main heavy chain classes (or isotypes) which ine the functional activity of an dy molecule: lgM, lgD, lgG, lgA and lgE. Each chain contains ct 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 le regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The nt region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, ment g, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The icity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which er define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1- H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, es six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
As used herein, the term "antibody" denotes both conventional (full-length) antibodies and fragments thereof, as well as single domain antibodies and fragments f, in particular variable heavy chain of single domain antibodies. Fragments of ntional) antibodies typically comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody, and retain the ical function of the conventional antibody. Examples of such fragments include Fv, Fab, F(ab')2, Fab', dst, , scFv, sc(Fv)2 and diabodies.
The function of the antibody is to direct the biologically active compound as a cytotoxic compound towards the biological target. o aryl group: a cyclic aromatic group containing between 5 to 10 carbon atoms. Examples of aryl groups e phenyl, tolyl, xylyl, naphtyl; o biological target: an n (or group of ns) preferably located at the surface of cancer cells or stromal cells associated with this ; these antigens may be, for example, a growth factor receptor, an oncogene product or a mutated "tumor suppressant" gene product, an angiogenesis-related molecule or an adhesion molecule; 0 conjugate: an antibody-drug conjugate or ADC, i.e. an antibody to which is ntly attached via a linker at least one molecule of a cytotoxic compound; 0 cycloalkyl group: a cyclic alkyl group comprising between 3 and 6 carbon atoms engaged in the cyclic structure. Examples that may be mentioned include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups; 0 DAR (drug-to-antibody ratio): an average number of cytotoxic molecules attached via a linker to an dy; 0 halogen: any of the four elements fluorine, chlorine, bromine and iodine; o heteroaryl group: an aryl group containing between 2 to 10 carbon atoms and between 1 to 5 heteroatoms such as nitrogen, oxygen or sulphur engaged in the ring and connected to the carbon atoms forming the ring. Examples of aryl groups include pyridyl, pyrimidyl, thienyl, imidazolyl, triazolyl, indolyl, o-pyridyl, pyrazolyl; o heterocycloalkyl group: a cycloalkyl group containing between 2 to 8 carbon atoms and between 1 to 3 heteroatoms, such as nitrogen, oxygen or sulphur engaged in the ring and connected to the carbon atoms forming the ring. Examples include inyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, ydrothiofuranyl, tetrahydropyranyl, azetidinyl, oxetanyl and pyranyl; o linker: a group of atoms or a single bond that can covalently attach a cytotoxic compound to an antibody in order to form a conjugate; 0 fragment: simpler molecules that allow the total synthesis of cryptophycin compounds; 0 payload: a cytotoxic compound to which is covalently attached a linker; o reactive chemical group: group of atoms that can promote or undergo a al reaction.
Abbreviations ADC: dy-drug conjugate; AcOH: acetic acid; AIBN: azobisisobutyronitrile; ALK: (C1- C12)alkylene group, more particularly )alkylene, more particularly of the form -(CH2)n-, n being an integer from 1 to 12 and preferably from 1 to 6; aq.: aqueous; Ar: argon; AUC: area under the curve; BEMP: 2-tert-butyliminodiethylamino-1,3-dimethylperhydro-1,3,2- diazaphosphorine; BF3: boron trifluoride; Boc20: t—butyl-dicarbonate; BuLi: butyl lithium; CAN: ceric ammonium nitrate; Cbz: carboxybenzyl; CHCI3: chloroform; CH3CN: acetonitrile; CH3I: methyl iodide; C02: carbon dioxide; CL: clearance; m—CPBA: m-chloroperbenzoic acid; CR: te response; crypto denotes the unit of formula R10"? RRliixRRHNlQGR R7 RE R5 R4H , crypto especially denoting one of the D1-D19 cryptophycin compounds described later or a cryptophycin compound of an example; D1 refers to an ADC with a DAR of 1, D2 to an ADC with a DAR of 2, etc.; d: day; DAR: o-antibody ratio; DBU: abycyclo[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: ropylazodicarboxylate; DIEA: N,N- diisopropylethylamine; DMA: dimethylacetamide; DMAP: 4-(dimethylamino)pyridine ; DME: dimethoxyethane; DMEM: co's Modified Eagle Medium; DMEM/F12: Dulbecco's Modified Eagle Medium Nutrient Mixture F-12; DMF: dimethylformamide; DMSO: dimethylsulfoxide; DPBS: co’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-ethoxycarbonylethoxy— 1,2-dihydroquinoline; ELSD: evaporating light scattering detector; eq.: equivalent; ES: electrospray; EtOAc: ethyl acetate; EtZO: diethyl ether; EtOH: ethanol; Ex.: e; FCS: foetal calf serum; Fmoc: 9-fluorenylmethoxycarbonyl; FmocOSu: N-(9-fluorenylmethoxycarbonyloxy) succinimide; GI: electroinductive group; Grubbs l: benzylidene- bis(tricyclohexylphosphine)dichlororuthenium; Grubbs ll: (1,3-bis(2,4,6—trimethylphenyl) imidazolidinylidene)dichloro(pheny|methylene)(tricyclohexylphosphine)ruthenium; h: hour; H20: water; Hal: halogen; HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate; HCI: chlorohydric acid; HEPES: 4-(2-hydroxyethyl)—1- zineethanesulfonic acid; HIC: hydrophobic interaction chromatography; HOAt: oxy—7- azabenzotriazole; HOBt: 1-hydroxy—benzotriazole; HPLC: high performance liquid chromtography; HRMS: high resolution mass spectrometry; |C50: median inhibitory concentration; i.e.: id est meaning that is; IEC: ion exchange chromatography; iPrOH: anol; iPrZO: diisopropyl ether; i.v.: intravenous; K2003: potassium carbonate; LDA: lithium diisopropylamide; LiOH: lithium hydroxide; marg.: marginally; MeOH: methanol; MeTHF: 2—methyl-tetrahydrofuran; MgSO4: magnesium sulfate; min: minute; MNBA : 2—methylnitrobenzoic anhydride ; MTBE: methyl tertbutyl ether; MTD: maximum ted dose; NaH: sodium hydride; : sodium bisulfate; Na28203: sodium thiosulfate; NaCl: sodium chloride; Nchog: sodium hydrogen carbonate; NaH803: sodium bisulfite; NaHSO4: sodium hydrogen sulfate; NaOH: sodium hydroxide; NH4CI: ammonium chloride; NHS: N-hydroxysuccinimide; NMP: 1-methylpyrrolidone; NMR: nuclear magnetic resonance; P2—Et: l-2,2,4,4,4-pentakis(dimethylamino)-2)\5,4)\5- 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 amount what is needed; Q-Tof: quadrupole time-of-flight; quant.: quantitative yield; RCG: reactive chemical group; RT: room temperature; sat.: saturated; s.c.: subcutaneously; SCID: severe combined immunodeficiency; SEC: size exclusion chromatography; TBAF: tetrabutylammonium fluoride; tBuOK: potassium tert—butoxide; TCEP: tris(2-carboxyethyl)phosphine hloride; TEA: triethylamine; TEMPO: (2,2,6,6-tetramethylpiperidiny|)oxy|; TFA: trifluoroacetic acid; TFS: tumor-free survivor; THF: tetrahydrofuran; THP: ydropyran; TLC: thin layer chromatography; t1/2: half-life; tR: retention time; : para-toluenesulfonic acid; UPLC: Ultra Performance Liquid Chromatography; UV: ultra-violet; VSS: apparent volume ofdistribution at steady state.
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 tion 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-231 xenograft in SCID mice Figure 13: In vivo PK profile of ADC1 following a single i.v. administration in SCID mice Figure 14: In vivo PK profile of ADC1 following a single i.v. administration in non-rodent species Figure 15: In vivo PK profile of EX.3 following a single i.v. stration in SCID mice Figure 16: In vivo PK profile of EX.7 following a single i.v. administration in SCID mice Figure 17: HRMS spectrum of deglycosylated ADC1 at 96 h following a single i.v. administration in SCID mice Figure 18: HRMS spectrum of ADC2 light chain at 96 h following a single i.v. administration in SCID mice Figure 19: HRMS um ofdeglycosylated ADC1 at 6 d following a single i.v. administration in non-rodent species Figure 20: HRMS um of EX.3 at 96 h following a single i.v. administration in SCID mice Figure 21: HRMS um of EX.7 at 96 h ing a single i.v. administration in SCID mice Figure 22: HRMS spectrum of Ex.10 at 96 h following a single i.v. administration in SCID mice Figure 23: HRMS spectrum of Ex.14 at 96 h following a single i.v. stration in SCID mice Figure 24: HRMS spectrum of EX.20 at 96 h following a single i.v. administration in SCID mice Figure 25: HRMS spectrum of EX.23 at 96 h following a single i.v. administration in SCID mice Detailed description of the invention The ion relates to phycin compounds of formula (I): R10"? RRliixR7RlR5 wherein: - R1 represents a (C1-Cs)alkyl group; - R2 and R3 represent, independently of each other, a en atom or a (C1-Cs)alkyl group; or alternatively R2 and R3 form together with the carbon atom to which they are attached a (C3- Ce)cycloalkyl or a )heterocycloalkyl group; - R4 and R5 ent, independently of each other, a hydrogen atom or a (C1-Cs)alkyl group or a (C1-Ce)alkyl-NH(R12) group or a (C1-Cs)alkyl-OH group or a (C1-Ce)alkyl-SH group or a (C1- Cs)alkyl-COZH group; or alternatively R4 and R5 form together with the carbon atom to which they are attached a (C3- Ce)cycloalkyl or a (Cs-Cs)heterocycloalkyl group; - R6 represents a hydrogen atom or a (C1-Cs)alkyl group; - R7 and R8 represent, ndently of each other, a hydrogen atom or a )alkyl group or a (C1-Cs)alkyl-COZH group or a (C1-Cs)alkyl-N(C1-Ce)alkyl2 group; or alternatively R7 and R3 form together with the carbon atom to which they are attached a (C3- Ce)cycloalkyl group or a (Cs-Cs)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-Ce)alkyl or - N(C1-Ce)alkyl2 or -NH(C1-Cs)cycloalkyl or (Cs-Cs)heterocycloalkyl group; - R10 represents at least one substituent of the phenyl nucleus chosen from a hydrogen atom and a group (C1-C4)alkyl; - W represents 0 (C1-Cs)alkyl-NH(R11), more particularly NHR11; o (C1-Ce)alkyl-OH, more particularly (CH2)nOH; o (C1-Ce)alkyl-SH, more particularly (CH2)nSH; o COZH or C(=O)NH2; o (C1-Cs)alkyl-COZH or (C1-Ce)alkyl-C(=O)NH2; or o (C1-Ce)alkyl-N3.
W is positioned in an ortho (0), meta (m) or para (p) position of the phenyl nucleus; - R11 and R12 represent, independently of each other, a hydrogen atom or a group (C1-Cs)alkyl, more ularly a methyl group; - n represents an integer between 1 and 6.
The cryptophycin compound may be one of the following D1-D19: Wm Mu \ to 1le \ *0 ?g" OMe OMe HNVN \o H HNVN \oH 3 3 "HAN o OMe NVN o OMe H H H H wherein: - W and R12 are as defined in formula (I); Or the cryptophycin compound may be an equivalent unit described in one of the examples.
Among the compounds of formula (I) that are subject matter of the ion, a first group of compounds is composed of the nds for which R1 represents a (C1-Ce)alkyl, in particular a methyl group.
Among the compounds of formula (I) that are subject matter of the invention, a second group of nds is ed of the compounds for which each of R2 and R3 represents a hydrogen atom.
Among the compounds of a (I) that are subject matter of the invention, a third group of compounds is composed of the compounds for which one of R2 and R3 represents a (C1-Ce)alkyl, in particular a methyl group, and the other one represents a hydrogen atom.
Among the compounds of formula (I) that are subject matter of the invention, a fourth group of compounds is composed of the compounds for which R2 and R3 form together with the carbon atom to which they are attached a (Cg-Ce)cycloa|ky| group, in particular a cyc|opropy| group.
Among the nds of formula (I) that are subject matter of the invention, a fifth group of compounds is composed of the compounds for which each of R4 and R5 represents a (C1- Ce)alkyl, in ular a methyl group.
Among the compounds of formula (I) that are subject matter of the invention, a sixth group of compounds is composed of the compounds for which R6 represents a hydrogen atom.
Among the nds of formula (I) that are subject matter of the invention, a seventh group of compounds is composed of the compounds for which R7 and R8 represent independently of each other an hydrogen atom or a (C1-Cs)a|ky| group.
Among the compounds of formula (I) that are subject matter of the invention, an eighth group of compounds is composed of the compounds for which R9 represents two substituents selected from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-CI and 4-methoxy.
Among the compounds of formula (I) that are subject matter of the invention, a ninth group of compounds is composed of the nds for which R10 represents a hydrogen atom.
Among the compounds of formula (I) that are t matter of the invention, a tenth group of compounds is composed of the compounds for which W is positioned in the para position of the phenyl nucleus.
Among the compounds of formula (I) that are subject matter of the ion, a eleventh group of compounds is composed of the compounds for which W is selected from (C1-Ce)alkyI-NHR11, in particular (C1-Cg)alkyl-NHR11, particularly a CH2-NH2 group, (C1-Ce)alkyl-OH, in particular (C1- Cg)alkyI-OH, ularly a CH2-OH group, (C1-Ce)alkyI-SH, in particular (C1-Cg)alkyI-SH and (C1- C5)alkyl-COZH, in particular (C1-C3)alkyl-COZH.
Among the nds of formula (I) that are subject matter of the invention, a twelfth group of compounds is composed of the compounds for which R1 represents a (C1-Ce)alkyl, in particular a methyl group, each of R2 and R3 represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, more particularly, the phenyl nucleus comprises two tuents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-CI and 4-methoxy and R10 represents a hydrogen atom.
Among the compounds of formula (I) that are subject matter of the invention, a thirteenth group of compounds is composed of the compounds for which R1 represents a (C1-Ce)alkyl, in particular a methyl group, R2 and R3 ents a (C1-Cs)alkyl, in particular a methyl group, and the other one ents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, more particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-CI and 4- methoxy and R10 represents a en atom. atively, W is selected from (CH2)nNHR11, (CH2)nSH from (CH2)nOH, wherein n represents an r between 1 and 6.
Among the compounds of formula (I) that are t matter of the invention, a fourteenth group of compounds is composed of the compounds of the following structure (beta epoxide configuration): All these sub-groups taken alone or in combination are part of the invention.
Among the compounds of formula (I) that are the subject matter of the invention, mention may be made in particular of the following compounds: NwN o OMe o OMe HNwN H H H HO O\ O O N3 0\ O O HNj Um HNL\©[CI o OMe OMe HNVN NVN *0 H H H H2N 0% O O :CI N3 0% O O HNl\©[CI o OMe OMe HNVNH HNJ>(LN \oH stereomer1 / /O / /O , , HZN o\ O O o HN1\\\©:CI HO 0\ o HN1\\\©:CI H H H H stereomer 1 stereomer 1 / /O / /0 N3 0\ o CI O HN N%No HNL HZN o\ o \CECI o OMe OMe H H HN%N *0H stereomer 1 stereomer 1 O O / O / O , 7 H0 010 o HN CI HO 0\ O NVN \o HN1\©[CI OMe NVN \o OMe H H H H HO 0% o HJ5¥Hl?d N N o OMe >6HNkNHNk?/VEECIo 0M9 / /O HZN 0% o o HRHN HNl\\\©:C| N O OMe wherein: - R1, R2, R3, R4, R5, R6, R7, R7, R9 and R10 are as defined in formula (I); - Y represents (C1-Ce)alkyl-NR11 or (C1-Ce)alkyl-O or (C1-Cs)alkyl-S; or alternatively Y represents C(=O)O, C(=O)NH, (C1-C5)alkyl-C(=O)O or (C1-Ce)alkyl-C(=O)NH; (C1-C6)a|kyI/N%+ or alternatively Y represents (C1-C5)alkyl-triazole- like _ Y is positioned in an ortho (0), meta (m) or para (p) position of the phenyl nucleus; - R11 represents a hydrogen atom or a )alkyl group; . L ents a linker; - RCG1 ents a reactive chemical group present at the end of the linker, RCG1 being ve towards a reactive chemical group present on an antibody.
Among the compounds of a (II) that are subject matter of the invention, a first group of compounds is composed of the compounds for which R1 represents a )alkyl, in particular a methyl group.
Among the compounds of formula (II) that are subject matter of the invention, a second group of compounds is composed of the compounds for which each of R2 and R3 represents a hydrogen atom.
WO 76998 2016/076603 Among the compounds of a (II) that are t matter of the invention, a third group of compounds is composed of the compounds for which one of R2 and R3 represents a (C1-Ce)a|ky| group, in particular a methyl group and the other one represents a hydrogen atom.
Among the compounds of formula (II) that are subject matter of the invention, a fourth group of compounds is composed of the compounds for which R2 and R3 form together with the carbon atom to which they are attached a (Cg-Ce)cycloa|ky| group, in particular a cyclopropyl group.
Among the compounds of a (II) that are subject matter of the ion, a fifth group of compounds is composed of the compounds for which each of R4 and R5 represents a (C1-Ce)a|ky| group, in particular a methyl group.
Among the compounds of formula (II) that are t matter of the invention, a sixth group of compounds is composed of the compounds for which R6 ents a hydrogen atom.
Among the compounds of formula (II) that are subject matter of the invention, a seventh group of compounds is ed of the compounds for which R7 and R8 ent independently of each other an hydrogen atom or a (C1-Cs)a|ky| group.
Among the compounds of formula (II) that are subject matter of the invention, an eighth group of compounds is composed of the compounds for which R9 represents two substituents ed from a methoxy group and a chlorine atom. More particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-CI and 4-methoxy.
Among the compounds of formula (II) that are subject matter of the invention, a ninth group of compounds is composed of the compounds for which R10 represents a hydrogen atom.
Among the compounds of formula (II) that are subject matter of the invention, a tenth group of compounds is composed of the compounds for which Y is positioned in the para position of the phenyl nucleus.
Among the compounds of formula (II) that are subject matter of the invention, an eleventh group of compounds is composed of the compounds for which Y represents (C1-Ce)alkyI-NR11 in particular (C1-Cg)alkyl-NR11 more particularly CH2-NH.
Among the compounds of formula (I) that are subject matter of the invention, a twelfth group of compounds is composed of the compounds for which R1 represents a (C1-Ce)alky|, in particular a methyl group, each of R2 and R3 represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a chlorine atom, more particularly, the phenyl nucleus ses two substituents in positions 3 and 4 on the phenyl nucleus, preferably, it is 3-Cl and oxy and R10 represents a hydrogen atom.
Among the compounds of formula (I) that are subject matter of the invention, a thirteenth group of compounds is composed of the nds for which R1 represents a (C1-Ce)alkyl, in particular a methyl group, R2 and R3 represents a (C1-Cs)alkyl, in particular a methyl group, and the other one represents a hydrogen atom, R6 represents a hydrogen atom, R9 represents two substituents selected from a methoxy group and a ne atom, more 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.
Among the compounds of formula (II) that are subject matter of the invention, a fourteenth group of compounds is ed of the compounds of the following ure (beta epoxide configuration): Rromo $2;th0 RR R7RlR5R4" All these sub-groups taken alone or in combination are part of the invention.
Among the compounds of formula (II) that are the t matter of the invention, mention may be made in particular of the following compounds: G + l \\ O O A 0 D3 0 OMe HNWNH O O \\ o o A 0 NWN 0 We \\ N/O NVMNij 0% o HN Cl C H « H 1 a \\o o o A o N%N x0o OMe stereomer2 O O H H \ /o NQK JYN o\ o HN \\\\\\ CI N W H \ O l \\o O A o‘ H H \o OMe 0 Him 0 \f%iNH H0N OMe NwNIUCIo OMe The invention also s to conjugates of formula (III): m .
A - -L-Y i R8 $2 % R9 R N N O R6 R5 R4 H (lll) 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 product of reaction between RCG1, a reactive chemical group present at the end of the linker and RCGZ, an orthogonal reactive chemical group present on the antibody (Ab); - Ab ents an dy.
Among the compounds of formula (III) that are subject matter of the invention, a first group of compounds is composed of the compounds for which R1 represents a (C1-Ce)alkyl, in particular a methyl group.
Among the compounds of formula (III) that are subject matter of the invention, a second group of compounds is composed of the compounds for which each of R2 and R3 represents a hydrogen atom.
Among the compounds of formula (III) that are t matter of the invention, a third group of compounds is composed of the compounds for which one of R2 and R3 represents a (C1-Ce)alkyl group, in particular a methyl group and the other one represents a hydrogen atom.
Among the compounds of formula (III) that are subject matter of the invention, a fourth group of compounds is composed of the compounds for which R2 and R3 form together with the carbon atom to which they are attached a (Cg-Ce)cycloalkyl group, in ular a cyclopropyl group.
Among the compounds of formula (III) that are subject matter of the invention, a fifth group of compounds is composed of the compounds for which each of R4 and R5 represents a (C1-Ce)alky| group, in particular a methyl group.
Among the nds of formula (III) that are subject matter of the ion, a sixth group of compounds is composed of the nds for which R6 represents a hydrogen atom.
Among the nds of formula (III) that are subject matter of the invention, a seventh group of compounds is composed of the nds for which R7 and R8 represent independently of each other an hydrogen atom or a (C1-Cs)alkyl group.
Among the compounds of formula (III) that are subject matter of the invention, an eighth group of compounds is composed of the compounds for which R9 represents two substituents selected from a methoxy group and a ne atom. More ularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl nucleus. Preferably, it is 3-CI and 4-methoxy.
Among the compounds of formula (III) that are subject matter of the invention, a ninth group of compounds is composed of the compounds for which R10 represents a hydrogen atom.
Among the compounds of formula (II) that are subject matter of the invention, a tenth group of compounds is composed of the compounds for which Y is positioned in the para position of the phenyl nucleus.
Among the compounds of formula (III) that are subject matter of the invention, an eleventh group of compounds is composed of the compounds for which Y represents (C1-Ce)alkyI-NR11 in ular (C1-Cg)alkyl-NR11 more particularly CH2-NH.
Among the nds of formula (I) that are subject matter of the invention, a twelfth group of compounds is composed of the nds of the following structure (beta epoxide configuration): R1omO RRLW0 RR R7RlR5R4" All these sub-groups taken alone or in combination are part of the invention.
Among the compounds of formula (III) that are the subject matter of the invention, mention may be made in particular of the following compounds: Hml?Me H H H _R35-747N NJk JYN M n l o o A o hU2H11_R35-74*NM u \ O O /\ stereomer 1 0 JV" h2H11R3574HU ' ’MH — \ l l N l o o A o lg stereomer1 h2H11R3574HU 7 _ H O O /\ hu2H11_R35-74*N3 l a O O A O O O hu2H11_R35-74"WM N?h‘NNYOH 0% 00 ml» 7 o C:OlleCI 0 O HNwNH hu2H11_R35N NVN o OMe H H The ment between the cryptophycin payload of formula (II) and the antibody, in order to obtain the ate of formula (III), is produced by means of a reactive chemical group RCG1 present on the payload that is reactive towards a reactive group RCGZ present on the dy.
The reaction n RCG1 and RCGZ ensures the attachment of the compound of formula (II) to the antibody by formation of a nt bond. In the conjugate of a (III), parts of RCG1 and R062 can remain forming the attachment between the linker and the antibody.
Examples of RCG1 that may be mentioned include: (i) the -C(=O)—ZaRa reactive group for which Za represents a single bond, 0 or NH, more particularly 0, and Ra represents a hydrogen atom or a (C1-Cs)a|kyl, (Cs-C7)cycloa|kyl, alkenyl, aryl, heteroaryl or (Cg-Cy)heterocycloa|ky| group. The aryl group may be substituted by 1 to 5 groups chosen from halogen, in particular F, alkyl, alkoxy, nitro and cyano groups; (ii) one of the following reactive groups: the maleimido group; the haloacetamido XNVBrorl group with R13 representing a hydrogen atom or a )alky| group, more particularly Me; -C|; -N3; -OH; -SH; -NH2; -CECH or an activated CEC such as a cyclooctyne moiety like ; an O-alkyl hydroxylamine or a Pictet-Spengler reaction substrate such as described in AganNal P., et al., Bioconjugate Chem 2013, 24, 846-851.
More particularly, -ZaRa may represent -OH, -OCH3, -OCH20H=CH2, (O-NHS) or // M=H or cation where cation represents for example sodium, potassium or cesium or \ ,N:N or the group in which GI represents at least one electroinductive group such as -N02 or -Hal, especially -F. They may be, for example, the following groups: 2 F F or . Another type of -C(=O)ZaRa group is the following: . More ularly, RCG1 may be chosen from one of those bed in the es.
Examples of R062 that may be mentioned include (Garnett MC, et al., Advanced Drug Delivery Reviews 2001, 53, 171-216): (i) s—amino groups of lysines borne by the side chains of the lysine residues that are present at the surface of an antibody; (ii) or-amino groups of N-terminal amino acids of antibody heavy and light chains; (iii) the saccharide groups of the hinge region; (iv) the thiols of cysteines generated by ng intra-chain disulfide bonds or the thiols of engineered cysteines; (v) amide groups borne by the side chains of some glutamine residues that are present at the surface of an antibody; (vi) aldehyde groups uced using glycine generating enzyme.
More recently, other conjugation ches have been considered, for instance the introduction of cysteines by mutation (Junutula J.R., et al., Nature Biotechnology 2008, 26, 2), the introduction of ral amino acids allowing other types of try (Axup J.Y., et al., PNAS 2012, 109, 40, 16101-16106) or the conjugation on antibody glycans (Zhou Q., et al., Bioconjugate Chem. 2014, 25, 510-520). Another approach for site-specific cations 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, 165). For a review of site-specific conjugation strategies, see AganNal P. and Bertozzi C.R., Bioconjugate Chem 2015, 26, 2. These conjugation technologies may also be applied to cryptophycin payloads described in the present invention.
It is also possible to chemically modify the antibody so as to introduce novel reactive chemical groups RCGZ. Thus, it is well known to those skilled in the art how to modify an antibody with the aid of a modifying agent introducing for example activated disulfide, thiol, maleimido or haloacetamido groups (see especially W02005/077090 page 14 and W02011/001052). The modification makes it possible to improve the conjugation reaction and to use a wider variety of groups RCG1.
More particularly, in the case where RCG1 is of the type (ii) above, it is possible to chemically modify the antibody using an adequate modifying agent or to introduce one or more unnatural amino acids so as to introduce the adequate functions RCGZ. For example: - when RCG1 represents a oxysuccinimidyl ester, RCG2 represents a -N H2 group; - when RCG1 represents a maleimido or haloacetamido function or a -Cl group, RCG2 may be a -SH group; - when RCG1 represents a -N3 group, RCGZ may be a -CECH group or an activated CEC such as a cyclooctyne moiety; - when RCG1 represents a -OH or -NH2 group, RCGZ may be a carboxylic acid or amide function; - when RCG1 represents a -SH group, RCGZ may be a maleimido or haloacetamido function; - when RCG1 represents a -CECH function or an activated CEC, RCGZ may be a -N3 group; - when RCG1 represents a yl ylamine on or a Pictet—Spengler reaction substrate, RCGZ may be an de or ketone function.
Examples of G that may be mentioned include: km N The invention relates to cryptophycin payloads of formula (II) and to conjugates of formula (III): in which the linker L is of formula (IV): +L2 k(AA)wL1+ (IV) in which: - L1 represents . a single bond or a NR15(hetero)aryI-CR15R14-O-C(=O) group if Y = (C1-Ce)alkyl- N(R11)§ a NR1g-(Cz-Ce)alkyl-NR17-C(=O) group or a NR15(hetero)ary|-CR15R14-OC (=O)NR1g-(C2-Cs)alkyI-NR17-C(=O) group if Y = (C1-Ce)alkyl- O- or (C1-Ce)alkyl- . a NR1e(hetero)aryI-CR15R14 group if Y = C(=O)O, C(=O)NH, (C1-Ce)alkyI-C(=O)O or (C1-Cs)alkyI-C(=O)NH; - R11, R14, R15, R16, R17 and R13 represent, ndently of each other, H or a )alky| group; - (AA)w represents a sequence of w amino acids AA connected er via peptide bonds; - w represents an r ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3; - L2 represents a single bond or a (C1-Cs)alkyl group or a (C1-Cs)alkyl-(OCHZCH2)i group or a (C1-Cs)alkyl-(OCHZCH2)i-O(C1-Ce)alkyl group or a (CHZCH20)i(C1-Cs)alkyl group or a CH(SOsH)-(C1-Ce)alkyl group or a )alkyl-CH(SOgH) group or a )alkyl-cyclohexyl group or a NR1g-(C1-Cs)alkyl group or a NR20-(CHZCH20)i(C1-Ce)alkyl group or a NR21-aryl group or a NR21-heteroaryl group or a (C1-Cs)alkyl-NR220(=O)-(C1-Ce)alkyl group or a(C1- Cs)alkyl-NR220(=O)-(C1-C5)alkyl-(OCHZCH2)i group. More particularly L2 represents a (C1- CG)alkyl group or a (C1-CG)alkyl-(OCHZCH2)i group or a CH(SOBH)—(C1-CG)alkyl group; - R19, R20, R21 and R22 represent, independently of each other, H or a (C1-Cs)alkyl group; - i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50).
AA denotes a natural or unnatural amino acid, of configuration D or L, more particularly chosen from: alanine (Ala), B-alanine, obutyric acid, 2-aminocyclohexylacetic acid, 2—amino phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), (x,(x—dimethyl-y-aminobutyric acid, B,B-dimethyl-y—aminobutyric acid, glutamine (Gln), glutamic acid (Glu), glycine (Gly), ine (His), iso|eucine (lle), |eucine (Leu), lysine (Lys), 2- acetyl-lysine ), methionine (Met), ornithine (Orn), phenylalanine (Phe), e (Pro), serine (Ser), ine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val). More particularly, AA is chosen from alanine (Ala), citrulline (Cit), glutamine (Gln), glycine (Gly), g-acetyl-lysine (AcLys), valine (Val).
The sequence (AA)W has the formula: H O *JVMA‘LKJLLM R23 in which R23 represents the side chain of one of the amino acids described above. Examples of ces are as follows: Gly—Gly, Phe—Lys, Val-Lys, Val-AcLys, Val-Cit, Phe—Phe—Lys, D-Phe—Phe—Lys, e—Lys, Ala-Lys, Val-Ala, Phe—Cit, Leu-Cit, lle-Cit, Trp-Cit, Phe—Ala, Ala-Phe, Gly—Gly—Gly, a-Phe, Gly—Val-Cit, Gly—Phe—Leu-Cit, Gly—Phe—Leu-Gly, Ala- Leu-Ala-Leu.
For Y = (C1-Ce)alkyl-N(R11) and more particularly CH2NH, RCG1-L may be one of the following (lV1-17): RCG1 Examples of RCG1-L O O o \ gr t 0 AO/N ‘o ALKA(AA)W*(IV1) O O g‘i/oAALK40")i(AA)wI)(v2 $00OWLKMA |(V3) soH QMALKAHO (IV4) 9 o o QNALK'foMQALKkMAM (IV5) /N? O o 40 C (AA)wi (IV6) / N\ALK O O &:/ALK$(AA)W (IV7) \ SO3H IorBrQHAANE (IV8) I or B r NRIEALKJK(AA)W*(IV9) )K/I or Br O I . or Bryk"'ngOllAl-KJHAAM (IV10) o MKWok2 I B k M (AA)w4IV11) 0r erNRmMAf H2N\ALKA(AA)W * (IV12) "Z"N01ALKK N3 ALKA(AA)W* (IV14) Na/NOMALKJK.
(AA)wi (IV15) \00NJKALK'NR22OALm4)IV16 NJKALK'NRZZALK4ONJHAA) (|V)17 in which: - (AA)w represents a ce of w amino acids AA connected er via peptide bonds as described above; - w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3; - i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50); - M1, M2, M3 and M4 are chosen, independently of each other, from CR24 and N; - R19, R20, R21, R22 and R24 represent, independently of each other, a hydrogen atom or a (C1- Ce)alkyl group, more particularly a hydrogen atom or a methyl group.
For Y = (C1-Ce)alkyI-O or (C1-Ce)alkyI-S and more particularly CHZO or CHZS, RCG1-L may be one of the following (lV18—34): RCG1 Examples of RCG1-L $1 A 0 0 ‘o ALKA(AA)WNR18ALKNR#(IV18) go 0 AO/N KdONiOHAA)wNR8ALKNR?i ()Iv19 o o N\O)K(ALKf(' AA)W NR ALK NRk IV20) o SOSH O \ yo 0 /N \ALKA(AA)W NR ALK NRJK IV21) O o f o o QN ALK (OW" I O\ 7 ALKJK(AA)WNR18ALKNR1J7K(IV22)l o o @:0 QAMAMNRmALKNR?KUVZB) // \ALK' o o o @/ALK'%(AA)WNREALK7NR# \ \ SOSH o o I or BrQHAAMNRmALKNR?K (IV25) o o o | or BrQ?JRWALK-A(AA)WNR18ALKNR§K (IV26) )K/ O Ior Br O O I B ' or erNRz/ONO?AL?k(AA)wNR18ALKNR# (IV27) O M? 2% K IorBr$N kM/Ms (AA)W*NREALK*NR17 (IV28) R21 4 o o 2 ALKA(AA)WNR18ALKNR1J7K(IV29). 0 o HzNMO)ALKk(AA)wNR18ALKNR1J7Ki (IV30) o o N3\ALKA(AA)WNRmALKNR"k (IV31) o o N?wOlALKk(AA)wNR18ALKNR?K (IV32)i O o 0 0 NkALK'iNR22 ALKAmAMREALWNR?K (IV33)1 O o o o O \\ NJKALK'NR?EALKdONHkMAMNRmALKNR?K (IV34) in which: - (AA)w represents a sequence of w amino acids AA connected together via peptide bonds as described above; - w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3; - i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50); - 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-CG)aIkyI group, more particularly a hydrogen atom or a methyl group.
For Y = (C1-Ce)alkyI-N(R11) and more particularly CH2NH, RCG1-L may also be one of the following (lV35-51): RCG1 Examples of RCG1-L O J 15R 4 g ? i JL V40k / 2\ ‘o ALK (AA)wNR16Jf‘J3 o R R 0 0 CK C1 i 0 WNW AO/N I ‘o ALIHO I (AA) *NR16Jf‘J3 R R O /O 514k QNDJYALKfIAA)/ O 1K O (IV37) *NR16Jf 3 O SOBH o O JR15R14 0 r: I mm\ALK R16Jf3 v 0 / R R O o // O J1/J2\15 IV39) ‘ N’ALK'mMQALKkI/IAI *NRtJfJB 0 8i(IV40) ¢ OAmAMwNRtJfJ3N‘ALK J 15 14 Q 0 (a okavm) CN/ALngmmwiNkag16\ 4 \\O SO3H RSR O /J 1 14 I orBrQOK JR 3%k (IV42) (M)W*NR16J4 3 R R O O O J /J2\15 Ior Br$NR19ALKAK 11 O K / (IV43) o (M)W*NR16JfJ3 X/Ior Br IorBr? NRszVOJIIALKkmAMNR:' J1/JE15R1gkav44) O JR 0 M¢M2 (AA)wNR16J4(£5140k (IV45) | or BIQKNREMAMS HN\ A J/ngJBUJOKO (|)V46 2 ALK & (AA)wwNR6)st J 4 J1/ 2Vf’léok (IV47) 16 4 O J/Jf?RMJOK (IV48) N A 1K o 3 ALK J (AA) *NR16Jf 3 O J/JEHRMJK mo) 4 k A O ("’49) N3 ALK (AA) ; 3 R R O O J 15 14 O O i J1 2} okavso) NJKALKPNR ALK (AA) *NR16Jf3 D J R15R14 O J/2\ k NkALK'NR22OALKRONI?HA/tw O (IV51) 11wNR16JfJ3 in which: - (AA)w represents a sequence of w amino acids AA connected together via peptide bonds as bed above; - w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3; - i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50); - 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 en atom or a (C1-Cs)alkyl group, more particularly a hydrogen atom or a methyl group.
For Y = )alkyI-O or (C1-Ce)alkyI-S and more particularly CHZO or CHZS, RCG1-L may also be one of the following (IV52-68): RCG1 Examples of RCG1-L o R R O o O O J/J2\15 14* N\ k -A L o NR17 (IV52) 6 0 ALK (AA)wNR16 JfJ3 R15R10 o O /Oo 0 \ J/J2\5 4k AK kALiHoleAA 0 NREALRNR17 (1v53) AO/N MWNRKS R R O o /J2\15 14 k 1 §N\OJYALK'/W\)WNR16 J:o 1 2:40 NRgALKiNRWKUVM) O SO3H 0 o o R R O o 1514 i \ // \ / 0 /J J1 2\ o NRiALieNRK N 1 18 17 (IV55) o R151R O o CONALKHOMO oK J/J25 OWNRRALKNRWKUWES) (AA)wNRJ4/J3 R15R14 O O 2240KNRmALKNR#(IV57) q 0%)11ALK' R R O O J 15 14 O 0 J1/ \ CN/ALKWAMAMNRR\ l 1 2%OXNR18ALKNR1J7K (IV58) Jf‘J3 \\o sogH R R O o O J1/J2\15 1gkNNRALKNRk K K /J 17 ()(V59 (AA)wiNR16 J4/ 3 O O J/J2R15R14? i ""8er K 1K O NR1§ALK7NR17 (IV60) NREALK' O (M)W*NR16 J¢3 )K/IorBr O O J//2JR15R14JOK JOK IorBrQKNRNmiALKN K/J o NR1’8ALK7NR17 (IV61) (AA)wWNR16 J;3 R R O O J 15 14 J11/ 2\ OkNRmALKNR1J7KUV62) IorBrQK KOM/MZPOKMA/MAA)VH\JR16J4/3 NR1 M O /J2R15R14Ji J11 o i NR1§ALK7NR17()|V63 H2N\ 1 . A ALK NR16J4/3 O 5R14JDK O i K/J NR11TALK7NR17 (IV64) HN"0 kALK' (AA)wNR16 J4/J3 O J/JEHRMJOK )1 -N3 1113\wa 11 o NRgALLeNR17 (IV65) RR 0 0 /2J 15 14 o J k NAyoliALKt 1 0 l NR?ALK’NR?kUWES) (AA)wwNRtJ:3 o o o o NREALIQNR17 (IV67) N ALK ’NRZZ ALK..
L 'i 123J/ t \\ (AA)WNR16 J4//3 O O 00 J RR14 NA VHAA J,/ A OXNRTBALKiNR# (IV68) ALK"NR22ALHo AA)wNR?J/Js in which: - (AA)w ents a sequence of w amino acids AA connected together via peptide bonds as described above; - w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3; - i represents an integer n 1 and 50 and preferably n 1 and 10 (i may take all the 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 , 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-Cs)alkyl group, more particularly a hydrogen atom or a methyl group.
For Y = C(=O)O, C(=O)NH, (C1-Ce)alkyI-C(=O)O or (C1-Cs)alkyI-C(=O)NH and more particularly C(=O)O or CH2-C(=O)O, RCG1-L may be one of the following (IV69-85): RCG1 Examples of RCG1-L ADO/p ONO EK(|)V69 o? o o J/JLSBZ |(V70) N XALHO%(AA)WNR:J4/J3(. / /J2j1:& (N71) NO ALKW((A)AwNRkaJ3 O 4 G A// o aM/2\ // \ALK (AA)WNR16 J: 3 o 5/sz I N ALKIOMo k K J (IV73) \ALK (AA)WNR16 If 3 o\ 0 /Q\ o "5% o //0 ( L/J (IV74) / (AA)W’NR16J4/3 / \ALK C\) O J/J2R155R14 N $(AA)WNwR16J4/3& Ef?m) \ SO3H O J/JR15R14 I or r¢m~wNR1A6J/JB (N76) J 15 14 O O J/ 2% 1 (IV77) )K/I or Br 0 o J:1R 5R14 J/ 2% IorBr (IV78) J 15 14 O / 2\ M J) WW) IorBryok Mk M (AA)W7NR:J4//J3 NR21 M; 3 HZNNOJJALKAKH 00 /J2R1 5R141A NAALK'NR22OALKfOVkHAAAA)wNR? (IV85) J4//J3 in which: - (AA)w represents a ce of w amino acids AA connected together via peptide bonds as described above; - w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3; - i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50); - 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-Cs)alkyl group, more particularly a hydrogen atom or a methyl group.
For Y = C(=O) or (C1-Ce)alkyI-C(=O), the ion also s to cryptophycin payloads of formula (II) and to conjugates of formula (III): in which the linker L is of formula (V): +L3/NR"(A/Ami (V), in which: - (AA)’w ents a ce of w amino acids AA connected together via peptide bonds; - w represents an integer ranging from 1 to 12, preferably from 1 to 6 and more particularly 2 or 3; - R25 represents a hydrogen atom or a (C1-Cs)a|ky| group; - L3 represents a (C1-Cs)alkyl group or a (CHZCHZO)i-(C1-Ce)alkyl group or a (C1-Cs)alkyl- (OCHZCHZ) group or a CH(SOsH)-(C1-Cs)alkyl group or or a (C1-Ce)aIkyI-(OCHZCH2)i-O(C1- Ce)alkyl group or a NR19-(C1-Cs)alkyl group or a NRzo-(CHZCH20)iCHZCH2 group or a (C1- y|-cyc|ohexy|-C(=O)NR19-(C1-Cs)alkyl group or a (C1-Cs)a|ky|-cyc|ohexy|-C(=O)NR20- ZO)i-CHZCH2 group or a NR21-aryI-C(=O)NR19-(C1-Ce)alkyl group or a NR21- heteroaryI-C(=O)NR19-(C1-Cs)alkyl group or a (C1-Cs)alkyI-NR220(=O)(CHZCH20)i-(C1- Ce)alkyl group; - R19, R20, R21, R22 and R25 represents a hydrogen atom or a (C1-Cs)a|ky| group; - RCG1 represents a reactive chemical group that is reactive towards a reactive chemical group present on the antibody, as defined above; - R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined above.
AA denotes a natural or ral amino acid, of configuration D or L, more particularly chosen from: alanine (Ala), ine, y—aminobutyric acid, 2-aminocyclohexylacetic acid, 2—amino phenylacetic acid, arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), ne (Cys), (x,(x-dimethyI-y-aminobutyric acid, B,B-dimethyl-y—aminobutyric acid, glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), iso|eucine (||e), |eucine (Leu), lysine (Lys), 2- acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), ne (Tyr), valine (Val). More particularly, AA is chosen from alanine (Ala), citrulline (Cit), glutamine (Gln), glycine (Gly), g-acetyl-lysine (AcLys), valine (Val) The sequence (AA)’W has the formula: *AALK'H?W’k R26 in which R26 represents the side chain of one of the amino acids described above. Examples of sequences are as follows: GIy—Gly, Lys-Phe, Lys-Val, AcLys-Val, Cit-Val, Lys-Phe-Phe, Lys-Phe-DPhe, Lys-Phe-Gly, Lys-Ala, Ala-Val, Cit-Phe, u, e, Cit-Trp, Ala-Phe, Phe-Ala, Gly—Gly—Gly, Phe-Ala-Gly, Cit-Val-Gly, Cit-Leu-Phe-Gly, Gly—Leu-Phe-Gly, Leu- Ala-Leu-Ala.
For Y = C(=O) or (C1-Cs)alkyI-C(=O) and more particularly C(=O) or =O), RCG1-L may be one of the following (V86-101): RCG1 Examples of RCG1-L OgkkOJKALKNRZAAAYWO (V86) A0}?O O\ 0 N\OAALK-(O/\’)1’NR2?(AA)IW_ (V87) 0 O 6/ N\OJK(ALKNR25(AA)'W (V88) Q0/ \ALKiNRZg(AA)'W* (V89) o\ // /Q QNALWOMQALKNRMAMW (v90) 0 o / N/ NszVOMNRZ?AAmi (V92) \ALK' IorBrJ1 NREALK—NR2—5(AA)'w— (V93) 0 Ior Br? 0 i up) or Br NRZON A)W*(V94), o mf'v'z ""3er k /M NRgALKeNRZgAAmr 0/95) NR21 M4 3 HZNiALKiNRg(AA)'W* (V96) HZNNO%ALK7NR2;(AA)'W* (V97).
N;ALK*NRg(AA)'wi (V98) NB/NOiiALKiNRZ?AAmi (V99) N ALK*NR2§(AA)'W* (V100) N ALK’NRZZWO?ALK'iNRZ?AAYW’ (V101) in which: - (AA)’w represents a sequence of w amino acids AA connected together via peptide bonds as described above; - w represents an integer ranging from 1 to 12, ably from 1 to 6 and more particularly 2 or 3; - i represents an integer between 1 and 50 and preferably between 1 and 10 (i may take all the values between 1 and 50); - 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-Cs)alkyl group, more ularly a hydrogen atom or a methyl group.
The linker L may also be chosen from the illustrated compounds.
In accordance with the invention, the compounds of general formula (I), (II) and (Ill) can be prepared by the following processes. s for re arin thec to h cin com ounds General route A for the re aration of the com ounds of formula I in the case where W=CH2N3 or CHzNHz 0 OH R1 R1 0 / o lO?/Jrl?tR ll \ O X g NAG . :Ns 0\ 0 0 0\ OH 7 0 Re‘ R10 R8 R10 A D O R, NH AD1 R3 R2 0 H Y \ M Y Q 0 ~ 0 NH2 + \ R9 RSR2 j R R5 R4 9 HO \O HO N %0 C B R5 R4 BC R1 R1 / /O / \ \ COZtBu N o HN o N o o BocHN 3 o\ 3 R10 R1 R3 R2 1 R9 R10 RI 0 R3 R2 1 GR, .31 E NMN *0 E NMN So 7RgR5R4H 7RgR5R4H R1 R1 / ¢O / /0 N3 0% 0 SN (iv) H N o\ 0 0R 2 \ 0R SN R10 R 3 2 ©R R10 R 3 2 \\@R 9 9 P f3 NMNLO P f3 NMN]\O 2 7Rl R5 R4H 3 7Rl R5 R4H 6 6 Fragments A, B, C and D allow the preparation of cryptophycin compounds P1 to P3 with the aid of the steps detailed below: M: ic coupling between nts AD1 and BC in the ce of coupling reagents such as, for example, HOAt and HATU; Step (iii: deprotection in acidic conditions using for example TFA and macrocyclization in the presence of coupling reagents such as, for example, HOAt and HATU; Step {iii}: oxidation of the olefine to form the epoxide using, for example, m—CPBA; Step (v): reduction of the azido group using, for example, TCEP.
Fragments A and B were prepared according to the synthesis described below. Fragments C protected as methyl esters are commercially available for R4=H, R5=Me (R), R2=R3=H (CAS number [925357]); R4=H, R5=Me (S), R2=R3=H (CAS number [1181380]); R4=R5=Me, R2=R3=H (CAS number [25307-82—8]); R2=H, R3=Me (R), R4=H, R5=Me (R) (CAS number - 92-5]); R2=H, R3=Me (S), R4=H, R5=Me (R) (CAS number [865446]); R2=H, R3=Me (R), R4=R5=Me (CAS number [1315052—25-5]); R2=H, R3=Me (S), R4=R5=Me (CAS number [1315050- 96-41); R2=H, R3=iPr (R), R4=R5=Me (CAS number [13149998]); R2=H, R3=iPr (S), R4=R5=Me (CAS number [13150541]); R2=R3=R4=R5=Me (CAS number [908866]); R4 and R5 form together with the carbon atom to which they are attached a cyclopropyl group (CAS number id="p-9142269" id="p-9142269" id="p-9142269" id="p-9142269" id="p-9142269" id="p-9142269" id="p-9142269"
[9142269]). They may also be ed as described in the es. Non-commercially ble fragments C were prepared as described in the examples. All fragments D are commercially available: L-Fmoc—Ala-OH (CAS number [356613]); L-Fmoc-Val-OH (CAS number [68858—20-8]); L-Fmoc—tert—Leu-OH (CAS number [1326847]); L-Fmoc—Leu-OH (CAS number [356610]); L-Fmoc—NMe-Leu-OH (CAS number [103478-62—2])§ —3- dimethylamino-Ala-OH (CAS number [5878802]); L-Fmoc—(Oallyl)Asp-OH (CAS number id="p-1469823" id="p-1469823" id="p-1469823" id="p-1469823" id="p-1469823" id="p-1469823" id="p-1469823"
[1469823]); L-Fmoc—4-methyl-Leu-OH (CAS number [1395519]). Building blocks AD1 and BC were prepared according to the synthesis described below.
General route B for the re aration of the com ounds of formula I in the case where W=CH20H or CH2N3 or CHzNHz O\ OH R1 R1 0 / 0 R1 o \ 2 l /lo ANAO . :H0 o\0 of 0\ OH 0 7 Rel R10 R8 R10 A D 0 R7 RhllH AD2 C B R5 R4 BC R1 R1 \ /O / \ COZtBu HO O O HN (ii) HO O O BocHN § 0 V \ R R3 R2 0 R R3 R2 m Re M R9 10 R9 \ R8 "5% s N N o N N o R R 7RlR5R4H 7RlR5R4H R10 3 2 H\©LR R10 3 2 9 "\GLR F3 NMN \o ? NMN \o 9 4 7Rg R5 R4'" 2 7R6‘ R5 R4'" / /O HZN 0% O OR |:g-IN R10 R 3 2 kQR p3 R NMN O 7Ré R5 R4H Fragments A, B, C and D allow the preparation of cryptophycin compounds P2 to P4 with the aid of the steps detailed below: M: peptidic coupling between nts AD1 and BC in the presence of coupling reagents such as, for example, HOAt and HATU; Step (ii): deprotection in acidic conditions using for example TFA, macrocyclization in the presence of ng reagents such as, for e, HOAt and HATU and acetate hydrolysis at pH 6-7 using for example NaOH in a e of water and AcOEt; Step {iii}: benzylic alcohol protection as a silyl ether using for example chlorotriisopropylsilane in the presence of a base such as, for example, imidazole; oxidation of the olefine to form the epoxide using, for example, ; deprotection of the si|y| ether using, for example, a TBAF solution.
Step (iv): azidation in the presence of DPPA and a base such as, for example, DBU; Step (v): reduction of the azido group using, for example, TCEP.
Fragments A and B were prepared according to the synthesis described below. Fragments C were either commercially available as reported in the previous section or prepared as described in the examples. All nts D are commercially available as reported in the previous section.
Building blocks AD2 and BC were prepared according to the synthesis described below.
General route C for the re aration of the com ounds of formula I in the case where W= CHon or CH2N3 or CHzNHz 0\ OH R1 PMBOW + I: N o o% O OH R; . R8 Sakurai alcohol D 0 R7 [\‘IH AD3 l R6 Oi R3 R2 H 0 N HN \0 NH. + K lGRQ: 0m] QR R5 R4 9 HO \O HO N %o c B R5 R4 H BC N N N R R 7 R; R5 R4 H 7 R; R5 R4 H J (iii, iv) R1 R1 0\ / /O / ; /O 3 2 R9 3 2 R 10 R9 8 R8 N N O _ N N \o R X' N3! P2 R7 R‘ Rl H R R 7 H R R 5 4 4 6 X = OH, P4 6 Scheme 3 i l and fragments B, C and D allow the preparation of cryptophycin compounds P2 to P4 with the aid of the steps detailed below: Step (i): peptidic coupling between fragments AD3 and alternative BC in the presence of coupling reagents such as, for example, HOAt and HATU; Step (ii): macrocyclization by ring closing esis in the presence of a catalyst such as, for example, Grubbs l st; Step (iii): deprotection of the p—methoxybenzyl ether in acidic conditions such as, for example, % 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 tric Corey-Chaykovsky on using appropriately substituted isothiocineole-derived chiral sulfonium in the presence of a base such as, for example, phosphazene base P2-Et; Step (vi): reduction of the azido group using, for example, TCEP.
Sakurai alcohol and fragment B were prepared according to the synthesis bed below.
Fragments C were either commercially available as reported in the previous section or prepared as described in the examples. All fragments D are cially available as reported in the previous section. Building blocks AD3 and alternative BC were prepared according to the synthesis described below.
Pre aration of the com ounds of a I in the case where W=CHZSH R1 R1 HO 0\ o N 0 CI 0% o R10 R10 Rail :\L\©»R9N R8 @ 1%\G‘Rg R8 R N N o N N o R5 P R P 4 7Rg R4H 5 7R4 R5 R4H R10, R1 0% \ I / O H o 0 ss 0 o N o/ 8 N N N N \o R g H R4 RRB‘ 7 dlmer 7R6l R5 R4H (iii) HSW00% o F? N o R R10 R 3 2 ©R P F? NMN \o 6 7Rg R5 R4H Scheme4 P4 allows the preparation of other cryptophycin compounds P5 and P6 with the aid of the steps detailed below: M: 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 thyldisilathiane according to Hu J., et a/., J. Org. Chem. 1999, 64, 4959-496: in the course of this reaction, the intermediate dimer depicted in Scheme 3 is usually formed; Step (iii): ion of the dimer using a phosphine such as, for e, TCEP.
Schemes 1, 2, 3 and 4 describe the cases n=1 but may also apply for the ation of compounds of formula (I) in the case where n>1 starting from P2, P3 or P4 with the aid of the steps detailed below and described in Scheme 2 of W02011/001052: 1 OH R1 0%OORRHN, (I). WWHO R R 0%OORRHN‘, 3 2 3 2 R8 M R 10 \ 9 R8 M \ R9 N N \o N N \o W=CH2N3,P2 R, ‘ R R H R, ‘ R R H Re 5 4 Re 5 4 W=CH2NH2,P3 w = CHZOH, P4 out 7R; R5 R4H 7R6‘ R5 R4H Scheme 5 Step (i1: opening of the epoxide ring in acidic medium so as to obtain the diol function; concentrated perchloric acid may be used, for example; Step (ii): oxidative cleavage of the diol using, for example, sodium periodate; Step (iii): Wittig reaction using a suitable phosphonium halide, for example a bromide, and a strong base, for instance BuLi; Step (iv): oxidation of the olefine to form the epoxide using, for example, m—CPBA; Step (v1: deprotection of the silyl ether using, for e, a TBAF solution.
Preparation of the compounds of formula (ll in the case where W=COzH Scheme 6 P4 allows the preparation of other cryptophycin compounds P7 and P8 with the aid of the steps detailed below: M: oxidation using the Dess-Martin reagent; Step (iii: oxidation of Pinnick type in the presence of 2 methylbutene (Pinnick H.W., Tetrahedron 1981, 37, 2091-2096).
Scheme 6 bes the case n=0 but may also apply for the preparation of compounds of formula (I) in the case where W=(CH2),,COZH starting from an analog of P4 bearing a (CH2)n+1OH group. Schemes 1, 2, 3, 4, 5 and 6 are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other nds of formula (I), especially D1-D19. s for re arin thec to h cin a loads The compounds of formula (II) might be prepared according to Scheme 7 starting with a cryptophycin compound of formula (I) and a linker precursor (LP): R R1 WW0 O 1 o O / /0 RCG1-L-YW LP :13"R7RlR5RHGR7 1%EDGR,R7R65R R (I) ("l Scheme 7 W represents 0 (C1-Cs)alkyl-NH(R11), more particularly (CH2),,NHR11; o (C1-Ce)alkyl-OH, more particularly (CH2),,OH; o (C1-Ce)alkyl-SH, more ularly (CH2),,SH; o COZH; o (C1-Cs)alkyl-COZH, more particularly (CH2),,COZH; or o (C1-Ce)alkyl-N3. in which R11 represents a hydrogen atom or a group (C1-Cs)alkyl, more particularly a methyl group.
The linker precursor LP has the function of introducing a precursor of the linker L into the cryptophycin compound after reaction between the group W and a al function present on ln Scheme 7, several steps and/or reactions may be necessary to prepare the cryptophycin payload (ll) starting from the cryptophycin compound (I). For example, in the case where ZaRa = - O-NHS, a linker L for which ZaRa = -O-allyl may be introduced using the corresponding linker precursor, followed by deprotecting the ester function and introducing -O-NHS. Deprotection may be performed by treatment with a palladium catalyst, for example Pd(PPh3)4 in the presence of a scavenger amine, for e morpholine; the activation may be performed with DSC in the presence of a base such as, for example, DIEA or with NHS in the presence of a coupling agent such as, for example, DCC. This conversion of a group ZaRa into r group ZaRa (e.g. yl 9 -O-NHS) may be applied to obtain other groups ZaRa, ally those described previously.
Scheme 7’ similarly illustrates the preparation of a cryptophycin compound comprising a linker bearing respectively, a ido, a haloacetamido, an amino or an azido group (L* represents a fragment of a linker such that L: leimido or L: -L*—haloacetamido or L: -L*—NH2- or L: - L*-N3 ). @o 2 \L'7Crypto // N 0 N3 0 O \IinkerK/ 3\Iinker?? ONHS ONHS FmocHN Br or IdN Elinker%o linkerxO ONHS ONHS g0N\ Bror IQL HZN N3\ 0 pto NR?9'3CFYDtO \L**Crypto L**Crypto (Ila) (Ilb1)9 (Ilc) (Ild) Scheme 7’ These compounds are obtained by reaction between a cryptophycin compound comprising a linker L’ comprising an amino group and a modifying agent for ucing, respectively, a maleimido, a haloacetamido, an amino or an azido group.
The compounds of a (II) might alternatively be prepared according to Scheme 8 starting with the same cryptophycin compound of formula (I) and another linker precursor (LP’): W 1 0 O L'-Y: R7Rl O R5 RH R7 R R R H (I) (la) (II) with L=L'-L" Scheme 8 In Scheme 8, several steps and/or ons may be necessary to arrive at the cryptophycin payload (ll) starting from the cryptophycin compound (I). For example, in the case where ZaRa = - O-NHS, a protected linker precursor L’ may be introduced, followed by deprotection and coupling to a linker precursor L" to introduce the ylic acid function that may be directly ted to ZaRa = -O-NHS or first deprotected and subsequently activated (L’ and L" represent fragments of linker such that L=L’-L"). Deprotection may be performed by treatment with a base, for example, piperidine; coupling to a linker L" may proceed through opening of a cyclic anhydride, for example, glutaric anhydride; the activation may be med with DSC in the presence of a base such as, for example, DIEA or with NHS in the presence of a coupling agent such as, for example, DCC.
An example of reaction between the group W and a chemical function present on LP is an amidation either between a linker precursor LP bearing a carboxylic acid function and W=(CH2),,NH2 or between a linker precursor LP bearing an amine function and W=COZH or (CH2),,COZH: this reaction may be performed in the presence of a coupling agent such as, for example, EDCI or HOBt. It is also le to react a linker precursor LP bearing an amine function and W’=(CH2),,O-C(=O)—O-(4-nitrophenyl) obtained from ),,OH and p—nitrophenyl chloroformate (activation of the alcohol in the form of carbonate) according to the scheme below (R17 = H or (C1-Ce)alkyl): -NHR17 + crypto-(CH2),,O-C(=O)—O-(4-nitrophenyl) 9 -(CH2)nO-C(=O)-NR17- The same kind of reaction can also be performed between a linker precursor LP bearing an alcohol function activated as a carbonate and W=(CH2),,NH2. Another example of reaction is an esterification between a linker precursor bearing an alcohol function and W=COzH or (CH2),,COZH: this reaction may be performed in the presence of a coupling agent such as, for example, MNBA. Another e of on is a copper-catalyzed alkyne cycloaddition n a linker precursor bearing an alkyne function and W=(CH2)N3: this reaction may be performed in the presence of copper sulfate and sodium ascorbate.
Preparation of the compounds of formula (II) in the case where )nNH2 and L=(IV11 with n=1I R15=H and ALK=(CH2l3 based on Scheme 7 Scheme 9 M: peptidic coupling in the ce of coupling reagents such as, for example, EDC and HOBt, and a base such as, for example, DIEA; Step (ii): deprotection of the allyl ester in the presence of a st such as, for example, tetrakis- (triphenylphosphine)palladium; Step liiil: activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Preparation of the compounds of formula (II) in the case where W=(CHg)nNH2 and L=(lV1l with n=1, R11=H and ALK=(CH2l3 based on Scheme 8 fmoci(AA)wi 1111,13 Scheme 10 Step (i): peptidic coupling in the ce of coupling reagents such as, for example, EDC and HOBt and a base such as, for example, DIEA; Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine; Step {iii}: ng to glutaric anhydride; Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for e, DIEA.
Schemes 9 and 10 describe the case L(|V1) with n=1, R11=H and ALK=(CH2)3 but they may also apply to other linkers L with RCG1=C(=O)ONHS, namely the case L(|V1) with n¢1 and/or R11¢H and/or ALK¢(CH2)3 and the cases L(|V2) and L(|V3). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W= CH2 nNHz with n=1 R11=H and aleimido haloacetamido NH2 N3 or c clooct ne namel linkers L |V4 to L1 |V1 7[ §NM(AA)WH/ O W0 o R ,0 OENVORSRRzglJQR,R7 (i) R‘ R5 R4 (0 IQk(AA)W*NH ; P3 —’ o\ O HN R10 R81 )9? kGRgo N N O 7 R‘ R5 R4 H (i) 6 / O NMMAWNH o\ o HN R10 R87[ W 1\ O R R N N \0 (i) R7 Rg R5 R4 H R1 7RE‘3 R5 R4H FmocHN (AA)W’N O\ O HN R10 R1 0 R3 R2 kGRg E NMN o (H)__ k 7 R; R5 R4 H 0 / /O MH : H2N (AA wiN o% 6 HN R; 0 W2 AGRQ8 NMN 7 R; R5 R4 H Scheme 11 Step (i): peptidic coupling in the ce of coupling reagents such as, for example, EDC and HOBt and a base such as, for example, DIEA; Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine.
Scheme 11 describes the cases L(|V4) with n=1, R11=H and ALK=(CH2)5, L(|V8) with n=1 and R11=H, L(|V12) with n=1, R11=H and ALK=(CH2)4, L(|V14) with n=1, R11=H and ALK=(CH2)3 and L(|V16) with n=1, R11=H, ALK=(CH2)3 and ALK’=(CH2)2 but it may also apply to other linkers L(|V4) to L(|V17) with RCG1=maIeimido, iodoacetamido, NH2, N3 or ctyne. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group.
It is given for a linker in the para on, but may identically apply for the ortho or meta positions.
Similarly, it is given for a cryptophycin nd, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W= CH2 nX with X=O or S and L= |V18 with n=1 R17 and R18=H and ALK= CH2 3 based on Scheme 7 P4 or P6 0/ 1/O /O / O O O HN OZN R: 0 R10 R3 R2 P10 8 NMN *0SN\©LR R5 R4H Scheme 12 M: tion of the benzylic l or benzylic thiol as a p—nitrophenyl-(thio)carbonate by treatment with p—nitrophenyI-chloroformate in the presence of a base such as, for example, DIEA; Step (ii): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA; Step (iii): deprotection of the allyl ester in the ce of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium; Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Pre aration of the com ounds of formula II in the case where W= CH2 nX with X=O or S and L= |V18 with n=1 R17 and R18=H and ALK= CH2 3 based on Scheme 8 (DJ R1 / O fmoc (AA)w, /\/H X 0\ O HN , hi Y \ R10 0 R3 R2 0 R8 M 9 ("l R 7 R6All R H R4 H-(AA)W*NN TX 0% O HN R O R R H 10 O R8 W R9 N N O ("01 R 7 R1; R5 R4 H 0 o (AA)WH7 NN x o\ o HN HO \g R10 R\ 0 R3 R2 ] GRQ E NMN \0\ (iv) REL R5 R4 H N H x o o HN / \o (AA)W*NN Y S R o R R O H 10 3 2 O R8 R9 7 R6All R R H \O 4 Scheme 13 Step (i): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA; Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, dine; Step (iiil: coupling to ic anhydride; Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Schemes 12 and 13 describe the case L(|V18) with n=1, R17 and R18=H and ALK=(CH2)3 but they may also apply to other linkers L with RCG1=C(=O)ONHS, namely the case L(|V18) with n¢1 and/or R17, R18¢H and/or ALK¢(CH2)3 and the cases L(|V19) and L(|V20). They are given for a linker in the para on, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the ation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W= CH2 nX with X=O or S and RCG1=maleimido haloacetamido NHz N3 or c clooct ne namel linkers L |V21 to / O NW H WAAwiN/VN X a < i H E RzglGR7 l R5 R4 (0 RR6 (I) | N O R8 R9 N N O R5 R4H 0) R1 (I) H 2 ("NH ll R 0 R3 2 l R O R8 9 N N \o R7 l R R H 0) R6 5 4 0 o o o / MW H 2 X 0 O HN N /\/N i l t i GN El; NM \0 (inj 7R6l R5 R4H M*N/\/N X 0 O HN HN AA \ 2 < t R81 LGRQ R NVN7Rl R5 R4H 6 Scheme 14 Step (i): formation of the (thio)carbamate, the reaction is performed in the presence of a base such as, for example, DIEA; Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine.
Scheme 14 describes the case L(|V21) with n=1, R17 and R18=H and ALK=(CH2)5, the case ) with n=1 and R17 and R18=H, the case L(|V29) with n=1, R17 and R18=H and ALK=(CH2)4, the case L(|V31) with n=1, R17 and R18=H and ALK=(CH2)3 and the case L(|V33) with n=1, R17, R18 and R22: H, ALK=(CH2)3 and ALK’=(CH2)2 but it may also apply to other linkers L(|V21 to 34) with RCG1=maleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is ed for an iodoacetamido reactive group but may identically apply for a bromoacetamido ve group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. rly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of a (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W= CH2 nNHz and L= IV35 with n=1 R11= =J2=J3=J4=CH and ALK= CH2 3 based on Scheme 7 0 (UK WtR RKNR7 0 0 (int R5 HOWCA‘AW W WN\©VOON\H/ /0 0 (not Raw MOWMm / R3 k0R9 Scheme 15 Step (i): formation of the carbamate, the reaction is med in the presence of a base such as, for example, DIEA; Step (ii): deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis- (triphenylphosphine)palladium; Step {iii}: activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Pre aration of the com ounds of formula II in the case where W= CH2 nNHz and L= |V35 with n=1 R11=R14=R15=R16=H J3=J4=CH and ALK= CH2 3 based on Scheme 8 Scheme 16 Step (i): formation of the carbamate, the reaction is performed in the presence of a base such as, for example, DIEA in the presence of a suitable p-nitrophenylcarbonate reagent; Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine; Step {iii}: coupling to glutaric anhydride; Step (iv): activation of the carboxylic acid as a NHS ester by ent with DSC in the ce of a base such as, for example, DIEA.
Schemes 15 and 16 describe the case L(|V35) with n=1, R11=R14=R15=R16=H, J1=J2=J3=J4=CH, a para-benzylic alcohol and ALK=(CH2)3 but they may also apply to other linkers L with RCG1=C(=O)ONHS, namely the case L(|V35) with n¢1 and/or R11, R14, R15, R15¢H and/or J1, J2, J3, J4¢CH and/or an ortho—benzylic alcohol and/or ALK¢(CH2)3 and the cases L(|V36) and L(|V37). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially .
Pre aration of the com ounds of formula II in the case where W= CH2 nNHz with n=1 R11=R14=R15=R16=H J1=J2=J3=J4=CH and RCG1=maleimido haloacetamido NH2 N3 or c clooc ne namel linkers L |V38 to L |V151 / o NM H R / (AA)W’Nm0 / O O H o o HN T R10 Ff ORgRZNk 0i R 7 RTIJQ5$24R4 P3 AA)WH\©VOW a) RR7 Rl R5 R Hk RR7RNJ>84 Wk(AA)wiN\©VW Hm MN Scheme 17 Step (i): formation of the carbamate, the reaction is performed in the presence of a base such as, for example, DIEA in the presence of a suitable p-nitrophenylcarbonate reagent; Step (ii): deprotection of the Fmoc amine in the presence of a base such as, for example, piperidine.
Scheme 17 describes the cases L(|V38) with n=1, R11=R14=R15=R15=H, J3=J4=CH, a para- benzylic alcohol and ALK=(CH2)5, L(|V42) with n=1, R11=R14=R15=R15=H and J1=J2=J3=J4=CH and a para-benzylic alcohol, L(|V46) with n=1, R11=R14=R15=R15=H, J1=J2=J3=J4=CH, a para- benzylic alcohol and ALK=(CH2)4, L(|V48) with n=1, R11=R14=R15=R15=H, J1=J2=J3=J4=CH, a para-benzylic alcohol and ALK=(CH2)3 and L(|V50) with n=1, R11=R14=R15=R15=R22=H, J1=J2=J3=J4=CH, a para-benzylic alcohol, ALK=(CH2)3 and CH2)2 but it may also apply to other linkers L(|V38) to L(|V51) with RCG1=maleimido, iodoacetamido, NH2, N3 or ctyne. It is given with a para-benzylic alcohol but it may also apply to an ortho—benzylic alcohol. It is depicted for an iodoacetamido ve group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may cally apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W= CH2 nX with X=O or S "/52 Wlth n=1 R14=R15=R16=R17=R13=H J3=J4=CH and ALK= CH2 3 based on Scheme 7 (DJ R1 0 O VOWMAMNO" W" R 10 R, RTNLGa O RI O 9 E NMN o H 7 .. k Rl R5 R4 H (II) 6 N/\/ TX o o HN o o o \ R o R R W H 10 3 2 o R8 R, H0 (AA)W*N \o H R NMN 7 Rl R5 R4 H (In) \ //0 o o o OXNNNYX o\ R10 o R RHN W H 2 3 0 R R9 \0 N H E NMN \o O 7 Rl R5 R4 H Scheme 18 Step (i): formation of the (thio)carbamate by reacting with an amine in the presence of a base such as, for example, DIEA; Step (ii): deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium; Step {iii}: activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Pre aration of the com ounds of formula II in the case where W= CH2 nX with X=O or S "/52 Wlth n=1 R14=R15=R16=R17=R13=H J1=J2=J3=J4=CH and ALK= CH2 3 based on Scheme 8 0)] R1 / /O A H x o o HN O N/\/ T \ O R R H R 3 2 O 10 R8 R9 fmoci(AA)wiN N N \O H (ml R 7 R‘ R5 R4 H / O 0 E i NNH x o o HN H O N O " H t a; R, R2 pom MANN" 0 R NMN (ml 7 R; R5 R4 H / O 0 i o o N/\/ TN x o o o S o R W R R H 10 O R8 W R9 HO (AA)wiN N N o H (iv)‘ R 7 R‘ R5 R4 H // k H N/V Tx o o HN o o o \ o R R W H R10 3 2 l 0 R81 Re \0 (AA)wiN O R NMN O H 7 Rl R5 R4 H Scheme 19 Step (i): formation of the (thio)carbamate by ng 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, dine; Step {iii}: coupling to glutaric anhydride; Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Schemes 18 and 19 describe the case L(|V52) with n=1, R14=R15=R16=R17=R18=H, J1=J2=J3=J4=CH, a para-benzylic alcohol and ALK=(CH2)3 but they may also apply to other linkers L with RCG1=C(=O)ONHS, namely the case L(|V52) with n¢1 and/or R14, R15, R16, R17, R18¢H and/or J1, J2, J3, J4¢CH and/or an benzylic alcohol and/or ALK¢(CH2)3 and the cases L(|V53) and ). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. rly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W= CH2 nX with X=O or S and RCG1=maleimido etamido NHz N3 or c clooct ne namel linkers L |V55 to §|V68l H 2 qNM// o o N/VN X o\ o HN H \[g R10 R8 (AA)W*N N R JBRBQIZZ :\J\\©»R9N o o 7R6l R5 R4H (I) / /O o X o\ o R FE-IN (I) IQK H O 10 R8 W R9 P10 (AA)W’N N N O 7R; R5 R4H (I) / /o H , 0 OX NN X o\ o HN (I) H R10 R3 R2 kgRg N E R 3 (AA)wiN E NMN o 7R; R5 R4H / /O )k H O M N X o o HN O ©?O N/\/ Wof R83:\ O ‘ H R10 R3 R2 k R9 N H (AA)W*N N N o H R R 7R‘ R5 R4H (")1 Rl R5 R4 H / O NN X o o HN "20k go H 1; R10 R\ OR3R2 k\©LRg HzN (AA)W*N E NMN O 7 R‘ R5 R4 H Scheme 20 Step (i): formation of the (thio)carbamate by reacting with an amine in the ce of a base such as, for example, DIEA; Step (ii): ection of the Fmoc amine in the presence of a base such as, for example, piperidine.
Scheme 20 describes the case L(|V55) with n=1, R14=R15=R15=R17=R18=H, J1=J2=J3=J4=CH, a para-benzylic alcohol, ALK=(CH2)2 and ALK’=(CH2)5, the case L(|V59) with n=1, R14=R15=R15=R17=R18=H, J1=J2=J3=J4=CH, a para-benzylic alcohol and ALK=(CH2)2, the case L(|V63) with n=1, R14=R15=R16=R17=R18=H, J1=J2=J3=J4=CH, a para-benzylic alcohol, ALK=(CH2)2 and ALK’=(CH2)4, the case L(|V65) with n=1, R14=R15=R16=R17=R18=H, J3=J4=CH, a para-benzylic alcohol, H2)2 and ALK’=(CH2)3 and the case L(|V67) with n=1, R14:R15=R15=R17=R18=R22=H, J3=J4=CH, a para-benzylic alcohol, ALK=(CH2)2, ALK’=(CH2)5 and ALK"=(CH2)2 but it may also apply to other linkers L(|V55 to 68) with aleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a phycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W=C =0 0 and L= |V69 with R14=R15=R16=H J1=J2=J3=J4=CH and ALK= CH2 3 based on Scheme 7 R7 R'lq O R5 R4 H Scheme 21 M:esterification in the presence of a coupling reagent such as, for example, MNBA and a base such as, for example, DMAP and DIEA; M: deprotection of the allyl ester in the presence of a catalyst such as, for example, tetrakis- (triphenylphosphine)palladium; Step {iii}: tion of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Pre aration of the com ounds of formula II in the case where W=C =0 0 and L= |V69 with R14=R15=R16=H J1=J2=J3=J4=CH and ALK= CH2 3 based on Scheme 8 Scheme 22 Step (i): esterification in the presence of a ng reagent such as, for example, MNBA and a base such as, for example, DMAP and DIEA; Step (ii): ection of the Fmoc amine in the presence of a base such as, for example, piperidine; Step {iii}: coupling to glutaric anhydride; Step (iv): activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Schemes 21 and 22 describe the case L(|V69) with R14=R15=R16=H, J1=J2=J3=J4=CH, a para- benzylic alcohol and ALK=(CH2)3 but they may also apply to other linkers L with RCG1=C(=O)ONHS, namely the case L(|V69) with R14, R15, R16¢H and/or J1, J2, J3, J4¢CH and/or an ortho—benzylic alcohol and/orALK¢(CH2)3 and the cases L(|V70) and ). They are given for a linker in the para position, but may identically apply for the ortho or meta positions.
Similarly, they are given for a cryptophycin nd, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W=C =00 with R14=R15=R16=H J1=J2=J3=J4=CH and RCG1=maleimido haloacetamido NH2 N3 or c clooc ne namel linkers L |V72 to L |V85 O RBINMNKJGRQR 7 R: R5 R4 H 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): ection of the Fmoc amine in the presence of a base such as, for example, piperidine.
Scheme 23 describes the cases L(|V72) with n=1, R14=R15=R16=H, J1=J2=J3=J4=CH, a para- benzylic alcohol and ALK=(CH2)5, L(|V76) with n=1, R14=R15=R16=H and J1=J2=J3=J4=CH and a para-benzylic alcohol, L(|V80) with n=1, 5=R16=H, J1=J2=J3=J4=CH, a para-benzylic l and ALK=(CH2)4, L(|V82) with n=1, R14=R15=R16=H, J1=J2=J3=J4=CH, a para-benzylic alcohol and ALK=(CH2)3 and ) with n=1, R14=R15=R15=R22=H, J1=J2=J3=J4=CH, a para- benzylic alcohol, H2)3 and ALK’=(CH2)2 but it may also apply to other linkers L(|V72) to L(|V85) with aleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is given with a para- benzylic alcohol but it may also apply to an ortho—benzylic alcohol. It is depicted for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions.
Similarly, it is given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W=C =0 0 and L= V86 with R25=H and ALK=§CH2l7 Scheme 24 Step (i1: activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA; Step (ii): peptidic coupling in the presence of a base such as, for e, DIEA; Step {iii}: activation of the carboxylic acid as a NHS ester by treatment with DSC in the presence of a base such as, for example, DIEA.
Scheme 24 describes the case L(V86) with R25=H and ALK=(CH2)7 but they may also apply to other s L with RCG1=C(=O)ONHS, namely the case L(V86) with R25¢H and/or ALK¢(CH2)7 and the cases L(V87) and L(V88). They are given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, they are given for a cryptophycin compound, but may also apply to the preparation of other compounds of formula (I), especially D1-D19.
Pre aration of the com ounds of formula II in the case where W=C =0 0 with R30=H and aleimido haloacetamido NHz N3 orc clooct ne namel linkers L V89 to L V101 Scheme 25 WO 76998 2016/076603 M: peptidic coupling in the presence of a base such as, for example, DIEA; Step (ii): reduction of the azido by treatment with a reducing agent such as, for example, TCEP.
Scheme 25 describes the cases L(V89) with R25=H and ALK=(CH2)5, L(V93) with R25=H and ALK=(CH2)3, L(V96) with R25=H and ALK=(CH2)4, L(V98) with R25=H and ALK=(CH2)3 and L(V100) with R22=R25=H, H2)3 and ALK’=(CH2)2 but it may also apply to other linkers L(V89) to L(V101) with aleimido, iodoacetamido, NH2, N3 or cyclooctyne. It is ed for an iodoacetamido reactive group but may identically apply for a bromoacetamido reactive group. It is given for a linker in the para position, but may identically apply for the ortho or meta positions. Similarly, it is given for a cryptophycin compound, but may also apply to the ation of other compounds of a (I), especially D1-D19. s for re arin thec to h cin con'u ates The compounds of formula (III) might be prepared according to Scheme 26 starting with a cryptophycin payload of formula (II) and an antibody (Ab): Ab+RCG1-L-YWO 1 O 1 o o AbG|__YW " ° G7 p113 Scheme 26 in which: - Y represents (C1-Ce)alkyl-NR11 or (C1-Ce)alkyl-O or (C1-Cs)alkyl-S; or alternatively Y represents C(=O)O or O (C1-Cs)alkyl-C(=O)O; (C1-C6)alkyI/N% or alternatively Y represents (C1-C5)alkyl-triazole- like _ Y is positioned in an ortho (0), meta (m) or para (p) position of the phenyl nucleus; - R11 represents a hydrogen atom or a (C1-Cs)alkyl group; - L represents a linker positioned in an ortho (0), meta (m) or para (p) position of the phenyl nucleus, as d above; - RCG1 represents a reactive chemical group that is reactive towards a reactive chemical group present on the antibody, as defined above; - Ab represents an antibody.
Process for preparing the building blocks for the synthesis of cryptophycin compounds of formula I General synthesis of fragment A R1 MeO R R HO OMe (i) m (ii) (m) O o (w). 1 R1 R1 R1 HO / /0 ? PMBOWO ? PMBOW OH OtBu OH OtBu (3H (Viol Sakurai alcohol Scheme 27 Fragment A may be prepared in 12 steps starting from riate hydroxy esters with the aid of the steps detailed below: M:protection of the alcohol as a p—methoxybenzyl ether using p—methoxybenzyl alcohol activated as a oroacetimidate in the presence of a tic amount of acid such as, for example, p—TsOH ; Step (ii): reduction of the ester using a reducing agent such as, for example, m 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 ll catalyst; Step (vi): ection 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 the alcohols as silyl ethers using chlorotriethylsilane in the presence of a base such as, for example, ole; 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 le 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): oxidation of the benzylic 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.
Hydroxy esters are commercially available for R1=Me (CAS number [726579]), Et (CAS number [726040]) and iPr (CAS number [726041]). Preparation of phosphonium halides is described in W02011/001052. l 5 nthesis of fra ment B H2N BocHN BocHNkGRQ HO O Scheme 28 Fragment B may be prepared in 2 steps starting from appropriate amino acids with the aid of the steps ed below: M: protection of the amine by ent with Boc20 in the presence of a base such as, for example, TEA; Step (ii): saponification of the methyl ester using a base such as, for example, LiOH.
Amino acids protected as methyl esters are commercially available for R9=3-Cl, 4-OMe (CAS number [7048702]); 3-Cl, 4-OEt (CAS number [12569639]); 3-Cl, 4-OPr (CAS number id="p-12597099" id="p-12597099" id="p-12597099" id="p-12597099" id="p-12597099" id="p-12597099" id="p-12597099"
[12597099]); 3-Cl, 4-OMe, 5-F (CAS number [1213670-98—4]); 3-Cl, 4-OMe, 5-Cl (CAS number [1259701-22-];) 3-Cl, 4-OMe, 5-OMe (CAS number [12128204--2]), 3-Cl, 4-OH, 5-OMe (CAS number [12134267]); 3-Cl, 4-NH2 (CAS number [12136720]); 2-Cl, 3-Cl, 4-NH2 (CAS number 07-8-];) 3-Cl, 4-NH2, 5-F(CAS number [1213400--8;]) 3-Cl, 4-NH2, 5-Cl (CAS number [12134808]).
General 5 nthesis of buildin block AD1 w,are,Wt NHFmoc WY9(in)W?/ RwENHFmoc R;0NHFmoc R R 7 7 \ / 0 AD1 R# R NHz Scheme 29 Fragments A and D allow the preparation of building block AD1 with the aid of the steps detailed below: M: esterification of fragment D with fragment A in the presence of a coupling reagent such as, for example, MNBA and a base such as, for example, DMAP and DIEA; Step (ii): reduction of the aldehyde into an alcohol using a reducing agent such as, for e, sodium trimethoxyborohydride; Step {iii}: activation of the alcohol as a mesylate by treatment with esulfonyl chloride in the presence of a base such as, for example, TEA; substitution 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 5 s of buildin block AD2 R1 0\ OH \ / (i) 7 O ‘ O?/WgNHFmoc O\ OH O R, R1o A D (iii) HO O\ O O ‘7 HO Scheme 30 nts A and D allow the preparation of building block AD2 with the aid of the steps detailed below: M: esterification of fragment D with fragment A in the presence of a coupling reagent such as, for example, MNBA and a base such as, for example, DMAP and DIEA; Step (ii): reduction of the aldehyde into an alcohol using a reducing agent such as, for example, sodium hoxyborohydride; Step {iii}: deprotection of the amine by treatment with a base such as, for example, piperidine. l synthesis of building block AD3 R1 R 0 OH s PMBOW PMBOM R ' " / o 6 PMBOM + L s L o\ 5 i Ea, "#me R R\ OH R6 E3 l\‘lFmoc Ea NH 7 R6 7 Re; Sakurai alcohol D Scheme 31 Step (i): esterification of nt 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): ection of the amine by treatment with a base such as, for example, piperidine.
General 5 nthesis of buildin block BC R3 R2 BocHN RE’EORHN \ R9 (ii) 0 90) BoggHNl R9 R5R ?HOR Scheme 32 Fragments B and C allow the preparation of building block BC with the aid of the steps detailed below: M: peptidic coupling of fragment B with fragment C in the ce of coupling ts 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 5 nthesis of an alternative buildin block BC R2 BocHN HolGRQ(i) K©~? (II).. lg \ 0 NH2 + N R5R ORR4 H Scheme 33 nts B and C allow the preparation of an alternative ng block BC with the aid of the steps detailed below: Step (i): peptidic coupling of fragment B with fragment C in the presence of coupling reagents such as, for example, HOBt and EDC and a base such as, for example, DIEA; Step (ii): deprotection of the Boc group in acidic conditions using, for example, TFA; Step (iii): formation of the acrylamide by treatment with acryloyle chloride in the presence of a base such as, for example, DIEA; Step (iv): saponification of the methyl ester using a base such as, for example, tBuOK.
Preparation of the linker precursors LP LP may be one of the following; LP1 to LP101 are described using L amino acids but may also apply to D amino acids. They are given for w=2 but may similarly apply to w>2 by repeating as many times as required the peptide coupling step. 0 R O RbeOC-ALKXNWNQkOH27 H O R,: LP1 28 prepared according to the scheme below: R27 0 R o R o Fm°CHNJY :? . 27 .. 27 o\ (I) H (n) ’ FmocHNW yLOH ?’ HZNJY JonH 0 HZNEJLOH E 3 0 0 O R25 R25 o o o o (III) VOJ‘ALKJLOH ?’ VOJLALKiLO/Q (N). o 0 R2, o VOJLALKiN/‘YNQKOHH H 0 R58 M: peptide coupling between Fmoc—L-amino NHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH e in the presence of a base such as, for example, dine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 on is performed at RT in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; the diacids monoprotected as allyl esters are commercially available for n=2 llyl succinate) or may be prepared by transesterification of the methyl or tert-butyl monoesters, which are commercially available for n=2 to 6.
ALK-(OCHZCHZMANWN?OH27 H R28 LP2 prepared according to the scheme below: R27 0 R o o b 27 27 o (I) H FmocHN/Sf QLOH (n) H Fm°CHN N % o HZNJ?f QLOH O / OH 0 O RES 0 R28 O 0 R O vOiALK/fONiJLNWNQLOHHH M: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium onate; Step (ii): deprotection of the amine; the reaction is performed in a polar t such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar c solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, ted DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; the PEG diacids monoprotected as allyl esters may be prepared ing to the scheme below: when ALK=CH2C_H2 Kojv + Hod/VOJ?H L VOJEH L HOMONO?F o o J0") HOMO/\?ovigfo?/ 0 «? \oMoA/kov?fo?/ L\OMOMOH O O VOMOA’EOWOH VOMONOWOY L when ALK¢CH?g o F AOJOKALKiHaI + HfOAi/OTHP L $o?AL?oA/komp L HOXALK{*OAiO?F Hal = Br, I O O HOJKALK£OA$OWO¥ ? \okALKEo ,OWOY L\oiALK%O OH 0 (I) l (v) O O VOXALK o O O L VOXALK o O OH Step (i): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic solvent such as THF or DMF by treatment of an unsaturated protected acid with the alkoxyde ted by the action of sodium in catalytic amount; Step (ii): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid. In the latter case, trifluoroacetate of the alcohol function present on the structure may 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 performed in a polar aprotic solvent such as MeOH, by treatment with trimethylsilyldiazomethane; Step (iv): saponification of the methyl ester; the reaction is performed in a mixture of polar solvents such as a 0 mixture in the presence of LiOH; Step (v): protection of the carboxylic acid as an allyl ester; the reaction is performed in a polar aprotic solvent such as DCM in the presence of allyl l, a ng agent such as, for example, EDC and a base such as, for example, DMAP; Step (vi): elongation of the PEG chain; the reaction is med in an anhydrous polar aprotic solvent such as THF or DMF by treatment of a halogenated ester with the alkoxyde of the PEG diol monoprotected as a THP ether. The preparation of this type of monoprotected PEG diol is well bed in the literature: see, for example, d A., et al., Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou E.G., et al., Tetrahedron 2003, 59, 9083-9090.
The ng PEG diols are commercially available for i=3 to 12.
RbeocYALKim?’Y\H?OH soH o Rga LP3 prepared according to the scheme below: R27 0 R27 O FmOCHNW bO 0' H (H)ll —‘ FmocHN/'\[f JOHN ‘> N O HZN;:~LOH 0 R O 2:8 HNgofHdLOH 8380H 0 (m SOH o /\/OW/kALKiOH ?> /\/OW/kALKQ (iv) NOW/kALKiNijN\JLOH M: peptide ng between Fmoc—L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the ce of a base such as, for e, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar solvent such as a mixture DMF/H20 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 performed at RT in a polar aprotic solvent such as a sCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; the sulfo diacids monoprotected as allyl ester may be prepared ing to the scheme below: H038 HO 5 o HOW/AmifiN WOWALK7:N WOVALK€N WOW/LALKJLOH3 0), (n).. (m) 0 o alternative when ALK=CH2C_H2 Br Br SAC SOSH SOSH mm LWWW ?/NOM L/NOM L"WOMOH. .
N N N N O O O O O 0 Step (i): tion of the carboxylic acid as an allyl ester; the reaction is performed in a polar aprotic solvent such as DCM in the ce of allyl alcohol, a coupling agent such as, for example, EDC and a base such as, for example, DMAP; Ste ii : or-sulfonation of the ylic acid; the reaction is performed at 75°C in a polar aprotic t like 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 performed by treatment with sodium hydroxide; Step (iv): substitution of the bromide by a thioacetyle; the reaction is med at -20°C in a polar aprotic solvent such as THF using thioacetic acid in the presence of a base such as, for e, DIEA; Step (v1: formation of the sulfonic acid moiety; the reaction is performed at RT by treatment with hydrogen peroxide and acetic acid.
Cyano carboxylic acids are commercially available for n=1 to 12. q 0 / R27 O N\ALKANJYNQkOH O H o R2 LP4 28 prepared according to the scheme below: R27 0 R27 o R27 o FmocHN/Koro‘b —’HN?0 FmocHNJiD‘fNRgLOH ?’ HEN/KOWNELOHH (..) H O O o W/ / i V r N\ALK a IV OH N\ALK o/N / / o o o / R27 o N\ i N / ALK "W QkOH o o R; M: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO e in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for e, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 performed in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; the maleimido carboxylic acids are commercially available for n=1 to 12. dNALKxOMRALKiNJVNQLOHO R O 27 H \\ H 0 (‘3‘ R LP5 prepared accor mgd' t0 the SC emeh below R27 0 R o R o Fm°°HNW D/ (i) 27 H (ii) 27 o H —> FmocHNW JOHN M» N o HZN/‘?f QLOH O HZNEJLOH Fag, o Fag, o o iiNAl-K'dONP‘ALKJLOH E»o o l NALK'%O/\/)P\ALKJ\O/Q (iv) o O QN/ eALKHo/\/)PALKJYNQLOH M: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the on is performed in a polar t such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is med in a polar solvent such as a DCM/MeOH mixture in the ce of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar aprotic solvent such as DCM by treatment with NHS in the presence of a ng agent such as, for example, supported DCC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; the maleimido acids are cially available for K’=CHZCH2 and i=1 to 7 and may be othenNise prepared according to the scheme below: when ALK=ALK’=CHgC_Hg and i>7 O\ /O $0JOV/ HOW MMVM éiwovo0M0% &omMOH when ALK¢CH?g and ALK’=CHgC_Hg AOJ‘LALK7HaIo o i(v) + "(0% momp"?/Ro?ALK{OMOm \/\OH \/\OHTP ?»WALK{77W0 HaI=Br,l (ii) IOU/00 gT/VOVOJrALKJLOH ‘L CNOVVoirALKioJF when ALK=CH2C_H2 and ALK’¢CHZC_H2 OTHP (vi) f HOALK'OH OTHP OTHP (viii) OTHP —>Ho7ALK' Bno7ALK' (vii) FOA?OWO?/(X‘7 BnoALKJFOyoWW YBnoiALK'{7OAi/OH ii) Josgo O o dNALK'{7OAi/OWO% ?,E) O ifNALK-jfoA/kOWOHDE) o WgC—Hz OTHP?)(vi) (:SfEOAfomp (:iLiK'Ho OH (viii) HOiALKijAfOTHP BnOALKJFOAJFOTHP OiALK Hal lUX) 0\ 0 HI Br I BnO*ALK'%OAi/I OH Ho7A7I_'OKJFW0HALK7f O?/eBno7ALKjfoA/k ALKj‘ ?/<—a 0 (iv) (ii) 0W0 O O QNALK'iowiqALKTfOYL/\ O QNALK'JrOAleWKfOHo O 0 Step (i): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic solvent such as THF or DMF by treatment of an unsaturated protected acid with the alkoxyde generated by the action of sodium in catalytic amount; Step (ii): Mitsunobu reaction on maleimide; the reaction is performed in a polar aprotic t such as THF by treatment of ide by the PEG hydroxy acid in the presence of PPh3 and DIAD; Step (iii): deprotection using a solution of hydrochloric acid (for example solution in e) or of trifluoroacetic acid; Step (iv): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic solvent such as THF or DMF by treatment of a halogenated ester with the alkoxyde of the PEG diol monoprotected as the THP ether. The preparation of this type of monoprotected PEG diol is well described in the literature: see, for example, Richard A., et a/., Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou E.G., et al., Tetrahedron 2003, 59, 9083-9090; Step (v): selective deprotection of THP ether; the reaction is performed in a polar protic solvent such as MeOH using a catalytic amount of acetyl chloride; Step (vi): activation of the alcohol as a te; the on is performed 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 med in a polar aprotic solvent such as DMF in the ce of a base such as for example sodium hydride; Step (viii 1: protection of the alcohol as a benzyl ether; the reaction is performed 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 solution of hydrochloric acid (for example solution in dioxane); Step (x): deprotection of the alcohol by enolysis in the presence of a catalyst such as, for example, palladium on carbon.
The starting PEG diols are commercially available for i=3 to 12. The starting ALK diols are commercially available for n=1 to 12.
/ O‘HJ’NVN?OH ?N\ALK O R 0 LP6 prepared according to the scheme below: FmOCHNE'LgODbl" R27 R27 O H H " HNWNEQLOH o HNFmocHNJ?fQLOH O 0 RES .40"V, W: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a F/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the ylic acid as a NHS ester; the reaction is performed in a polar solvent such as a mixture DMF/H20 by ent 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 performed in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of —amino acids are commercially available; the maleimido cyclohexanecarboxylic acid is commercially available for ALK=CH2 and may be othenIvise ed according to the scheme below: When n=2 to 4 o o (i) (II) (III) OH 9 OH H OH H o CszN CszN n, HZN X n n n with n'=n-1 o o iUV) o o O O o / OH (VI) U / /?o o N N (v) / n / n HEN K o 0 When n>4 C?‘"C?xmn?xV' ?x M: hydrogenation; the reaction is performed at 60°C in acetic acid in the presence of a catalyst such as platinum oxide under pressure of hydrogen, for example 60 psi; Step (ii): tion of the amine; the reaction is performed in a polar protic t such as a dioxane/HZO 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 performed 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 performed by hydrogenolysis in a polar protic solvent such as MeOH in the presence of a st such as, for example, palladium; Step (v1: introduction of the maleimido moeity; the reaction is performed in a polar aprotic solvent such as DMF by ent with maleic ide in the presence of NHS and a coupling reagent such as, for example, EDC; Step (vi): ection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid; Step (vii): palladium-catalyzed coupling; the reaction is performed in a polar aprotic solvent such as THF by treatment with the bromo-ALK-cyano derivative in the presence of lithium chloride, zinc and hylchlorosilane and a catalyst such as, for example, tetrakis(triphenylphosphine)palladium.
The suitable ALK-cyano derivatives are commercially available for n=1 to 12.
RMWN0 R 27 u" \ SOHH 0 R \o 3 28 LP7 prepared according to the scheme below: R27 R27 R FmocHN/‘wf bO lo (i) H —, ?. N o FmocHNkrHdLOHN O O/ HZNEAOH O '42ng QLOHR: \\ N/ALKAOH LO\SZDALKWJkOH?(iv) Q SOH O jiALKjAo0:90,) @MwwSOH M: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the ce of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for e, piperidine; Ste iii : or-sulfonation of the carboxylic acid; the reaction is med at 75°C in a polar c solvent like 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 a NHS ester; the reaction is performed at RT in a polar solvent such as a mixture DMF/HZO by ent with N,N'-disuccinimidyl carbonate in the ce of a base such as, for example, DIEA; Step (v): peptide coupling between the dipeptide and the NHS ester; the reaction is performed at RT in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; the maleimido acids are commercially available for n=1 to 12. kaVMOH LPg prepared according to the scheme below: R o R o Fm°°HNJWT bo (i) 27 H (n).. 27 H —> FmocHN/‘?f QLOHN a :NW €LOHN o / H2N\)LOH o Ria R: R27 O l or BrQLN/‘?fNQLOH 0 R5, M: peptide ng between Fmoc—L-amino acid-ONHS and o acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture 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 performed in a polar aprotic solvent such as a sCN mixture.
NHS esters of Fmoc—L-amino acids are commercially ble; N-succinimidyl bromo- and iodo- acetates are cially available. 0 0 O IorBerNR19ALKAN R27 N%0HH 0 R28 LPg prepared according to the scheme below: R o FmOCHN/klfo(3me 27 H (ii) H o HFmocHN/Sf JOH‘) HNWNdL0H RES 0 R28 O O RWHN ALKJLOH | or Br Ior BrQL:Q; AL iv ( ) (iii) ALK 0/"? | BQLor r NR1?ALK0"]?ngULOH M: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the on is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): coupling and tion of the carboxylic acid as a NHS ester; the coupling of N- succinimidyl bromo- and iodo-acetate with the amino carboxylic acid is performed at RT 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): e coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; N-succinimidyl bromo- and iodo- acetates are commercially available, the amino carboxylic acids also for n=1 to 12 and the N- ated amino carboxylic acids for n=1 to 7.
O R O - H O R23 LP10 8 prepared according to the scheme below: it; 0 R" 0 R" o (i) H H Fm°CHN ?. HN/Kf QLOHN H N —, N o FmocHN/Kf QLOH 2 O HN\)L ‘ E ‘ 3 2 OH 0 0 2 R28 IorBrwop — O \ O NHR/K/OliALKAOH20 I or B r , JNRzo/NO\AILKJ\o/N' (IV). (iii) l IorBrQ?RZO/NO\KLKXN%(QQLOHHR M: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as a NHS ester; the coupling of N- succinimidyl bromo- and iodo-acetate with the amino ylic acid is performed 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 performed in a polar aprotic solvent such as a sCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; N-succinimidyl bromo- and iodo- e are cially ble; in the case where ALK=CHZCH2 and R20=H, the amino PEG ylic acids are commercially available for i=1 to 6 and othenNise may be prepared from tertbutyl acrylate and the corresponding amino-PEG-alcohol; in the case where ALK¢CHZCH2, they may be prepared according to the schemes below: in the case where ALK= CHgC_H2_and R?? o o o HOMOh/OVNHZ (I). HOMOb/OVNHBOC (II).. XOMOb/OVNHBOC J (iii) 0 0 HOMOWOVNHRZD ‘L XOMOWOVNRZDBOCiv in the case where ALKiCHgC_Hg in the case where Ra? XOiALKiHaIo 0 + O?i\/\‘N a") XOiALKioh/OVN (iv) \OkALKiHaI +HO+\/0\L\/\N i o LY) ALK’OWOMN ?’. \okALKio+\/o?\/\NHZ HHHH 0 0 0 0 (i) O O 0 HOiALK’Oh/OMNRZOBOC B(vi) \OJLALKwd’VOVNRmBoc (iii) \okALKi?’Vodr/WHBOC 1 (iv) HokALKiob/OVNHRZO Step (i): protection of the amine; the reaction is performed 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 performed in a polar aprotic solvent such as DCM by treatment with tert—butanol in the presence of a ng reagent such as, for example, EDC and a base such as, for example, DMAP; Step (iii): alkylation of the en atom; the reaction is performed in an anhydrous polar aprotic t such as THF by treatment with a base such as sodium hydride in the presence of a reagent bearing a leaving group such as a halide; Step (iv): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of oroacetic acid; Step (v): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic solvent such as THF or DMF by treatment of a halogenated ester with the alkoxyde of a henone-imine-PEG-alcohol ted via the action of sodium hydride or potassium naphthalenide as described in W02007/127440; Step (vi 1: saponification of the ester; the reaction is performed by reacting the ester with LiOH in the presence of H20.
The amino-PEG-alcohols are commercially available, for example for i=3, 4, 7, 8 or may be prepared from the PEG diols, which are commercially available for i=3 to 12, according to the procedure described in US7230101. The protection of the amine function with benzophenone may be performed by opic dehydration in the presence of a Lewis acid such as BF3 etherate.
O R O M 27 H o M? ZQXNJYNQkOH lorBerNRknw/M3 H O R; LP11 21 4 28 prepared according to the scheme below: M: peptide coupling between Fmoc—L-amino acid-NHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine Fmoc; the reaction is performed in a polar solvent such as a DCM/MeOH e in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as a NHS ester; the coupling of N- succinimidyl bromo- and iodo-acetate with the amino carboxylic acid is performed at RT in a polar c 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 performed at RT in a polar aprotic solvent such as a sCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; N-succinimidyl bromo- and iodo- acetate are commercially available; the amino-(hetero)aryl-carboxylic acids are commercially available, like for example obenzoic acid, 6-amino—3-pyridine ylic acid, 5-amino— 2—pyrazine carboxylic acid, 2—amino—5-pyrimidine carboxylic acid or 6-amino-1,2,4,5-tetrazine carboxylic acid.
LP12 prepared according to the scheme below: R27 R27 R27 /'\/0\5‘5 (I). H H FmOCHN ’ ll FmocHN/'\‘/NdL0H \‘/NdL0H / HN 2 \AOH 0 R28 0 R28 o o \ALKJKH/'\H/NEQKOHH o R: Step (i): peptide coupling n Fmoc—L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar c solvent such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (iv): e coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc-L-amino acids are commercially available; the amino carboxylic acids are commercially available for n=1 to 11. 0 R27 O FmocHNJfV )ALKkNWNQkOHH0' 0 R28 LP13 prepared according to the scheme below: R27 R27 FmOCHN/kl/O()beHFmocHNJYN¢LOH( \FN¢LOHH H 0 R28 0 R28 i (m) FmocHN/NOJiALK N/No))iJOL N (')IV OH ALK o O R o , H FmocHNNOJALKJkN/[YNQLOH 0 RE M: peptide ng between Fmoc-L-amino acid-ONHS and o acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar aprotic solvent such as DCM by ent with NHS in the presence of a ng agent such as, for example, supported DCC; Step (iv): e coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture.
NHS ester of Fmoc-L-amino acids are commercially ble; in the case where ALK=CHZCH2, Fmoc-protected amino PEG carboxylic acids are commercially available for i=1 to 6 and othenNise may be prepared from tert—butyl acrylate and the corresponding amino-PEG-alcohol; in the case where ALK¢CHZCH2, they may be prepared according to the schemes described for linker precursor LP10. Protection of the amine function with a Fmoc group may be ed by treatment with FmocOSu (CAS number [829111]) in the presence of a base such as, for example, DIEA. 0 R27 O o R: LP14 28 prepared Ing to the scheme below.. _ R27 O O FmOCHN/Sfoblo R" 2’ (I) (u) H o FmocHN/HfNQkOHH HEN/KW QkOH o/ HZNEJLOH 0 R35 0 R35 M: peptide coupling between Fmoc—L-amino acid-ONHS and L-amino acid; the on is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): uction of the azido group; the reaction is performed in a polar solvent such as an acetone/H20 mixture by ent with sodium azide; Step (iv): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 performed in a polar aprotic solvent such as a sCN mixture.
NHS esters of Fmoc-L-amino acids are commercially available; bromo carboxylic acids are commercially available for n=1 to 11. 0 R27 O XVOI H N3 ALKikN/‘?HNVLLOH 0 R28 LP15 prepared according to the scheme below: R27 0 R27 o R27 o FmOCHN/Voj? —o’ FmocHNJYNQLOH0 H (..) HZNJYNQLOHH O 0/ HZN\EJLOH 0 R28 0 R28 \ALKJ\OH?>Na/F\/O\ALK. i. (iii) (W). o/N 0 R27 o Na/NQALKJKHJWNQLOH. H o Rig Step (i): peptide coupling between -amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO e in the presence of a base such as, for e, sodium bicarbonate; Step (ii): deprotection of the amine; the on is performed in a polar solvent such as a DCM/MeOH mixture in the ce of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 performed in a polar c solvent such as a DCM/CHsCN mixture.
NHS esters of Fmoc—L-amino acids are commercially available; azido PEG acids may be prepared according to the schemes below: when 20_H2 %o%W 4’ KOMWIW "'4 M when ALK¢CH_2CH2 \00ALKHal OTHPS()v (iv) \oiALKfOA/kOTHP \ XALK OH 0 if Rf.0 Hal: Brl J0" (vi) jL (iii) \ N )1 OMs ALK ALK 3 ALK%OW Step (i): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic solvent such as THF or DMF by treatment of an unsaturated protected acid with the alkoxyde generated by the action of sodium in catalytic amount; Step (ii): activation of the alcohol function; the reaction is med in a polar solvent such as DCM using methanesulfonyl chloride in the presence of a base such as, for example, TEA; Step (iii): substitution of the mesylate by an azido group; the reaction is performed in a polar solvent such as an acetone/H20 mixture by treatment with sodium azide; Step (iv): deprotection using a solution of hydrochloric acid (for example solution in dioxane); Step (v): elongation of the PEG chain; the reaction is performed in an anhydrous polar c solvent such as THF or DMF by treatment of a halogenated ester with the alkoxyde of the PEG diol monoprotected as a THP ether, alkoxyde 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 E.G., et al. Tetrahedron 2003, 59, 9083-9090; Step (vi ): saponification of the methyl ester; the reaction is performed at RT in a mixture of polar solvents such as a THF/HZO mixture in the presence of LiOH.
The starting PEG diols are cially available for i=3 to 12; the bromo methyl esters are commercially available for n=1 to 12. prepared ing to the scheme below: R27 /C/) R" O O /Bf0 (i) H 2’ (n) H O FmocHN ‘ N\EJLOH HzN/‘\ll/ \EAOH 0/ ~LOH 0 R38 0 Rig o Cfoi i O O N\ O O O O O O NJkALK-HWR22 (4/ O ALK OH O O NkALK'iNRikALKAOH (iv) NkALK'iNRgALKAO/p H —.\\ ?’ \\ 0 ()V (iii) M: peptide coupling between -amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): cyclooctyne coupling; the reaction is med in a polar t such as DCM; Step (iv): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 performed in a polar aprotic solvent such as DCM.
NHS esters of Fmoc—L-amino acids are commercially available; cyclooctynes are commercially available for n’=1, 2, 3 and 5; diacids monoactivated as NHS ester are commercially available for n=1 to 3 and for n=4 to 10 they may be prepared by activating commercially available s with NHS in the presence of a coupling agent such as, for example, DCC and a base such as, for example, DMAP.
N ALK' NR22ALHOVkN/YNdkOH LP17 prepared according to the scheme FmOCHN/ZkgoOb (—o’ FmocHN‘)?/BKJLOHR27 0 ("l H HNWNEJLOH HZNJOH o REa g\OJ\ALK%ON?lkORo 1.(iii) o O i o O\ N ALK'iNHRZZO \O NJRLK'NRZALKAo"/#(10H;Vl OMAR LK' NR22OALK40"4‘IlLo/N \l —, ('lVI 2. (iv) if R = [err-butyl O R = H for ALK=CHZCH2 HO R = ten-butyl for other ALK OHS ° " H? LKNR22iiALK40"/#LNHN/I?f OH o R; M: e coupling between Fmoc—L-amino acid-ONHS and o acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for e, piperidine; Step (iii): cyclooctyne coupling; the reaction is performed in a polar solvent such as DCM; Step (iv): deprotection using a solution of hydrochloric acid (for example solution in dioxane); Step (v): activation of the cyclooctyne-PEG carboxylic acid as a NHS ester; the reaction is performed in a polar c t such as DCM by treatment with NHS in the presence of a coupling agent such as, for example, EDC; Step (vi 1: peptide coupling between the dipeptide and the NHS ester; the on is performed in a polar aprotic solvent such as DCM.
NHS esters of -amino acids are commercially available; ctynes are commercially available for n’=1, 2, 3 and 5; the PEG diacids mono-activated as NHS ester are prepared according to the scheme below: when ALK=CH2C_H2 40% O + Wt HOMONOWOH 1 (iii) g‘OfK/TONOWOH when ALK¢CH?g o/EOiALKeHaE + HEOAJEOTHF".('V?/EOiALKWOTHP i. HoiALKJfo?OW/EFF Hal=Br | )V EM MME0"EO"O‘E EALEKE0"EO"OE ‘—m\OEALKEokaH o E gwEEyE M:elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic solvent such as THF or DMF by treatment of an unsaturated ted acid with the alkoxyde generated by the action of sodium in catalytic amount; Step (ii): deprotection using a solution of hydrochloric acid (for e solution in dioxane) or of oroacetic acid; Step (iii): mono-activation of one acid as a NHS ester; the reaction is performed 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 e, DMAP; Step (iv): tion of the PEG chain; the reaction is performed in an anhydrous polar aprotic solvent such as THF or DMF by treatment of a halogenated ester with the alkoxyde of the PEG diol monoprotected as a THP ether. The ation 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 E.G., et al., Tetrahedron 2003, 59, 9083-9090; Step (v): tion of the carboxylic acid as a methyl ester; the reaction is performed in a polar aprotic solvent such as MeOH, by treatment with trimethylsilyldiazomethane; Step (vi): saponification of the methyl ester; the reaction is performed in a mixture of polar solvents such as a THF/H20 mixture in the presence of LiOH; Step (vii): activation of the acid as a NHS ester; the reaction is performed 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 e, DMAP.
The starting PEG diols are commercially available for i=3 to 12.
RbeOC-ALK'ANJ\fNQENR18ALKNHR1727 H LP18 is prepared according to the scheme below: R27 /0 R 0 FmocHN/'\11/ 27 (.) H o\ I N —O> WNQLOH —> NR187ALK7NR17Boc \JL 5 NR1B77ALKNRBBC U 0 H2N 0 R: FmocHNR/'7\(1)‘/NR 0 OH 28 RES Rim) o 0 R27 0 R27 0 , N/'\11/NHQkNR?WALKNRBOGé HZNWNQLNRW77ALKNR800H VOJLALK" 0 R58 VOiALK'iLO/Q 0 0 o R58 To") 0 r 0 \/\o\ ALK'1"W LK7NHR17N o o o R;8 VOJLALKJLOH M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar aprotic solvent such as DCM by ent 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 performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of -amino acids are commercially available; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other. The diacids otected as a||y| esters are commercially available for n=2 (monoallyl succinate) or may be ed by transesterification of the methyl or tert—butyl monoesters, which are commercially available for n’=2 to 6.
ALK'dON?kafN?NR8ALKNHR17 LP19 prepared according to the scheme below: FmocHN/Sf:N FmocHN/SfatN —H>FmOCHN/'?fNHQLOH m: i NRKALKiNR?BOC O Rés Jok 0 R27 R27 0 ALK'JroAing/‘YNJ)3 NRKALKiNR?Boc‘?HzN/Sf(iv) "Qt NR1BOC o Rga 0 Rig mo "HMO,\ if N lo) \ o 0 R27 o 1(iii) kALK'?LO/WAm/‘Y QLNRHTALKiNHRWH O o N "0 ALK’OMOH\ o Rgs M: peptide coupling; the reaction is performed in a polar solvent such as a F/H20 mixture 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, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a OH e in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar c 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 performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of -amino acids are commercially available; the mono-protected diamines are cially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the PEG s monoprotected as a||y| esters are prepared according to the schemes described for linker precursor LP2. 803H 0 A R ALK'lkN/l\FNQkNRwALKNHR1727 H RbeOC LP20 is prepared according to the scheme below: R27 0 R 0 FmocHN/Sf (1). 27 H o\ / F HN NQLOH —> FmocHN NR1B"ALKNR Bot: OMNRiALKiNR Bee moc 18 17 0 \i 5 O/ H2N 23 Rj:ii) 303H 0 R27 0 R27 0 WOWKKALKJkNH/Kll/NHQLNR?ALKNRBOC<—O\H2N/'\H/NQLNR§ALKNR800(w) H o 0 R38 AOijffK-EOQ 0 R28 imo o o SOH 1"") / YNRKALKiNHR? AOT‘AALKAOH M: e coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for e, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar aprotic solvent such as DCM by ent with NHS in the ce of a coupling agent such as, for example, EDC; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is performed in a polar c solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a on of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the rotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the sulfo diacids monoprotected as a||y| ester are prepared according to the schemes described for linker precursor LP3.
N 1 H \ 9 ' "JVN$ 6/ ALK ; NREALKiNHRW R§8 LP21 prepared according to the scheme below.. .
R27 0 OH —‘ R27 O / H FmocHN O‘N (I). l HzN?OH FmOCHN OH FmocHN NQLNRiALKiNR 800 NRm"ALKNRBoc i 18 17 o 0/ o Rgs 0 Jun ?ll/\ALK?ka/NQLNRBALKNRnBOc‘?HzN/[W/ $NR18WALKNR1BOC0 R27 0 R o / 27 H (iv) H / O \\ o o R3 0 R2; i M28 CECEALKALONQ /0 To") gMLKAH/‘YNQLNRWALKNHRN/ 0 R27 o /0 H g‘ALK'JLOH/ O o o R: M: e coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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, TEA.
Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the ylic acid as a NHS ester; the reaction is performed 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 med in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the maleimido carboxylic acids are commercially available for n=1 to 12.
// O R O Q ALKlkH/‘VNQkNRmALKNHRW27 H NiALK" oi Mo .
O O Rig LP22 prepared according to the scheme below: R27 0 R 0 R27 0 FmocHNWO‘D (—2)) WNQLOH m:/ 27 i H H i JYNEQLNRWALKiNR?BOC O H2N\;)LOH 0 RES 0 R28 0 Jan KEN 0 R 0 R27 O JL 27 H (iv) H -- N , \ , N ALK %O/\/)P ALK NW LKiNRWBOC(— 3 NRWALKiNR?Boc O HzN/KH/ H O o R3 o \ o R’£2; 0 28 l NHALKHo/V'rOxALKJLOjN? l \ o To") 00 / o R27 0 H l N’ALK"(O/\%O\ALK.JLOH N ALK do")?.. \ JL l , ALK, N/Kf QLNRWALKiNHRWN E: \ H O M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 e 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, TEA; Step (ii): ection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH e in the ce of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hydrochloric acid (for example on in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the rotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the ido PEG acids are commercially available for ALK=ALK’=CH2CH2 and i=1 to 7 and are othenNise prepared according to the s described for linker precursor LP5. 0 R27 O C// m/KF ?NRwALKNHRwN N\ 0 R28i // ALK' LP23 prepared according to the scheme below: R27 0 R 0 R27 0 FmocHN/k‘/Ob —O> FmocHN/HfNQLOHm FmocHNWNyLNRWALKiNRWBOCo 2’ H H (i) (i) ‘8 ‘7 0/ H2N\2LOH 0 R2; o Rge O R O 27 2’ nu . 0 <~> nu / NW ; NRWALKiNRWBoc‘—O HZN ‘ NRfAl—K’NRWBOC N‘ 0 0 0 I R28 0 Rée l V g GAO El <4 r) o 'ALK' 0 R27 o H To") 0 O EQkNRwiALKiNHR17 O HN/KW \ALK'OLOH N\ O R3 CE/ / ALK' 28 O M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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 ide and a base such as, for e, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): tion of the carboxylic acid as a NHS ester; the reaction is performed 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): e ng between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hydrochloric acid (for example on in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the maleimido cyclohexanecarboxylic acid is commercially available for ALK=CH2 and may othenIvise be prepared according to the schemes described for linker precursor LPG.
CN/ALK#H II ; NREALIeNHR17 \\ SOBH 0 R58 LP24 prepared according to the scheme below: R27 0 R 0 R27 0 FmocHNWO‘b 0—2)» FmocHNWNkaHL FmocHNJYNQkNRTALKiNRWBocg/ . 27 H . H NRmiALKiNRWBoc : 0/ HZNEJLOH 0 RES 0 R58 R38 jun..
GOALKWANJYNHdeRWALKiNR?BOC<—O N NRWALKiNR?Boc SOH OiwiRie \o\ :7? 0 Rée Q/ALK0:15:30," SALALKijNWN?NRfALKiNHR."R27 lo SOH OCEOALKWACM(iii))QALKJLOHSOH O M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture in the ce of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a ng reagent such as propylphosphonic anhydride and a base such as, for example, TEA; Step (ii): deprotection of the amine; the reaction is med in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Ste iii : (x-sulfonation of the carboxylic acid; the on is performed at 75°C in a polar aprotic solvent like DCE by treatment with sulfonic acid in the presence of a base such as, for e, DIEA; Step (iv): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar aprotic solvent such as DCM by treatment with NHS in the ce of a coupling agent such as, for example, EDC; Step (v): peptide ng between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (vi): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the maleimido acids are commercially available for n=1 to 12. lorBr$WNQkNR 0 R28 LP25 prepared according to the scheme below: R27 0 o\ / (i) FmocHN N OH —>FmocHN Boc o FmocHN 18 17 NR"ALKNRBoc O/ H 0U OMNRiALKiNR O R o RJaOi) BA 27 nu H J 5 NRFALKiNRWBoc HN O R: 3MNRWALKiNRWBoc lorBr\)Lo)OEIQ J (iv) o 0 R27 o | or Bryk NQL H J : NRWALKiNHR17 0 R2, M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a coupling t such as phosphonic anhydride and a base such as, for example, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step liiil: peptide coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (iv): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; N- succinimidyl bromo- and iodo-acetates are commercially available. lor Brdo i H NR ALK, NkaQkNR ALK NHR o R3 LP26 28 prepared according to the scheme below: 3&0 /0 R" O (i) H FmocHN M (JJ 0‘35 m» FmocHNJYON:LOH—’NRALKNRBOG—>FmocHN/'?fNR NRKALKiNR?BOC 23 RJ(ii) o 0 R27 0 R o H 2’ H I or FQLNRKALK' "W B i N (iv) ALK NR800 < O O HEN/[\W LKNR,800N 0 R28 IorBrJNR’ALl/Q‘OJZ?o 2 0 R28 J(v) 19 t (iii) IOFBFQL RJgHNiALK OH NR,g'ALKjNngfNHQLNRgALK’NHR? O\ lorBrQLOJQO M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture in the presence of a base such as sodium bicarbonate or in a polar t such as DCM in the presence of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as a NHS ester; the coupling of N- succinimidyl bromo- and iodo-acetate with the amino ylic acid is performed 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 performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hydrochloric acid (for e solution in dioxane) or of trifluoroacetic acid.
NHS esters of -amino acids are commercially ble; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; N- succinimidyl bromo- and iodo-acetates are commercially ble; the amino carboxylic acids are cially available for n=1 to 12 and the N-methylated amino carboxylic acids for n=1 to 7.
IorBrQH R27 O ( I 1| ALK M ‘ g NREALKiNHR17 R28 LP27 ed according. to the scheme below: R27 0 R 0 R27 0 FmocHN/l?fo‘N 2’ n H H l—> FmocHNk/N¢LOH l—*n FmocHN NQLNRiALKiNR Bee 0 ll 2 NRmiALKiNRWBoc i ‘5 ‘7 o/ HZNEJLOH 0 R5,, 0 R25 REE Jan 0 0 R27 0 0 ('V).
I B - H or rQLNRSOVOURLKHH"WNQHNRFALKiNRWBoc H ‘?HgN/K? \EJHNRFALKiNRWBOC 0 R2:8 lorBrJ O \\ O R?a 1M NRkOIALKHO‘N? i ("01m LOHO O 0 R2, 0 I or rykNRSO\/O\RLK'J\NWNQLNRKALK7NHRWB - H 0 R58 lorBr\)LO/:\> M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar t such as a DCM/MeOH mixture 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 bromo- and iodo-acetate with the amino carboxylic acid is performed in a polar aprotic solvent such as a DCM/DMF mixture followed by the on of N,N’-disuccinimidyl ate and a base such as, for example, DIEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the on is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a on of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; N- succinimidyl bromo- and iodo-acetate are commercially available; in the case where ZCH2 and R20=H, the amino PEG carboxylic acids are commercially available for and i=1 to 6 and othenNise may be prepared from tert—butyl acrylate and the ponding amino-PEG- alcohol; in the case where ALK¢CHZCH2, they may be prepared according to the schemes described for linker precursor LP10. 0 R27 0 MK 2%N/‘YNQkNR18ALKNHR17 lorBr¢NRfM/MB H 0 R28 LP28 prepared according to the scheme below: R27 0 R 0 R27 0 FmocHN/'W N15 —O> FmocHN/'WNQLOHm FmocHN. 27 . o\ (I) H (I) NQ\NRKALK7NR17BOC‘ g 7 7 oc : O HERA 18 W O// 0 R28 O Réa 28 J00 R O R O M?Mz???Ni/N? 0") HZN/'\H/"dL 1 3% A; 11 NR?ALKNRBoc<—O ; NRKALKiNRnBOC or r NR21MQM3 O R: O \ 0 RE O MfMZ Op 28 28 R1 (1/) lorBr\)L1\1RJZ\\111/14M3 Cl) WNagy? H? (in) "1 QAOH KNHR1R21HN M1Ms lorBr j\\ NR1M,;M3 0 R28 OCR lorBr\)J\O 1 M: peptide coupling; the reaction is performed in a polar t such as a DME/THF/H20 mixture in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the presence of a ng reagent such as propylphosphonic anhydride and a base such as, for example, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): coupling and activation of the carboxylic acid as a NHS ester; the coupling of N- succinimidyl bromo- and iodo-acetate with the amino ylic acid is performed in a polar aprotic solvent such as a DCM/DMF e followed by the addition of N,N’-disuccinimidyl carbonate and a base such as, for example,DlEA; Step (iv): peptide coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the rotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; N- succinimidyl bromo- and iodo-acetate are commercially available; the (hetero)aryl- carboxylic acids are commercially available, like for example 4-aminobenzoic acid, 6-amino pyridine carboxylic acid, 5-aminopyrazine carboxylic acid, 2-amino—5-pyrimidine carboxylic acid or 6-amino—1,2,4,5-tetrazine carboxylic acid. 0 R O FmocHN\ALK'k 27 "db iii ; NREALWNHR17 LP29 28 ed accordIng. to the scheme below: R27 0 R 0 R27 0 2’ H H o\ (i) (i) FmocHN i N —‘ JVNQk —‘ FmOCHN OH FmocHN/‘VNQLNRiALKiNRii Boc 0 ll 18 17 3 NRmiALKiNRWBoc ; O O// H2N\:)J\OH 0 R28 0 Rita R5, km) 0 R27 0 R27 O ALKl'kH , H \ ‘NQkNRFALKiNRWBOf—OH (Iv) HZN/‘\H/ QLNRWALKiNRWBocN O Rée \ 0 O Rita i") FmocHN\ALK,JkO/N \ O R o FmocHN\ i ALK' HQL ("0T 0 H ‘ é NRWALKiNHRW FmocHN AK 0 Rge \ALK' OH M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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 ide and a base such as, for example, TEA; Step (ii): deprotection of the amine; the on is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 performed in a polar aprotic t such as a DCM/CHsCN e; Step (v): deprotection using a solution of hydrochloric acid (for example on in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the rotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the amino ylic acids are commercially ble for n=1 to 12.
FmOCHNkcmlAI-K'ikN/‘?fHQkNRALKNHRR27 18 17 o R§8 LP30 prepared according. to the scheme below: R27 0 R27 0 R27 O o\ / (i) H (i) FmocHN N —* FmocHN NQLOH —* FmocHN NQLNRiALKiNRH l Boc 3 NRmiALKiNRWBoc 18 17 0/ H2N\:)LOH 0 R28 0 Rée R; Jan 0 R27 0 R27 0 H (IV) H Fmocit"‘l/(/\/O‘/)\ILK H JNRWALKiNRWBoc ‘—o HZN l EQLNRKALKiNRnBOC o Rge \ 0 Rée J") FmocHNA/O‘RLK'J‘LO/N EL R27 O O H T mO\AL)k'- (iii) H ‘ NQLNRWALKiNHRW . o R: FmocHNA/O‘» M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the ce of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed 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 ng between the dipeptide and the NHS ester; the on is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc-L-amino acids are commercially ble; the mono-protected 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 cially available for and i=1 to 6 and ise may be prepared from tert—butyl te and the corresponding amino-PEG-alcohol; in the case where ALK¢CHZCH2, they may be prepared according to the schemes for linker precursor LP10. Protection of the amine function with a Fmoc group may be realized by treatment with FmocOSu (CAS number [829111]) in the presence of a base such as, for example, DIEA.
LP31 is prepared according to the scheme below: R27 R27 FmocHN/'\/o\ (I). NQL —)>H /'\/H H N —O> FmocHN OH FmocHNWNRiALKiNR Boc 18 17 NR "ALKNR Box: 18 17 HZN\i 0 R28 O Réar E OH 28 low 0 R 0 R27 0 N\3 k H (-V N NdkH ALK l 7 2 NRmALKi N HzN NRwBOC HJV‘ QkNRwiALKiNRWBoc‘N—l 0 0 R28 R28 1 (V) \ALKA O R o 27 H N\3 k N ALK "W $NRWALKNHR17 N3\11ALK' O Rae M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar aprotic t such as DCM by treatment with NHS in the presence of a coupling agent such as, for e, EDC; Step (iv): e coupling between the dipeptide and the NHS ester; the on is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of -amino acids are commercially ble; the rotected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; the azido carboxylic acids may be prepared according to the schemes described for linker precursor LP14. o R 0 m0) H N3 ALKANi?NQkNRmALKNHRnI 0 R28 LP32 prepared according to the scheme below: R27 0 FmocHN OM —), N? HN OH FmOCHN ‘ é NRWALKiNRWBOC NR8WALKNRBOC 0 R58 0 R27 0 R27 0 H (IV), H NS/(Vo\|)ALK' "W QLNRKALK’NRWBOC <—O, JL N HZN/Hi/ QkNRWALK’NRWBOCN O RS2; \\ 0 R28 i") N/N0ALK' o"AOL N \\ NW0‘)ALK'ONJZLOWNHQkNRWALKiNHRW To") O N/No)ALK' OH M: peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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 phosphonic anhydride and a base such as, for example, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for example, piperidine; Step (iii): activation of the carboxylic acid as a NHS ester; the on is performed 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 ide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (v): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the mono-protected es are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; azido PEG carboxylic acids may be prepared according to the schemes described for linker precursor LP15.
\ONi 0 22LALKiWN?NRwALKNHRW LP33 prepared according to the scheme below: R27 0 R 0 R27 0 / 27 FmocHN/K/O‘N Li NQL ¥>H i l OH FmocHN LKiNRH ii Fm°CHN i Boc 0 l ; NR?fALKiNRWBoc E ‘8 ‘7 O/ HZNJOH 0 RES 0 R58 23 JO" 0 O O o R O R O k k 27 JL 2’ ALKiNRZZ.
N ALK HQL H ‘ E NRgALKiNR?Boc HzN : NRWALKiNRWBoc ll 0 R58 0 0 o O O Rgs k k i lav) /N N ALK'iNRZZ ALK o \\ ii 0 O o o 0 R2, O k k k WNykNREALK’NHR?H O ll 0 st M: peptide ng; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture in the presence of a base such as sodium bicarbonate or in a polar solvent such as DCM in the ce of a coupling reagent such as propylphosphonic anhydride and a base such as, for example, TEA; Step (ii): deprotection of the amine; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a base such as, for e, dine; Step liiil: peptide ng n the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (iv): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of Fmoc—L-amino acids are commercially available; the mono-protected diamines are commercially available for n=2 to 4 and R17 and R18 = H or Me independently of each other; cyclooctyne linkers activated as NHS esters may be prepared according to the schemes described for linker precursor LP16.
Pit, OK 0 R27 o LK'~NR22 ALHONIJkNWNQkNRMALKNHRW 0 R58 prepared according to the scheme below: R27 0 R 0 R27 0 27 H FmocHN/KWo\ (I). H (I). N N —O- FmocHN/K?N\)LOH —- FmocHN ‘ \ANRFALKiNRWBoc ; NRmiALKiNRHBoc 2 0/ HZN\A 0 0 R5, : OH Fag, 0,0 Jun 0 R27 0 H R27 0 . NQK ,K. NR22ALK’(O"A (iii) I "W 7NRWBDC HO NRlsAl-Kp E < HZNW \ANRWALKiNRWBocN 0 R2, O 0 OR: Jok 28 i O lav) N ALK'iNRHALKAOA/ELo/N \\ o O o k O N ALK'NR22ELALk?o"?JLNHigngk \\ NRmALKNHR,7 Step (i): peptide coupling; the reaction is performed in a polar solvent such as a DME/THF/H20 mixture 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, TEA; Step (ii): deprotection of the amine; the reaction is med in a polar solvent such as a DCM/MeOH e in the presence of a base such as, for example, piperidine; Step (iiil: peptide coupling between the dipeptide and the NHS ester; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (iv): deprotection using a solution of hloric acid (for example solution in dioxane) or of trifluoroacetic acid.
NHS esters of -amino acids are commercially available; the mono-protected diamines are commercially ble for n=2 to 4 and R17 and R18 = H or Me independently of each other; cyclooctyne linkers activated as NHS esters may be prepared according to the schemes described for linker precursor LP17.
RbeOC-ALKXHW QkNRtJ/lRO LP35 is prepared according to the scheme below: RbeOCALKjiNHJYN?NRJEJ/Aa Step (i1: e ng between the linker precursor LP1 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH e in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of the benzylic alcohol as a p-nitrophenyl-carbonate by treatment with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially available such as, for example, 4-(hydroxymethyl)- aniline (CAS number [6231]), 4-(1-hydroxyethyl)-aniline (racemic (CAS number 5]) or enantiopure (R) (CAS number [2107549]) or (8) (CAS number [5002295])), 4-amino- owe-dimethyl-benzene—methanol (CAS number 1]), 4-amino-(x-methoxy-(x-methyl- benzenemethanol (CAS number [1379318—81-6]), 4-amino-(x-methyl-(x-trifluoromethyl- benzenemethanol (CAS number [851652—56-7]), 2-amino-benzenemethanol (CAS number [5344- , 2-amino-or—methyl-benzenemethanol (racemic (CAS number [105177]) or enantiopure (R) (CAS number [32058]) or (8) (CAS number [32058])), 6-aminopyridinemethanol (CAS number [1132933]), o-d-methylpyridinemethanol (CAS number [1335054 ]), 6-amino-d-ethylpyridinemethanol (CAS number [13552252]), 6-amino-d,d-dimethyl pyridinemethanol (CAS number [8436468]), 5-aminopyridinemethanol (CAS number 14]), 2-aminopyridinemethanol (CAS number [236129]), 2-amino-d-methyl pyridinemethanol (racemic (CAS number [8695679]) or opure (R) (CAS number id="p-9367183" id="p-9367183" id="p-9367183" id="p-9367183" id="p-9367183" id="p-9367183" id="p-9367183"
[9367183]) or (8) (CAS number [9367182])), 2-amino-d-ethylpyridinemethanol (CAS number 38]), 2-amino-d,d-dimethylpyridinemethanol (CAS number [213666-96—7]), 3-amino—4-pyridinemethanol (CAS number [1523985]), 3-amino-d-methylpyridinemethanol (CAS number [1242470-88—7]), 3-amino-d,oc-methylpyridinemethanol (CAS number [13357 8]), 4-aminopyridinemethanol (CAS number [1381164]), 4-amino-d-methyl pyridinemethanol (CAS number [7412234]), 4-amino-d,(x-methylpyridinemethanol (CAS number [13390131]), opyridinemethanol (CAS number [523789]), 3-amino-d- methylpyridinemethanol (CAS number [9542401]), 3-amino-d,oc-methylpyridinemethanol (CAS number [8994384]).
RbeOC-ALK—(OCHZCHZNAHJYNQkNFji0 R27 o H J/JZB15R314?o/Q/2J4/J LP36 R28 prepared according to the scheme below: J1, 1:3H 0 R27 O H R HN J¢J3 J1/JL15 R14 RbeOC-ALK-(OCHZCHZNAH?n]:LOH %’ RbeOC-ALK-(OCH22)CHiiNj7YN$NRJGKijfOI—I it") NO N 3?;£00 RbeOC-ALK-(OCH22CHMANWH?NRj:J//J3 M: e coupling between the linker precursor LP2 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Ste ii : activation of the benzylic alcohol as a p-nitrophenyl-carbonate by treatment with p- nitrophenyI-chloroformate in the presence of a base such as, for example, DIEA.
Many e tives are commercially available such as, for example, those listed for LP35.
R R 0 N02 0 R27 O H J1/J2\1514i 0 RbeOC ALKJK WNQk K ¢J3 O O Y H NR16 J4 U337 SOSH o Rgs prepared according to the scheme below: J R15R14 J/ A124 1‘ OH )\J¢J3 R15 R /JA 14 ALKJLngNHQLOHRHN—.16 4 RbeOCY ALKiNKKN?NR RbeOCY J//J3 803H 0) LP3 soH i (ii) R15 R14 000 ALKji "We RbeOCY NWNQLNRkJ/As SO3H Step (i1: peptide coupling between the linker precursor LP3 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a ng agent such as, for example, EEDQ; Ste ii : activation of the benzylic alcohol as a p-nitrophenyl-carbonate by ent with p- nitrophenyI-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline tives are commercially available such as, for example, those listed for LP35.
Cimowm?fNO0N\ N x /J / ALK N?r?: : NR16 J/3 LP38 28 prepared according to the scheme below: /O OH / R27 HQL J/Jix N A \ALK H/KfNR: OH of?MM?JA3(ii) No2 go"JONJYH%N,:xJ/ixo : M: peptide coupling between the linker precursor LP4 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Ste ii : activation of the benzylic alcohol as a p-nitrophenyl-carbonate by ent with p- nitrophenyI-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially available such as, for example, those listed for LP35. 0 N02 0 /J2R15R14JK Q p9R27HOJ1,\ 00 l N’ALKtOM \AL?NJYNQkNRkJ/AS \\ 16 4 O 0 R28E LP39 prepared ing to the scheme below: /J2R15R14 ONJ1‘EXOH O JRléRM / f3 / 0 R27 O Jl/A H OH NALKjOA/mALKONEKJNHQLOHR6HN(_—)4‘ Q"ALK'dONP‘ALKJLQJYNQkNR?JfJB I \ o R:: 0 LP 25 0 (ii) J E) o M: peptide coupling between the linker sor LP5 and an aniline derivative; the reaction is performed 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 the ic alcohol as a ophenyl-carbonate by treatment with p- henyI-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially available such as, for example, those listed for LP35.
O R" iw :0 mtg N\ALK O RiB LP40 0 prepared according to the scheme below: R’lSR’lA 0 R27 o ?4 OH 0 o H Aé‘Ja R27 H J,‘/2\ OH N\ 0 RE . N\ o R: ALK 25 (I) ALK 25 O LP6 O J (ii) 0 Step (i1: peptide coupling between the linker precursor LPG and an aniline derivative; the reaction is performed in a polar t such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): activation of the benzylic alcohol as a ophenyl-carbonate by treatment with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many e derivatives are commercially available such as, for example, those listed for LP35.
J 15 14 O 0 R27 J1/2\ oio LP41 0 prepared according to the scheme below: J 15 14 )\J:\/l) N NWN¢LRH15 3 OH \ SOEHH 0 R255 0 :N/OALKWJkN577?;HQLNRJE‘FJ//J3SOBH LP, RR, ?og J, in QALK2%,,WENRy,SOBH M: peptide coupling between the linker precursor LP7 and an aniline derivative; the reaction is performed in a polar t such as a DCM/MeOH mixture in the presence of a coupling agent such as, for e, EEDQ; Step (ii): activation of the benzylic alcohol as a p-nitrophenyl-carbonate by ent with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially ble such as, for example, those listed for LP35.
RR 0 Q J 1514 0 R27 o H J,/2\ 0A0 lorBrQK NQK K/A N l NR16J4 3 LP42 R5, prepared according to the scheme below: J 15 M ,,/ AWN R27 H? M R HNAJj/JB /2\J 15 lorBrQOL N"kg : OH—>lorBrQkH10"H31";J\///J R? (i) J LPS fan N02 RMR 0 R27 "gin"?J,/J2\5 0* OO ' 3%or r NWNR 3 R,8J\JA/J Step (i1: peptide coupling between the linker precursor LP}; and an aniline derivative; the reaction is performed 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 the benzylic alcohol as a p-nitrophenyl-carbonate by ent with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many e derivatives are commercially available such as, for example, those listed for LP35. o No2 J 15 14 Q 0 O R27 0 J1‘/ 2\ | or BerNRwiALKJk W %H 0&0 N NRtJZ/J3 O R: LP43 28 prepared according to the scheme below: R15R14 0 k SXOH R27 0 R HN JfJ3 o 0 R27 0 OH 0 R25 M: peptide coupling between the linker precursor LPg and an aniline derivative; the reaction is performed 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 the benzylic alcohol as a p-nitrophenyl-carbonate by treatment with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many e tives are commercially available such as, for example, those listed for LP35.
J 15 14 IorBrJ R27 0 J/ 2\ i RWO)ALKJOkN/FV&N%NRJ16\J4//J3HH 1 O O o Rag LP44 prepared according to the scheme below: J RlSRM 0 R27 0 RleHN/KJIT/Js O O /2§15R14 IorBerNRR/O)AiLKJkarNQLOHE IOFBRANRNQKLKL iii/Ni J1, OH H 0‘ (0 20 NR16J4 LP10 RES (‘3‘ R; R R 0 IorBrQLNRK/oAiLKjkW J«WAQ o o J‘\//1)3 Step (i): peptide coupling between the linker precursor LP10 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the ce of a coupling agent such as, for example, EEDQ; Step (ii): activation of the benzylic l as a p-nitrophenyl-carbonate by treatment with p- nitrophenyI-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially ble such as, for example, those listed for LP35.
RR 0 Q J1514 O R O MZgikN/i\o/N\s/M\NR*\J¢%27 H J,‘/2\ 0A0 0 Mg? IorBrQKNRKWVI/M3 H (Bl R; 16 4 LP45 2‘ 4 28 prepared according to the scheme below: JRlSRM JWXOH‘ O R15 R14 R O ¢J3 MMMFJkN 27 "\A RwHN J4 0 R27 J/J2%OH IorBrQLNR)\\M M3 H/KO‘K O : O —’H Ml/Z 4J3 R58 (i) lorBerNRJZTMX?/iLmng:LNRJEJ LP" l(ii) N02 R R 0 0 R27 0 J,/2\ Mi 0 o o o M? 2% l K/ H IorBrQLNR?fMA/MS NHJWOl/ : NR16 JA/ Step (i1: peptide coupling between the linker precursor LP11 and an aniline derivative; the reaction is med 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 the benzylic alcohol as a p-nitrophenyl-carbonate by treatment with p- nitrophenyI-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially ble such as, for e, those listed for LP35.
R15 R14 0 m, o Am??J\/MVJW§:yJOH J/J O?\oj::f FmocHN\ LP46 28 prepared according. to the scheme below: J/J2\R’l§RM R15 R14 OH R27 NjkJJ//3 /J RH" J4 J FmocHNALKjL U jXOH NRJNR OH —‘\FmocHNALKJL)?/NR;J\J4//J3 RRM o R ORR](H) 0 J/JA oi g F HN HQK moc ALK "W% E J\JfJ3 o Rga Step (i1: e coupling between the linker precursor LP12 and an aniline derivative; the on is performed 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 the benzylic alcohol as a p-nitrophenyl-carbonate by treatment with p- 1 1 1 nitrophenyl-chloroformate in the ce of a base such as, for example, DIEA.
Many aniline derivatives are commercially available such as, for example, those listed for LP35. t o 2 O FmOCHN%\eot|ALK,i\%7 HJYNQKNR?sz/JsH l O O o R3 LP47 28 prepared according to the scheme below: /J2\ 15 14 o R 0 j‘\ )JXOH J 2524 ALKLNWNEJLoH —‘. /k\/O‘RLKJ\N/'\‘(H\E)kr\jR),;\J4//J327 H RlBHN J4/3 0 R27 0 J’l/ 2\ FmOCHN/NO\)' . OH 0 R55 (I) H o R; LP13 25 R R 0 J 15 M O R o J/ ?x i O 27 H FmocHN/k\/O ALKJkN/‘Wf O O , NR; J? 3 H 3 0 R55 Step (i1: peptide coupling between the linker precursor LP13 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for e, EEDQ; Step (ii): activation of the ic alcohol as a p-nitrophenyl-carbonate by treatment with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially available such as, for example, those listed for LP35. 14 ji &, O H JfJL odko N ‘ 3\ALK "WNQkNRth/‘k/ LP48 R3 prepared according to the scheme below: /JR15RM O R27 O jkAEXOH 3\ALKJkN/‘Y QLOHH RwHN J?3 0 R27 0 J,/2\JSQA _ 3\ALKJkNJ\WHQkNRJ,;\J,,//J3 0 (0 R25 H o R: LP" 25 0 R27 H ‘ (JARWRgiog ALKANWN%NRJ,6\J4¢J3H 0 R255 Step (i1: peptide coupling between the linker precursor LP14 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the ce of a coupling agent such as, for example, EEDQ; Step (ii): activation of the ic l as a p-nitrophenyl-carbonate by treatment with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline tives are commercially available such as, for example, those listed for LP35. - 27 H 1 H 16 4 LP49 R28 prepared according to the scheme below: J/JASQAOH 0 R27 O Ns/R/O‘RLKlkN/‘WNQLOH—c Na/R/OKLKiNngdLNRJKJéJB. H R HNAJ¢J3 0 R o J/JASQAOH 0 R255 (i) H is 4 O R; LP15 25 M: peptide coupling between the linker precursor LP15 and an e derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agents such as, for example, EEDQ; Step (ii): activation of the benzylic alcohol as a p-nitrophenyl-carbonate by ent with p- nitrophenyI-chloroformate in the presence of a base such as, for example, DIEA. 27 H 1‘ O O \ \ NJKALK'NRiEALKkNWN?NRtJst o R; LP50 prepared according to the scheme below: 0 o o o o m o o o o o NkALK'iNRikALKiNWNQLOHH H ‘ OH R "N Jst k k i W" /J H H 0 R58 O . H Rés O O J (ii) Step (i1: e coupling between the linker precursor LP16 and an aniline derivative; the reaction is performed in a polar solvent such as a DCM/MeOH mixture in the presence of a coupling agent such as, for example, EEDQ; Step (ii): tion of the benzylic alcohol as a p-nitrophenyl-carbonate by treatment with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially available such as, for example, those listed for LP35.
O J 1514 O J1/2\ 0A0 NkALK'NR22ALK%ON)J£NJYN%NRtJ4//J3 \\ R28 LP51 prepared according to the scheme below: R15R14 \OO 0 J1/JA1240H o NJKA R27 0 R16HN/KJ4// 351kl J 1/X4OH LK'NR2,ALKAO"A| "\A "Ar-{Z7 u 0H ALK'NRZZlkALKAo l N Qthg/mem {LN/J3 O R: \\ 28 (I) O 0 J00 N02 0 O 0 0 k R o J/JARmRMJOL O 27 H 1 o \\ ALK"NR£EALK4(ON%J\NJ\WN\E)LNRJ16\J4¢J3 o R; Step (i1: peptide coupling between the linker sor LP17 and an aniline derivative; the reaction is performed in a polar t such as a DCM/MeOH mixture in the presence of a coupling agent such as, for e, EEDQ; Step (ii): activation of the benzylic alcohol as a p-nitrophenyl-carbonate by treatment with p- nitrophenyl-chloroformate in the presence of a base such as, for example, DIEA.
Many aniline derivatives are commercially ble such as, for example, those listed for LP35.
ALKANWNQkNRtJé‘k27 H 1, o NREALKeNI-IR17 LP52 28 prepared according to the scheme below: R5R 0 J 4 0 R27 o J/ R12402400 (I) R27 /J2:?15R14OJOL RbeOC-ALKJkN/I\H/NE¢kNRJ16\JA//J3H NRFALKiNHR? KNR1Boc ALKJOLNH/KOfN$NRJ/J3 0 R58 Step (i): formation of the carbamate between the linker precursor LP35 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
RR 0 J11 271/40ANR18ALKNHR171514 / A 27 Hi RbeOC-ALK-(OCHzCH2)i MW NR16 Jf 3 LP53 R58 prepared according to the scheme below: R15 R14 R15 R14 J/5240i000 O (0 EXA O NRTWALKNHR17 NRALKNRBoo RbeOC--ALK(OCH22)CHijiWNRJ1\6J: RbeOC_ALK_1OCH2CH21)jiN/HfN"£11;ijJ//J3 0 RE Step (i): formation of the carbamate between the linker precursor LP36 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for e, DIEA; ection using a solution of hydrochloric acid (for example solution in e) or of trifluoroacetic acid.
RR 0 J1514 O R27 o H J1/2\ i RbeOC?/ALKA JYNQk O J\¢J3 NRTBALK’NHR17 H l ' NR16 J4 80 H O R LP54 3 28 prepared accordIng_ to the scheme below: N02 0 R R 0 J ‘5 1" 14 Ami J/JA CAL O 0) O Ami J1/ {ilXOANRTALKNHR1 RbeOCY NJW/N\JLNRJ\J//J Nng’ALKiNRWBoc RbeOCY NWNRUwJEN;J3 SOH 30H Step (i): formation of the carbamate between the linker precursor LP37 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
// J 15 14k / 0 R27 0 V A H o NRiALKiNHR N l 18 17 LP55 58 prepared according to the :0»; ONQK /J 15 M /JAJR15RM ()1 J12\ OXO©—‘ J o NR1€7ALK7NHR17 J\/JRJ18\J¢J3 AL NR18J1/ 3 A'— NR13ALKNR17Boo 4 Step (i): formation of the ate between the linker precursor LP38 and a otected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example on in e) or of trifluoroacetic acid. 0 R15R 0 J 14 J1/ A OXNRiALKiNHR l 18 17 NWALK'iO/VIOALKNJYNQLNRkJéJ \ 16 LP56 prepared ing to the scheme below: 0 R11 0 J QN’ALK?o/MloiALK WNR? O /JA1 0 J1/Ngj?zwCog/NO(—_i) NEEHZL\JLNRJEf/J3J 1X00NR17,1ALK7NHR17 NR1¥77ALKNRBoc QNRALKWON'QJOLALK Step (i): formation of the carbamate between the linker precursor LP39 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hloric acid (for example solution in dioxane) or of trifluoroacetic acid.
R15R14 0 J1//2J R27 0 "L O NR18ALK NHR17L , / NJVNMNRtJst N\ 0 ALK R28 LP57 prepared according to the scheme below: N02 O 0 14 0 2\R15R1‘1 R27 ELMO J1/JkgioiNR?ALK7NHR1 q:L?HNW NR1E"ALKNR1Boc R25 C:ALKOdk? \ANRBJ\J/J3 Step (i): formation of the carbamate between the linker precursor LP4o and a monoprotected e; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
J 15 14 k 0 0 R27 0 H J1/ A 0 NR7 LKiNHR \ N/ALKLJLNJyNQLNRthl/JS1 18A 17 \o SOSHH o Rgg LP58 prepared according to the. scheme below: OJLO©/—_N02 O 1 0 15 14 12%;}:ka 3: R27 J/J:1X0ANR17 9L"W"uNRL 7ALK7NHR1 ALK ALK NR W1 803H SOBH 2BLP‘11 Step (i): formation of the carbamate between the linker precursor LP41 and a otected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
RR 0 /2JVéokNRTBALKeNI-IR1715 14 IorBrWNQRJfJ LP59 prepared according to the scheme below: R R O 15 14 J 15 14 szjx i O , / U) J NRF H HALKNHR1 ERJNQKNRJN/gH O O —. WNQkNRj?J//J3 NR1,¢ALK7NR1,Boc Step (i): formation of the carbamate n the linker precursor LP42 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the ce of a base such as, for example, DIEA; deprotection using a solution of hloric acid (for example solution in dioxane) or of trifluoroacetic acid.
IorBerNREN-Kik )VNQkNRk/ASR27 H J1/J2\15R1:DXNR ALK NHR 18 17 LP60 R58 prepared according to the scheme below: R R O 4" JL (0 1/2124iJRlSRMO 0 NRALK’NHR O O —‘ lor Br\)L 17 Ior Br\)k NR19ALKNJ\11/N\ARJ16\J//JSNR1E*ALK*NR17BOC NR19ALKJL N \ANRk /J1sJ/3 Step (i): formation of the carbamate between the linker precursor LP43 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
I or Br NRZO/fVOJALKJk WN?NRtJZ/JSi 27 H 11 O NRiALK’NHR 1s 17 LP61 58 prepared according to the scheme below: Ji/JLWM 0/0/ O (i) 0 R27 O Ju/JASJZOJOKNR;WALKNHR IorBrQiR2mm"M?aw:NRxJ31X0R27 0 $R5N°\RLKKNJWNQRRNf’sH- H 0 R258 LZBPM Step (i): formation of the carbamate between the linker precursor LP44 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for e solution in dioxane) or of oroacetic acid. 0 0 J12\/ XJ2 R27 0'";szleNWNngLJI/J:H O NR18ALK7 7NHR17 IorBrQ4NRkm"M3 0 R LP62 58 prepared according to the scheme below: R15 R14 O / —_ (*124OiNRTALKNHR lorBrJNR?MZB/bWNR6J\J/J3NR1E’ALK*NRWBOC I0rBr\)LNRj\\M4M3)W/MRJ‘\J2‘JsMgM2% Ste i : formation of the carbamate between the linker precursor LP45 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
R R O R27 i/éokNRwALKNHR1715 14 / 2\ FmocHN \ALK MY1 "204 i NR. ,423 LP63 Rég prepared ing to the scheme below: J RiSRM i ONOZ Ji/K (i) 0 R27 0 :324 ii\ 0 R27 O H o \ALKJLN/'\WNQLNR1;H o o NRFALKiNHRW J4/2J3 NR?fALKiNRWBoc FmOCHN‘ALkaH/KWNEQLNRJngJ3 0 R58 0 Rés Step (i): formation of the carbamate between the linker precursor LP46 and a monoprotected diamine; the on is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
R R O J 15 14 J1/LWXOXNREALK’NHRW2\ FmocHNAVONLKkNWNNR1:J4/J3 LP64 prepared according to the scheme below: R R O E I 5R (i) A FmocHN/NoAmiNW £JA15 MAL"2240 0 —‘NRALK NR Boo FmocHN’NOJAiLA"WNRUNijjjjéojiNR?ALKNHR7 Step (i): formation of the carbamate n the linker precursor LP47 and a monoprotected diamine; the on is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
"JR5 0 o NRTBALKRNHRN 3\ALKWN?NRtJéJ LP65 R28 prepared according to the scheme below: 000/N02 R R 0 R15 R14 J 15 14k N3\ALKjiNHigh;N€OLNRijJgJ1/JA OJL (i) 0 J1/ A NRiALKiNHR o . l 18 17 16 4 NRmiALKiNRWBm:[\‘3\ALKj1N|_|/'\WNH:VXLNF{J16\J4//‘J3 0 R58 LP,la Step (i): ion of the carbamate between the linker precursor LP48 and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a on of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
RR 0 o R, J/2\ 1514*o NRTBALieNI-IR17 N3Xvol'ALKNNJYN ONRszl/J/ LP66 R28 prepared according to the scheme below: No2 R R O 0 J "515R" AOL g (i) 1l 124O O —> N/NONALKJk N mfj:q§<4ALNR15 ALKi ,NHR17 XVONAN M N3 ALK NWN NR?JA/?s NRmiALKiNRWBoc LP,19 Step (i): formation of the carbamate between the linker sor LP49 and a monoprotected e; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for example, DIEA; deprotection using a solution of hydrochloric acid (for e solution in dioxane) or of trifluoroacetic acid.
R R O O O /J\15 14 O 0 R27 0 H J1 l 2%OANRTBALK7NHR17 LK'iNRikALK NW éQkNR16 Jf 3 \ H 0 R58 LP67 prepared according to the scheme below: 2 O O ONXALK'NRJKALKANWH\)LNJ\J¢1J? O O 0 0 0 R2, 0 JKJLHMJL O R27 O NRHALK7 , 0 NHR" (ii H$NRJ1§JfJ3 22 A H 0 O R; NR,;ALK*NRHBOC \\ R233 0 0 Step (i): ion of the carbamate n the linker precursor LP5o and a monoprotected diamine; the reaction is performed in a polar solvent such as CchN in the presence of a base such as, for e, DIEA; deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid.
\ONi R R O ? N91 R27 Hi J1/J2\15 14*o K J NR?ALicNHR17 ALK"NR22ALlHO i NW NR16J4//3 o R;8 LP63 prepared according to the scheme below: \ONXALK'NRgALKAoquHQLNR?JQJSXO 0 R RM O JKJAR‘SRMJL O0 O O O 0 O 0 R27 0 R27 0 o Jl/szgé i0 NRlaALK7 ,NHRn \ NkALK'~NR:LALKAoVku/SfHSANR?JfJa O Rig NR‘fALKiNRWEoc 0 Rig 0 0 Step (i): formation of the carbamate; the reaction is performed in a polar solvent such as CchN in the ce of a base such as, for e, DIEA; deprotection using a solution of hydrochloric acid (for example on in dioxane) or of trifluoroacetic acid.
R15R14 i R27 O J1/J2\ OH RbeOC-ALK HJYNQkNRtJI/J3H LP69 R58 prepared according to the 1St step described for linker precursor LP35. 0 R27 O H Jl/J2\15 1:)H RbeOC-ALK—(OCHZCHZNAMJVNQkNR?Jf/‘gl LP70 R58 prepared according to the 1St step described for linker precursor LP36. 0 /J§15R14 R27 0 H J1‘ OH RbeOC YALKJKNWNQKNRkJ/AS 16 4 LP71 SOSH 0 R28 prepared according to the 1St step bed for linker precursor LP37.
/ A0 R" O H Jn/JLR15R14OH O H/IYNQkNRtJAf/J3 0 : LP72 R28 ed according to the 1St step described for linker precursor LP33.
O R‘I5R‘I4 - (‘3 R27 O H J‘I/J2\ OH l NALK'ONOJkLKKNNNQKNRKJ/As H E 16 4 0 R28 LP73 O prepared according to the 1St step described for linker precursor LP39. 0 R27 0 J1/J2\1514 /0 H OH / J‘\ /J / N l NR16 J4/ 3 N H / \ALK 28 LP74 0 prepared according to the 1St step described for linker precursor LP40.
R15R14 o 0 R27 0 H J1/JZj/éOH \\ ‘ /ALK NQK //J3 N N l NR16 J4 \ SOsHH o Rge LP75 0 prepared according to the 1St step described for linker sor LP41.
J 15 14 0 R27 0 H J1/ 2\ OH LP76 prepared according to the 1St step described for linker precursor LP42.
O 0 o IOFBerNRwALKJkN/K‘/N%NRJ16\J4//J3R27 H J1/J2\15 12H LP77 R28 prepared according to the 1St step described for linker precursor LP43.
O J 15 14 O R O J/ 2\ I or rQRRzo/(VOJIALKJkNJYNQKNRKJ/AgB ' 27 H 1 OH H : 16 LP78 R28 prepared ing to the 1St step bed for linker precursor LP44. o R o M 27 H J1/J2\15 "SH o M? Z?NJYNQkNRkJ/As IorBrykNRkwl/M3 H 0‘ RE 16 4 LP79 21 4 28 prepared according to the 1St step described for linker precursor LP45.
O R O 15R14 OH 27 FmocHN H 1‘ \ALKJk HJYN NRtJst "’80 R3 prepared according to the 1St step described for linker precursor LP46.
/J2R15R14 R" O \ FmocHNNON J" OH ALK? N N ‘ : NRJ16\J4//J3 LPs1 R28 prepared according to the 1St step described for linker precursor LP47. 0 R27 O H Jl/J2\15 18H l 16 4 "’82 R28 ed according to the 1St step described for linker precursor LP43.
O R O J/J2R15R14 XVOQi l 27 H 1‘ OH LP83 R58 prepared according to the 1St step described for linker precursor LP49.
Oi i R2 o N ALK'7NR22 ALKiNWNQkNRtJ/A \\ H 0 R58 described for linker precursor LP50.
O /J2R15R14 k H%OJ1‘ iXOH N ALK'7\NR2O2ALK%O%N NQk \\ ‘ NR16 JfJS LP35 ed according to the 1St step described for linker precursor LP51.
O O R HOi7ALI l l R29 0 CF322COH LP86 ed accordIng to the scheme below:_ LNHFmoc o O o O .
OH (I) R29 + db k/NHFmoc a okALK7NR25Fmoc —- OXALKiNRK , . /\ a HO ALKiNR Fmoc resIn O 25 (ii ’ iii) . R resIn O O 29 resIn Wang resIn_ (ii, iii) HOTKLw,H O fmoc R30 R30 R30 kALKNR (iv) (ii) 2 NH2 ?o?ALK7NR NH 2 A/QAOAKALK7NR2 2 fir—I 0 CF3CO2 H \O EH0 RHO fmoc resinA0 M: coupling of the acid on Wang resin by esterification; the reaction is performed 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 performed in a polar solvent such as DMF by treatment with a base such as, for example, piperidine; Step {iii}: peptidic ng; the reaction is performed in a polar solvent such as DMF in the presence of coupling reagents such as, for example, HOBt and HATU and a base such as, for example, DIEA; Step (iv): cleavage of the resin; the on is performed in a polar solvent such as a DCM/HZO mixture in the presence of an acid such as, for example, TFA.
Fmoc protected alkyl-amino acids are commercially available for n=1 to 9; Fmoc L-amino acids are commercially available.
O 29 HOJkALK%O/\V?,NR25H NkNH 0 CFHCOH LPs7 R30 ed according to the scheme below: 0 g 0 R29 ?+ 0 OH (I). k R29 NR _ NRZSFmoc k )L O 25 NR2Fmoc a ALK40M —‘ o ALK40M NHFmoc \o HO ALK (OM (II, III) resinA0 resinA0 Wang resIn_ R (ii, iii) HOWKNH O fmoc 0 R29 0 fmoc 2::220722422\2 2242At2NH2 23L:" NR 21:220?22422\22J\Jk/NR NH o R resin o a" resin o HOiALKAOMNR279:"XZCFfmoH M: coupling of the acid on Wang resin by esterification; the reaction is performed 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 med in a polar solvent such as DMF by treatment with a base such as, for example, piperidine; Step {iii}: peptidic coupling; the reaction is performed in a polar solvent such as DMF in the presence of coupling reagents such as, for example, HOBt and HATU and a base such as, for example, DIEA; Step (iv): cleavage of the resin; the reaction is performed in a polar solvent such as a DCM/HZO mixture in the presence of an acid such as, for example, TFA.
Fmoc-protected amino PEG carboxylic acids are commercially available for i=1 to 6 and othenNise may be prepared from tert—butyl acrylate and the corresponding amino-PEG-alcohol; in the case where ALK¢CHZCH2, they may be prepared according to the schemes bed for linker sor LP10. Protection of the amine function with a Fmoc group may be realized by treatment with u (CAS number [829111]) in the presence of a base such as, for example, DIEA; Fmoc L-amino acids are commercially available.
HOJYALKNR25/kNJVNH 803H 7% CIH LPss 30 prepared accordIng to the scheme below:.
R30 0 o NHSOj7/LNHBOC 0 R30 LKiNHR25 0 R29 JRNH2 0—, k"\C7)7/kNHBoc —>(ii)Ho503H 2577/LNkNHBoc HO HO R R 29 29 SOH 7(iii) MALKNR257/E9NEEC": Step (i): peptidic coupling between amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium onate; Step (ii): ic coupling with sulfo amino acids; the reaction is performed in a polar solvent such as CchN in the presence of coupling reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for e solution in dioxane).
L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; sulfo amino acids are commercially available for n=1 and 2 or othen/vise may be prepared according to the scheme below: o o o o \OJKKALKiBr (i) ALKi Br (ii) ALKiBr (iii) ALKiN \0 \0 \o 3 Br SAc 303" 303" o o ALKiNHZ (V) ALKiNa HO HO 803H 803H M: substitution of the bromide by a etyle; the reaction is performed in a polar aprotic solvent such as, for e, 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 performed by treatment with hydrogen peroxide and acetic acid; Step (iii): substitution of the bromide by an azido; the reaction is performed in a polar solvent such as, for example, DMA by ent with sodium azide; Step (iv): methyl ester deprotection; the reaction is performed in acidic conditions such as, for example, a mixture of HCI and AcOH; Step (v): reduction of the azido; the reaction is performed by hydrogenolysis in a polar solvent such as H20 in the presence of a catalyst such as palladium on carbon.
Dibromo derivatives are commercially available for n=3, 4 and 9.
/ O H R30 / N\ALK*NR25 ; NTKKNHZ 0 R29 0 CIH prepared according to the scheme below: R O // NHSO / 0 0C 0 R30 H /NALKNHR25 km R30 , kw 0—. "0 "O NHBOC (i) E i ALKNR NHBoc R29 R29 0 LRIO ([29(iii) R30 ALKNR25J:N M: peptidic coupling between Boc-L-amino acid-ONHS and L-amino acid; the reaction is med in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium onate; Step iii): peptidic coupling with maleimido amines; the reaction is performed in a polar solvent such as CchN in the ce of coupling reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): ection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
L-amino acids are commercially available; NHS esters of otected L-amino acids are commercially available; ido amines are commercially available for n=2 to 6.
I? o H R30 QNALK'mMOMLKNR25 ; N?/kNH\ F} o CIH O 29 LP90 prepared according to the scheme below: o NHSOm/CONHBoc 0 R30 KléNALK'jONP‘ALKygql—T25 o 0 R30 HokNl—b+ HOJJYHWKLNHBW O?’E:NALK'WOWNO‘ALKNR25 2 ' " "\H/kNHBoc R R o R o 29 O 29 o in") I?"ALK'IOdOA/iALKNR§%NWKKNH*/ R30 M: peptidic coupling between Boc-L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for e, sodium bicarbonate; Step iii): peptidic ng with maleimido PEG amines; the reaction is performed in a polar solvent such as CchN in the presence of coupling reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; maleimido PEG amines are commercially available for ALK=ALK’=CHZCH2 and i=0 and 1 or otherwise may be prepared according to the scheme below: when ALK=ALK’=CHgC_Hg and i>1 NR25W QEZA/po?oNNRZSEO?/ giwviogwf,\ when ALK¢CH_2CH2 and HgCHg ii @NWW HoiALKiNstBOC 2’ /©/S\C\;O ALK NRQSBOC > (iv) @OVOWOiALKiNstBOC H M] @MOW0777ALKNHR25 N O /0 (" gut/W0W0 77ALKNRBoc HOMOWO7ALK7NRZSBM HCI if when ALK=CH2C_H2 and ALK’¢CHZC_H2 H?oyo\/\NRZ5Boc( 05’]ch HOALK'OH / Ai/O\/\NRZSBOC—,.) ‘EOyo\/\NR25Boc o o QN ALK{~O $21";st ‘7 NA'OLK{~yo\ANstBOC o 0 when ALK and ALK’iCHgC_Hg (III) Wm 7NRQSBoc?.0: \Ovo ALK NR2 Bee5 0W077ALKNRBoc Ho7ALK-70H HO7ALK,f WO7ALK7NRESBoco O//s\\{/ W0\ 0 (vi) iALKiNRESBoc Hl/ WO7ALK7NRESBoco (vii) 0 k UHN (i) o\ /o ‘ (")H OWO7ALK7NRxBocv ‘ \NiALKJFO\/?i\o7ALK7NHR25v CIH O O Step (i1: Mitsunobu reaction on maleimide; the reaction is performed in a polar c solvent such as, for example, THF by treatment of malemide by the PEG hydroxy acid in the presence of PPh3 and DIAD; Step (ii): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid; Step (iii): tion of the alcohol as a tosylate; the reaction is performed in a polar aprotic solvent such as, for example, DCM by treatment with tosyl chloride in the presence of a base such as, for example, TEA; Step (iv): substitution of the tosyl group; the reaction is performed in a polar aprotic solvent such as, for example, THF, in the presence of a base such as, for example, sodium hydride.
Step (v): deprotection of the benzyl group; the reaction is performed by hydrogenolysis in a polar t 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 performed in a polar aprotic solvent such as, for example, DCM by treatment with esulfonyl chloride in the presence of a base such as, for e, TEA; Step (vii): nucleophilic substitution; the reaction is performed in a polar aprotic solvent such as, for example, DMF in the presence of a base such as sodium hydride; The starting 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 Boc20 for i=5 to 8; the starting ALK amino alcohols are commercially available for n=1 and 3 to 10; the starting PEG diols monoprotected with a benzyl group are commercially available for i=1 to 9; the ng ALK’ diols are commercially available for n=1 to 12.
/ NREALK=NR25Co , N\ALK, R29 0 CNIH LP91 ed according to the scheme below: / NR,ALKNHR25 CIH R30 O , NHBOC O R20 HOkNH2 H E N\,,/LNHBoc (i) HokNj/LNHBocO i 5 ‘ ft," R29 R29 0 ON:L/©iNRw,ALKNR(ill) NR,ALKNR E' H2 Step (i): peptidic coupling between Boc-L-amino NHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic coupling with maleimido amines; the reaction is performed in a polar solvent such as CchN in the presence of ng reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in e).
L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; maleimido amines may be prepared according to the scheme below: gAOLK/OJLOHL) Egg/1&0OEQ—MHFViALKNR253°C gAOL/OleRmWALKNR5Boc(ii) (iii) / NRKTALKiNHRZS N CIH Step (i): activation of the carboxylic acid as a NHS ester; the on is performed in a polar solvent such as a mixture DMF/HZO by treatment with N,N'-disuccinimidyl ate in the presence of a base such as, for example, DIEA; Step (ii): peptide coupling; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
The maleimido cyclohexanecarboxylic acid is commercially available for ALK=CH2 or othenNise may be prepared according to the schemes described for LPG; the diamines monoprotected with a Boc group are commercially available for n=1 to 10. o 0 R30 //0 NRNOM MVNVKN NR25 ll NH2 N\ALK' R29 0 CIH LP92 prepared ing to the scheme below: R30 I NHSO N\ deR?/ MNHRK’CIH NHBDC 0 R30 o 0 R30 ALK' §29 (i) (ii) R29 0 $0 OAR"MRRUHAWN‘ALK' g:OAR"MRUaimCIH Step (i): peptidic coupling between Boc-L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar t such as a F/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic coupling with maleimido PEG amines; the reaction is performed in a polar solvent such as CchN in the presence of ng reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are cially available; maleimido PEG amines may be prepared according to the scheme below: 0 0/3?0\ O 70 mom 0 / OH L /O NHR20 NR253°° QM / NRfOVOMNR?Boc N 0 (ii) Nx ALK .
\ALK ALK o o o (in) o . rr ,0"waN\ CIH Step (i): activation of the carboxylic acid as a NHS ester; the reaction is performed in a polar solvent such as a mixture DMF/HZO 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 performed in a polar c solvent such as a DCM/CHsCN e; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in e).
The maleimido cyclohexanecarboxylic acid is commercially ble for ALK=CH2 or othenNise may be prepared according to the schemes described for LPG; the PEG diamines monoprotected with a Boc group are commercially available for i=1 to 9. 0 O R IorBrQK H 30 NREALKiNR25 ; NH2 F} o CIH LP93 29 prepared according to the scheme below: NHSO I or erNRwiALKMNHRZSB 0 ll NHBOC o R30 o o R30 HOJRENHZ —.’ HOJVENW/LNHBOCf’H H 0 CIH | or erNRwiALKMNRjiVENWKLNHBOCB R29 (I) R29 (H) 0 R29 0 1 (iii) 0 o R30 | or FQLNRWMALKiNRkNWKLNHZB R29 0 CIH M: peptidic coupling between Boc-L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic coupling with halogenoacetamido PEG amines; the reaction is performed in a polar solvent such as CchN in the presence of coupling reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example on in dioxane).
L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; noacetamido amines are commercially available for n=2 to 4 and 8 or may be ed according to the scheme below: NHRiALKNR25Boc 0 (ii) 0 IorBrQL:Q—’| or Br\ANRTALKiNRZSBOC 8 | or BrQLNRTALKiNHRZS Step (ii): peptide coupling; the on is performed in a polar aprotic solvent such as a DCM/CHsCN e; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
N-succinimidyl bromo- and iodo-acetates are commercially available; the diamines monoprotected with a Boc group are commercially available for n=1 to 10.
Ior BrJNRNOMNR: E NVKONH R o CN'H LP94 29 prepared accordIng to the scheme below:.
R30 0 o NHSOm/LNHBoc o R '°rBerNRi\/OMNHR o H 30 20 25 HokNHZ 0—’ HokNm/LNHBM CIH IorBr\)LNRNOMNEHj/kNHBoc § (0 lie (ii) IorBrjkNRéyOMNRJVNSEQ? M: peptidic coupling between Boc-L-amino acid-ONHS and o acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic coupling with halogenoacetamido PEG amines; the reaction is med in a polar solvent such as CchN in the presence of coupling reagents such as, for e, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in e). o acids are commercially available; NHS esters of Boc-protected L-amino acids are cially available; halogenoacetamido amines are commercially available for n=2 to 4 and 8 or may be prepared according to the scheme below: NHR Boc (ii) Iorsryt:Q—> NRNOMNRBoc?lorBrJNRNOMNRR CIH25 Step (ii): peptide coupling; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
N-succinimidyl bromo- and iodo-acetates are commercially available; the PEG diamines monoprotected with a Boc group are commercially available for i=1 to 9.
O O R M H 30 o M 29) IorBer & M3 NRWALK NR25§, 7 7 NYK 0 CW2 LP95 21 4 29 prepared according to the scheme below: /M NHSO o MK 29) l NHB°° o 0 R30 IorBr\)L )\\ NRVALK’NHst /M3 o M H HO , , HO NHBOC—‘ KNR25 : NHBOC ; ; c‘) (u).. lorBr )\\ /M : : : \ANRZ M4 3 ’ R29 O Oj?ii) R30 0 W?)[VIZ/RX ""3er x M, NRvALKNR : NRZM4 LS0 g: M: peptidic coupling n Boc-L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO e in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic ng with halogenoacetamido amines; the reaction is performed in a polar solvent such as CchN in the presence of coupling reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
L-amino acids are commercially available; NHS esters of otected o acids are commercially available; halogenoacetamido amines can be prepared according to the scheme below: 0 O\\ IorBr\ANRJ1/\\V9)j\dNV/Vz ALKiNRZSBoc o Msz?k (H) o ?’ IorBr\)LNR;\\M;M3 7 Msz?k —‘(i)IorBr\)LNR;\\M;M3 NR", ALK NRZSBOC NR", ALKi ,"(1:525 Step (ii): peptide coupling; the reaction is performed in a polar aprotic solvent such as a DCM/CHsCN mixture; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in e).
NHS esters of halogenoacetamides may be prepared as described for linker precursor LP11; the diamines monoprotected with a Boc group are commercially available for n=1 to H R30 0 CIH LP96 & LP98 9 prepared according to the scheme below: o NHBoc o R N3HALKNR25 H CIH25 —> —>3NHALKNR NHBoc 0 (H) liVHO lRZQ(iii) NHALKNRkHjANI—I M: peptidic coupling between Boc-L-amino acid-ONHS and L-amino acid; the on is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic coupling with azido amines; the reaction is performed in a polar solvent such as CH3CN in the presence of ng reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane). o acids are commercially available; NHS esters of Boc-protected L-amino acids are cially available; azido amines are cially available for n=2 to 8 and 10.
LP97 & LP99 29 2prepared according to the scheme below: o \?/'\NHBocO ‘)FALKNHR25 kNH C'H Ho Ho NHBoc—> N/f\/O%ALKNRJ3V 3 AQR'O NHBoc l(iiio)R29 N/f\/O+LALKN£%QN\H/‘\NH M: peptidic coupling between Boc-L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic coupling with azido PEG amines; the reaction is performed in a polar solvent such as CchN in the presence of coupling ts such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
L-amino acids are commercially available; NHS esters of Boc-protected L-amino acids are commercially available; for ALK=CHZCH2, azido PEG amines are commercially available for i=1 to 8 and 10, for ALK¢CHZCH2, azido PEG amines may be prepared according to the scheme below: o (i) \ /0 (ii) N H/ iNRZSBOC //S\\ WO%ALK7NRZSBOC % 3WO%ALK7NRZSBOC O O l (iii) N3VO$~ALKiNHRZS M: activation of the alcohol as a mesylate; the reaction is performed in a polar aprotic solvent such as, for e, DCM by treatment with methanesulfonyl chloride in the presence of a base such as, for example, TEA; Step (ii): substitution of the mesylate by an azido group; the reaction is performed in a polar solvent such as an acetone/H20 mixture by treatment with sodium azide; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
The starting PEG amino ls may be prepared as described for linker precursor LPgo.
O )1 0 N ALKiNRzzé ?ANH2 \\ R29 0 CIH LP1oo prepared according to the scheme below: 0 0 NkALKiNHRZZ R, ll L NHBoc 0 R30 R30 0 O O H LNH o‘ LH NkALKiNRibg YLNHBoc HO HO NHBoc : é 0 . : ‘ .. \\ 29 R29 0) R29 0 l") O JO") 0 o o R30 NJ\ALK7NR£VENWJ\NH2H \\ fig, W M: peptidic coupling between Boc-L-amino acid-ONHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): peptidic coupling with cyclooctyne amines; the on is performed in a polar solvent such as CchN in the presence of ng reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for e solution in dioxane).
Cyclooctyne amines are commercially available for n=1, 2, 3 and 5. 0LALKNR O?ALK'NRLip; \\ "TN 25R29 0 CIHH prepared according to the scheme below \l ALK’NRHWO?iALK'iNHRK R20 CIH NHso O o R O \H/LNHBDC O R O k N H N ALKiNR O?rALK- O .7 k/H 22 NR25; ‘ NHBoc NH N HO : HO : , NHBoc—>\\ 5 (ii) 0 R29 R29 R29 0 0 Jam N ALK NR22 \\ uWO?r/XLKNR25R;; NH M: peptidic coupling between Boc-L-amino NHS and L-amino acid; the reaction is performed in a polar solvent such as a DME/THF/HZO mixture in the presence of a base such as, for example, sodium bicarbonate; Step (ii): ic coupling with cyclooctyne PEG amines; the reaction is performed in a polar solvent such as CchN in the presence of coupling reagents such as, for example, DCC and HOBt and a base such as, for example, DIEA; Step (iii): deprotection of the Boc group using a solution of hydrochloric acid (for example solution in dioxane).
Cyclooctyne PEG amines may be prepared according to the scheme below: 0 O HOiALKiNstBoc L /(‘ST\\07ALK7NR Beam ?/OWOWOJFALKiNRZSBoc O 25 (ii) (V) Howof?olTALKiNsteoc LHOWOVolALKNHR30 O O kALKeNHRZZ O O O 0 NkAI-K’NRHWO?iALK'iNRZSBoc (iii) ll ?’ \l NkALK’NRzz?VO?iALK'iNHRZSCIH O 0 o o Step (i): activation of the alcohol as a te; the reaction is performed in a polar c solvent such as, for example, DCM by treatment with methanesulfonyl chloride in the ce of a base such as, for example, TEA; Step (ii): nucleophilic substitution; the reaction is performed in a polar aprotic solvent such as, for example, DMF in the presence of a base such as sodium hydride; Step liiil: ection using a solution of hydrochloric acid (for example solution in dioxane) or of trifluoroacetic acid; Step (iv): protection of the amine with a Boc group; the reaction is performed in a polar solvent such as, for example, DCM, by treatment with BoczO in the presence of a base such as, for example, TEA; Step (v): peptidic coupling; the reaction is performed in a polar solvent such as, for e, DCM, in the ce of coupling reagents such as, for example, HOBt and EDC.
The starting ALK amino alcohols are commercially available for n=1 to 10; cyclooctynes are commercially available for n=1, 2, 3 and 5.
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IJO?Z 23 HQ ow M o 3 13me mNW—m 0 $323333 33oémmz¢33< 2qu wbcsoquo 3323333233 33233312 3.33:8)3233 0 36:3 £9.90 mZ 00 : m_n_wwoq : .choquo w_ 8:— 3.. 3.. mal— 02.. 8F t N .T :5 IZ N3.32 ?x 0% Eoquabo :0 I 60:68 ZJ?Zé/ZZ O lg < .0ng 0 5 < 5:30qu I 30$ 9: 3333 m 38 993 00 z $20 9m 520 : 03 $5888 ._ 3 ._ 8n... wan—n. nan—I— can... SF My n... 2%... 9: E F N Process for re arin the con'u ates of formula III The conjugates of the present invention can obtained via the process comprising the steps that t in: (i) placing in contact and leaving to react an optionally buffered aqueous solution of an dy, nally first modified by means of a ing agent, and a on of the cryptophycin d of formula (II), the chemical group RCG1 of the compound of formula (II) being reactive s the chemical groups RCGZ t on the antibody especially towards the amino groups present on antibodies, the said chemical groups RCGZ having been introduced, where appropriate, by the modifying agent, so as to attach the compound of formula (II) to the antibody by formation of a covalent bond; (ii) and then in ally separating the conjugate formed in step (i) from the cryptophycin payload and/or from the unreacted antibody and/or from any aggregates formed.
According to one variant and more particularly, in step (ii) the conjugate from step (i) is separated only from the unreacted cryptophycin payload and from any aggregates formed, and any unreacted antibody is left in the solution.
The function of the placing in contact is to react the chemical groups RCG1 and RCGZ in order to ensure attachment of the cryptophycin payload to the antibody by formation of a covalent bond; preferably, - when RCG1 represents —ZaRa, the reaction preferably takes place on the amino functions of the dy, especially the o groups borne by the side chains of the lysine (Lys) residues of the antibody and the no groups of N-terminal amino acids of antibody heavy and light chains. A conjugate of the following formula is obtained in this case: mAb-[NH-C(=O)—L*—Crypto]d with L* = fragment of a linker L comprising RCG1:- C(=O)— ZaRa and such 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 thiol chemical groups; - when RCG1 represents an azido group, the antibody may comprise a -CECH moiety or an activated triple bond such as a cyclooctyne; - when RCG1 represents -NH2, the reaction may take place on amide function of the antibody using an enzymatic catalysis, especially the amide groups borne 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*—Crypto]d with L* = fragment of a linker L comprising RCG1=-NH2 and such that L: -L*NH2 and d representing the drug-to-antibody ratio or DAR; - when RCG1 represents -CECH or an activated CEC such as a cyclooctyne moiety, the dy may comprises azido groups.
The term "aggregates" means associations that may form between two or more antibodies, the antibodies possibly having been modified by conjugation. Aggregates are liable to form under the nce 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 d drugs and their hydrophobic nature, the ature (see the references cited in the introduction of J. Membrane Sci. 2008, 318, 311—316), the influence of some of them, r, having 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 may be determined via known techniques such as SEC (see in this respect ical Biochemistry 1993, 212 (2), 469-480).
The s solution of the antibody may be buffered with buffers such as, for example, ium phosphate or HEPES or a mixture of buffers such as buffer A bed 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.
The reaction takes place at a temperature generally of between 20°C and 40°C. The reaction time may vary between 1 and 24 hours. The reaction between the dy and the cryptophycin payload may be monitored by SEC with a refractometric and/or ultraviolet detector and/or HRMS in order to determine its degree of progress. If the degree of substitution is insufficient, the reaction can be left for longer and/or cryptophycin compound can be added. Reference may be made to the example section for further details regarding particular conditions. ular embodiments are described in Examples 3, 7, 10, 14, 20 and 23.
A person skilled in the art has at his disposal various chromatographic techniques for the separation of step (ii): the conjugate may be purified, for example, by steric exclusion chromatography (SEC), by adsorption chromatography (for instance ion exchange, lEC), by hydrophobic interaction chromatography (HlC), by affinity chromatography, by chromatography on mixed supports such as ceramic yapatite, or by HPLC. Purification by dialysis or diafiltration may also be used.
After step (i) or (ii), the solution of the conjugate may undergo an ultrafiltration and/or diafiltration step (iii). After these steps, the ate in aqueous solution is thus obtained.
Antibody The antibody can be a monoclonal antibody selected from the group consisting of a murine, chimeric, a humanized and a human antibody.
In one embodiment, the antibody is a monospecific antibody, i.e. an antibody specifically binding to one single target. Alternatively, it might be a specific)[RCPnantibody In one embodiment, the antibody is a lgG antibody, for instance an lgG1, an lgG2, an lgG3 or an lgG4 antibody.
The antibody ing to the invention specifically binds to a target, thereby directing the biologically active compound as a cytotoxic compound towards said target, As used herein, fically binds" or "binds specifically to" or "binds to" or the like, means that an antibody or antigen-binding fragment thereof forms a x with an antigen that is relatively stable under logical conditions. Specific g can be characterized by an equilibrium dissociation nt (KB) of at least about 1x10'8 M or less (e.g., a smaller KD denotes a 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 terized, for example, by their specific binding to target and/or target antigen using surface plasmon resonance, e.g., BIACORETM.
The target typically corresponds to a protein expressed at the cell surface, e.g. a n expressed at the surface of tumour cells.
In one embodiment, the target is the EphA2 receptor. fl‘he EphA2 receptor is an Ephrin receptor, and is also referred to as "EPH receptor A2" or "Epithelial Cell Receptor Protein-Tyrosine kinase" (see e.g. OMIM Entry *176946, available at the omimorg/entry/176946 world wide web site, version updated on July 21, 2016)][RCP2]. The antibody specifically binding to the EphA2 receptor might for instance correspond to one of the antibodies described in W02008/010101 or W02011/039724.
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 alos referred to as "B-lymphocyte antigen CD19" (see e.g. OMIM Entry 5, available at the rg/entry/107265 world wide web site, version last updated on May 11, 2015). The antibody specifically binding to CD19 might for instance correspond to imab, i.e. the antibody part (moiety) of Coltuximab Ravtansin.
The antibody may optionally be modified with a modifying agent so as to promote the attachment of the cryptophycin payloadas previously bed. 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 EphA2 receptor. The sequence of hu2H11_R3574 is described in W02011/039724 (SEQ ID NO: 18 for the heavy chain and SEQ ID NO: 16 for the light .
Coniugate A ate generally comprises from about 1 to 10 cryptophycin compounds ntly attached to the antibody (this is the degree of grafting or "drug-to-antibody ratio" or "DAR"). This number varies as a function of the nature of the antibody and of the cryptophycin compound, and also of the operating conditions used in the conjugation process (for example the number of equivalents of cryptophycin compound relative to the antibody, the reaction time, the nature of the solvent and of any ent). Placing of the antibody and the cryptophycin compound in contact leads to a mixture comprising several conjugates that are individually distinguished from each other by different DARs; optionally the ted antibody; optionally aggregates. The DAR that is determined on the final solution thus corresponds to an average DAR. The DAR may be calculated from the olution of the SEC-HRMS spectrum of the conjugate. The DAR (HRMS) is preferably greater than 0.5, more particularly between 1 and 10 and even more particularly between 2 and 7.
The conjugate may be used as an anticancer agent. Owing to the presence of the dy, the conjugate is made highly selective towards tumor cells rather than healthy cells. This makes it possible to direct the cryptophycin compound in an environment similar thereto or directly therein; (in this respect, see the following publications that describe the use of monoclonal antibody conjugates in cancer treatment: "Antibody-drug conjugates for cancer y" 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 le to treat solid or liquid cancers. The conjugate may be used alone or in combination with at least one other anticancer agent.
The conjugate is formulated in the form of a buffered aqueous solution at a concentration generally of between 1 and 10 mg/mL. This solution may be injected in perfusion form per se or may be rediluted to form a perfusion solution.
The examples which follow be the preparation of certain compounds in ance with the ion. These es are not limitative, and merely illustrate the t invention.
Analytical methods used Hi hPressure Li uid Chromato ra h —MassS ectrometr LCMS Method A1 Spectra have been obtained on a Waters UPLC-SQD system in positive and/or negative electrospray mode (ES+/—) using ELSD and UV (210-400 nm) detection. Chromatographic conditions were the following: column: ACQUITY BEH C18 — 1.7 pm — 2.1 x 50 mm; solvents: A: H20 (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% of B in 1.8 min; 2.4 min: 100% of B; 2.45 min: from 100 to 5% of B in 0.05 min.
Method A2 Spectra have been obtained on a Waters XeVo-QTof system in positive electrospray mode (ES+) using ELSD and UV (210-400 nm) detection. Chromatographic conditions were the following: column: ACQUITY BEH C18 — 1.7 pm — 2.1 x 100 mm; solvents: A: H20 (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% of B in 8.3 min; 8.6 min: 97% of B; 9 min: from 97 to 5% of B in 0.7 min and 5% of B during 2 min.
Method A3 Spectra have been obtained on a Waters XeVo-QTof system in positive electrospray mode (ES+). Chromatographic conditions were the following: column: ACQUITY BEH C18 — 1.7 pm — 2.1 x 100 mm; solvents: A: H20 (0.1 % formic acid), B: CH3CN (0.1 % formic acid); column ature: 45°C; flow rate: 0.6 mL/min; gradient (5.3 min): % of B during 0.3 min; from 5 to 100% of B in 3.7 min; 4.6 min: 100% of B; 5.3 min 5% of B.
Method A4 Spectra have been obtained on a Waters UPLC-SQD system in positive and/or negative electrospray mode (ES+/-) using ELSD and UV 00 nm) ion. Chromatographic conditions were the following: column: ACQUITY BEH C18 — 1.7 pm — 2.1 x 50 mm; ts: A: H20 (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% of B in 8.6 min; 9.6 min: 100% of B; 9.8 min: 5% of B.
Method A5 Spectra have been obtained on a Waters QD system in positive and/or negative electrospray mode (ES+/-) using ELSD and UV (210-400 nm) ion. Chromatographic conditions were the following: column: ACQUITY CSH C18 — 1.7 pm — 2.1 x 50 mm; solvents: A: H20 (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% of B in 1.8 min; 2.4 min: 100% of B; 2.45 min: from 100 to 5% of B in 0.05 min. 1H nuclear magnetic resonance (NMR) The 1H NMR spectra were acquired on a Bruker Avance spectrometer, either of model DRX—300, 0 or DRX—500. The chemical shifts (6) are given in ppm.
Size exclusion chromato ra h — hi h resolution mass s ectrometr SEC-HRMS The chromatographic analysis was performed on an Agilent HP1100 machine and a Waters BEH SEC 200 1.7 pm (2.1 x 150 mm) column at 30°C with a flow rate of 0.5 mL/min and an isocratic elution of (A) 25 mM ammonium formate + 1% formic acid/(B) CH3CN + 0.1% formic acid 70/30 for 15 minutes. The mass spectrometry was performed on a Waters QTOF-II machine with electrospray ionization in positive mode (ES+). The mass a were deconvoluted with the Waters MaxEnt1 software.
Analytical Size Exclusion Chromatography (SEC) The analysis was performed on a Waters Alliance HPLC system or a Hitachi Lachrom system equiped with a photodiode array or and a Tosoh Bioscience TSngI G3000 SWXL 5 pm (7.8 X 300 mm) column with a flow rate of 0.5 mL/min and an isocratic n of 30 minutes with a pH 7 buffer containing 0.2 M of KCI, 0.052 M of KH2PO4, 0.107 M of K2HPO4 and 20% by volume of isopropanol.
Buffers o Buffer A (pH 6.5): NaCl (50 mM), potassium phosphate (50 mM), EDTA (2 mM) 0 Buffer B (pH 6.5): NaCI (140 mM), potassium and sodium phosphate (9.6 mM) 0 PBS (pH 7.4): KH2PO4 (1.06 mM), NaCI (155.17 mM), Na2HPO4-7H20 (2.97 mM) 0 DPBS: KCI (2.67 mM), KH2PO4 (1.47 mM), NaCI (136.9 mM), Na2HPO4 (8.10 mM) adjusted at pH 6.5 with HCI 5N (1 mL per 1000 mL of buffer) General method used for the ation of antibody-drug conjugate (ADC) A solution of antibody 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 at imatively 10 mM of cryptophycin payload in DMA such that the final antibody concentration is 3 mg/mL and the percentage of DMA in the aqueous buffer is 20%. After stirring for 2 hours, the mixture was analysed by SEC- HRMS to determine the DAR on the tion of monomeric antibodies. If the DAR was found icient, the mixture was treated with a r excess (1 to 5 equivalents) of cryptophycin solution in DMA for 2 additional hours at RT under stirring. The mixture was purified by gel filtration using a ex 200 pg matrix (HiLoad 16/60 or 26/60 desalting colum, GEHeaIthcare) pre-equilibrated in aqueous buffer pH 6.5 (buffer B or DPBS) containing 10 to 20% of NMP. The fractions containing the monomeric conjugated antibody were pooled and concentrated on Amicon Ultra-15 (10k or 50k Ultracel membrane, Millipore) to a concentration of between 2 and 5 mg/mL. A buffer exchange or a dilution in the appropriate buffer was then performed to formulate the ate in the final buffer. In the case of a buffer exchange, it was realized by gel filtration using a SephadexTM G25 matrix , , NAP-25/PD-10 or Hiprep 26/10 desalting columns, GEHeaIthcare) pre-equilibrated with the final aqueous buffer whose composition and pH are suited to each conjugate. The conjugate was finally ed through a Steriflip® filter unit (0.22 pm Durapore® PVDF membrane, Millipore). The final conjugate was assayed by UV spectrometry or SEC-HPLC so as to e the conjugate tration, by SEC-HPLC so as to determine the monomeric purity and by SEC-HRMS so as to determine the DAR from the deconvolution of the mass spectrum of the conjugate.
S s of fra ment A: 2E 58 6R 7E -tert-but l rm l hen lh drox methylocta—ZJ-dienoate , PMBOMO ?» HOMO OW E E OH OtBu OH OtBu 0H 1 2 Sakurai l \ o\Mj /\SiOMOY j 3 HO 0H OtBu 0\ 0H OtBu A Compound 1: (2E,5S,6R)—tert—butyl 5-hydroxy((4-methoxybenzyl)oxy)methy|heptenoate Under argon, to a solution of (2R,3S)—1-[(4-methoxypheny|)methoxy]methylhexenol or Sakurai alcohol (CAS number [2039260], 100 g, 399.5 mmol) in DCM (350 mL) were added tert—butyl acrylate (354.7 mL, 2.435 mol) and Grubbs catalyst (4.849 g, 2.435 mol). After stirring for 17 h at RT, the reaction mixture was filtered on 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 (gradient elution heptane/EtOAc) to give 140.8 g of a brown oil. The oil was dissolved in ous DCM (550 mL), 10 g of quadraPureTM TU resin was added to remove the excess of 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 give 135.75 g of compound 1 as a brown oil (97%).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.85 (d, J = 7.0 Hz, 3 H); 1.42 (s, 9 H); 1.72 (m, 1 H); 2.18 (m, 1 H); 2.30 (m, 1 H); 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, 1 H); 3.73 (s, 3 H); 4.35 (s, 2 H); 4.65 (d, J = 5.8 Hz, 1 H); 5.76 (td, J = 1.5 and .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-dihydroxymethylhept—2-enoate To a solution of compound 1 in CH3CN (910 mL), H20 (90 mL) was added and the mixture was cooled at 12°C before adding in 25 min a solution of CAN (196.7 g, 358.9 mmol) in H20 (300 mL).
Stirring was continued for 1 h at RT. Additional CAN (18.83 g, 34.36 mmol) ved in a mixture of CH3CN (70 mL) and H20 (30 mL) was added and stirred for 1 h at RT to complete the reaction.
The mixture was quenched with NaCl until saturation of the aq. layer, then MTBE (400 mL) was added. After decantation, the aq. layer was extracted with MTBE (2 x 80 mL). The ed organic layers were washed with a 3:1 e of 5% NaHC03/HZO (3 x 200 mL), sat. brine (2 x 130 mL), dried over MgSO4, filtered and concentrated. The aq. layer was acidified with 2N HCl until pH 4, then saturated with NaCl and extracted with MTBE (2 x 150 mL). This second organic layer was washed with 5% NaHC03 (60 mL), sat. brine (60 mL), dried over MgSO4, concentrated and combined with the first organic layer to give an brown orange oil.
The e was dissolved in MeTHF (300 mL), then were added 1,2-ethanedithiol (22 mL, 0.260 mol) and p—TsOH (1.651 g, 8.590 mmol). After stirring at 60°C for 5 h, MTBE (200 mL) was added to the reaction mixture and it was washed with 5% NaHC03 (2 x 120 mL), sat. brine (120 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 520 g of silica gel (gradient n DCM/MTBE) to give 29.48 g of compound 2 as a yellow oil (75%). nd 3: (2E,5S,6R)—tert—butyl 6-methyl-5,7-bis((triethylsilyl)oxy)heptenoate Under argon to a on of compound 2 (39.38 g, 0.171 mol) in anhydrous DCM (500 mL) was added imidazole (51.540 g, 0.749 mol). The yellow mixture was cooled at 0°C, then was added triethylchlorosilane (63.99 mL, 0.374 mol) and the solution was allowed to warm up to RT overnight. The reaction mixture was quenched with sat. NH4C| (400 mL), MTBE (300 mL) and ice.
The pH was adjusted at 4 with 2M , the layers were separated after vigorous stirring. The aq. layer was extracted with MTBE (200 mL). The combined organic layers were washed with half-sat. NH4C| (2 x 250 mL), phosphate buffer pH 7 (150 mL), sat. brine (150 mL), dried over MgSO4, ed, concentrated in vacuo and purified by flash chromatography on 490 g of silica gel (gradient elution heptane/EtZO) to give 74.52 g of compound 3 as a pale yellow oil (95%).
Compound 4: (2E,5S,6S)—tert—butyl 6-methyloxo—5-((triethylsilyl)oxy)heptenoate Under argon, to a solution of DMSO (102.400 mL, 1.427 mol) in DCM (300 mL) was dropwise added at -75°C oxalyl chloride (63 mL, 0.719 mol) in 1 h and the stirring was continued for 15 min before adding, at -75°C, a solution of compound 3 in DCM (150 mL) while keeping the temperature below -70°C. The reaction e was allowed to warm up to -40°C and the stirring continued for 2 h. The reaction mixture was cooled again at -75°C, then DIEA (425 mL, 2.432 mol) was added in 75 minutes while keeping the temperature below -65°C. The reaction mixture was allowed to warm up to RT and then quenched with MTBE (500 mL), ice, sat. NH4C| (200 mL) and 2M NaHSO4 to reach pH 4. The layers were separated after vigorous stirring. The aq. layer was extracted with MTBE (200 mL). The combined organic layers were washed with NH4C| (3 x 300 mL), phosphate buffer pH 7 (300 mL), sat. brine (300 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 1.25 kg of silica gel (gradient elution heptane/EtZO) to give 51.74 g of compound 4 as an orange oil (93%).
Compound 5: (2E,5S,6R,7E)—tert—butyl 5-hydroxy(4-(hydroxymethyl)phenyl)methylocta-2,7- dienoate Under argon, to a suspension of phosphonium e (synthesis described in W02011/001052, 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 up to -40°C and the stirring was continued for 15 min. After g to RT, the red mixture was stirred for 1 h. The reaction medium was cooled at -70°C before adding a on of compound 4 (51.63 g, 126.6 mmol) in THF (200 mL) while keeping the temperature below -65°C. When the addition was complete, the reaction mixture was allowed to warm up to RT and the stirring was continued overnight. The reaction mixture was filtered to remove the insoluble that was washed with MTBE (500 mL). The filtrate was partially trated at 42°C (1/3), then quenched with ice, NH4C| (500 mL), MTBE (500 mL) and 2M NaHSO4 to reach pH 4. The layers were separated and the aq. layer was extracted with MTBE (200 mL). The ed organic layers were washed with at. NH4C| (250 mL), phosphate buffer pH 7 (250 mL), sat. brine (250 mL), dried over MgSO4, filtered and half-concentrated in vacuo until 250 mbar. The suspension was diluted with 50:50 heptane/MTBE (600 mL) cooled with an ice bath, ed off and washed with 50:50 heptane/MTBE. The filtrate was concentrated in vacuo to give 100.05 g as a brown oil.
The crude compound was dissolved in anhydrous THF (320 mL), then was added TBAF trihydrate (90 g, 282.40 mmol) and the reaction mixture was stirred for 3 h. After concentration in vacuo, the crude was d with 10:1 MTBE/THF (770 mL), washed with a half-sat. NH4C| (3 x 200 mL), 1M NaHSO4 (200 mL), phosphate buffer pH 7 (200 mL), sat. brine (200 mL), dried over MgSO4, filtered and concentrated. The aq. layer was extracted with MTBE. After decantation, the organic layer was washed with phosphate buffer pH 7 (30 mL), sat. 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 (gradient elution heptane/Et20) to give 36.91 g of nd 5 as a brown-red oil (88 %).
Fragment A: (2E,5S,6R,7E)—tert—butyl 8-(4-formylphenyl)hydroxymethylocta-2,7-dienoate 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 the on mixture was stirred for 4 h at 35°C. The oxidant was filtered through a plug of celite and washed with warm aceton. The filtrate was concentrated to give 30.43 g of a red-orange oil.
Under argon, the oil was diluted with benzene (800 mL) and refluxing in the presence of AIBN (880 mg, 4.58 mmol), and thiophenol (2.81 mL, 27.51 mmol) for 1 h. Additional AIBN (530 mg, 0.03 eq) and thiophenol (1.03 mL, 10.087 mmol) were added and refluxing continued for an additional 1 h. onal AIBN (530 mg, 0.03 eq) and thiophenol (1.03 mL, 10.087 mmol) were added and refluxing continued for 3 h. Additional AIBN (353 mg, 1.834 mmol) and thiophenol (468 pL, 4.58 mmol) were added and refluxing continued for 1 h. The conversion E/Z was 92/8, 352.6 mg of AIBN was added and refluxing ued for 1 h. The reaction mixture was cooled to RT and concentrated in vacuo to give 28.03 g of fragment A as an orange 0" (92%) with a E/Z ratio superior to 98/2. 8 nthesis of fra ment B: R tert-butox carbon lamino 3-chloromethox - hen | ro anoic acid 0130 : :OMe OHoio : :OMe To a solution of methyl ((tert—butoxycarbonyl)amino)(3-chloromethoxyphyl)- propanoate (CAS number [1624653], 30 g, 87.26 mmol) in THF (225 mL), were added H20 (30 mL) and LiOH monohydrate (4.4 g, 104.85 mmol). The reaction medium was stirred 2 h at RT. After this time, the on medium was diluted with H20 (200 mL) then acidified at pH 2 with 5N HCI (20 mL) and extracted by EtOAc (2 x 250 mL). The combined organic phases were washed with H20 (500 mL), dried over MgSO4, filtered, trated in vacuo, d with EtZO and concentrated in vacuo to give 26.9 g of fragment B as a white solid (94%).
RMN 1H (300 MHz, 6 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, 3 H); 4.04 (m, 1 H); 6.99 to 7.09 (m, 2 H); 7.18 (dd, J = 2.3 and 8.7 Hz, 1 H); 7.29 (d, J = 2.3 Hz, 1 H); 12.60 (broad m, 1 H).
S nthesis of fra ment C1: meth l3-amino-2 2-dimeth | ro anoateh drochloride O o o HOJYNHBOC % \okpNHBoc ’ \OJYNHZHCI 6 c1 Compound 6 : methyl 3-((ten‘-butoxycarbonyl)amino)-2,2-dimethylpropanoate Under argon, to a solution of ert-butoxy)carbony|]amino)-2,2-dimethylpropanoic acid (CAS number [1801816], 250 mg, 1.09 mmol) in DCM (6 mL) and MeOH (2 mL) was added, dropwise at 0°C, (trimethylsilyl)diazomethane (819.86 pL, 1.64 mmol) until yellow persistent color.
Then AcOH was added until complete discoloration. The reaction mixture was concentrated in vacuo then diluted with H20 and extracted with DCM twice. The combined organic phases were washed with sat. brine, dried over MgSO4, filtered and concentrated in vacuo to give 260 mg of compound 6 as a colorless oi| (quant.).
Fragment C1: methyl 3-amino-2,2-dimethylpropanoate hloride To a solution of compound 6 (260 mg, 1.12 mmol) in 1,4-dioxane (10 mL) was added 4N HCI in oxane (2.81 mL, 11.24 mmol). The on mixture was stirred at RT overnight then concentrated in vacuo to give 200 mg of fragment C1 (quant.).
S nthesis of fra ment C2 : meth | 3-amino-2 2-dimeth lbutanoate h drochloride 2 Li 2\ HOM 2 Li NH2 HO NHBoc \o NHBoc OjYLNHBOC 7 8 9 , HCI \OWNHBOC c2 10 Compound 7: rt—butoxycarbonyl)amino)butanoic acid To a solution of minobutyric acid (CAS number [54102], 18.72 g, 176.09 mmol) in H20 (96 mL) were added di-tert—butyl dicarbonate (39.62 g, 176.09 mmol) and NaOH (8.03 g, 200.74 mmol) in H20 (76 mL) then tert—butyl alcohol (132 mL). The on mixture was stirred at RT overnight. After this time, the reaction medium was concentrated in vacuo then diluted with EtOAc (200 mL) and acidified at pH 3 with diluted HCI. After settling, the aq. phase was extracted with EtOAc (200 mL). The combined organic phases were dried over MgSO4, filtered and concentrated in vacuo to give 34.5 g of compound 7 as a colorless oil (82%).
RMN 1H (300 MHz, 8 in ppm, CDCIs—d1): 1.25 (d, J = 7.0 Hz, 3 H); 1.45 (s, 9 H); 2.55 (m, 2 H); 4.04 (m, 1 H); 4.93 (broad m, 1 H); 8,41 (broad m, 1 H).
Compound 8: methyl 3-((tert-butoxycarbonyl)amino)butanoate 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 ed 3 h then evaporated in vacuo to give 10.8 g of compound 8 as a pale yellow oil (quant.).
RMN 1H (300 MHz, 8 in ppm, CDCIs—d1): 1.20 (d, J = 7.0 Hz, 3 H); 1.45 (s, 9 H); 2.50 (m, 2 H); 3.69 (s, 3 H); 4.03 (m, 1 H); 4.90 (broad m, 1 H).
Compound 9: methyl 3-((tert—butoxycarbonyl)amino)methy|butanoate 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 1 h at -75°C then iodomethane (5.79 mL, 92.05 mmol) was added dropwise at -70°C. The reaction mixture was stirred 3 h at -75°C then at RT overnight. After this time, an aq.
WO 76998 2016/076603 solution of 20% NH4C| (100 mL) and Et20 (125 mL) were added. After settling, the organic phase was washed with sat. NaHC03 (80 mL) then sat. NaCl (80 mL). The combined aq. phases were extracted with EtZO (125 mL). The ed organic phases were dried over MgSO4, filtered and concentrated in vacuo to give 5.49 g of crude oil. Cristallization with pentane (15 mL) gave 2.65 g of compound 9 after drying in vacuo. The pentane solution was concentrated in vacuo and purified by flash tography on 100 g of silica gel (gradient elution heptane/Et20) to give 1.98 g of compound 9. The two batches were pooled (87%) and used as such in the following step. nd 10: methyl 3-((tert—butoxycarbonyl)amino),2-dimethylbutanoate Under argon, THF (46 mL) was cooled at -72°C then were added LDA (2M in THF, 22 mL, 44.0 mmol) and dropwise at -72°C (+/- 2°C) a solution of compound 9 (4.60 g, 19.89 mmol) in THF (64 mL). The on mixture was stirred 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 RT. After this time, an aq. solution of 20% NH4C| (50 mL) and EtZO (80 mL) were added. After settling, the organic phase was washed with sat. NaHCOs (50 mL) and sat. brine (50 mL). The ed aq. phases were extracted with EtZO (80 mL). The combined c phases were dried over MgSO4, filtered and concentrated in vacuo to give 6.5 g of crude oil that was purified by flash chromatography on 200 g of silica gel (gradient elution heptane/iPrZO) to give 1.9 g of compound 10 as a colorless oil (39%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.92 (d, J = 7.1 Hz, 3 H); 1.02 (s, 3 H); 1.04 (s, 3 H); 1.48 (s, 9 H); 3.58 (s, 3 H); 3.88 (m, 1 H); 6.62 (broad 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 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 RT overnight then concentrated in vacuo. The crude solid obtained was precipitated in iPr20 (15 mL), filtered and dried to give 210 mg of fragment CZ as a white solid (95%).
LCMS (A1): ES m/z = 146 [M+H]+; tR = 0.8 min. 8 nthesis of fra ment C3: eth |2aminoc clo ro |-2 2-dimeth | ro anoate o o "0%d" A0 NH2 Under argon, to a solution of ethyl 2-cyano—2-methylpropionate (CAS number [15721], 2 g, 13.88 mmol) in EtZO (48 mL) was added titanium (IV) isopropoxide (4.63 g, 15.97 mmol). The WO 76998 2016/076603 reaction e was d 10 min at RT then cooled at -5°C. A solution of ethylmagnesium bromide 3M in EtZO (9.72 mL, 29.16 mmol) was added dropwise at -5°C-0°C in 25 min, the reaction mixture was then stirred 40 min without the cooling bath. At this time, a TLC showed than the reaction was complete. The reaction medium was cooled at 0°C and boron trifluoride diethyl etherate (3M in EtZO, 3.6 mL, 29.16 mmol) was added dropwise at 0°C. The reaction mixture was stirred 30 min without the cooling bath. After this time, 1N HCI was added at 0°C until pH 1-2 (8 mL) then 2N NaOH until pH 8 (28 mL), the reaction mixure 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 crude yellow oil that was purified by flash chromatography on 70 g of silica gel ent elution DCM/MeOH) to give 915 mg of fragment C3 as a pale yellow oil (39%).
RMN 1H (400 MHz, 6 in ppm, CDCI3-d1): 0.52 (m, 2 H); 0.70 (m, 2 H); 1.11 (s, 6 H); 1.27 (t, J = 7.2 Hz, 3 H); 1.64 (broad s, 2 H); 4.18 (q, J = 7.2 Hz, 2 H).
S nthesis of fra ment C4: meth | 3-amino h drox meth |meth | ro anoate hydrochloride 0 o o H0J\fNH2 i HokaHBoc ’ \okaHBoc 11 12 o o \o NH2 9 \o NHBoc HO PMBO c4 13 nd 11: 3-((tert—butoxycarbony|)amino)methy|propanoic acid To a solution of DLaminoisobutyric acid (CAS number [105699], 5 g, 47.52 mmol) in 2 N NaOH (24.7 mL) was added dropwise a solution of Boc20 (11.73 g, 53.22 mmol) in THF (75 mL) while keeping the reaction medium temperature below 30°C with a cold water bath. The reaction medium was stirred at RT for 18 h then concentrated in vacuo, diluted in H20 (75 mL) and washed with MTBE (3 x 150 mL). The aqueous phase was acidified to pH 3 by addition of citric acid at 100 g/L (150 mL) and extracted with EtOAc (3 x 150 mL). The combined organic phases were washed with H20 (3 x 45 mL), dried over MgSO4, filtered and concentrated in vacuo to give 9.4 g of compound 11 as a colorless oil (97%).
RMN 1H (400 MHz, 6 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). nd 12: methyl 3-((tert—butoxycarbonyl)amino)methylpropanoate To a solution of nd 11 (9.4 g, 46.25 mmol) in acetone (300 mL) were added K2CO3 (16.14 g, 115.63 mmol) and CH3| (13.26 g, 92.5 mmol). The reaction medium, a yellow suspension, was d at RT for 20 h then filtered over Clarcel. The cake thus obtained was washed with acetone, the filtrate concentrated in vacuo, diluted with DCM (100 mL), filtered over l, the cake 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%).
RMN 1H (400 MHz, 6 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)(((4-methoxybenzyl)oxy)methy|) propanoate To a solution of DIEA (3.2 mL, 22.45 mmol) in THF (10 mL) cooled at -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 was added dropwise at -75°C a on of compound 12 (2 g, 9.21 mmol) in THF (16 mL) and the reaction mixture was stirred at -75°C for 10 min. A solution of 1- ((chloromethoxy)methy|)methoxybenzene (1.72 g, 9.21 mmol) in THF (16 mL) was then quickly added to the reaction mixture and the stirring carried on at -25°C for 4 h. The reaction medium was diluted with DCM (100 mL) before the addition of a citric acid solution at 100 g/L (50 mL) while keeping the temperature below 5°C. The reaction medium was stirred at RT for 15 min. The organic phase was washed with a citric acid solution at 100 g/L (2 x 50 mL), H20 (3 x 50 mL), dried over MgSO4, filtered and concentrated in vacuo to give 4.08 g of a yellow-orange oil that was purified by flash chromatography on 150 g of silica gel (gradient elution heptane/EtOAc) to provide 2.05 g of compound 13 as a colorless oil (60%).
RMN 1H (400 MHz, 6 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). nt C4: methyl 3-amino—2-(hydroxymethyl)methy|propanoate hydrochloride Compound 13 (1.5 g, 4.08 mmol) was treated with a 4 M HCI solution in dioxane (24 mL) at RT. for 1 h. The reaction medium was then concentrated in vacuo and co-evaporated in the presence of toluene to give 794 mg of fragment C4 as a viscous oil (quant.).
S nthesis of AD1: 2E 58 6R 7E -tert-bu | 5- S aminometh | entano lox 4- (azidomethyl)phenyl)methylocta-2,7-dienoate \ / O \ / O 0% OH ?/ 0\ OH O NHFmoc A 14 NHFmoc \ / ‘ O?/ , 0% O O NHFmoc (W) + \ / ‘ O?/ +N3 O\ O O AD1 NH nd 14: (2E,5S,6R,7E)—tert—butyl 5-(((S)((((9H-fluorenyl)methoxy)carbonyl)amino)—4- methylpentanoyl)oxy)—8—(4-formylphenyl)methylocta-2,7-dienoate Under argon, in a round bottom flask under magnetic stirring, eu-OH (4.9 g, 13.86 mmol) was introduced, ed 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 stirred overnight at RT. After this time, the reaction medium was washed with H20 (100 mL) and sat. brine (100 mL). The organic phase was dried over MgSO4, filtered and concentrated in vacuo to give 9.1 g of crude orange 0" that was purified by flash chromatography on 400 g of silica gel (gradient e|ution heptane/AcOEt) to give 5 g of compound 14 as a pale yellow oil (71%).
RMN 1H (400 MHz, 8 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, 4 H); 1.38 (s, 9 H); 2.39 to 2.63 (partially masked m, 2 H); 4.01 (m, 1 H); 4.13 to 4.31 (m, 3 H); 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, 3 H); 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: (2E,5S,6R,7E)—tert—butyl 5-(((S)((((9H-fluorenyl)methoxy)carbonyl)amino)—4- methylpentanoyl)oxy)(4-(hydroxymethyl)phenyl)methylocta-2,7-dienoate Under argon, to a solution of compound 14 (5 g, 7.51 mmol) in MeTHF (60 mL), was added sodium trimethoxyborohydride (1.2 g, 8.91 mmol) portionwise. The reaction medium was stirred h at RT. After this time, sat. NH4C| (100 mL) and acetone (20 mL) were added. The reaction medium was stirred 1 h at RT. After settling, the organic phase was washed with H20 then with sat. brine (50 mL), dried over MgSO4, filtered and concentrated in vacuo to give 5.3 g of crude yellow oil that was purified by flash chromatography on 300 g of silica gel (gradient elution heptane/AcOEt) to give 3.15 g of compound 15 as a white semi-solid (63%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.80 (m, 6 H); 1.02 (d, J = 7.0 Hz, 3 H); 1.35 to 1.66 (m, 4 H); 1.38 (s, 9 H); 2.35 to 2.56 ally masked m, 2 H); 4.02 (m, 1 H); 4.15 to 4.32 (m, 3 H); 4.43 (d, J = 5.5 Hz, 2 H); 4.92 (m, 1 H); 5.12 (t, J = 5.5 Hz, 1 H); 5.80 (d, J = 15.9 Hz, 1 H); 6.10 (dd, J = 8.8 et 16.2 Hz, 1 H); 6.39 (d, J =16.2 Hz, 1 H); 6.69 (m, 1 H); 7.20 (d, J = 8.4 Hz, 2 H); 7.28 to 7.33 (m, 4 H); 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+HC02H]'; tR = 1.84 min.
Compound 16: (2E,5S,6R,7E)—tert—butyl 5-(((S)((((9H-fluorenyl)methoxy)carbonyl) amino)- 4-methylpentanoyl)oxy)—8—(4-(azidomethyl)phenyl)methy|octa-2,7-dienoate Under argon, compound 15 (3.15 g, 4.72 mmol) and DCM (50 mL) were introduced in a round bottom flask. At 0°C, TEA (987 pL, 7.08 mmol) was added, followed by methanesulfonyl chloride (438 pL, 5.66 mmol), the reaction medium was stirred 1 h at 0°C and 13 h at RT. After this time, DCM (50 mL) and water (50 mL) were added. After settling, the organic phase was washed with sat. 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 on medium was stirred ght at RT. After this time, DMF was concentrated in vacuo and AcOEt was added. The mixture obtained was washed with 0.1N HCI (25 mL), with sat. NaHCOs (25 mL) and sat. brine (25 mL), dried over MgSO4, filtered and concentrated in vacuo to give 3.3 g of crude that was ed by flash chromatography on 200 g of silica gel (gradient elution heptane/AcOEt) to give 1.8 g of compound 16 as a colorless gum (55%) and 740 mg of compound AD1 (33%).
LCMS (A2): ES m/z = 715 [M+Na]+; tR = 8.95 min.
Compound AD1: (2E,5S,6R,7E)—tert—butyl 5-(((S)—2-aminomethylpentanoyl)oxy)—8—(4- (azidomethyl)phenyl)methy|octa-2,7-dienoate 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 RT. After this time, it was washed with 1N HCI then with sat. , sat. brine, dried over MgSO4, filtered and concentrated in vacuo to give 2 g of crude that was purified by flash tography on 130 g ofsilica gel (gradient elution heptane/AcOEt) to give 1.48 g of compound AD1 as a pale yellow oil (quant.).
LCMS (A3): ES m/z = 471 ; tR = 2.65 min. 8 nthesis of AD2: 2E 58 6R 7E -tert-but | 5- S amino-4 4-dimeth | entano | ox 4- (hydroxymethyl)phenyl)methy|octa-2,7-dienoate hydrochloride \ / OAK . Q o\ o é 0% o 0\ 0H NHFmoc 17 NHFmoc \ / \ / 7 ‘ O?/ ‘ O?/ HO o\ 6 o %HO 0% o o NHZ, HCI NHFmoc Compound 17: ,6R,7E)—tert—butyl 5-(((S)—2-((((9H-fluorenyl)methoxy)carbonyl)amino)- 4,4-dimethylpentanoyl)oxy)(4-formylphenyl)methylocta-2,7-dienoate Under argon, in a round bottom flask to a solution of compound A (1.633 g, 4.94 mmol) in DCM (60 mL) were 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 h at RT, the on medium was diluted with H20 (50 mL) and extracted twice with DCM. The combined organic phases were washed with citric acid (2 x 50 mL), sat. NaHC03 (50 mL) and sat. brine (50 mL), dried over MgSO4, filtered and trated in vacuo to give 3.24 g of crude compound 17 used directly in the subsequent reduction (96%).
Compound 18: (2E,5S,6R,7E)—tert—butyl 5-(((S)—2-((((9H-fluorenyl)methoxy)carbonyl)amino)- 4,4-dimethylpentanoyl)oxy)—8—(4-(hydroxymethyl)phenyl)methylocta-2,7-dienoate 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 h at RT, additional sodium trimethoxyborohydride (304 mg, 2.38 mmol) was added and stirred for 2 h. Then the reaction medium was cooled at 0°C, diluted with acetone (18 mL) and sat. NH4CI (36 mL) and extracted with AcOEt. The combined c phases were washed with sat. brine, dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 100 g of silica gel (gradient Heptane/AcOEt) to give 2.61 g of compound 18 as a colorless amorphous solid (80%).
RMN 1H (400 MHz, 6 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, 9 H); 2.37 to 2.58 (partially masked m, 2 H); 4.04 (m, 1 H); 4.16 to 4.33 (m, 3 H); 4.45 (d, J = 5.7 Hz, 2 H); 4.91 (m, 1 H); 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, 1 H); 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+HC02H]'; tR = 1.85 min. nd AD2: (2E,5S,6R,7E)—tert—butyl 5-(((S)amino-4,4-dimethylpentanoy|)oxy)(4- (hydroxymethyl)phenyl)methy|octa-2,7-dienoate hydrochloride 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 h at RT. Then it was concentrated and extracted with AcOEt. The combined organic layers were washed with 1N HCI, H20 and sat. brine, dried over MgSO4, filtered and concentrated in vacuo.
The e was diluted with iPrZO (50 mL) and stirred for 40 h at RT. The crude product was filtered, washed with iPrZO and dried in vacuo 3 h at 40°C to provide 1.706 g of nd AD2 as a colorless amorph solid (90%).
RMN 1H (400 MHz, 6 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 (partially masked m, 2 H); 3.92 (t, J = 5.5 Hz, 1 H); 4.47 (d, J = 5.7 Hz, 2 H); 5.02 (m, 1 H); 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 (broad m, 3 H). LCMS (A1): ES m/z = 460 [M+H]+; tR = 0.95 min. 8 nthesis of AD3: 2R 3S 4-methox benz lox meth lhexen | S amino-4 4- 0H PMBO PMBO\/J\V/A\¢? \V/L\//\\// o\\ o o\\ o PMBO / \ \ ; NHFmoc ?’ 0H NHFmoc NH2 19 AD3 Compound 19: (2R,3S)—1-((4-methoxybenzyl)oxy)methy|hexenyl ((((9H-fluoren yl)methoxy)carbonyl)amino)-4,4-dimethylpentanoate To a solution of Sakurai alcohol (1.02 g, 4.08 mmol) and (S)—2-((((9H-f|uoren hoxy)carbonyl)amino)-4,4-dimethy|pentanoic acid (1.5 g, 4.08 mmol) in THF (15 mL) were added 2,4,6-trichlorobenzoyl de (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 RT for 5 h, then cooled using an ice bath before the addition of 1N HCI (60 mL) while keeping the temperature below °C. The resulting medium was stirred at RT for 15 min and extracted with EtOAc (3 x 50 mL).
The combined organic phases were washed with sat. NaHC03 (15 mL), sat. brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and ed by three successive flash chromatographies on silica gel (150 g, gradient elution heptane/EtOAc; 150 g and 20 g, gradient elution DCM/MeOH) to give 1.78 g of compound 19 as a colorless oil (72%).
RMN 1H (400 MHz, 6 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+HC02H]; tR-— 1. 86 min.
Compound AD3: (2R,3S)—1-((4-methoxybenzyl)oxy)—2-methy|hexenyl (S)—2-amino-4,4- dimethylpentanoate To a on of compound 19 (1.78 g, 2.98 mmol) in DCM (63 mL) was added dropwise dine (1.77 mL, 17.9 mmol). The reaction medium was stirred at R.T. for 4 h, diluted with DCM (150 mL), washed with 1 N HCI (2 x 20 mL), sat. NaHCOs (20 mL), sat. brine (3 x 20 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 50 g of silica gel (gradient elution heptane/EtOAc) to give 644 mg of compound AD3 as a colorless oil (57%).
RMN 1H (400 MHz, 6 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 ( broad s, 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. 8 nthesis of BC1: 3- R tert-butox carbon lamino 3-chloromethox hen l- ro anamido-2 2-dimeth | ro anoic acid 0 BOCHN \0km" kg%OBOCHNlUCI 0 BocHNi~\©[CI HO% \0 OMe nd 20: methyl 3-((R)—2-((tert—butoxycarbonyl)amino)(3-chloromethoxyphenyl)- propanamido)—2,2-dimethylpropanoate Under argon, in a round bottom flask were introduced fragment C1 (981.45 mg, 4.00 mmol) and DCM (50 mL), 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 RT. After this time, the reaction medium was diluted with H20 (30 mL) and extracted twice with DCM. The combined organic phases were washed with sat. brine, dried over MgSO4, filtered and concentrated in vacuo to give 2.5 g of crude oil that was purified by flash chromatography on 110 g of silica gel (gradient elution heptane/AcOEt) to give 1.06 g of compound 20 as a white meringue (66%).
RMN 1H (400 MHz, 6 in ppm DMSO-d6): 1.06 (s, 3 H); 1.07 (s, 3 H); 1.30 (s, 9 H); 2.65 (dd J = 11.6and 13.9 Hz, 1 H) 282 (dd J: 4.0and 13.9 Hz, 1 H) 3.18(dd, J: 6.1 and 134 Hz 1 H) 3.29 (partially masked m, 1 H); 3. 60 (s, 3 H), 3. 81 (s, 3 H); 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). nd BC1: 3-((R)—2-((tert—butoxycarbonyl)amino)(3-chloro—4-methoxyphenyl)- propanamido)—2,2-dimethylpropanoic acid Under argon, in a round bottom flask were introduced compound 20 (1.06 g, 2.39 mmol) and THF (25 mL) followed by LiOH (70.18 mg, 2.87 mmol) and H20 (1 mL). The reaction medium was stirred few hours before adding 70 mg of LiOH and stirred overnight at RT. After this time, Amberlit resin was added until pH 4, filtered then washed with THF and concentrated in vacuo to give 1 g of compound BC1 as a white solid (97%).
S nthesis of BC2: 3- R tert-butox carbon lamino 3-chloromethox hen l- ro anamido -2 2-dimeth lbutanoic acid £6BOCHNKUm a BC2, stereomer1OMe a BC2, stereomer 2 21, stereomer 1 21, stereomer 2 Compound 21: methyl 3-((R)—2-((tert—butoxycarbonyl)amino)(3-chloro—4-methoxyphenyl)- propanamido)—2,2-dimethylbutanoate Under argon, in a round bottom flask were introduced fragment B (1.02 g, 2.78 mmol) and DCM (25 mL), followed by EDC (592 mg, 3.03 mmol) and HOBt (478 mg, 3.03 mmol). The on medium was stirred 15 min then nt C2 (0.5 g, 2.75 mmol) and DIEA (1.7 mL, 9.73 mmol) were added. The reaction medium was stirred overnight at RT. After this time, the reaction medium was concentrated in vacuo then diluted with AcOEt. The organic layer was washed with sat. brine, dried over MgSO4, filtered and concentrated in vacuo to give 1.45 g of crude oil that purified by flash chromatography on 100 g of silica gel (gradient elution heptane/AcOEt) to give 845 mg of nd 21 as a white foam (67%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): diastereoisomer mixture 50:50; 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, 1.5 H); 1.07 (s, 3 H); 1.31 (s, 9 H); 2.58 to 2.87 (m, 2 H); 3.59 (s, 1.5 H); 3,60 (s, 1.5 H); 3.81 (s, 3 H); 3.99 to 4.20 (m, 2 H); 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): diastereoisomer mixture 50:50; ES m/z = 457 [M+H]+; m/z = 479 [M+Na]+; tR = 3.19-3.2 min.
Compound 21 stereomers 1 and 2: methyl 3-((R)—2-((tert—butoxycarbonyl)amino)(3-ch|oro methoxyphenyl)-propanamido)-2,2-dimethylbutanoate stereomers 1 and 2 Under argon, in a round bottom flask to a solution of fragment B (1.17 g 3.55 mmol ) in DCM (30 mL) were added EDC (741.75 mg, 3.87 mmol), HOBt (592.57 mg, 3.87 mmol). After stirring min at RT, fragment C2 (639 mg, 3.52 mmol) and DIEA (2.17 mL, 12.31 mmol) were added.
The reaction medium was stirred for 4 h, then concentrated and diluted with AcOEt (100 mL). The c layers were washed with H20 (2 x 10 mL) and sat. 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 (gradient n heptane/AcOEt) to give 1.306 g of mixture of diastereoisomers (81%) that were separated by two sive flash chromatographies, the first one on 100 g of silica gel (gradient elution DCM/MeOH) to give 376 mg of compound 21 stereomer1 (23%), the second one on 70 g of silica gel (gradient elution DCM/MeOH) to give 181 mg of compound 21 stereomer 1 (11%), 279 mg of compound 21 stereomer 2 (17%) and 476 mg of mixture of diasteroisomers.
Compound 21 stereomer 1 RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.85 (d, J = 6.8 Hz, 3 H); 1.07 (s, 6 H); 1.31 (s, 9 H); 2.57 (m, 1H); 2.80 (dd, J = 5.2 and 13.8 Hz, 1 H); 3.60 (s, 3 H); 3.81 (s, 3 H); 4.05 (m, 1 H); 4.16 (m, 1 H); 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.
Compound 21 stereomer 2 RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.95 (d, J = 6.8 Hz, 3 H); 1.00 (s, 3 H); 1.03 (s, 3 H); 1.31 (s, 9 H); 2.55 (m, 1H); 2.80 (dd, J = 5.0 and 14.3 Hz, 1 H); 3.59 (s, 3 H); 3.81 (s, 3 H); 4.09 to 4.22 (m, 2 H); 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.
Compound BC2: 3-((R)—2-((tert—butoxycarbonyl)amino)(3-chloromethoxyphenyl)- amido)—2,2-dimethylbutanoic acid Compound 21 (0.845 g, 1.85 mmol) and MeOH (20 mL) were introduced in a round bottom flask followed by 1.8 mL of 10M NaOH. The on was stirred and heated at 50°C overnight. The reaction medium was evaporated in vacuo then diluted with H20 (20 mL) and neutralized with 5N HCI. The on was extracted twice with AcOEt. The combined organic layers were washed with sat. brine, dried avec MgSO4, filtered and concentrated in vacuo to give 800 mg of compound BC2 as a white foam (97%).
RMN 1H (400 MHz, 8 in ppm, DMSO-d6): diastereoisomer 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, 3 H); 1.03 (s, 1.5 H); 1.07 (s, 1.5 H); 1.31 (s, 9 H); 2.57 (m, 1 H); 2.83 (m, 1 H); 3.81 (s, 3 H); 4.01 to 4.16 (m, 2 H); 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 (broad m, 1 H). LCMS (A1): diastereoisomer mixture 50:50; ES m/z = 387; m/z = 443 [M+H]+; tR = 1.20-1.21 min.
Compound BC2 stereomer 1: 3-((R)—2-((tert—butoxycarbonyl)amino)(3-chloromethoxy phenyl)propanamido)-2,2-dimethylbutanoic acid stereomer 1 Compound 21 stereomer 1 (0.325 g, 1.85 mmol) and MeOH (8 mL) were uced in 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 vacuo then diluted with H20 (20 mL) and ted with AcOEt (3 x 5 mL). The aq. layers were acidified with 5N HCI and extracted with AcOEt (3 x 30 mL). The combined organic phases were washed with sat. brine (5 mL), dried over MgSO4, filtered and concentrated in vacuo to give 303 mg of compound BC2 stereomer 1 as a white foam (96%) used directly in the subsequent reaction.
Compound BC2 stereomer 2: 3-((R)—2-((tert—butoxycarbonyl)amino)(3-chloromethoxy )propanamido)-2,2-dimethylbutanoic acid stereomer 2 Compound 21 stereomer 2 (1.094 g, 2.39 mmol), THF (5 mL) and H20 (5 mL) were introduced in a round bottom flask followed by LiOH (301 mg, 7.18 mmol ). The solution was stirred for 44 h at RT. The on was not complete, LiOH (301 mg) was added. The mixture was stirred for 48 h, then 301 mg of LiOH was added in THF (10 mL) and H20 (5 mL) and the reaction medium d 40 h at 60°C. The reaction medium was evaporated in vacuo. 1M citric acid was added until pH 2 and the mixture was extracted with AcOEt (2 x 20 mL). The combined organic layers were washed with H20, dried over MgSO4, filtered and concentrated in vacuo to give 1.096 g of compound BC2 stereomer 2 as a white amorph solid (quant.) used ly in the subsequent reaction. 8 nthesis of BC3: eth | 2- R 2- tert-butox carbon lamino 3-chloro methox hen l- ro anamido c clo ro lmeth | ro anoic acid HoigmBocHNl\©:CIO OMe Compound 22: ethyl 2-((R)—1-(2-((tert—butoxycarbonyl)amino)(3-chloromethoxyphenyl)- propanamido)cyc|opropy|)methy|propanoate Under argon, in a round bottom flask were introduced fragment C3 (1.2 g, 7.01 mmol) and THF (16.5 mL), followed by fragment B (2.54 g, 7.71 mmol), HOBt (1 .779, 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 h at RT. After this time, the reaction medium was diluted with H20 (25 mL) and extracted with AcOEt (250 mL).
The organic layers were washed with H20 (2 x 25 mL), sat. brine (2 x 25 mL), dried over MgSO4, filtered, concentrated and purified by flash chromatography, the first one on 200 g of silica gel (gradient e|ution heptane/AcOEt) to give 2.16 g of compound 22 as a colorless foam (64%) and 343 mg of mixture ning the expected compound that was further ed on 30 g of silica gel (gradient e|ution e/AcOEt) to give 160 mg of compound 22 as a colorless foam (4.7%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.62 (m, 1 H); 0.74 to 0.99 (m, 3 H); 1.03 (s, 3 H); 1.10 (s, 3 H); 1.24 (t, J = 7.2 Hz, 3 H); 1.42 (s, 9 H); 2.90 (m, 2 H); 3.88 (s, 3 H); 4.08 (m, 1 H); 4.10 (q, J = 7.2 Hz, 2 H); 4.96 (m, 1 H); 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-chloromethoxyphenyl)- propanamido)cyc|opropy|)methy|propanoic acid Compound 22 (2.09 g, 4.33 mmol), THF (10 mL) and H20 (8 mL) were introduced in a round bottom flask followed by LiOH (726.33 mg, 17.31 mmol). The solution was stirred and heated at 65°C. After 16 h, the on was not completed, 726.33 mg of LiOH in 10 mL H20 were added.
The e was stirred for 48 h at 65°C. After cooling, the reaction medium was diluted with H20 (20 mL) then extracted with AcOEt (3 x 40 mL). The organic layers were washed with H20 (2 x mL), dried over MgSO4, ed and concentrated in vacuo to give 1.29 g of mixture ester/acid.
The aq. layer was acidified with 5N HCI until pH 3, then extracted with AcOEt (3 x 50 mL). The organic layers were washed with H20 (2 x 10 mL), dried over MgSO4, filtered and concentrated in vacuo to give 787 mg of compound BC3 as a beige foam (40%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.50 to 0.90 (m, 4 H); 1.03 (s, 6 H); 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, 3 H); 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, 1 H); 12.11 (broad m, 1 H).
S nthesis of BC4: S Rac lamido3-chloromethox hen | ro anamido -2 2- dimethylbutanoic acid o 0 o H 0%NH2 kg CI 0 \ + a HO 0 OMe We 0%"l"\©EC'o B 23 Compound 23: methyl ((R)—2-((tert—butoxycarbonyl)amino)(3-chloro—4-methoxyphenyl)- propanamido)—2,2-dimethylbutanoate 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 RT for 15 min then were added methyl (S)—3-amino-2,2-dimethy|butanoate (881 mg, 6.06 mmol) and DIEA (1.53 mL, 9.10 mmol). The reaction medium was stirred at RT for 4 h, concentrated in vacuo and diluted with EtOAc (100 mL) and H20 (20 mL). The aqueous phase was ted with EtOAc (20 mL); the combined organic phases were washed with sat. brine (2 x 20 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash tography on 150 g of silica gel (gradient elution heptane/EtOAc) to give 2.21 g of compound 23 as a colorless lacquer (79%).
RMN 1H (400 MHz, 6 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-chloromethoxyphenyl)propanamido)—2,2- dimethylbutanoate 2,2,2-trifluoroacetate 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 RT overnight, concentrated in vacuo and co- evaporated in the ce of toluene to provide 2.0 g of compound 24 as a diastereoisomer mixture (88%).
RMN 1H (400 MHz, 6 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)((R)acrylamido(3-chloromethoxyphenyl)propanamido)—2,2- dimethylbutanoate To a solution of nd 24 (2.0 g, 4.25 mmol) in DCM (20 mL) were added acryloyl chloride (536 uL, 6.37 mmol) and DIEA (2.5 mL, 12.74 mmol). The reaction medium was stirred at RT for 2 h then diluted with H20 (20 mL). The aqueous phase was extracted with DCM (2 x 20 mL), the combined organic phases were washed with sat. brine (2 x 20 mL), dried over MgSO4, ed, concentrated in vacuo and purified by flash chromatography on 100 g of silica gel (gradient e|ution e/EtOAc) to give 850 mg of compound 25 as a 85:15 diastereoisomer e (68%).
RMN 1H (400 MHz, 6 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)((R)acry|amido(3-chIoromethoxyphenyl)propanamido)—2,2- dimethylbutanoic acid To a solution oftBuOK (1.11 g, 9.86 mmol) in THF (4 mL) cooled at 0°C were added H20 (47 uL) and compound 25 (450 mg, 1.10 mmol). The reaction medium was stirred at RT for 3 h, then acidified with 1N HCI (5 mL). The aqueous phase was extracted with DCM (2 x 20 mL); the combined organic phases were washed with H20 (30 mL), sat. 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 pm column (isocratic n at 85/15 COZ/[MeOH + 0.1% TEA] to give 385 mg of compound BC4 as an ous solid (89%).
RMN 1H (400 MHz, 6 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 (broad s, 1H). LCMS (A5): ES m/z = 395 [M-H]'; m/z = 397 [M+H]+; tR = 0.92 min.
S nthesis of BC5: S R tert-butox carbon lamino 3-chloromethox hen l - Compound BC5 was prepared starting from methyl (3S)—3-amino-2,2-dimethylbutanoate 9256689) and following general synthesis of ng block BC depicted in Scheme 32 and described for compounds BC1 and BC2.
RMN 1H (400 MHz, 6 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 (broad s, 1H). LCMS (A1): ES m/z = 441 ; m/z = 443 [M+H]+; m/z = 883 [2M-H]' ; tR = 1.16 min. 8 nthesis of BC6: 3- Rac lamido3-chloromethox henl ro anamido h drox meth lmeth | ro anoic acid 0 BocHN CI BocHN Cl 0 1 NH2 + kg 9 \OJWN *0 0Me HO 0 OM6 HO H0 26 HZNj CI 0 0 \OJWH *0 0Me \OWN *0HZN].«\©[C|0Me 3330 2:3 H0 27 a»l a» HNj CI 9 Hij WW" *0 : :OMe HOW" *0 : :OMe Et3SiO 29 HO BC6 Compound 26: methyl 3-((R)—2-((tert—butoxycarbonyl)amino)—3-(3-chloromethoxyphenyl)- propanamido)—2-(hydroxymethyl)methylpropanoate 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 on medium was stirred at RT for 48 h, concentrated in vacuo and purified by flash chromatography on 200 g of silica gel (isocratic elution heptane/EtOAc) to give 1.05 g of nd 26 as a colorless oil (39%).
RMN 1H (400 MHz, 6 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+HCOZH]'; tR = 1.1 min.
Compound 27: methyl 3-((R)—2-amino(3-chloromethoxyphenyl)propanamido)—2- (hydroxymethyl)methy|propanoate hydrochloride Compound 26 (1.05 g, 2.29 mmol) was treated with HCI 4M in dioxane (16 mL, 64 mmol) for 1 h at RT. The reaction medium was concentrated in vacuo and co-evaporated twice in the presence of toluene. The crude product was triturated with iPr20 (10 mL), filtered and washed twice with iPrZO (5 mL). The cake was then dissolved in DCM, filtered and concentrated in vacuo to give 809 mg of compound 27 as a white foam (90%) that was used without further purification.
RMN 1H (400 MHz, 6 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 (split d, J = 8.6 Hz, 1H); 7.19 (split dd, J = 2.0 and 8.6 Hz, 1H); 7.38 (split d, J = 2.0 Hz, 1H); 8.15 (broad s, 3H); 8.39 (m, 1H).
Compound 28: methyl 3-((R)—2-amino(3-chloromethoxyphenyl)propanamido)methyl (((triethylsilyl)oxy)methyl)propanoate 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 g the temperature below 4°C. Stirring at 4°C was carried on for 10 min then the on medium was d at RT for 20 h. Sat. brine (20 mL) and DCM were added to the medium that was stirred for 10 min. The organic phase was washed with sat. brine (3 x 10 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 70 g of silica gel (gradient elution DCM/MeOH) to give 706 mg of compound 28 as a pale yellow oil (73%).
RMN 1H (400 MHz, 6 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]'; tR = 0.97 min.
Compound 29: methyl 3-((R)—2-acrylamido(3-chloromethoxyphenyl)propanamido) methyl(((triethylsilyl)oxy)methy|)propanoate To a solution of compound 28 (704 mg, 1.49 mmol) in DCM (19 mL) cooled with an ice/acetone bath were added DIEA (780 pL, 4.46 mmol) and dropwise acryloyl chloride (181 uL, 2.23 mmol).
The reaction medium was stirred at 0-5°C for 1 h then EtOAc was added (38 mL) and the medium washed with 1N HCI (5 mL), sat. NaHCOs (5 mL), sat. brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 30 g of silica gel (gradient elution DCM/MeOH) to give 742 mg of compound 29 as a colorless oil (94%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.52 (split q, J = 8.0 Hz, 6H); 0.89 (split t, 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 (split dd, J =10.3 and 17.3 Hz, 1H); 7.02 (d, J = 8.5 Hz, 1H); 7.18 (split dd, J = 2.0 and 8.5 Hz, 1H); 7.31 (split d, 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+HC02H]'; tR = 1.54 min.
Compound BC6: 3-((R)—2-acrylamido(3-chIoromethoxyphenyl)propanamido)—2- (hydroxymethyl)methy|propanoic acid To a suspension of tBuOK (1.42 g, 12.65 mmol) in THF (7 mL) cooled with an ice/acetone bath was added H20 (50 pL), the medium was stirred for 10 min before the addition of a solution of compound 29 (741 mg, 1.41 mmol) in THF (7 mL). Stirring was carried on at 0°C for 10 min then at RT for 1 h. The on medium was cooled with an ice bath before the addition of 1N HCI (16.9 mL). After 15 min of stirring, the reaction medium was extracted with DCM (3 x 25 mL). The combined organic phases were washed with sat. brine (2 x 15 mL), H20 (15 mL), dried over MgSO4, filtered and concentrated in vacuo to give 656 mg of compound BC6 as a yellow foam (quant.).
RMN 1H (400 MHz, 6 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 , 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 (broad s, 1H). LCMS (A5): ES m/z = 397 [M-H]'; m/z = 399 [M+H]+; tR = 0.77 min.
Synthesis of Examples 1 to 3 : benzylic amine of aza-C52, NHS ester of yI-Val-Ala- 2 benzylic amine and corresponding ADC \ / o 1 AK BocHN Cl COtBu N3 0\ O O + i?iU aN3 BocHNl OMe AD1H2 Ho??N \o Hiram0:57 WNW HN?NJYNQ/?ka? / O H H HO N N o\ o HNperCl M l o» 0 0 0 /\ 34 ")5?" o OMe / /O O O H H 2 \\ 0 Nu N o o HN Cl N/M \ 0 LU O O /\ O Example2 NW" 0 OMe hu2H11 _R35—74NMNQk NW0NJYN eii/E:O)5?!"NimZMB Compound 30: (6R,13S)—(1E,3R,4S,6E)—1-(4-(azidomethyl)phenyl)(tert—butoxy)—3-methyl-8— oxoocta-1 ,6-dienyl 6-(3-chloromethoxybenzyl)isobutyl-2,2,1 0,10-tetramethyl-4,7,1 1- trioxooxa-5,8,12-triazatetradecanoate Under argon, in a round bottom flask were introduced compound BC1 (899.55 mg, 2.10 mmol) in DMF (25 mL), HATU (861 mg, 2.20 mmol) and HOAt (302 mg, 2.20 mmol). The mixture was d for 30 minutes at RT. After that, compound AD1 (940 mg, 2 mmol) and DIEA (1.05 mL, .99 mmol) were added. The reaction medium was d for 4 h at RT. After this time, the reaction medium was diluted with H20 (50 mL) and extracted with AcOEt (2 x 40 mL). The organic layers were washed with sat. brine (25 mL), dried over MgSO4, filtered, concentrated and purified by flash chromatography on 80 g of silica gel (gradient n heptane/AcOEt) to give 1.011 g of compound 30 as a yellow semi-solid (57%).
RMN 1H (400 MHz, 8 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, 6 H); 1.39 (s, 9 H); 1.37 to 1.63 (m, 3 H); 1.41 (s, 9 H); 2.36 to 2.60 (partially masked m, 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, 3 H); 4.09 (m, 1 H); 4.26 (m, 1 H); 4.40 (s, 2 H); 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, 3 H); 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+HC02H]'; tR = 1.89 min.
Compound 31: (38,10R,16S,E)—16-((R,E)(4-(azidomethyl)phenyl)but—3-enyl)—10-(3-chloro— 4-methoxybenzyl)isobutyl-6,6-dimethyloxa-4,8,1 1-triazacyclohexadecene—2,5,9,12- ne Compound 31 was prepared in two steps.
SEM: in a round bottom flask were introduced compound 30 (1.011 g, 1.15 mmol) in DCM (10 mL). After cooling to 0°C, TFA (1.72 mL, 22.94 mmol) and 100 pL of H20 were added. The reaction medium was stirred for 72 h at RT. Upon completion, toluene was added to the medium and it was concentrated in vacuo to give 900 mg of acid intermediate (quant.).
RMN 1H (400 MHz, 8 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, 3 H); 1.08 (s, 3 H); 1.38 to 1.62 (m, 3 H); 2.35 to 2.58 (partially masked m, 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, 3 H); 4.03 (m, 1 H); 4.26 (m, 1 H); 4.40 (s, 2 H); 4.92 (m, 1 H); 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 (broad s large, 3 H); 8.10 (t, J = 6.4 Hz, 1 H); 12.2 (broad s, 1 H). LCMS (A1): ES m/z = 723 [M-H]'; m/z = 725 ; tR = 1.09 min.
M: in a round bottom flask, to a solution of amino/acid intermediate (840 mg, 1.16 mmol) in 200 mL of CH3CN were added DIEA (1.95 mL, 11.58 mmol), HOAt 3 mg, 1.16 mmol) and HATU (499.40 mg, 1.27 mmol). The reaction medium was stirred for 3 h at RT. After concentration in vacuo, the crude was diluted with AcOEt (200 mL), neutralized with 0.5M citric acid (12 mL) and 1N HCI (6 mL). The organic layer was separated, washed with sat. NaHSOs, sat. NaHC03, sat. brine, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 40 g of silica gel ent elution DCM/MeOH) to give 558 mg of compound 31 as a white solide (68%).
RMN 1H (400 MHz, 8 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, 3 H); 1.07 (s, 3 H); 1.09 (d, J = 7.0 Hz, 3 H); 1.15 (m, 1 H); 1.33 to 1.48 (m, 2 H); 2.24 (m, 1 H); 2.53 to 2.70 (m, 2 H); 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, 3 H); 4.18 (m, 1 H); 4.35 (m, 1 H); 4.40 (s, 2 H); 4.95 (m, 1 H); 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)oxiranyl)ethyl)— -(3-chloromethoxybenzyl)isobutyl-6,6-dimethyloxa-4,8,1 1-triazacyclohexadec—13-ene- 2,5,9,12-tetraone In a round bottom flask, to a solution of nd 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 RT, m—CPBA (130 mg) was added twice in 24 h. Once the reaction completed, the crude mixture was d for 1 h with sat. NaHCOs (15 mL) and sat. Na28203 (15 mL) then extracted with DCM (3 x 15 mL). The organic layers were washed with sat. brine (15 mL), dried over MgSO4, filtered and concentrated to give 440 mg of a mixture of alpha and beta epoxides as a yellow semi-solid. Alpha and beta epoxides were separated by chiral liquid chromatography that was carried out on a 76 x 350 mm column packed with 1.1 kg of 10 pm 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 were obtained as a white solid (44%) and 118 mg of the alpha epoxide were ed as a white solid (28%).
RMN 1H (400 MHz, 8 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, 3 H); 1.04 (d, J = 7.0 Hz, 3 H); 1.08 (s, 3 H); 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, 2 H); 2.85 (broad d, J = 13.2 Hz, 1 H); 3.00 (m, 2 H); 3.25 (dd, J = .2 and 13.2 Hz, 1 H); 3.80 (s, 3 H); 3.90 (d, J = 2.0 Hz, 1 H); 4.17 (m, 1 H); 4.35 (m, 1 H); 4.46 (s, 2 H); 5.12 (m, 1 H); 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.
Exam le 1: 3S 10R 168 E S 2R 3R 4- aminometh | hen | oxiran | eth | - - 3-chloromethox benz | isobut l-6 6-dimeth loxa-4 811-triazac clohexadec ene-2,5,9,12-tetraone In a round bottom flask, to a solution of compound 32 (185 mg, 255.79 pmol) in DCM (6 mL), MeOH (6 mL) and H20 (0.8 mL), was added TCEP (81,47 mg, 281,37 pmol). The solution was stirred 16 h at RT. Once the reaction te, the crude e was diluted with sat. NaHCOs (15 mL) and extracted with DCM (2 x 30 mL). The organic layers were washed with sat. brine, dried over MgSO4, fitlered, concentrated and purified by two successive flash chromatographies, on 15 g of silica gel (gradient elution DCM/MeOH) to give 90 mg of example 1 as a white solid (51%).
RMN 1H (400 MHz, 6 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, 3 H); 1.03 (d, J = 7.1 Hz, 3 H); 1.07 (s, 3 H); 1.14 (m, 1 H); 1.42 to 1.57 (m, 2 H); 1.77 (m, 1 H); 1.92 (broad m, 2 H); 2.26 (m, 1 H); 2.64 (m, 2 H); 2.86 (broad 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, 2 H); 3.80 (s, 3 H); 3.84 (d, J = 2.0 Hz, 1 H); 4.18 (m, 1 H); 4.34 (m, 1 H); 5.11 (m, 1 H); 5.79 (dd, J = 1.7 and 15.6 Hz, 1 H); 6.38 (ddd, J = 41,116 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)isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexad ec-1 3- eny|)ethy|)oxiranyl)benzyl)amino)—1-oxopropany|)methy|butanamide Under argon, in a round bottom flask to a solution of example 1 (90 mg, 129.01 pmol) in DCM (20 mL), were added (S)—2-((S)—2-((((9H-fluorenyl)methoxy)carbonyl)amino)methy|butanamido )propanoic acid or FmocValAla (CAS number [1501 149], 79.47 mg, 193.61 pmol), EDC (34.27 pL, 193.61 pmol) and HOBt (20.93 mg, 154.9 pmol). The on medium was stirred overnight at RT. After this time, piperidine (129 pL, 1.29 mmol) was added and d for 2 h. The solvent was removed and the crude residue was purified by flash chromatography on 15 g of silica gel (gradient elution DCM/MeOH) to give 50 mg of compound 33 (45%) as a white solid.
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 0.75 (m, 9 H); 0.88 (d, J = 7.1 Hz, 3 H); 0.96 (s, 3 H); 1.03 (d, J = 7.1 Hz, 3 H); 1.08 (s, 3 H); 1.18 (m, 1 H); 1.23 (d, J = 7.0 Hz, 3 H); 1.42 to 1.57 (m, 2 H); 1.70 (broad m, 2 H); 1.79 (m, 1 H); 1.92 (m, 1 H); 2.26 (m, 1 H); 2.63 (m, 2 H); 2.86 (broad d, J = 13.0 Hz, 1 H); 2.99 (m, 3 H); 3.22 to 3.33 (partially masked m, 1 H); 3.80 (s, 3 H); 3.86 (d, J = 2.0 Hz, 1 H); 4.18 (m, 1 H); 4.28 (d, J = 6.3 Hz, 2 H); 4.33 (m, 2 H); 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, 4 H); 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 (broad 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+HC02H]'; tR = 0.88 min.
Compound 34: 5-(((S)—1-(((S)—1-((4-((2R,3R)—3-((S)—1-((3S,10R,16S,E)—10-(3-chloro—4- methoxybenzyl)isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexad ec-1 3- eny|)ethy|)oxiranyl)benzyl)amino)—1-oxopropanyl)amino)methy|oxobutan no)oxopentanoic acid Under argon, in a round bottom flask, to a solution of compound 33 (54 mg, 57.64 pmol) in DMF (5 mL) was added glutaric anhydride (8 mg, 69.17 pmol). The reaction medium was d for 3.5 h at RT. After this time, the solvent was removed and the crude residue was ed by flash chromatography on 1.8 g of silica gel (gradient elution DCM/MeOH) to give 51 mg of compound 34 as a white solid (90%).
RMN 1H (500 MHz, 6 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, 3 H); 1.03 (d, J = 7.2 Hz, 3 H); 1.05 (s, 3 H); 1.15 (m, 1 H); 1.22 (d, J = 7.2 Hz, 3 H); 1.43 to 1.52 (m, 2 H); 1.70 (m, 2 H); 1.77 (m, 1 H); 1.97 (m, 1 H); 2.17 (m, 2 H); 2.24 (m, 1 H); 2.55 to 2.68 (m, 2 H); 2.85 (broad 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 (partially masked m, 1 H); 3.80 (s, 3 H); 3.85 (d, J :18 Hz, 1 H); 4.22 to 4.38 (m, 4 H); 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 et 8.7 Hz, 1 H); 7.23 (broad s, 4 H); 7.28 (d, J = 2.0 Hz, 1 H); 7.37 (broad d, J = .7 Hz, 1 H); 7.87 (d, J = 8.7 Hz, 1 H); 7.93 (d, J = 9.2 Hz, 1 H); 8.06 (m large, 1 H); 8.37 ( broad m, 1 H); 8.41 (broad m, 1 H); 12.03 (broad m, 1 H). LCMS (A1): ES m/z = 979 [M-H]'; m/z = 981 [M+H]+; tR = 1.17 min. methyloxobutanyl)amino)oxopentanoate Under argon, in a round bottom flask, to a solution of compound 34 (30 mg, 30.56 pmol) in THF (5 mL) were added DIEA (5.34 uL, 30.56 pmol) and DSC (16.31 mg, 61.13 pmol). The reaction medium was stirred for 2 h at RT. After this time, the solvent was removed and the crude residue was purified by flash chromatography on 1.3 g of silica gel (gradient elution DCM/iPrOH) to give 9 mg of example 2 as a white solid. A second batch containing the expected compound as well as an ty was diluted with MeTHF, washed twice with H20, sat. brine, dried over MgSO4, ed and concentrated to give 15 mg of example 2 as a white solid (global yield of 73%).
RMN 1H (500 MHz, 6 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, 3 H); 1.03 (d, J = 7.2 Hz, 3 H); 1.07 (s, 3 H); 1.15 (m, 1 H); 1.23 (d, J = 7.2 Hz, 3 H); 1.43 to 1.56 (m, 2 H); 1.78 (m, 1 H); 1.81 (m, 1 H); 1.97 (m, 1 H); 2.20 to 2.33 (m, 3 H); 2.58 to 2.69 (m, 4 H); 2.80 (s, 4 H); 2.85 (broad 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 (partially masked m, 1 H); 3.80 (s, 3 H); 3.85 (d, J = 2.1 Hz, 1 H); 4.15 (m, 2 H); 4.22 to 4.38 (m, 4 H); 5.11 (m, 1 H); 5.79 (d, J = 15.1 Hz, 1 H); 6.38 (ddd, J = 39,112 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 (broad 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+HC02H]'; tR = 1.23 min.
Example 3: mAb-Ex2 The general method described previously was used for the preparation of e 3. 60 mg of hu2H11_R35-74 were reacted with 161 pL of a 9.96 mM solution of example 2 in DMA (1.733 mg, 4 eq.) for 2 h. At that time, 121 uL of the solution of example 2 (3 eq.) were added and the medium stirred for 2 h. At that time, 121 uL of the solution of example 2 (3 eq.) were added and stirred for 2 h. After purification on Superdex 200 pg in DPBS pH 6.5 + 20% NMP, concentration on Amicon Ultra-15, buffer exchange on PD-10 in buffer B pH 6.5 + 5% NMP and filtration on Steriflip, 39.9 mg of example 3 were ed as a colorless limpid solution at a concentration of 2.28 mg/mL with a DAR of4.6 (HRMS), a monomeric purity of 99% and a global yield of 66%.
SEC-HRMS: um 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). 8 nthesis of Exam les 4 to 7: benz lic amine of S neo ent -C52 NHS ester of luta l-Val-Ala- S neo ent le-aza-C52 benz lic amine and corres ondin ADC \ / OAK \ / l BocHN Cl COZtBu HO 0% O o 0 km HO 0% o BocHN CI HOJYN O o OMeH A02 km NH2 H 35 "V" o OMe BC1 J \ / /O \ / 2 COZH HO 0\ o HN .~\©:CI HO 0\ o o o 37 NWN \0 OMe 36 NWNHZNl~\©:CI0 OMe H H H H \ / /O / O TIPSO o\ o HN \CECI Ho 0\ o HN gel o o 38 NWN \0 OMe Example4 NWN \0 OMe H H H H / /O / /O Example 5 Compound 35: (6R,13S)—(1E,3R,4S,6E)—8—(tert—butoxy)—1-(4-(hydroxymethyl)phenyl)—3-methyl-8— oxoocta-1 nyl 6-(3-chloromethoxybenzyl)-2,2,10,10-tetramethylneopentyl-4,7,1 1- trioxooxa-5,8,12-triazatetradecanoate Under argon, in a round bottom flask, to a solution of fragment BC1 (1.32 g, 3.08 mmol) in DCM (60 mL) were added DIEA (1.53 mL, 9.25 mmol), HOAt (503.75 mg, 3.70 mmol) and HATU (1.41 g, 3.70 mmol). The yellow suspension was stirred for 30 min at RT, then fragment AD2 (1.7 g, 3.08 mmol) was added. The reaction medium was stirred for 2 h at RT. After this time, 1.53 mL of DIEA were added and stirred for 1 h. 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 , H20, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 100 g of silica gel (gradient elution heptane/AcOEt) to give 1.475 g of compound 35 as a colorless solid (57%).
Compound 36: (2E,5S,6R,7E)—5-(((S)(3-((R)amino(3-chloro—4-methoxyphenyl)- propanamido)—2,2-dimethylpropanamido)-4,4-dimethylpentanoyl)oxy)(4-(hydroxymethyl)— phenyl)—6-methylocta-2,7-dienoic acid In a round bottom flask, to a solution of nd 35 (1.475 g, 1.69 mmol) in DCM (20 mL), were added TFA (8 mL, 52.27 mmol) and H20 (2 mL). The reaction medium was stirred for 5 h at RT. The solvent was d. The residue was diluted with H20 (20 mL) and AcOEt (20 mL) and treated with 2M NaOH (2 mL) for 2 h at RT. The organic layer was separated and the aq. layer was extracted with AcOEt (2 x 10 mL), dried over MgSO4, ed and concentrated to give 1.24 g of a colorless solid. The solid was dissolved in AcOEt (10 mL) and H20 (10 mL) and treated with 2M NaOH (400 pL) for 2 h at RT. The organic layer was separated, the aq. 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 (gradient elution heptane/AcOEt) to give 990 mg of compound 36 as a white solid (82%).
RMN 1H (400 MHz, 8 in ppm, DMSO-d6): 0.80 (s, 9 H); 0.99 (s, 3 H); 1.02 (d, J = 7.0 Hz, 3 H); 1.05 (s, 3 H); 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 (partially masked m, 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, 3 H); 4.29 (m, 1 H); 4.45 (s, 2 H); 4.90 (m, 1 H); 5.12 (broad 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, 3 H); 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 ; tR = 0.94 min.
Compound 37: (38,10R,16S,E)—10-(3-chloromethoxybenzyl)—16-((R,E)(4-(hydroxymethyl)- phenyl)but—3-enyl)—6,6-dimethylneopentyloxa-4,8,1 1-triazacyclohexadec—13-ene-2,5,9,12- Under argon, in a round bottom flask, were introduced compound 36 (990 mg, 1.39 mmol) and CH3CN (150 mL), heated with a water bath at 50°C until complete solubilization and stirred for min. After that, DIEA (687.20 pL, 4.16 mmol), HOAt (207.51 mg, 1.52 mmol) and HATU (579.7 mg, 1.52 mmol) were added and stirred for 30 min at RT. The reaction medium was neutralized with 1M citric acid (30 mL). The solvent was removed and the aq. layer was extracted with AcOEt (2 x 40 mL). The organic layers were washed with 1M NaHSO4, H20, dried over MgSO4, filtered and concentrated. The crude solid was diluted with H20 (200 mL) and stirred for 1 h. The solid was ed then d with AcOEt, dried over MgSO4, filtered and concentrated to give a mixture of compound 37 and HATU. The solid was stirred with MeTHF (50 mL) and H20 (50 mL). The organic layer was separated, washed with H20 (4 x 20 mL), dried over MgSO4, filtered, concentrated and ed by flash chromatography on 10 g of silica gel (gradient elution heptane/AcOEt) to give 680 mg of compound 37 as a colorless solid (70 %).
RMN 1H (400 MHz, 8 in ppm, DMSO-d6): 0.68 (s, 9 H); 0.98 (s, 3 H); 1.03 (s, 3 H); 1.09 (d, J = 6.9 Hz, 3 H); 1.19 (broad d, J = 14.5 Hz, 1 H); 1.58 (dd, J = 10.3 and 14.5 Hz, 1 H); 2.23 (m, 1 H); 2.52 to 2.70 (m, 3 H); 2.86 (broad d, J = 12.7 Hz, 1 H); 3.00 (dd, J = 3.3 and 14.8 Hz, 1 H); 3.22 to 3.33 (partially masked m, 1 H); 3.80 (s, 3 H); 4.18 (m, 1 H); 4.42 (m, 1 H); 4.46 (d, J = 6.0 Hz, 2 H); 4.91 (m, 1 H); 5.13 (t, J = 6.0 Hz, 1 H); 5.85 (broad d, J = 15.0 Hz, 1 H); 6.05 (dd, J = 8.4 and 15.9 Hz, 1 H); 6.40 (m, 2 H); 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 min.
Compound 38: (38,10R,16S,E)—10-(3-chloromethoxybenzyl)-6,6-dimethylneopentyl ((R,E)(4-(((triisopropylsilyl)oxy)methyl)phenyl)but—3-enyl)—1-oxa-4,8,1 1-triazacyclohexadec— 13-ene—2,5,9,12-tetraone Under argon, at 0°C in a round bottom flask, to a solution of compound 37 (680 mg, 0.976 mmol) in CHCI3 (10 mL) were added 1H-imidazole (305.84 mg, 4.49 mmol) and chlorotriisopropylsilane (480.13 pl, 2.25 mmol). The reaction medium was stirred for 5 h at RT then diluted with sat. NH4C| and MTBE (30 mL). The organic layer was washed with 1M NaHSO4, sat. NaHC03, sat. brine, dried over MgSO4, filtered and trated to give 970 mg of compound 38 as an orange amorph solid (quant.).
RMN 1H (500 MHz, 8 in ppm, DMSO-d6): 0.63 (s, 9 H); 0.87 a 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, 3 H); 2.86 (broad d, J = 13.0 Hz, 1 H); 3.00 (dd, J = 3.2 and 14.9 Hz, 1 H); 3.24 to 3.35 (partially masked m, 1 H); 3.80 (s, 3 H); 4.18 (m, 1 H); 4.41 (m, 1 H); 4.76 (s, 2 H); 4.91 (m, 1 H); 5.87 (broad d, J = 15.4 Hz, 1 H); 6.06 (dd, J = 8.9 and .9 Hz, 1 H); 6.40 (m, 2 H); 7.03 (d, J = 8.5 Hz, 1 H); 7.18 (broad 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 C02H]'; tR = 2.15 min.
Exam le 4: 3S 10R 168 E 3-chloromethox benz | S 2R 3R 4- h drox - meth | hen |oxiran leth l-6 6-dimeth o ent loxatriazac clohexadec- 13-ene-2,5,9,12-tetraone Example 4 was prepared in 2 steps.
Sim: 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 h at RT then diluted with DCM (10 mL) and stirred for 15 min with sat. NaHC03 (30 mL) and Na28203 (30 mL). The c layer was separated, washed with sat. brine, dried over MgSO4, filtered and trated to give 1.1 g of mixture of alpha and beta epoxides as a colorless foam (quant.).
M:the mixture of alpha and beta epoxides was diluted in THF (30 mL) and 1M TBAF (952.32 pL) was added. After stirring for 2 h, 952 pL of TBAF were added. After stirring for 1 h, the mixture was diluted with H20 (50 mL) and extracted with AcOEt (3 x 50 mL). The organic layers were washed with sat. brine, dried over MgSO4, ed and concentrated to give 770 mg of a mixture of alpha and beta epoxides as a yellow solid (quant.). Alpha and beta epoxides were separated by chiral liquid chromatography that was carried out on a 76 x 350 mm column packed with 1.1 kg of 10 pm Chiralpak AD (amylose tris-3,5-dimethylphenylcarbamate coated on a silica gel support, Chiral Technologies ) using isocratic elution with 75:25 heptane/EtOH. After concentration, 190 mg of example 4 were obtained as a white solid (31%) and 125 mg of the alpha epoxide were obtained as a white solid (20%).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.81 (s, 9 H); 0.98 (s, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.05 (s, 3 H); 1.26 (m, 1 H); 1.71 (dd, J = 10.4 and 14.5 Hz, 1 H); 1.82 (m, 1 H); 2.26 (m, 1 H); 2.52 (m, 2 H); 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 (partially masked m, 1 H); 3.81 (s, 3 H); 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 min.
Compound 39: (3S,10R,16S,E)—16-((S)—1-((2R,3R)—3-(4—(azidomethyl)phenyl)oxiranyl)ethyl)— -(3-chloromethoxybenzyl)-6,6-dimethylneopentyloxa-4,8,1 1-triazacyclohexad ec-1 3- ene-2,5,9,12-tetraone Under argon, in a round bottom flask, to a solution of example 4 (100 mg, 140 pmol) in THF (5 mL) were added DPPA (156.82 pl, 701.98 pmol) and DBU (110.22 pl, 701.98 pmol). The solution was d for 6 h at RT then diluted with H20 and extracted with AcOEt (3 x 30 mL).
The organic layer was separated, washed with sat. brine, dried over MgSO4, filtered, concentrated and ed on 15 g of silica gel (gradient n DCM/iPrOH) to give 100 mg of nd 39 as a white solid (40 %).
RMN 1H (400 MHz, 8 in ppm, DMSO-d6): 0.80 (s, 9 H); 0.97 (s, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3 H); 1.28 (d, J =14.5 Hz, 1 H); 1.69 (dd, J = 10.7 and 14.5 Hz, 1 H); 1.84 (m, 1 H); 2.28 (m, 1 H); 2.62 (m, 2 H); 2.84 (d, J = 12.8 Hz, 1 H); 2.93 to 3.03 (m, 2 H); 3.22 to 3.34 (partially masked m, 1 H); 3.80 (s, 3 H); 3.92 (broad s, 1 H); 4.17 (m, 1 H); 4.40 to 4.49 (m, 3 H); 5.10 (m, 1 H); 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, 1 H); 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 min.
Exam le 5: 3S 10R 168 E S 2R 3R 4- aminometh | hen | oxiran | eth | - - 3-chloromethox benz l-6 th lneo ent loxatriazac clohexadec- 13-ene-2,5,9,12-tetraone In a round bottom flask, to a solution of compound 39 (100 mg, 122.07 umol) in DCM (2.5 mL) and MeOH (2.5 mL) was added dropwise a solution of TCEP (38.88 mg, 134.28 umol) in H20 (500 uL). The reaction medium was stirred for 24 h at RT. The reaction mixture was diluted with H20 and sat. NaHC03, ted with DCM (3 x 10 mL). The organic layer was separated, washed with sat. brine, dried over MgSO4, filtered and concentrated to give 73 mg of example 5 as a white solid (84%) used without further purification in the next step.
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.82 (s, 9 H); 0.97 (s, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3 H); 1.29 (d, J = 14.4 Hz, 1 H); 1.70 (dd, J = 10.2 and 14.4 Hz, 1 H); 1.80 (m, 1 H); 2.02 (broad m, 2 H); 2.25 (m, 1 H); 2.62 (m, 2 H); 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 ally masked m, 1 H); 3.71 (s, 2 H); 3.80 (s, 3 H); 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, 1 H); 5.09 (m, 1 H); 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 min.
Compound 40: (9H-fluorenyl)methyl ((S)—1-(((S)—1-((4-((2R,3R)((S)—1-((3S,10R,16S,E)—10- (3-chloromethoxybenzyl)-6,6-dimethylneopentyl-2,5,9,12-tetraoxooxa-4,8,1 1- triazacyclohexadec—13-enyl)ethyl)oxiranyl)benzyl)amino)—1-oxopropanyl)amino)—3- methyl-1 -oxobutanyl)ca e Under argon, in a round bottom flask were introduced example 5 (73 mg, 82.10 umol) and DMF (1 mL), followed by FmocValAla (50.55 mg, 123.16 umol), HOBt (17.75 mg, 131.37 umol), DCM (10 mL) and EDC (14.53 pl, 82.10 umol). The solution was stirred for 4 h at RT then diluted with H20 (10 mL) and extracted with DCM (3 x 20 mL). The organic layer was separated, washed with H20, dried over MgSO4, filtered, concentrated and purified on 15 g of silica gel (gradient elution DCM/iPrOH) to give 90 mg of nd 40 as a colorless solid (99 %).
RMN 1H (500 MHz, 6 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, 3 H); 1.02 (d, J = 7.1 Hz, 3 H); 1.04 (s, 3 H); 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, 1 H); 1.99 (m, 1 H); 2.25 (m, 1 H); 2.60 (m, 2 H); 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, 1 H); 3.80 (s, 3 H); 3.88 (d, J =1.9 Hz, 1 H); 3.90 (m, 1 H); 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/z =1147[M-H+HC02H]';tR = 1.71 min.
Compound 41: (S)—2-amino-N-((S)—1-((4-((2R,3R)—3-((S)—1-((3S,10R,16S,E)—10-(3-chloro methoxybenzyl)-6,6-dimethylneopentyl-2,5,9,12-tetraoxooxa-4,8,1 1-triazacyclohexad ec-1 3- enyl)ethy|)oxiranyl)benzyl)amino)—1-oxopropanyl)methylbutanamide Under argon, in a round bottom flask, were introduced compound 40 (104 mg, 76.65 umol) in DCM (5 mL) followed by piperidine (138.87 pl, 1.40 mmol). The solution was stirred for 5 h at RT then concentrated and purified on 10 g of silica gel (gradient elution DCM/MeOH/HZO) to give 50 mg of compound 41 as a colorless solid (60 %).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.76 (d, J = 7.0 Hz, 3 H); 0.81 (s, 9 H); 0.88 (d, J = 7.0 Hz, 3 H); 0.97 (s, 3 H); 1.02 (d, J = 7.1 Hz, 3 H); 1.04 (s, 3 H); 1.22 (d, J = 7.3 Hz, 3 H); 1.29 (d, J = 14.6 Hz, 1 H); 1.66 (broad m, 2 H); 1.70 (dd, J = 10.6 and 14.6 Hz, 1 H); 1.81 (m, 1 H); 1.92 (m, 1 H); 2.25 (m, 1 H); 2.60 (m, 2 H); 2.83 (d, J = 13.0 Hz, 1 H); 2.93 (dd, J = 1.9 and 7.4 Hz, 1 H); 2.99 (m, 2 H); 3.28 (dd, J = 10.4 and 13.0 Hz, 1 H); 3.80 (s, 3 H); 3.89 (d, J = 1.9 Hz, 1 H); 4.16 (ddd, J = 3.2 and 8.2 et 11.8 Hz, 1 H); 4.29 (d, J = 6.0 Hz, 2 H); 4.34 (m, 1 H); 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 .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, 4 H); 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 [M+2H]2+; m/z = 879 ; m/z = 881 ; m/z = 925 [M-H+HC02H]'; tR = 0.99 min.
Compound 42: 5-(((S)—1-(((S)—1-((4-((2R,3R)—3-((S)—1-((3S,10R,16S,E)—10-(3-chloromethoxy— benzyl)-6,6-dimethylneopentyl-2,5,9,12-tetraoxooxa-4,8,1 zacyclohexadec—13-en yl)ethy|)oxiranyl)benzyl)amino)oxopropanyl)amino)methyloxobutanyl)amino)—5- oxopentanoic acid Under argon, in a round bottom flask, were introduced compound 41 (50 mg, 51.05 umol) in DCM (10 mL) followed by glutaric anhydride (10.48 mg, 91.89 umol). The reaction medium was stirred for 2 h at RT, concentrated and purified on 10 g of silica gel (gradient elution DCM/MeOH/HZO) to give 42 mg of compound 42 as a colorless solid (82 %).
RMN 1H (500 MHz, 6 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, 3 H); 1.02 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3 H); 1.23 (d, J = 7.2 Hz, 3 H); 1.29 (d, J = 14.2 Hz, 1 H); 1.70 (m, 3 H); 1.81 (m, 1 H); 1.96 (m, 1 H); 2.18 to 2.25 (m, 5 H); 2.61 (m, 2 H); 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, 3 H); 3.89 (d, J = 2.0 Hz, 1 H); 4.16 (m, 2 H); 4.25 to 4.31 (m, 3 H); 4.42 (m, 1 H); 5.09 (m, 1 H); 5.79 (dd, J :19 and 15.5 Hz, 1 H); 6.39 (ddd, J = 3.8, 11.6 et15.5 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, 4 H); 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, 2 H); 8.35 (t, J = 6.1 Hz, 1 H); 8.39 (d, J = 8.0 Hz, 1 H); 12.04 (broad 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 min. methyloxobutanyl)amino)oxopentanoate Under argon, in a round bottom flask, were introduced compound 42 (23 mg, 23.10 umol) in DCM (5 mL), followed by DSC (8.29 mg, 32.34 pmol) and DIEA (5.63 pL, 32.34 umol). The reaction medium was stirred for 2 h at RT. After this time, 2 mg of DSC, 1pL of DIEA and DCM (2 mL) were added and stirred for 1 h at RT. The solvent was removed and the crude e was ed by flash chromatography on 10 g of silica gel (gradient elution DCM/iPrOH) to give 20 mg of example 6 as a colorless solid (79%).
RMN 1H (500 MHz, 6 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, 3 H); 1.02 (d, J = 7.0 Hz, 3 H); 1.04 (s, 3 H); 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, 3 H); 1.97 (m, 1 H); 2.21 to 2.32 (m, 3 H); 2.61 (m, 2 H); 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 et 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, 3 H); 3.88 (d, J = 2.0 Hz, 1 H); 4.16 (m, 2 H); 4.23 to 4.31 (m, 3 H); 4.42 (m, 1 H); 5.10 (m, 1 H); 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, 4 H); 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+HCOZH]'; tR = 1.34 min.
Example 7 : mAb-Ex6 The general method described previously was used for the preparation of e 7. 60 mg of hu2H11_R35-74 were reacted with 233 uL of a 10.6 mM solution of example 6 in DMA (5 eq.) for 2 h. After purification on Superdex 200 pg in DPBS pH 6.5 + 20% NMP, concentration on Amicon Ultra-15, buffer exchange on NAP-25 in buffer B pH 6.5 + 5% NMP and filtration on Steriflip, 39 mg of example 7 were obtained as a ess limpid on at a concentration of 1.98 mg/mL with a DAR of 4.1 (HRMS), a monomeric purity of 100% and a global yield of 66%.
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 : benzylic amine of 7-Me-aza-C52 stereomer1, NHS ester of g|uta[yl-Val-AlaMe-aza-C52 benzylic amine mer1 and corresponding ADC m1/OESH/Hz OOYHOOBocHNk cotBu I-|Bc2 3}:j\?lHkBocHNk Me \ / 0 N3 0\ o HN Cl 44: stereomer 1 1%U O OMe 45: stereomer ZEHJVgH N3 03%" \O["1"ij 8N3 0% OHNI\\©:CI O 46 ENV N \ OMe H H stereomer 1 stereomer 1 FmOCHNQkN/IYNMg‘7EM \0 ExampleB/EH stereomer 1 stereomeHr 1 NHYHp??l? 0WeHNH?n stereomer 1 hu2H11_R35—74FNWNQNNJYNWExample10/E\:;j>(L stereomer1 Compound 43: (6R,13S)—(1E,3R,4S,6E)—1-(4-(azidomethyl)phenyl)(tert—butoxy)—3-methy|—8— oxoocta-1,6-dieny| 6-(3-chIoromethoxybenzyl)isobutyI-2,2,9,10,10-pentamethyI-4,7,11- trioxooxa-5,8,12-triazatetradecanoate Under argon, in a round bottom flask, were introduced fragment BCZ (1.10 g, 2.48 mmol) in DMF (5 mL), followed by HATU (950 mg, 2.5 mmol) and HOAt (340 mg 2.5 mmol). The mixture was d for 30 min at RT, then fragment AD1 (1.24 g, 2.18 mmol) and DIEA (1.2 mL 6.87 mmol) were added. The yellow solution was stirred for 16 h at RT, quenched with H20 and extracted with AcOEt (3 x 30 mL). The organic layers were washed with H20, sat. brine, dried over MgSO4, filtered, concentrated and purified on 200 g of silica gel (gradient elution heptane/AcOEt) to give 1.55 g of compound 43 as a white meringue (77%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 55/45 diastereoisomer e; 0.82 to 0.89 (m, 6 H); 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 ally masked m, 3 H); 2.68 (m, 1 H); 2.84 (m, 1 H); 3.79 (s, 1.35 H); 3.81 (s, 1.65 H); 4.00 to 4.18 (m, 2 H); 4.30 (m, 1 H); 4.40 (broad s, 2 H); 4.92 (m, 1 H); 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 (broad 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 min.
Compounds 44 & 45: (3S,10R,16S,E)—16-((R,E)(4-(azidomethyl)phenyl)butenyl)(3- methoxybenzyl)isobutyl-6,6,7-trimethyloxa-4,8,1 1-triazacyclohexadec—13-ene- 2,5,9,12-tetraone Compounds 44 & 45 were obtained in two steps.
Sim: 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 were added TFA (2.6 mL, 34.65 mmol) and 100 uL of H20. The mixture was stirred for 15 min at 0°C and at RT for 6.5 h. The reaction medium was then evaporated in vacuo and porated in the presence of toluene to give 1.7 g of deprotected intermediate as an orange solid.
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 55/45 diastereoisomer mixture; 0.70 to 0.80 (m, 7.65 H); 0.90 (s, 1.35 H); 0.95 (m, 3 H); 1.01 to 1.09 (m, 6 H); 1.38 to 1.68 (m, 3 H); 2.40 to 2.60 (partially masked m, 3 H); 2.80 to 3.02 (m, 2 H); 3.81 (s, 1.35 H); 3.82 (s, 1.65 H); 3.96 to 4.08 (m, 1 H); 4.18 (m, 1 H); 4.28 (m, 1 H); 4.41 (s, 2 H); 4.91 (m, 1 H); 5.83 (d, J =15.7 Hz, 0.45 H); .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, 1 H); 7.08 to 7.20 (m, 2 H); 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, 2 H); 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 (broad m, 3 H); 12.22 (broad m, 1 H). LCMS (A1): 55/45 reoisomer mixture; ES m/z = 737 [M-H]'; m/z = 739 [M+H]+; tR = 1.07-1.09 min.
M: in a round bottom flask, to a solution of deprotected ediate (1.43 g, 1.68 mmol) in 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 h at RT. Then the solvent was removed, the medium diluted with AcOEt (200 mL), neutralized with 0.5 M citric acid and HCI.
The organic layer was separated, washed with sat. NaH803, sat. NaHC03, sat. brine, dried over MgSO4, filtered, concentrated and purified on 100 g of silica gel (gradient elution DCM/MeOH) to give 640 mg of nds 44 & 45 as a yellow solid (53%).
Diastereoisomers at C7 were separated by chiral liquid chromatography that was carried out on a 76.5 x 350 mm column packed with 1.1 kg of 10 pm Whelk 01 SS (4-(3,5-dinitrobenzamido) tetrahydrophenanthrene, Regis Technologies) using isocratic elution with 50:50 heptane/EtOH.
After concentration, 210 mg of compound 44 (stereomer 1)were obtained as a white solid (17%) and 236 mg of compound 45 (stereomer 2) were obtained as a white solid (19%).
Compound 44 (stereomer 1): RMN 1H (500 MHz, 8 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, 3 H); 1.05 (d, J = 7.0 Hz, 3 H); 1.10 (d, J = 7.0 Hz, 3 H); 1.14 (s, 3 H); 1.18 (m, 1 H); 1.40 to 1.54 (m, 2 H); 2.24 (m, 1 H); 2.48 to 2.61 (partially masked m, 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, 1 H); 3.81 (s, 3 H); 4.19 (m, 2 H); 4.40 (s, 2 H); 4.96 (m, 1 H); 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 min Compound 45 (stereomer 2): RMN 1H (8 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, 3 H); 1.10 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3 H); 1.21 (m, 1 H); 1.49 to 1.60 (m, 2 H); 2.23 (m, 1 H); 2.46 to 2.61 (partially masked m, 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, 1 H); 3.80 (s, 3 H); 4.05 (m, 1 H); 4.11 (ddd, J = 3.7, 7.6 and 11.3 Hz, 1 H); 4.40 (s, 2 H); 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 = 52,103 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 min.
Compound 46: (38,10R,16S,E)—16-((S)—1-(3-(4-(azidomethy|)phenyl)oxiranyl)ethy|)(3- methoxybenzyl)isobutyl-6,6,7-trimethyloxa-4,8,1 1-triazacyclohexadecene- 2,5,9,12-tetraone At 0°C, under argon, in a round bottom flask to a solution of compound 44 (154 mg, 213.51 pmol) in DCM (10 mL) was added m—CPBA (80 mg, 324.51 pmol). The on medium was stirred at RT for 5 d. The reaction mixture was diluted with DCM (15 mL) and stirred 15 min with sat. NaHC03 (6 mL) and Na28203 (6 mL). The organic layer was ted, washed with sat. brine (2 x 3 mL), dried over MgSO4, filtered and concentrated to give 150 mg of nd 46 as a white solid (quant.).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 60/40 reoisomer 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, 1.8 H); 1.18 (s, 1.2 H); 1.20 (m, 0.6 H); 1.38 (m, 0.4 H); 1.59 to 1.62 (m, 2 H); 1.80 (m, 0.6 H); 1.89 (m, 0.4 H); 2.25 (m, 0.6 H); 2.40 to 2.71 (partially masked m, 2.4 H); 2.96 to 3.08 (m, 2 H); 3.54 (m, 1 H); 3.80 (s, 3 H); 3.82 (d, J = 1.9 Hz, 0.4 H); 3.92 (d, J :19 Hz, 0.6 H); 4.14 to 4.29 (m, 2 H); 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); .95 (dd, J = 2.0 and 15.5 Hz, 0.4 H); 6.38 (m, 1 H); 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 min.
Exam le 8: 3S 10R 168 E S 3- 4- eth | hen | oxiran | eth | 3- chloromethox benz | isobut l-6 6 7-trimeth |oxa-4 811-triazac clohexadec ene-2,5,9,12-tetraone At 0°C, in a round bottom flask, to a solution of compound 46 (60 mg, 81.38 pmol) in DCM (3 mL) and MeOH (3 mL) was dropwise added a solution of TCEP (38.9 mg, 134.3 pmol) in 1 mL of H20. The reaction mixture was stirred at RT for 35 h, then diluted with DCM (15 mL) and sat.
NaHC03. After stirring for 10 min, the organic layer was separated, washed with sat. brine, dried over MgSO4, filtered, concentrated in vacuo and purified on 5 g of silica gel (gradient e|ution DCM/MeOH) to give 23 mg of example 8 as a white solid (40 %).
RMN 1H (500 MHz, 8 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, 1.8 H); 1.00 (s, 1.2 H); 1.02 to 1.09 (m, 4.8 H); 1.17 (s, 1.8 H); 1.19 (s, 1.2 H); 1.22 (m, 0.6 H); 1.40 (m, 0.4 H); 1.51 to 1.64 (m, 2 H); 1.78 (m, 0.6 H); 1.89 (m, 0.4 H); 2.28 (m, 0.6 H); 2.40 to 2.71 (partially masked m, 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, 1 H); 3.70 (s, 1.2 H); 3.72 (s, 1.8 H); 3.78 (d, J = 2.2 Hz, 0.4 H); 3.81 (s, 3 H); 3.88 (d, J = 2.2 Hz, 0.6 H); 4.12 to 4.30 (m, 2 H); 5.12 (m, 1 H); 5.84 (dd, J = 1.8 and 15.6 Hz, 0.6 H); 5.96 (dd, J :18 and 15.6 Hz, 0.4 H); 6.39 (m, 1 H); 7.05 (d, J = 8.7 Hz, 1 H); 7.16 to 7.38 (m, 6 H); 7.90 (d, J = 8.1 Hz, 0.6 H); 7.93 (d, J = 8.1 Hz, 0.4 H); 8.28 (d, J = .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 ; tR = 0.86 min. nd 47: (9H-fluorenyl)methyl ((S)—1-(((S)((4-(3-((S)—1-((3S,10R,16S,E)—10-(3-chloro— 4-methoxybenzyl)isobuty|-6,6,7-trimethy|-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexad ec- 16-yl)ethyl)oxiranyl)benzyl)amino)—1-oxopropanyl)amino)methyloxobutan y|)carbamate Under argon, in a round bottom flask were introduced example 8 (110 mg, 154.65 umol) and DMF (2 mL), followed by FmocValAla (90 mg, 219 pmol), HOBt (30 mg, 222 pmol), DCM (10 mL) and EDC (35 pl, 197.52 pmol). The solution was stirred at RT for 3h30 then quenched with H20 (10 mL) and extracted by DCM (3 x 10 mL). The organic layer was separated, washed with sat.
NaHC03, sat. brine, dried over MgSO4, filtered, concentrated in the presence of toluene and purified on 15 g of silica gel (gradient elution DCM/MeOH) to give 49 mg of compound 47 as a white solid (46 %).
RMN 1H (500 MHz, 8 in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.76 (d, J = 7.0 Hz, 1.8 H); 0.80 (m, 3 H); 0.82 to 0.89 (m, 6.4 H); 0.95 (d, J = 7.0 Hz, 1.2 H); 0.98 (s, 1.8 H); 1.00 (s, 1.2 H); 1.02 to 1.09 (m, 4.8 H); 1.17 (s, 1.8 H); 1.19 (s, 1.2 H); 1.20 to 1.32 (m, 3.6 H); 1.40 (m, 0.4 H); 1.51 to 1.63 (m, 2 H); 1.78 (m, 0.6 H); 1.85 (m, 0.4 H); 2.00 (m, 1 H); 2.27 (m, 0.6 H); 2.40 to 2.72 (partially masked m, 2.4 H); 2.96 (m, 1 H); 3.04 (m, 1 H); 3.55 (m, 1 H); 3.77 (d, J = 2.2 Hz, 0.4 H); 3.81 (s, 3 H); 3.88 (d, J = 2.2 Hz, 0.6 H); 3.90 (m, 1 H); 4.15 to 4.38 (m, 6 H); 5.12 (m, 1 H); 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+HC02H]'; tR = .57 min. nd 48: -amino—N-((2S)—1-((4-((2R,3R)((1S)—1-((3S,10R,168,E)—10-(3-chloro—4- methoxybenzyl)isobutyl-6,6,7-trimethyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexadec enyl)ethy|)oxiranyl)benzyl)amino)—1-oxopropanyl)methylbutanamide In a round bottom flask, piperidine (60 pL, 600.6 pmol) was added to a solution of compound 47 (50 mg, 45.30 pmol) in DCM (5 mL). The resulting mixture was stirred for 24 h at RT, concentrated in vacuo and purified on 5 g of silica gel (gradient n DCM/MeOH) to give 70 mg of apha and beta epoxides.
RMN 1H (500 MHz, 8 in ppm, DMSO-d6): 60/40 diastereoisomer mixture; 0.75 (d, J = 7.0 Hz, 1.8 H); 0.80 (m, 3 H); 0.82 to 0.92 (m, 6.4 H); 0.96 (d, J = 7.0 Hz, 1.2 H); 0.98 (s, 1.8 H); 1.00 (s, 1.2 H); 1.02 to 1.09 (m, 4.8 H); 1.15 (s, 1.8 H); 1.19 (s, 1.2 H); 1.20 to 1.42 (m, 4 H); 1.50 to 1.70 (masked m, 2 H); 1.78 (m, 0.6 H); 1.85 (m, 0.4 H); 2.00 (m, 1 H); 2.25 (m, 0.6 H); 2.40 to 2.72 (partially masked m, 2.4 H); 2.92 to 3.08 (m, 3 H); 3.54 (m, 1 H); 3.79 (d, J = 2.1 Hz, 0.4 H); 3.81 (s, 3 H); 3.88 (d, J = 2.1 Hz, 0.6 H); 4.13 to 4.41 (m, 6 H); 5.12 (m, 1 H); 5.85 (d, J = 15.4 Hz, 0.6 H); 5.93 (d, J =15.4 Hz, 0.4 H); 6.38 (m, 1 H); 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+HC02H]'; tR = 0.92 min.
Alpha and beta epoxides were separated by chiral liquid tography that was d out on a 76 x 350 mm column packed with 1.1 kg of 10 pm Chiralpak AD (amylose tris-3,5— dimethylphenylcarbamate coated on a silica gel support, Chiral Technologies Europe) using isocratic elution with 70:30 heptane/EtOH. After concentration, 28 mg of nd 48 were obtained as a white solid (70%) and 19 mg of the alpha epoxide were obtained as a white solid.
RMN 1H (500 MHz, 8 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, 3 H); 1.03 (d, J = 7.0 Hz, 6 H); 1.17 (s, 3 H); 1.23 (d, J = 7.0 Hz, 3 H); 1.35 (m, 1 H); 1.50 to 1.68 (m, 2 H); 1.78 (m, 1 H); 1.91 (m, 1 H); 2.26 (m, 1 H); 2.58 to 2.70 (m, 2 H); 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, 3 H); 3.89 (d, J :19 Hz, 1 H); 4.10 to 4.23 (m, 3 H); 4.28 (d, J = 6.1 Hz, 2 H); 4.35 (m, 1 H); 5.11 (m, 1 H); 5.83 (dd, J :19 and 15.2 Hz, 1 H); 6.37 (ddd, J = 4011.2 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, 4 H); 7.29 (d, J = 2.4 Hz, 1 H); 7.90 (d, J = 8.2 Hz, 1 H); 8.08 (broad 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+HC02H]'; tR = 0.92 min.
Compound 49: 5-(((S)(((S)—1-((4-((2R,3R)—3-((S)—1-((3S,10R,16S,E)—10-(3-chloro—4- methoxybenzyl)isobutyl-6,6,7-trimethyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexadec enyl)ethy|)oxiranyl)benzyl)amino)—1-oxopropanyl)amino)—3-methyloxobutan yl)amino)—5—oxopentanoic acid Under argon, in a round bottom flask, a solution of ic anhydride (3.78 mg 32.44 pmol) in DCM (4 mL) was added to a solution of compound 48 (26 mg, 29.5 pmol) in DCM (9 mL). The resulting mixture was d 2 h at RT, concentrated in vacuo and purified on 2.5 g of silica gel (gradient elution DCM/MeOH/HZO) to give 18 mg of compound 49 as a white solid (61%).
RMN 1H (500 MHz, 8 in ppm, 6): 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, 3 H); 1.03 (d, J = 7.0 Hz, 6 H); 1.15 (s, 3 H); 1.23 (broad d, J = 7.0 Hz, 4 H); 1.49 to 1.61 (m, 2 H); 1.70 (m, 2 H); 1.78 (m, 1 H); 1.97 (m, 1 H); 2.20 (m, 4 H); 2.25 (m, 1 H); 2.62 (m, 2 H); 2.95 (m, 1 H); 3.02 (m, 1 H); 3.53 (m, 1 H); 3.80 (s, 3 H); 3.88 (s, 1 H); 4.10 to 4.22 (m, 3 H); 4.24 to 4.31 (m, 3 H); 5.11 (m, 1 H); 5.83 (d, J = 15.7 Hz, 1 H); 6.36 (m, 1 H); 7.03 (d, J = 8.7 Hz, 1 H); 7.19 (broad d, J = 8.7 Hz, 1 H); 7.23 (m, 4 H); 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, 2 H); 12.0 (broad m, 1 H). LCMS (A1): ES m/z = 993 ; m/z = 995 [M+H]+; tR = 1.21 min. methyloxobutanyl)amino)oxopentanoate Under argon, in a round bottom flask, to a solution of compound 49 (15 mg, 15.07 pmol) in DCM (5 mL) were added DSC (5.63 mg, 21.09 pmol) and DIEA (3.56 pL, 21.09 pmol). The resulting mixture was stirred for 1 h at RT, concentrated in vacuo and purified on 2.5 g of silica gel (gradient elution DCM/MeOH) to give 7.7 mg of example 9 as a white solid (47 %).
RMN 1H (500 MHz, 8 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, 3 H); 1.04 (d, J = 7.2 Hz, 6 H); 1.18 (s, 3 H); 1.26 (m, 4 H); 1.57 (m, 2 H); 1.79 (m, 1 H); 1.83 (m, 2 H); 1.99 (m, 1 H); 2.22 to 2.33 (m, 3 H); 2.60 a 2.71 (m, 4 H); 2.82 (s, 4 H); 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, 3 H); 3.89 (d, J = 2.1 Hz, 1 H); 4.19 (m, 3 H); 4.26 to 4.34 (m, 3 H); 5.12 (m, 1 H); 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, 4 H); 7.29 (d, J = 2.2 Hz, 1 H); 7.91 (m, 2 H); 8.09 (m, 1 H); 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+HCOZH]'; tR = 1.26 min.
Example 10: mAb-Ex9 The general method bed previously was used for the preparation of example 10. 60 mg of hu2H11_R35-74 were reacted with 198 pL of a 10.78 mM solution of example 9 in DMA (5 eq.) for 2 h. At that time, 120 pL of the solution of example 9 (3 eq.) were added and the medium stirred for 2 h. After purification on Superdex 200 pg in DPBS pH 6.5 + 20% NMP, concentration on Amicon Ultra-15, buffer exchange on PD-10 in buffer B pH 6.5 + 5% NMP and filtration on Steriflip, 46 mg of example 10 were obtained as a colorless limpid solution at a concentration of 2.23 mg/mL with a DAR of 4.7 (HRMS), a ric purity of 99.2% and a global yield of 78%.
MS: 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). 8 nthesis of Exam les 11 to 14: benz lic amine of 3-S-neo ent lMe-aza-C52 mer 1 NHS ester of luta l-Val-Ala S -neo ent lMe-aza-C52 benz lic amine stereomer1 and corresponding ADC H250 CO2tBu stereomer 1 VLUE HCOZH stereomer 1 H251 stereomer 1 E 0 TIPSO U\U51%0Example 11l o HN \CEC m\ NVN \oo oMe H H stereomer 1 stereomer 1 UEUVWE}U0Example 12 OMe stereomer 1 stereomer 1 QkNJYNo H /\ 0‘ QEWl UIUZ'M stereomer 1 HZNQKN/IyNO HW /\ 0‘ 56 "EU; HOM W0OOO HN O O Q 57 "V"H QUE; stereomer 1 N MnMan 0 O/\ O Example 13 0 VMOe stereomer 1 hu2H11_R35-7H4rNN¢§HWLHUU Example 14 stereomer 1 Compound 50: (6R,13S)—(1E,3R,4S,6E)—8—(tert-butoxy)—1-(4-(hydroxymethyl)phenyl)—3-methyl-8— oxoocta-1 nyl 6-(3-chIoromethoxybenzyl)-2,2,9,10,10-pentamethylneopentyl- 4,7,1 xooxa-5,8,12-triazatetradecanoate Under argon, in a round bottom flask, to a solution of fragment BCZ (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 30 min at RT. Then a solution of fragment AD1 (730 mg, 1.59 mmol) in DMF (10 mL) and DIEA (981 pL, 5.56 mmol) were added. The on medium was stirred for 24 h at RT. After this time, the reaction medium was diluted with ice (200 g), extracted with AcOEt (4 x 200 mL). The organic layers were washed with H20 (80 mL), sat. brine (2 x 80 mL), dried over MgSO4, ed, trated and purified by two successive flash chromatographies, the first one on 300 g of silica gel ent elution DCM/MeOH) and the second one on 70 g of silica gel (gradient elution OH) to give 428 mg of compound 50 as a colorless foam (30%).
Compound 51: (2E,5S,6R,7E)(((2S)—2-(3-((R)—2-amino(3-chloromethoxyphenyl)- propanamido)—2,2-dimethylbutanamido)-4,4-dimethylpentanoyl)oxy)(4-(hydroxymethyl)phenyl)— 6-methylocta-2,7-dienoic acid In a round bottom flask, were introduced compound 50 (428 mg, 483.87 pmol) and DCM (27 mL). The solution was cooled at 0°C then TFA (8 mL, 106.62 mmol )was added. The mixture was stirred for 1.5 h at RT. The solvent was removed and co-evaporated under reduced pressure with e (3 x 100 mL). The crude oil was diluted with 1:1 AcOEt/H20 (75 mL) and neutralized with 2M NaOH (250 pL) until pH 6-7. The mixture was stirred for 6 h at RT. The layers were separated. The aq. layer was extracted with AcOEt (3 x 50 mL). The organic layers were washed with sat. brine (2 x 15 mL), dried over MgSO4, filtered and concentrated to give 374 mg of compound 51 as a ess foam (quant.).
Compound 52: (38,10R,16S,E)—10-(3-chloro—4-methoxybenzyl)((R,E)(4-(hydroxymethy|)- phenyl)butenyl)-6,6,7-trimethylneopentyloxa-4,8,1 1-triazacyclohexadecene- 2,5,9,12-tetraone Under argon, in a round bottom flask, to a solution of compound 51 (352 mg, 483.31 pmol) in CH3CN (60 mL) were added HATU (208 mg, 531.64 mmol), HOAt (73.09 mg, 531.64 pmol) and DIEA (244.52 pL, 1.45 mmol). The reaction medium was stirred for 45 min at RT. After this time, the reaction medium was neutralized with 0.5 N citric acid until pH 4, concentrated partially in vacuo and extracted with AcOEt (150 mL). The organic layer was washed with sat. NaH003 (10 mL), sat. brine (3 x 10 mL), dried over MgSO4, filtered, concentrated and purified by flash chromatography on 15 g of silica gel (gradient elution DCM/MeOH) to give 188 mg of compound 52 as a colorless foam (54 %).
Compound 53: (38,10R,16S,E)—10-(3-ch|oromethoxybenzy|)-6,6,7-trimethylneopentyl ((R,E)(4-(((triisopropylsilyl)oxy)methyl)phenyl)butenyl)oxa-4,8,1 1-triazacyclohexadec- 13-ene-2,5,9,12-tetraone At 0°C, under argon, in a round bottom flask, to a solution of compound 52 (188 mg, 264.68 pmol) in DCM (7 mL), were added 1H-imidazole (83.72 mg, 1.2 mmol) and 1M chlorotriisopropylsilane (134.15 pl). The reaction medium was stirred for 5 h at RT, then quenched with sat. NH4CI and stirred for 15 min. The layers were separated. The aq. layer was extracted with DCM (3 x 25 mL). The organic layers were washed with 1M NaHSO4 (10 mL), sat.
NaH003 (10 mL), sat. brine, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 10 g of silica gel ent elution DCM/MeOH) to give 137 mg of compound 53 as a colorless foam (59 %).
Exam le 11: 3S 10R 168 E 3-chloromethox benz | S 2R 3R 4- jhydroxymethyl)phenyl)oxiranyl)ethyl)-6,6,7-trimethylneopentyloxa-4,8,11- triazacyclohexadecene-2,5,9,12-tetraone e 11 was prepared in two steps.
Sim: at 0°C under argon, in a round bottom flask, to a on of nd 53 (137 mg, 158.08 umol) in DCM (5.5 mL) was added a solution of m—CPBA (50.66 mg, 205.51 pmol) in DCM (2 mL) and the on medium was stirred for 2 h. Then 39 mg of m—CPBA were added twice after 2 h of stirring. After 16 h of stirring, the mixture was quenched with sat. NaHCOs (30 mL) and Na28203 (30 mL), stirred for 15 min and diluted with DCM (40 mL). The layers were separated. The aq. layer was extracted with DCM (2 x 20 mL). The organic layers were washed with sat. brine (2 x 8 mL), dried over MgSO4, filtered and concentrated to give 160 mg of alpha and beta epoxides as a colorless solid (quant.).
RMN 1H (500 MHz, 6 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, 0.6 H); 2.40 (m, 0.4 H); 2.55 to 2.79 (m, 2 H); 2.90 to 3.00 (m, 2 H); 3.44 (m, 1 H); 3.79 (d, J = 2.1 Hz, 0.4 H); 3.80 (s, 3 H); 3.90 (d, J = 2.1 Hz, 0.6 H); 4.03 to 4.22 (m, 2 H); 4.78 (s, 0.8 H); 4.80 (s, 1.2 H); 5.03 (m, 1 H); 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, 6 H); 7.85 to 7.93 (m, 2 H); 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+HCOZH]'; tR = 2.05-2.06 min.
M: at 0°C under argon, in a round bottom flask, to a solution of alpha and beta epoxides (177 mg, 200.53 pmol) in THF (7.5 mL) was dropwise added 1M TBAF (221 pL, 221 pmol). After ng for 2 h at RT, 50 pL of TBAF were added and stirred for 3.5 h. The reaction medium was diluted with H20 (9 mL), stirred for 10 min and extracted with DCM (3 x 25 mL). The combined organic layers were washed with sat. brine (3 x 8 mL), dried over MgSO4, filtered and concentrated to give 210 mg of alpha and beta epoxides as a colorless solid (quant.).
RMN 1H (500 MHz, 6 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, 2 H); 2.89 to 3.03 (m, 2 H); 3.44 (m, 1 H); 3.79 (d, J = 2.2 Hz, 0.4 H); 3.80 (s, 3 H); 3.90 (d, J = 2.2 Hz, 0.6 H); 4.04 to 4.22 (m, 2 H); 4.48 (d, J = 5.8 Hz, 0.8 H); 4.50 (d, J = 5.8 Hz, 1.2 H); 5.03 (m, 1 H); 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, 6 H); 7.83 to 7.94 (m, 2 H); 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/z = 726 [M+H]+; tR = 1.29 min.
Alpha and beta epoxides were separated by chiral liquid chromatography that was carried out on a 76 x 350 mm column packed with 1.1 kg of 10 pm Chiralpak AD (amylose tris-3,5- dimethylphenylcarbamate coated on a silica gel support, Chiral Technologies Europe) using isocratic elution with 75:25 heptane/EtOH. After concentration, 66 mg of e 11 were obtained as a white solid (45%) and 45 mg of the alpha epoxide were obtained as a white solid (31%).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.82 to 0.89 (m, 12 H); 0.99 (s, 3 H); 1.03 (d, J = 6.9 Hz, 3 H); 1.19 (s, 3 H); 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, 1 H); 2.61 (m, 1 H); 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, 1 H); 3.80 (s, 3 H); 3.91 (d, J = 2.1 Hz, 1 H); 4.03 to 4.14 (m, 2 H); 4.50 (d, J = 5.8 Hz, 2 H); 5.03 (m, 1 H); 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 = 47,109 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 min.
Compound 54: (38,10R,16S,E)—16-((S)—1-((2R,3R)—3-(4—(azidomethyl)phenyl)oxiranyl)ethyl)- -(3-chloromethoxybenzyl)-6,6,7-trimethylneopentyloxa-4,8,1 zacyclohexadec ene-2,5,9,12-tetraone Under argon, in a round bottom flask, to a solution of example 11 (66 mg, 90.87 pmol) in THF (5 mL) were added, at 0°C, DPPA (100.23 pl, 454.36 pmol) and DBU (69.27 pl, 454.36 pmol).
The solution was stirred for 5 h at RT then diluted with H20 and extracted with AcOEt (2 x mL). The organic layer was separated, washed with sat. brine (2 x 5 mL), dried over MgSO4, filtered, concentrated and purified on 15 g of silica gel (gradient n DCM/iPrOH) to give 64 mg of compound 54 as a colorless solid (94 %).
Exam le 12: 3S 10R 168 E S 2R 3R 4- aminometh | hen | oxiran | eth | - - 3-chloromethox benz l -6 6 7-trimeth lneo ent loxa-4 811- triazacyclohexadecene-2,5,9,12-tetraone In a round bottom flask, to a solution of compound 54 (64 mg, 85.18 pmol) in DCM (3 mL), MeOH (3 mL) and H20 (400 pL) was added TCEP (26.86 mg, 93.70 pmol). The reaction medium was stirred for 24 h at RT. The reaction mixture was diluted with DCM (15 mL) and sat. , stirred for 10 min and extracted with DCM (3 x 15 mL). The combined organic layers were washed with sat. brine, dried over MgSO4, filtered, concentrated and purified by flash chromatography on 4.5 g of propyl modified silica gel (gradient elution DCM/MeOH) to give 42 mg of example 12 as a white solid (68 %).
Compound 55: (9H-fluoreny|)methy| ((2S)—1-(((2S)—1-((4-((2R,3R)—3-((1S)—1-((3S,10R,16S,E)— -(3-chloromethoxybenzyl)-6,6,7-trimethylneopentyl-2,5,9,12-tetraoxooxa-4,8,1 1- triazacyclohexadecenyl)ethyl)oxiranyl)benzyl)amino)oxopropanyl)amino) methyl-1 -oxobutanyl)ca rbamate Under argon, in a round bottom flask, were introduced e 12 (42 mg, 57.91 pmol) and DMF (1 mL), followed by FmocValAla (34.44 mg, 86.86 pmol), HOBt (13.20 mg, 93.81 pmol), DCM (10 mL) and EDC (10.36 pl, 57.91 pmol). The reaction medium was stirred for 3 h at RT and then diluted with H20 (15 mL), stirred for 10 min at RT and extracted with DCM (3 x 20 mL).
The ed organic layers were dried over MgSO4, filtered and concentrated to give 83 mg of compound 55 as a white solid (quant.).
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-trimethylneopentyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexad ec- 13-enyl)ethy|)oxiranyl)benzyl)amino)oxopropany|)methy|butanamide In a round bottom flask, piperidine (57.8 pL, 579.10 pmol) was added to a solution of compound 55 (64.73 mg, 57.91 pmol) in DCM (10 mL). After stirring 5 h, 57.8 pL of piperidine were added and the medium was stirred ght at RT. The reaction medium was concentrated in vacuo and purified on 15 g of silica gel ent DCM/MeOH/HZO) to give 30 mg of compound 56 as a colorless solid (58%).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.86 (d, J = 7.0 Hz, 3 H); 0.82 to 0.90 (m, 15 H); 1.00 (s, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3 H); 1.24 (d, J = 7.0 Hz, 3 H); 1.34 (d, J = 14.6 Hz, 1 H); 1.66 (broad m, 2 H); 1.80 (m, 1 H); 1.91 (m, 1 H); 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, 3 H); 3.91 (d, J = 2.0 Hz, 1 H); 4.03 to 4.12 (m, 2 H); 4.29 (d, J = 6.2 Hz, 2 H); 4.34 (m, 1 H); 5.03 (m, 1 H); 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 (broad 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+HC02H]'; tR = 1.29 min.
Compound 57: 5-(((2S)—1-(((2S)—1-((4-((2R,3R)—3-((1S)—1-((3S,10R,16S,E)—10-(3-chloro—4- methoxybenzyl)-6,6,7-trimethylneopentyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexad ec- 13-enyl)ethyl)oxiranyl)benzyl)amino)oxopropanyl)amino)methyloxobutan y|)amino)oxopentanoic acid Under argon, in a round bottom flask, a solution of glutaric anhydride (4.29 mg 36.85 umol) in DCM (2 mL) was added to a solution of compound 56 (30 mg, 33.5 umol) in DCM (6 mL). The reaction medium was stirred for 2 h at RT, concentrated partially in vacuo and purified on 2.5 g of silica gel (gradient elution DCM/MeOH/HZO) to give 34 mg of compound 57 as a colorless lacquer (quant.).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.80 to 0.89 (m, 18 H); 0.99 (s, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3 H); 1.24 (d, J = 7.0 Hz, 3 H); 1.34 (d, J = 14.6 Hz, 1 H); 1.70 (m, 2 H); 1.80 (m, 1 H); 1.97 (m, 2 H); 2.19 (m, 4 H); 2.26 (m, 1 H); 2.60 (m, 1 H); 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, 1 H); 3.80 (s, 3 H); 3.91 (d, J = 2.2 Hz, 1 H); 4.03 to 4.18 (m, 3 H); 4.22 to 4.33 (m, 3 H); 5.03 (m, 1 H); 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 (broad d, J = 8.9 Hz, 2 H); 7.91 (d, J = 7.2 Hz, 1 H); 8.06 (broad d, J = 7.5 Hz, 1 H); 8.36 (broad t, J = 6.6 Hz, 1 H); 8.42 (d, J = 7.8 Hz, 1 H); 12.10 (broad m, 1 H). LCMS (A1): ES m/z =1007[M-H]';m/z =1009[M+H]+;tR = 1.21 min.
Exam le 13: 2 5-dioxo rrolidin | 511- 4- 2R 3R 1S 0R,16$,E)-1 0-(3-chloromethoxybenzyl)-6,6,7-trimethylneopentyl-2,5,9,12- xooxa-4 811-triazac clohexadecen l eth l oxiran l benz l amino oxopropan-Z-yl)amino)—3-methyloxobutanyl)amino)oxopentanoate Under argon, in a round bottom flask, to a solution of compound 57 (33 mg, 32.69 umol) in DCM (8 mL) were added DSC (11.72 mg, 45.76 umol) and DIEA (7.7 uL, 45.76 umol). The reaction medium was stirred for 1 h at RT, concentrated in vacuo and purified by two sive flash chromatographies on 2.5 g of silica gel (gradient elution DCM/iPrOH) to give 17.9 mg of example 13 as a colorlesss solid (49 %).
RMN 1H (500 MHz, 6 in ppm, DMSO-d6): 0.80 to 0.90 (m, 18 H); 1.00 (s, 3 H); 1.04 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3 H); 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, 2 H); 1.98 (m, 2 H); 2.26 (m, 1 H); 2.29 (m, 2 H); 2.60 (m, 1 H); 2.68 (t, J = 7.5 Hz, 2 H); 2.70 (dd, J = 11.2 and 14.6 Hz, 1 H); 2.80 (s, 4 H); 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, 1 H); 3.80 (s, 3 H); 3.90 (d, J = 2.2 Hz, 1 H); 4.06 to 4.15 (m, 2 H); 4.18 (dd, J = 6.9 and 8.7 Hz, 1 H); 4.21 to 4.33 (m, 3 H); 5.03 (m, 1 H); 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, 2 H); 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/z = 1106 [M+H]+; m/z = 1150 [M- H+HC02H]'; tR = 1.26 min.
Example 14: 13 The l method described previously was used for the preparation of example 14. 60 mg of hu2H11_R35-74 were reacted with 200 pL of a 10.05 mM solution of example 13 in DMA (5 eq.) for 2 h. At that time, 180 pL of the on of example 13 (4.5 eq.) were added and the medium stirred for 2 h. After purification on Superdex 200 pg in buffer B pH 6.5 + 10% NMP, concentration on Amicon Ultra-15, buffer ge on PD-10 in buffer B pH 6.5 + 5% NMP and filtration on Steriflip, 46 mg of example 14 were obtained as a colorless limpid solution at a concentration of 2 mg/mL with a DAR of 3.5 , a monomeric purity of 99.7% and a global yield of 77%.
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).
Exam le 15: 3S 10R 168 E 3-chloromethox benz | S 3- 4- h drox - 2,5,9,12-tetraone Example 15 Example 15 was prepared following general route B depicted in Scheme 2 and described for examples 4 and 11.
RMN 1H (500 MHz, 6 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, 0.65 H); 2.41 (m, 0.35 H); 2.55 to 2.70 (m, 2 H); 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 (partially masked m, 1 H); 3.77 (d, J = 2.1 Hz, 0.35 H); 3.81 (s, 3 H); 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 (broad d, J = 10.2 Hz, 0.65 H); 7.64 (broad 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 [M-H]'; m/z = 656 [M+H]+; tR = 1.06 min.
Exam le 16: 3S 10R 168 E 3-chloromethox benz | S 3- 4- h drox - meth l hen loxiran leth l-6 6-dimeth o ro loxatriazac clohexadec- 13-ene-2,5,9,12-tetraone HO o% O o NVNHN]_,.»\\©[CI*0 OMe H H Example 16 Example 16 was ed following general route B depicted in Scheme 2 and described for examples 4 and 11.
RMN 1H (500 MHz, 6 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, 1.8 H); 0.99 (d, J = 7.0 Hz, 1.8 H); 1.01 (s,1.2 H); 1.05 (d, J = 7.0 Hz, 1.2 H); 1.09 (s, 1.8 H); 1.11 (s, 1.2 H); 1.82 (m, 1 H); 1.91 (m, 0.6 H); 2.01 (m, 0.4 H); 2.30 (m, 1 H); 2.52 to 2.72 (m, 2 H); 2.88 to 3.04 (m, 3 H); 3.25 to 3.35 (masked m, 1 H); 3.79 (d, J = 2.2 Hz, 0.4 H); 3.80 (s, 3 H); 3.88 (d, J = 2.2 Hz, 0.6 H); 4.09 to 4.21 (m, 2 H); 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, 2 H); 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, 1 H); 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 [M-H]'; m/z = 684 [M+H]+; tR= 1.16 min.
Exam le 17: 3S 10R 168 E 3-chloromethox benz | S 3- 4- h drox - 13-ene-2,5,9,12-tetraone Example 17 Example 17 was prepared following general route B depicted in Scheme 2 and described for examples 4 and 11.
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 50/50 reoisomer mixture; 0.90 (s, 4.5 H); 0.91 (s, 4.5 H); 1.00 (d, J = 7.0 Hz, 1.5 H); 1.03 (s, 1.5 H); 1.05 (s, 1.5 H); 1.06 (d, J = 7.0 Hz, 1.5 H); 1.10 (s,1.5 H); 1.12 (s, 1.5 H); 1.85 (m, 1H); 2.32 (m, 1H); 2.55 to 2.72 (m, 2 H); 2.90 to 3.03 (m, 3 H); 3.25 to 3.35 (masked m, 1 H); 3.81 (s, 3 H); 3.90 (d, J = 2.2 Hz, 1 H); 4.15 (m, 1 H); 4.36 (d, J = .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); .15 (t, J = 5.9 Hz, 0,5 H); 5.18 (t, J = 5.9 Hz, 0.5 H); 5.29 (m, 1 H); 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, 1 H); 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, 6 H); 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 [M-H]; m/z= 698 [M+H]; tR-— 4. 13-4.16 min. 13-ene-2,5,9,12-tetraone stereomer 2 Example 18 Example 18 was prepared following general route A depicted in Scheme 1 and described for example 8.
RMN 1H (500 MHz, 6 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, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.19 (s, 3 H); 1.30 (m, 1 H); 1.50 to 1.90 (m, 5 H); 2.28 (m, 1 H); 2.55 to 2.76 (m, 2 H); 2.90 to 3.00 (m, 2 H); 3.48 (m, 1 H); 3.70 (s, 2H); 3.80(s, 3H); 3.89(s, 1H); 4.0.,2to415(m 2H), 5.0,6(m 1H); 5.8,8(d J= 15.,5Hz 1H); 6.47 (m, 1 H); 7.03 (d, J = 8.7 Hz, 1 H); 7.20 (m, 3 H); 7.31 (m, 3 H); 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 stereomer 2 e 19 was prepared as depicted in Scheme 3 and bed for examples 2, 6, 9 and 13.
RMN 1H (500 MHz, 8 in ppm, DMSO-d6): 0. 79 (d, J: 7. 0 Hz, 3 H), 0. 81 to 0. 89 (m, 18 H); 1.01 (s, 3 H); 1.03 (d, J = 7.0 Hz, 3 H); 1.20 (s, 3 H); 1.24 (d, J = 7.0 Hz, 3 H); 1.31 (m, 1 H); 1.60 to 1.74 (m, 2 H); 1.78 to 1.86 (m, 3 H); 1.96 (m, 1 H); 2.21 to 2.31 (m, 3 H); 2.55 to 2.72 (m, 4 H); 2.80 (s, 4 H); 2.96 (m, 2 H); 3.47 (m, 1 H); 3.80 (s, 3 H); 3.89 (d, J = 2.2 Hz, 1 H); 4.03 to 4.14 (m 2 H); 4.17 (dd, J = 6.8 and 8.6 Hz, 1 H); 4.21 to 4.33 (m, 3 H); 5.06 (m, 1 H); 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, 4 H); 7.32 (d, J = 2.3 HZ, 1 H); 7.80 (d, J = 8.3 HZ, 1 H); 7.89 (m, 2 H); 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/z = 1136 [M-H+HC02H]'; tR = 1.23 min.
Example 20: mAb-Ex19 hu2H11_R35-74*NM o 0A Example 20 stereomer 2 Example 20 was prepared in a similar way to examples 3, 7, 10 and 14. 45 mg of example 20 were obtained as a colorless limpid solution at a concentration of 2.55 mg/mL with a DAR of 4.4 (HRMS), a monomeric purity of 99.1% and a global yield of 78%.
MS: 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 Example 21 was prepared following general route A depicted in Scheme 1 and bed for examples 1 and 8. oxa-4 811-triazac clohexadecen l eth l oxiran l benz l amino oxo ro an l amino meth loxobutan l amino oxo entanoate 0 O H H C? M N \ l o \ o o A 0 ?g" 0 0 H">2"!H Example 22 Example 22 was prepared as depicted in Scheme 3 and described for examples 2, 6, 9 and 13.
RMN 1H (500 MHz, 8 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, 3 H); 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, 3 H); 1.11 (m, 1 H); 1.20 (m, 1 H); 1.23 (d, J = 7.0 Hz, 3 H); 1.43 to 1.54 (m, 2 H); 1.78 (m, 1 H); 1.82 (m, 2 H); 1.98 (m, 1 H); 2.22 (m, 1 H); 2.28 (m, 2 H); 2.65 (m, 3 H); 2.79 (m, 1 H); 2.81 (s, 4 H); 2.97 (dd, J = 2.2 and 7.9 Hz, 1 H); 3.80 (s, 3 H); 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, 1 H); 5.10 (m, 1 H); 5.79 (dd, J = 1.8 and 15.5 Hz, 1 H); 6.39 (ddd, J = 40,116 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, 4 H); 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 hu2H11_R35-74HMNQkNH 2 N o% o o o o A o HJ>¥H \0HNI\\\©[CIWe Example 23 Example 23 was prepared in a similar way to examples 3, 7, 10 and 14. 45 mg of example 23 were obtained as a colorless limpid solution at a concentration of 2.14 mg/mL with a DAR of 3.6 (HRMS), a monomeric purity of 100% and a global yield of 75%.
SEC-HRMS: um 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).
Exam le 24: 38 7S 10R 168 E S 2R 3R 4- azidometh l hen l l - eth l 3-chloromethox benz l -6 6 7-trimeth lneo ent a-4 811- triazacyclohexadecene-2,5,9,12-tetraone o o | NVNEHNIUCo OMe H H Example 24 was prepared following general route C depicted in Scheme 3 and described for example 32.
RMN 1H (500 MHz, 8 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); .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 ; m/z = 751 [M+H]+; m/z = 795 [M-H+HC02H]'; tR = 1.52 min.
Example 25 Example 25 was prepared as described for examples 1, 5, 8 and 11.
RMN 1H (400 MHz, 8 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 (broad 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 .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 [M-H]'; m/z = 725 [M+H]+; m/z = 769 [MH +HC02H]'; tR = 0.87 min. 8 nthesis of exam les 26 to 28: 3- S -neo ent l S a-C52 benz lic alcohol NHS ester of luta l-Val-Ala-EDA S -neo ent l S -Me-aza-052 benz lic alcohol and corresponding ADC PMBOW / R¢O PMBOVKJ/ K60 \ HN CI o 0% o HN CI NH2 HOVNkJCEOMeH HNj>@Nl\j$[on/leH AD3 BC4 58l HOMO PMBOMzo W» 6' My 2 OMe O H H HMy H OMe OTIPS CF3503- Br OH 64 63 62 TIPSO 0% 6 wk» CI Nj>?N O : :OMe H H HO 0% 5 HM)» CI N?w 8 CEH O OMe Example 26 Example 26 Example 27 o o o H H WW hu2H11_R35-74"W" 0% O CI ‘ N$?NNYO o 0 7 O :OMe Example 28 NJYN \O :HN:L~ H H Compound 58: (2R,3S)—1-((4-methoxybenzyl)oxy)—2-methylhexenyl (S)—2-((S)—3-((R)—2- acrylamido(3-chloromethoxyphenyl)propanamido)—2,2-dimethylbutanamido)—4,4- dimethylpentanoate To a solution of compound BC4 (365 mg, 919.7 pmol) in DMF (12 mL) were added HATU (402 mg, 1.06 mmol) and HOAt (144 mg, 1.06 mmol), the reaction medium was stirred at RT for min then were added a solution of compound AD3 (365 mg, 965.7 pmol) in DMF (5 mL) and DIEA (562 pL, 3.22 mmol). The reaction medium was stirred at RT for 4 h, then diluted with H20 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with H20 (15 mL), sat. brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by two sive flash chromatographies on 30 g of silica gel (gradient n OH and heptane/EtOAc) to give 564 mg of compound 58 as a colorless foam (81%).
RMN 1H (400 MHz, 8 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/z = 756 [M+H]+; m/z = 800 [M-H+HC02H]'; tR = 1.62 min.
Compound 59: (3S,7S,10R,16S,E)—10-(3-chloro—4-methoxybenzyl)((R)((4-methoxy- benzyl)oxy)propanyl)-6,6,7-trimethylneopentyloxa-4,8,1 1-triazacyclohexadec—13-ene— 2,5,9,12-tetraone To a solution of compound 58 (560 mg, 740.4 pmol) in DCM (56 mL) was added under Ar Grubbs l catalyst (31.0 mg, 37.02 pmol). The reaction medium was stirred at RT for 1 h 30 before the addition of 31 .0 mg of catalyst. Stirring was carried on at RT for 1 h 30 before the addition of 31.0 mg of catalyst. Stirring was carried on at RT for 1 h 30 before the addition of 31.0 mg of catalyst and the reaction medium was stirred at RT for 1 h 30. It was the concentrated in vacuo and ed by flash chromatography on 25 g of silica gel (gradient elution OH) to give 330 mg of nd 59 (61%) and 220 mg that were further purified by flash chromatography on g of silica gel (gradient elution DCM/MeOH) to give 204 mg of compound 59 (37%) as a mixture with lohexylphosphine oxide.
RMN 1H (400 MHz, 8 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+HCOZH]'; tR = 1.54 min.
Compound 60: (3S,7S,10R,16S,E)—10-(3-chloromethoxybenzyl)((R)hydroxypropan yl)-6,6,7-trimethylneopentyloxa-4,8,1 1-triazacyclohexadec—13-ene—2,5,9,12-tetraone Compound 59 (532 mg, 730.4 pmol) was treated with a solution of TFA (3.93 mL) in DCM (36 mL) at RT for 30 min. The reaction medium was poured on 9% aq. NaHCOs (130 mL) under magnetic stirring. Stirring was carried on for 30 min then the aqueous phase was extracted with DCM (2 x 70 mL).The combined organic phases were washed with H20 (3 X 20 mL), dried over MgSO4, ed, concentrated in vacuo and ed by flash chromatography on 30 g of silica gel (gradient elution DCM/MeOH) to give 299 mg of compound 60 as a white foam (67%).
RMN 1H (400 MHz, 8 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 min.
Compound 61: (S)—2-((3S,7S,10R,16S,E)—10-(3-chloromethoxybenzyl)-6,6,7-trimethyl neopentyl-2,5,9,12-tetraoxooxa-4,8,1 1-triazacyclohexadec—13-enyl)propanal To a solution of compound 60 (297 mg, 488.4 pmol) in DCM (6 mL) cooled with an ice/acetone bath were added at 0°C a solution of KBr (58.11 mg, 488.4 pmol) in H20 (1.18 mL), TEMPO (1.57 mg, 9.8 pmol) and se an aqueous solution of 1.56M sodium hypochlorite pH 9.5 (467.6 pL, 732.52 pmol). Stirring was carried on at 0°C for 15 min then was added at 0°C sat.
Na28203 (3.56 mL), the ice bath was removed and the reaction medium stirred for 10 min. It was then extracted with DCM (3 x 25 mL), the combined organic phases were washed with H20 (2 x mL), dried over MgSO4, filtered, trated in vacuo and purified by flash chromatography on 15 g of silica gel (gradient elution DCM/MeOH) to give 139 mg of compound 61 as a white lacquer (47%).
RMN 1H (400 MHz, 8 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 .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 [M- H]'; m/z = 606 [M+H]+; m/z = 650 C02H]'; tR = 1.28 min.
Compound 62: (4-(((triisopropylsilyl)oxy)methy|)phenyl)methanol To a solution of 1 ,4-benzenedimethanol (2 g, 14.33 mmol) in THF (100 mL) was added imidazole (1.13 g, 16.48 mmol). The reaction medium was stirred at RT for 15 min then was added triisopropylsilyl chloride (3.14 mL, 14.33 mmol) and stirring was carried on at RT overnight. EtZO (50 mL) was added to the reaction e and the organic phase was washed twice with sat. brine (100 mL), filtered over MgSO4, concentrated in vacuo, ved in DCM, filtered, concentrated in vacuo and purified by flash chromatography on 200 g of silica gel (isocratic elution 8:2 heptane/EtOAc) to give 1.9 g of compound 62 as a colorless oil (45%).
RMN 1H (400 MHz, 6 in ppm, DMSO-d6): 1.04 (d, J = 6,7 Hz, 18H); 1.15 (m, 3H); 4.48 (d, J = .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 To a suspension of N-bromosuccinimide (1.29 g, 7.16 mmol) in DCM (65 mL) cooled at 0°C was added dropwise dimethyl e (953 pL, 12.9 mmol). Stirring was carried on at 0°C for 10 min then the reaction medium was cooled at -20°C and d at -20°C for 10 min. A solution of compound 62 (1.9 g, 6.45 mmol) in DCM (30 mL) cooled at -20°C was then added dropwise.
Stirring was carried on at -20°C for 15 min then at 0°C for 15 min and at RT overnight. The reaction medium was washed twice with sat. brine (100 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by two successive flash chromatographies on 40 g of silica gel (gradient elution heptane/EtOAc). The fractions containing the expected compound were combined, concentrated in vacuo and dissolved in heptane (8 mL). The suspension was filtered, the filtrate concentrated in vacuo, dissolved in EtZO (5 mL), cooled at -20°C, filtered, concentrated in vacuo to give 1.18 g of compound 63 as a pale yellow oil (51%).
RMN 1H (400 MHz, 6 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 min.
Compound 64: (1 R,4S,5R,6S)—4,7,7-trimethyl(4-(((triisopropylsilyl)oxy)methyl)benzyl) thiabicyclo[3.2.1]octanium trifluoromethanesulfonate To (1R,4R,5R)—4,7,7-trimethylthiabicyclo[3.2.1]octane (CAS number [57182], 562.3 mg, 3.3 mmol) was added a on of compound 63 (1.18 g, 3.3 mmol) in DCM (3.4 mL). A solution of lithium trifluoromethanesulfonate (2.63 g, 16.51 mmol) in H20 (3 mL) was then added se and the on medium was stirred at RT overnight. H20 (15 mL) and DCM (15 mL) were then added to the reaction medium and stirring carried on at RT for 10 min. The aqueous phase was extracted with DCM (3 x 20 mL), the combined organic phases were washed with sat. brine (3 X 8 mL), dried over MgSO4, ed and concentrated in vacuo. The pale yellow oil was triturated in iPr20 (5 mL), the solid thus obtained was filtered, washed with iPr20 (2 x 5 mL) and dried under vacuum to give 1.125 g of compound 64 as a white solid (57%).
RMN 1H (400 MHz, 6 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 (broad 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).
WO 76998 2016/076603 Compound 65: (3S,7S,10R,16S,E)—10-(3-chloromethoxybenzyl)-6,6,7-trimethylneopentyl- 16-((S)—1-((2R,3R)—3-(4-(((triisopropylsilyl)oxy)methyl)phenyl)oxiranyl)ethyl)—1-oxa-4,8,1 1- triazacyclohexadec—13-ene-2,5,9,12-tetraone To a solution of compounds 61 (138 mg, 227.7 pmol) and 64 (149.5 mg, 250.4 pmol) in DCM (4 mL) cooled at -70°C was added dropwise BEMP (CAS number [980153], 90.2 pL, 296.0 pmol). The reaction mixture was stirred at -70°C for 2 h, sat. brine (7 mL) was added to the reaction e, the bath removed and stirring carried on vigorously up to 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 (gradien elution DCM/MeOH) to give 163 mg of compound 65 as a colorless oil (30%) and 60 mg of compound 61 (43%).
RMN 1H (500 MHz, 8 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 min.
Example 26: ,10R,16S,E)—10-(3-chloromethoxybenzyl)((S)—1-((2R,3R)—3-(4— (hydroxymethyl)phenyl)oxiranyl)ethyl)—6,6,7-trimethylneopentyloxa-4,8,1 1- cyclohexadec—13-ene-2,5,9,12-tetraone To a solution of compound 65 in THF cooled at 0°C was added TBAF. The reaction medium was stirred at 0°C for 1 h 30 then H20 (5 mL) was added and stirring carried on for 20 min. The reaction medium was extracted with DCM (3 x 20 mL), the combined organic phase were washed with sat. brine (3 x 5 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 15 g of silica gel (gradient elution EtOAc/MeOH/Hzo) to give 69 mg of example 26 as a white lacquer (91%).
RMN 1H (400 MHz, 8 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, .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 [M-H]'; m/z = 726 [M+H]+; m/z = 770 [M-H+HC02H]'; tR = 1.29 min.
Compound 66: 4-((2R,3R)—3-((S)—1-((3S,7S,10R,16S,E)—10-(3-chloromethoxybenzyl)-6,6,7- hylneopentyl-2,5,9,12-tetraoxooxa-4,8,1 1-triazacyclohexadec—13-enyl)ethyl)oxi ranyl)benzyl (4-nitrophenyl) carbonate To a solution of example 26 (68 mg, 93.6 pmol) in DCM (5 mL) were added bis (4- nitrophenyl)carbonate (118.7 mg, 374.5 pmol) and dropwise DIEA (49 pL, 281.0 pmol). The reaction medium was stirred at RT for 6 d then sat. brine was added and it 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 ent elution DCM/MeOH) to give 79 mg of compound 66 as a white lacquer (94%).
RMN 1H (400 MHz, 6 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, .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 ; m/z = 891 [M+H]+; m/z = 935 [M-H+HC02H]'; tR = 1.55 min.
Compound 67: (9H-fluorenyl)methyl ((S)—1-(((S)—1-((2-NBoc—aminoethyl)amino)—1-oxopropan- 2-yl)amino)—3-methyloxobutanyl)carbamate hydrochloride To a suspension of Fmoc—Val-Ala-OH (3.5 g, 8.53 mmol) in DCM (100 mL) were added TEA (3.6 mL, 25.58 mL) leading to complete dissolution of starting material, N-Boc—EDA (1.62 mL, .23 mmol) and a solution of propylphosphonic ide (6.51 g, 10.23 mmol) 50% in DCM.
The reaction medium was stirred at RT for 3 h then 1N NaOH (50 mL) was added and the suspension filtered, washed with H20 and DCM. The organic phase of the filtrate was washed with H20. The cake and the organic phase were ed, partially concentrated in vacuo, filtered, washed with DCM and dried to give 3.952 g of compound 67 as a white powder (84%).
RMN 1H (400 MHz, 6 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: uorenyl)methyl ((S)—1-(((S)—1-((2-aminoethyl)amino)—1-oxopropan yl)amino)—3-methyloxobutanyl)carbamate hydrochloride To as sion of compound 67 (100 mg, 159.5 pmol) in dioxane (3 mL) was added HCI 4N in dioxane (905 pL, 3.62 mmol). The reaction medium was stirred at RT for 20 h then concentrated in vacuo. The crude product was ended in iPrZO (3 mL), immersed in an ultrasonic bath and filtered. The cake thus obtained was washed with iPr20 (2 x 3 mL) and dried under vacuum to give 78 mg of compound 68 as a white solid (88%).
RMN 1H (400 MHz, 8 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-((3S,7S,10R,16S,E)—10-(3-chloromethoxybenzyl)-6,6,7- hylneopentyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexadecen-16yl)ethyl)oxiran 2-yl)benzyl (2-((S)—2—((S)—2-aminomethylbutanamido)propanamido)ethyl)carbamate To a suspension of compounds 66 (78 mg, 87.5 pmol) and 68 (51.4 mg, 105.0 pmol) in DCM was added DIEA (44.0 pL, 262.5 pmol). The reaction medium is stirred at RT overnight then were added compound 68 (24 mg, 49 poml) and DIEA (15 pL) and stirring was carried on at RT for 1 d. Piperidine (87.0 pL, 875.0 pmol) was added and ng d on at RT overnight. The reaction medium was concentrated in vacuo and purified by two successive flash chromatrographies on 5g of silica gel (gradient elution EtOAc/MeOH/H20) to give 57 mg of compound 69 as a white lacquer (66%).
RMN 1H (500 MHz, 8 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 = 45,104 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+HC02H]'; tR = 0.91 min.
Compound 70: (9S,12S)—1-(4-((2R,3R)—3-((S)—1-((3S,7S,10R,16S,E)—10-(3-chloro—4-methoxy— benzyl)-6,6,7-trimethylneopentyl-2,5,9,12-tetraoxooxa-4,8,1 1-triazacyclohexadecen yl)ethyl)oxiranyl)phenyl)-1 2-isopropylmethyl-3,8,1 1 ,14-tetraoxooxa-4,7,10,13- tetraazaoctadecanoic acid To a solution of compound 69 (56.0 mg, 56.8 pL) in DCM (8 mL) was added a solution of glutaric anhydride (7.27 mg, 62.5 pmol) in DCM (4 mL). The reaction medium was stirred at RT for 2 h, concentrated in vacuo and ed by flash chromatrography on 5 g of silica gel (gradient elution DCM/MeOH/HZO) to give 35.5 mg of compound 70 as a white lacquer (56%).
RMN 1H (500 MHz, 8 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 = .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 min.
Example 27: 2,5-dioxopyrrolidinyl (9S,12S)—1-(4-((2R,3R)—3-((S)—1-((3S,7S,10R,16S,E)—10-(3- chloromethoxybenzyl)-6,6,7-trimethylneopentyl-2,5,9,12-tetraoxooxa-4,8,1 1-triazacyclohexadec —13-eny|)ethy|)oxiranyl)phenyl)—12-isopropylmethyl-3,8,1 1 ,14-tetraoxooxa- ,13-tetraazaoctadecanoate To a solution of compound 70 (14.6 mg, 13.3 pmol) in DCM (5 mL) under Ar were added DSC (4.3 mg, 16.0 pmol) and DIEA (2.8 pL, 16.0 pmol). The reaction medium was stirred at RT for 3 h then were added DSC (1.5 mg, 5.6 pmol) and DIEA (1 pL, 5.7 pmol) and stirring was carried on at RT for 1 h. The reaction medium was concentrated in vacuo and purified by flash chromatography on 16.4 g of diol-modified silica gel (gradient elution DCM/iPrOH) to give 11.4 mg of example 27 as a white lacquer (72%).
RMN 1H (500 MHz, 6 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 .LCMS(A5):ES m/z = 311; m/z = 1191 [M- H]'; m/z = 1193 [M+H]+; m/z = 1237 [M-H+HC02H]'; tR = 1.26 min.
Example 28: mAb-Ex27 Example 28 was ed in a similar way to examples 3, 7, 10 and 14. 1.56 mg of example 28 were obtained as a colorless limpid solution at a concentration of 0.78 mg/mL with a DAR of 4 (HRMS), a monomeric purity of 100% and a global yield of 13%.
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). 8 nthesis of exam le 29: maleimido-mc-vc-PABA S -neo ent l S -Me-aza-C52 benzylic amine HZN o\\ O o HN:1:~ CI examp eI 25 OMe \\o HN/?:x<"\N H 0 o €1WN$NJ5IKN0 O H H \ g H o /\ o /o H o 6 HN 37 \ O O .
NJE%<\NH E o \o OMe example 29 Compound 71: 4-((S)—2-((S)—2—(6-(2,5-dioxo—2,5-dihydro—1H-pyrroIy|)hexanamido)—3- butanamido)—5—ureidopentanamido)benzy| (4-nitrophenyl) carbonate Compound 72 was prepared as described by Verma V.A., et a/., in Bioorg Med Chem Lett 2015, , 864-868.
RMN 1H (400 MHz, 6 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); .08 (s, 1H). LCMS (A5): ES m/z = 738 [M+H]+; m/z = 782 [M-H+HCOZH]'; tR = 1.1 min.
Examgle 29: 4-((S)—2-((S)—2-(6-(2,5-dioxo—2,5-dihydro—1H-pyrroIy|)hexanamido)—3-methy|— butanamido)—5—ureidopentanamido)benzy| (4-((2R,3R)—3-((S)—1-((3S,7S,10R,16S,E)—10-(3-chloro— 4-methoxybenzyl)-6,6,7-trimethylneopentyI-2,5,9,12-tetraoxo—1-oxa-4,8,1 zacyclohexad ec- 13-eny|)ethyl)oxirany|)benzy|)carbamate To compound 71 (16.0 mg, 21.7 pmol) were added a solution of example 25 (14.3 mg, 19.7 pmol) in DCM (2.5 mL) and DIEA (3.4 pL, 19.7 pmol). The reaction medium was stirred at RT overnight then was added DMF (1 mL) and stirring was carried on at RT for 1 d. H20 (8 mL) was added to the reaction medium, stirrring carried on for 15 min then the s 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 g on silica gel (gradient n OH) to give 15.8 mg of e 29 as a white solid (60%).
RMN 1H (400 MHz, 8 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 = 48,107 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 [M-H]'; m/z = 1324 [M+H]+; m/z = 1368 [M-H+HC02H]'; tR = 1.3 min.
Synthesis of examples 30 & 31: NHS ester of non-cleavable triazole(S)-neopentyl(S)- Me-aza-C52 and corresponding ADC O O o l N3 O :ENUCI + HOWO/ example 24 NV"! 0 OMe O H H 73 Nwm 0 OMG example 30 hu2H11_R35N example 31 Nwm 0 OMG Compound 72: tert—butyl 3-(propynyloxy)propanoate To a solution of gyl alcohol (1.362 mL, 23.4 mmol) in THF (23 mL) was added sodium (20.08 mg, 0.874 mmol), the reaction medium was heated at 60°C until complete solubilization of sodium then cooled down at RT before the addition of tert—butyl acrylate (2.286 mL, 15.6 mmol).
The on medium was stirred at RT overnight then H20 (25 mL) was added and the aqueous phase was extracted with EtOAc (3 x 25 mL). The combined organic phase were dried over NaZSO4, filtered and concentrated in vacuo to give 2 g of compound 72 as a colorless oil (70%).
Compound 73: 3-(propynyloxy)propanoic acid To a solution of compound 72 (2.0 g, 10.86 mmol) in DCM (50 mL) was added TFA (8.065 mL, 108.6 mmol). The reaction medium was stirred at RT overnight, concentrated in vacuo, co- evaporated with toluene and purified by flash chromatography on 80 g of silica gel ent elution DCM/MeOH) to give 1.1 g of compound 73 as an oil (80%).
RMN 1H (400 MHz, 6 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: 3-((1-(4-((2R,3R)((S)((3S,7S,10R,16S,E)—10-(3-chloromethoxybenzyl)- 6,6,7-trimethylneopentyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triazacyclohexadecen yl)ethy|)oxiranyl)benzyl)—1H-1,2,3-triazolyl)methoxy)propanoic acid To a solution of e 24 (50 mg, 66.6 pmol) in THF (0.5 mL) were added compound 73 (17.05 mg, 113.1 pmol), 0.1M aq. copper (ll) sulfate (266 pL, 26.6 pmol) and 0.2M aq. sodium ascorbate (266 pL, 53.2 pmol). The reaction medium was stirred at RT for 1 d then H20 (1 mL) was added and the aqueous phase was extracted with EtOAc (3 x 1 mL). The combined organic phases were washed with sat. brine, dried over MgSO4, filtered, concentrated in vacuo and purified by tion on Sephadex LH20 (elution DCM) to give 40 mg of an oil that was r purified by flash chromatography on 5 g of silica gel (gradient elution OH) to give 20 mg of compound 74 (35%).
RMN 1H (500 MHz, 6 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 = 48,108 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 min.
Example 30: 2,5-dioxopyrrolidinyl 3-((1-(4-((2R,3R)((S)((3S,7S,10R,16S,E)—10-(3- chloro—4-methoxybenzyl)-6,6,7-trimethylneopentyl-2,5,9,12-tetraoxo—1-oxa-4,8,1 1-triaza- cyclohexadecenyl)ethyl)oxiranyl)benzyl)—1H-1,2,3-triazolyl)methoxy)propanoate To a solution of compound 74 (20 mg, 22.7 pmol) in DCM (5 mL) were added DIEA (5.0 pL, 28.5 pmol) and DSC (6.34 mg, 23.8 pmol). The reaction medium was stirred at RT for 3 h then were added DSC (3 mg, 11.3 pmol) and DIEA (5 pL, 28.5 pmol) and stirring was carried on at RT for 2 h. The reaction medium was then concentrated in vacuo and purified by two successive flash chromatographies on 1.5 g of diol-modified silica gel ent elution DCM/iPrOH) to give 11.5 mg of example 30 (50%).
RMN 1H (500 MHz, 8 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 = 47,106 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+HC02H]'; tR = 1.29 min.
Example 31: mAb-Ex.30 Example 31 was prepared in a similar way to examples 3, 7, 10 and 14. 5.4 mg of example 31 were obtained as a colorless limpid solution at a concentration of 1.8 mg/mL with a DAR of 4 (HRMS), a ric purity of 98.1% and a global yield of 45%.
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).
S nthesis of exam le 32: 3- S -neo ent lMeCH20H-aza-C52 benz lic amine H H H Et3SIO 7s Et3SiO 79 Br N3 N3 KGVBI' REEVBI' K:jVSJrCF3SO3- 80 81 HZN 0% o HN "\CECI N3 0\ o HN o o example 32 "\CECI HNWN \o HNWN \0 H H HO Et3SiO nd 75: (2R,3S)—1-((4-methoxybenzyl)oxy)—2-methylhexenyl (2S)—2-((4-((R)—2- acrylamido(3-chloromethoxyphenyl)propanamido)—3-(hydroxymethyl)—3-methyl oxobutyl)amino)—4,4-dimethylpentanoate To a solution of compound BC6 (385.6 mg, 773.5 pmol) in DMF (12 mL) were added HATU (348.7 mg, 889.5 umol) and HOAt (122.3 mg, 889.5 umol). The reaction medium was stirred at RT for 30 min then were added a solution of compound AD3 (292 mg, 773.5 umol) in DMF (5 mL) and DIEA (475.2 uL, 2.71 mmol) and ng was carried on at RT for 4 h. H20 (50 mL) was added and the aqueous phase was extracted with EtOAc (3 X 50 mL). The combined organic phases were washed with H20 (15 mL), sat. brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 30 g of silica gel (gradient elution DCM/MeOH) to give 383 mg of compound 75 as a yellow oil (65%).
RMN 1H (500 MHz, 8 in ppm, 6): 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); .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 = 8.0 Hz, 0.45H); 7.84 (d, J = 8.0 Hz, 0.55H); 7.98 (t, J = 6.5 Hz, 0.45H); 8.00 (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+HCOZH]'; tR = 1.54-1.56 min.
Compound 76: R,16S,E)—10-(3-chloromethoxybenzyl)(hydroxymethyl)((R)—1-((4- methoxybenzyl)oxy)propanyl)methylneopentyloxa-4,8,1 1-triazacyclohexadec—13-ene— 2,5,9,12-tetraone To a on of compound 75 (380 mg, 501.1 pmol) in DCM (38 mL) under Ar was added Grubbs | catalyst (20.97 mg, 25.05 pmol). The reaction medium was stirred under Ar at RT for 4 h then was added Grubbs l catalyst (20.97 mg, 25.05 pmol). Stirring was carried on at RT for 3 h then was added Grubbs l catalyst (20.97 mg, 25.05 pmol) and stirring carried on at RT. After 24 h of reaction, Grubbs l catalyst (20.97 mg, 25.05 pmol) was added and stirring carried on for 4 h.
The on medium was trated in vacuo and purified by flash chromatography on 20 g of silica gel (isocratic elution heptane/EtOAc) to give 170 mg of compound 76 stereomer 1 (56%) and 138 mg of compound 76 stereomer 2 (37%). nd 76 stereomer 1 RMN 1H (400 MHz, 6 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 .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); 8.09 (d, J = 9.6 Hz, 1H); 8.38 (d, J = 8.0 Hz, 1H). LCMS (A4): ES m/z = 728 [M-H]'; m/z = 730 [M+H]+; m/z = 774; tR = 5.15 min.
Compound 76 stereomer 2 RMN 1H (400 MHz, 6 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 = 8.0 Hz, 1H). LCMS (A4): ES m/z = 728 [M-H]'; m/z = 730 [M+H]+; m/z = 774; tR = 4.79 min.
Compound 77: (38,10R,16S,E)—10-(3-chloromethoxybenzyl)((R)—1-((4-methoxy— benzyl)oxy)propanyl)methylneopentyl(((triethylsilyl)oxy)methyl)oxa-4,8,1 1-triaza- cyclohexadec—13-ene—2,5,9,12-tetraone To a on of compound 76 stereomer 1 (170 mg, 232.8 pmol) in DCM (3 mL) cooled with an ice bath were added at 4°C chlorotriethylsilane (159.3 pL, 931.1 pmol) and dropwise TEA (131.1 pL, 931.1 pmol). The reaction medium was stirred at 4°C for 30 min then at RT for 20 h. The reaction medium was diluted with DCM (50 mL) and brine (10 mL) and stirring was carried on for min. The organic phase was washed with sat. brine (2 x 10 mL), dried over MgSO4, filtered and concentrated in vacuo. The other reoisomer of compound 76 (138 mg, 189.0 pmol) was similarly ted, both batches were pooled and purified by flash chromatography on 20 g of silica gel (isocratic elution heptane/EtOAc) to give 139 mg of compound 77 stereomer 1 as a colorless foam (39%) and 107 mg of compound 77 stereomer 2 as a colorless foam (30%).
Compound 77 stereomer 1 RMN 1H (400 MHz, 6 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 .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 = 8.0 Hz, 1H). LCMS (A4): ES m/z 842 [M-H]'; m/z = 844 [M+H]+; m/z = 888 [M- H+HC02H]'; tR = 7.08 min.
Compound 77 stereomer 2 RMN 1H (400 MHz, 6 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 .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); 8.01 (d, J = 9.2 Hz, 1H); 8.41 (d, J = 8.0 Hz, 1H). LCMS (A4): ES m/z = 842 [M-H]'; m/z = 844 [M+H]+; m/z = 888 [M-H+HCOZH]'; tR = 6.88 min.
Compound 78: ,16S,E)—10-(3-chloromethoxybenzyl)((R)—1-hydroxypropanyl)—6- methylneopentyl(((triethylsilyl)oxy)methyl)oxa-4,8,1 1-triazacyclohexadec—13-ene- 2,5,9,12-tetraone To a solution of both diastereoisomers of compound 77 (245 mg, 290.1 pmol) in DCM (10 mL) and H20 (2.5 mL) cooled with an ice/acetone bath were added DDQ (339.4 mg, 1.45 mmol) and 2,6-di-tert-butylpyridine (171.7 mg, 870.3 pmol). The reaction medium was stirred at 0°C for 2 h then was d with aq. NaHCOs (10 mL) and DCM (10 mL) and stirring was carried on for min. The aqueous phase was ted with DCM (3 X 20 mL), the combined organic phases were washed with sat. brine (3 x 5 mL), dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 20 g of silica gel (gradient elution DCM/MeOH) to give 155 mg of compound 78 as a white foam (74%).
RMN 1H (400 MHz, 6 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, 0.1 1 H); 7.96 (d, J = 9,4 Hz, 0.52H); 8.00 (d, J = 9.4 Hz, 0.37H); 8.31 (d, J = 8.0 Hz, 0.63H); 8.42 (d, J = 8.4 Hz, . LCMS (A4): ES m/z = 722 [M-H]'; m/z = 724 [M+H]+; m/z = 768 [M-H+HC02H]'; tR = 5.68-5.78 min.
Compound 79: 28)—2-((3S,10R,16S,E)—10-(3-chloromethoxybenzyl)methylneopentyl- 2,5, 9, 1 2-tetraoxo(((triethylsilyl)oxy)methyl)-1 -oxa-4,8,1 1-triazacyclohexadecen yl)propanal To a on of compound 78 (154 mg, 212.6 pmol) in DCM (1.5 mL) cooled with an ice/ acetone bath were added at 0°C a solution of KBr (25.6 mg, 212.6 pmol) in H20 (612 pL), a solution of TEMPO at 5 mg/mL in DCM (137 pL, 4.25 pmol) and dropwise an aqueous solution of 1.56M sodium hypochlorite pH 9.5 (204 pL, 318.9 pmol). Stirring was carried on at 0°C for 15 min then was added at 0°C sat. Na28203 (1.9 mL), the ice bath was removed and the on medium stirred for 10 min. It was then extracted with DCM (3 x 20 mL), the combined organic phases were washed with H20 (2 x 10 mL), dried over MgSO4, ed, trated in vacuo and purified by flash chromatography on 15 g of silica gel ent elution DCM/MeOH) to give 36 mg of compound 61 (23%).
RMN 1H (400 MHz, 6 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 min.
Compound 80: 1-(azidomethyl)(bromomethyl)benzene To a solution of p—xylene dibromide (2.0 g, 7.2 mmol) in DMF (20 mL) was added in 20 min a solution of sodium azide (584.9 mg, 8.64 mmol) in DMF (20 mL). The reaction medium was stirred at RT for 20 h then poured on ice (100 g) and extracted with EtOAc (3 x 50 mL). The combined organic phase were washed with sat. brine (3 x 15 mL), dried over MgSO4, filtered, concentrated in vacuo, co-evaporated with toluene and purified by flash chromatography on 70 g of silica gel (elution EtZO) to give to give 773 mg of compound 80 as a colorless oil (47%).
RMN 1H (400 MHz, 8 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-trimethylthiabicyclo[3.2.1] octanium trifluoromethanesulfonate To (1R,4R,5R)-4,7,7-trimethylthiabicyclo[3.2.1]octane (CAS number [57182], 580.1 mg, 3.41 mmol) was added a solution of nd 80 (770 mg, 3.41 mmol) in DCM (2.2 mL). A solution of lithium trifluoromethanesulfonate (2.71 g, 17.03 mmol) in H20 2 mL) was then added se and the reaction medium was stirred at RT for 2 d. H20 (15 mL) and DCM (15 mL) were then added to the reaction medium and stirring carried on at RT for 10 min. The aqueous phase was extracted with DCM (3 x 15 mL), the ed organic phases were washed with sat. brine (3 X 8 mL), dried over MgSO4, filtered and trated in vacuo. The pale yellow oil was triturated in iPr20 (5 mL), the solid thus ed was filtered, washed with iPr20 (2 x 5 mL) and dried under vacuum to give 1.266 g of compound 81 as a white solid (79%).
RMN 1H (400 MHz, 8 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: (38,10R,16S,E)—16-((S)—1-((2R,3R)(4-(azidomethy|)phenyl)oxiranyl)ethy|)- 10-(3-chloromethoxybenzyl)methylneopentyl(((triethylsi|y|)oxy)methy|)oxa-4,8,1 1- triazacyclohexadec—13-ene-2,5,9,12-tetraone To a on of compounds 79 (35 mg, 48.5 pmol) and 81 (24.8 mg, 53.3 pmol) in DCM (1.5 mL) cooled at -70°C was added dropwise BEMP (CAS number [98015—45—3], 18.6 pL, 63.0 pmol). The reaction mixture was stirred at -70°C for 2 h 30, sat. brine (1.5 mL) was added to the reaction mixture, the bath removed and stirring carried on vigorously up to 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 (gradien elution DCM/MeOH) to give 10.2 mg of compound 82 as a colorless lacquer (24%).
RMN 1H (500 MHz, 8 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 = 8.0 Hz, 1H). LCMS (A5): ES m/z = 120; m/z = 865 [M-H]'; m/z = 867 [M+H]+; m/z = 911 [M-H+HC02H]'; tR = 1.87 min.
Example 32: (3S,10R,16S,E)—16-((S)—1-((2R,3R)—3-(4-(aminomethyl)phenyl)oxiranyl)ethyl)—10- (3-chloromethoxybenzyl)methylneopentyl(((triethylsilyl)oxy)methyl)—1-oxa-4,8,1 1- triazacyclohexadec—13-ene-2,5,9,12-tetraone To a solution of compound 82 (9.4 mg, 10.84 pmol) in DCM (1.3 mL) and MeOH (0.7 mL) was added a solution of TCEP (3.45 mg, 11.9 pmol) in H20 (0.7 mL). The reaction medium was stirred at RT overnight then was added aq. NaHCOs (2.5 mL) and stirring carried on for 10 min. The aqueous phase was extracted with DCM (3 X 10 mL), the ed organic phases were dried over MgSO4, filtered, concentrated in vacuo and purified by falsh chromatography on 5 g of silica gel (gradient elution DCM/MeOH/HZO) to give 4.55 mg of example 32 as a white lacquer (57%).
RMN 1H (500 MHz, 6 in ppm, 6): 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 .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); 8.08 (d, J = 9.4 Hz, 1H); 8.31 (d, J = 8.0 Hz, 1H). LCMS (A5): ES m/z = 725 [M-H]'; m/z = 727 [M+H]+; tR = 0.79 min.
Pharmacological results The compounds of the ion were ted to pharmacological tests for determining their antitumoral effect. The ADC of formula (III) were also assessed in terms of plasmatic stability.
For illustrative purposes, two ates, ADC1 and ADC2 depicted below, derived from WO2011/001052 were also tested.
H H hU2H11_R35-74*NMwN o 0A hu2H11_R35-74*NM O O Evaluation of the tion of proliferation of the HCT116 and MDA-MB-231 cell lines by the cryptophycin compounds of formula (I) 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 glutamine Gibco #25030 for the MDA—MB-231 cells; DMEM Gibco , % FCS Gibco #10500-056, 2 mM glutamine Gibco #25030 for the HCT116 cells). The cell suspension was seeded in Cytostar 96-well culture plates (GE Healthcare Europe, #RPNQ0163) in the whole culture medium containing serum at a density of 5000 cells/well. After incubation for 4 h, successive dilutions of the cryptophycin compounds were added to the wells at concentrations sing from 10'7 to 10'12 M (in triplicate for each concentration). The cells were cultured at 37°C in an here containing 5% C02 in the ce of the cryptophycin compounds for 3 d. On the 4th day, 10 pl of a 14C-thymidine solution (0.1 pCi/well, Perkin Elmer #NEC56825000) were added to each well. The incorporation of 14C-thymidine was measured 96 h after the start of the experiment with a eta radioactivity counter (Perkin Elmer). The data were expressed in the form of a percentage of survival by determining the ratio n the d count obtained with the cells treated with the cryptophycin compound and the count obtained with the cells of the control wells (treated with the culture medium alone).
The inhibitory activity is given by the concentration which inhibits 50% of the activity.
Table II Structural comment1 '33??? Mgk?gli?gm C52 8 epoxide 76 90 Ex.1 [3 epoxide 163 250 Ex.4 [3 epoxide 51 82 Ex.8 oc/B epoxide 3:7 518 564 Ex.11 B epoxide n.t. 102 Ex.15 oc/B epoxide 35:65 n.t. 511 Ex.16 oc/B epoxide 4:6 n.t. 2815 Ex.17 oc/B epoxide 5:5 n.t. 856 Ex.18 [3 epoxide 179 228 Ex.21 oc/B epoxide 3:7 n.t. 456 Ex.32 [3 epoxide n.t. 1777 1 Cryptophycin compounds of formula (I) were tested either as pure B epoxides or as a mixture of 0L and B epoxides as specified in the column entitled "structural t". It is known from the literature that the B epoxides are y 50 to 100 times more potent than the oc e (see for example, Al-Awar R.S., et al., J Med Chem 2003, 46, 2985-3007).
Cryptophycin compounds of formula (I) were found to inhibit the proliferation of HCT116 and MDA—MB-231 cell lines with IC50 ranging from 50 pM to 2.815 nM.
Evaluation of the inhibition of proliferation of the MDA-MB-231 cell line by the ADC of a (III) 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 in Cytostar 96-well culture plates (GE Healthcare Europe, #RPNQO163) in the whole culture medium containing serum at a density of 5000 cells/well. After incubation for 4 h, successive dilutions of the ADC are added to the wells at concentrations sing from 10'7 to 10'12 M (in triplicate for each concentration). The cells were cultured at 37°C in an atmosphere containing 5% C02 in the presence of the ADC for 3 d.
On the 4th day, 10 pl of a 14C-thymidine solution (0.1 pCi/well, Perkin Elmer #NEC56825000) were added to each well. The incorporation of ymidine was measured 96 h after the start of the ment with a microbeta radioactivity r (Perkin Elmer). The data were expressed in the form of a percentage of survival by ining the ratio between the reduced count obtained with the cells treated with the ADC and the count obtained with the cells of the control wells (treated with the culture medium alone). In n experiments, the naked antibody was added to the wells at a concentration of 1 pM at the start of the experiment and the inhibition of proliferation was measured as described previously.
|C50 (pM), MDA—MB-231 ADC1 42 4571 ADCZ 46 9377 Ex.3 20 3738 Ex.7 38 18042 Ex.10 35 10367 Ex.14 53 25887 Ex.20 43 1 1871 Ex.23 129 64270 Ex.31 72 31784 Cryptophycin ADC of formula (III), as well as ADC1 and ADC2, were found to inhibit the proliferation of MDA—MB-231 cell line with |C50 g from 20 pM to 130 pM and selectivity ratio ADC alone vs ADC + naked antibody between 185 and 500.
Determination of the MTD of the ADC of formula (III) following single i.v. administration in SCID mice MTD was determined as the maximal dose that does not induce 15% body weight loss during 3 consecutive days for an individual mouse or 20% body weight loss during 1 day or mortality. It was evaluated after a single intravenous (iv) bolus injection in 3 female SCID mice and during a period of 28 days post-treatment.
Table IV MTD (mg/kg) Ex.3 20 Ex.7 30 Ex.10 40 Ex.14 2 50 Ex.20 20 Ex.23 2 40 Cryptophycin ADC of formula (III) displayed MTD in SCID mice g from 20 mg/kg to 2 50 mg/kg.
Evaluation of the in vivo antitumor activity of ADC of formula (III) t MDA-MB-231 in SCID mice following single i.v. administration In vivo antitumor activity was evaluated at 3 dose-levels against measurable breast -231 xenografts implanted s.c. in female SCID mice. Control groups were left untreated. Conjugates were administered by a single iv. bolus injection, the day of the ent was indicated on each graph by an arrow (V).
For the evaluation of antitumor activity of conjugates, animals were weighed twice weekly and tumors were measured twice weekly by caliper. Animal body weights included the tumor weights.
Tumor volume were calculated using the a mass (mms) = [length (mm) x width (mm)2]/2.
The y efficacy end points were AT/AC, percent median regression, 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 day of first treatment (staging day) from the tumor volume on the specified observation day. The median AT was calculated for the treated group and the median AC was calculated for the control group. Then the ratio AT/AC was calculated and expressed as a percentage: AT/AC = (delta T/delta C)><100.
The percentage of tumor regression was defined as the % of tumor volume decrease in the d group at a specified observation day (t) compared to its volume on the first day of first treatment (t0). At a specific time point and for each animal, % sion was calculated. The median % regression was then calculated for the group. % regression (at t) = ((Volumeto — Volumet)/Volumeto)x100. Regressions were defined as l (PR) if the tumor volume decreased to 50 % of the tumor volume at the start of treatment and complete (CR) when tumor volume cannot be measured (0 mm3). Tumor free survivor (TFS) was defined as the animals with undetectable tumors at the end of the study (>100 days post last treatment).
Evaluation of the in vivo antitumor activity of Ex.3 against MDA-MB-231 in SCID mice following single i.v. administration Dose Median AT/AC Median % of Regressions Table V (mg/kg) . .
In % (day) regressuon pR CR TFS Control - - - - - - ADC2 2.5 < 0 (d53) 90% 4/6 3/6 0/6 1.25 18 (d49) - 0/6 0/6 0/6 Ex.3 2.5 < 0 (d53) 100% 6/6 5/6 0/6 1.25 9 (d53) - 2/6 0/6 0/6 0.625 44 (d42) - 0/6 0/6 0/6 tion of the in vivo antitumor activity of Ex.7 against MDA-MB-231 in SCID mice following single i.v. administration Dose Median AT/AC Median % of Regressions Table V ) . .
In % (day) regressuon pR CR TFS Control - - - - - - ADC2 2.5 < 0 (d53) 90% 4/6 3/6 0/6 1.25 18 (d49) - 0/6 0/6 0/6 Ex.7 2.5 < 0 (d53) 100% 6/6 6/6 1/6 1.25 < 0 (d53) 64% 5/6 1/6 0/6 0.625 42 (d42) - 0/6 0/6 0/6 Evaluation of the in vivo mor activity of Ex.10 against -231 in SCID mice following single i.v. administration Dose Median AT/AC Median % of Regressions Table VI (mg/kg) in % (day) regression PR CR TFS Control - - - - - - ADC2 2.5 < 0 (d42) 14% 0/6 0/6 0/6 1.25 6 (d42) - 0/6 0/6 0/6 Ex.10 2.5 < 0 (d59) 95% 6/6 3/6 0/6 1.25 < 0 (d46) 32% 0/6 0/6 0/6 0.625 16 (d42) - 0/6 0/6 0/6 Evaluation of the in vivo antitumor activity of Ex.14 against MDA-MB-231 in SCID mice following single i.v. administration Dose Median AT/AC Median % of sions Table Vll (mg/kg) in % (d34) regreSSIon pR CR TFS Control - - - - - - ADC2 2.5 21 - 0/6 0/6 0/6 1.25 48 - 0/6 0/6 0/6 Ex.14 5 < 0 100% 6/6 6/6 1/6 2.5 < 0 11% 1/6 0/6 0/6 1.25 39 - 0/6 0/6 0/6 Evaluation of the in vivo antitumor activity of Ex.20 against MDA-MB-231 in SCID mice following single i.v. administration Table Vlll Median AT/AC Median % of Regressions (mg/kg) In % (day 40) regreSSIon pR CR TFS Control - - - - - - ADC2 2.5 < 0 49% 4/6 0/6 0/6 1.25 14 - 0/6 0/6 0/6 Ex.20 5 < 0 100% 6/6 6/6 6/6 2.5 < 0 100% 6/6 4/6 1/6 1.25 < 0 3% 3/6 1/6 0/6 Evaluation of the in vivo antitumor activity of Ex.23 against -231 in SCID mice following single i.v. administration Dose Median AT/AC Median % of sions Table Vlll (mg/kg) in % (day 40) regreSSIon pR CR TFS Control - - - - - - ADC2 2.5 < 0 49% 4/6 0/6 0/6 1.25 14 - 0/6 0/6 0/6 Ex.23 5 < 0 81% 6/6 3/6 0/6 2.5 16 - 1/6 0/6 0/6 1.25 36 - 0/6 0/6 0/6 These results showed that all the tested es of the invention, as well as ADC2, displayed antitumor activity at doses ranging from 0.625 mg/kg to 5 mg/kg.
Determination of PK parameters of Ex.3 and Ex.7 following a single i.v. administration in SCID mice (5 mg/kg) In vivo cokinetics of ADC and total antibody were evaluated in female SCID mice following administration of the ate by a single iv. bolus injection. Female SCID mice (5-6 weeks of age, weight on average 20-25 g) were housed in a sterile room, under aseptic conditions in a r 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 and at each selected time point (i.e. 0.083, 0.25, 24, 72, 96, 168, 240, 336 h), 600 uL of blood were collected via cardiac puncture and blood samples were then centrifuged (15 min at 4°C and 3500 tr/min). fication of ADC and total antibody in plasma s was performed using immunoassays. Plasma concentration versus time profiles and PK parameters of conjugate and total dy in mouse were characterized using non-compartmental analysis (Phoenix, WinNonLin version 6.3) and are depicted in Figures 15 and 16. The area under the concentration-time curve, AUC (pg.day/mL) was estimated by the trapezoidal rule. Concentration at t=0 Co (ug/mL), Clearance CL (L/(day.kg), and terminal elimination half-life t1/2 (day) were derived and calculated from the curve. c0 AUClast tlast AUC CL Vss t1/22 Table IX Analytes (Hg/mL) (Hg-day/mL) (day) (Hg-day/mL) (L/(day-kg» (L/kg) (day) Ex.3 ADC 62.8 191 14 217 0.023 0.14 4.8 total 74.9 455 14 7918 8 0.108 118 Ex.7 ADC 81.8 235 14 288 0.019 0.12 4.8 total 77.4 368 14 741b 0.0087b 0.14b 18b a: informative data only as AUCext >> 30% (i.e. 42%); b: informative data only as AUCext >> 30% (i.e. 50%).
Evaluation by HRMS of plasmatic stability of cryptophycin ADC of formula (III) following single i.v. administration in SCID mice at 5 mg/kg Plasma samples from the PK study were also analyzed by LC-HRMS. The chromatographic analysis was performed on a Waters Acquity | Class and a Waters Mass Prep Micro Desalting um (2.1 x 5 mm) column at 80°C with a gradient elution of (A) water + 0.1% formic acid/(B) CH3CN + 0.1% formic acid described below.
Time (min) %B Flow (ml/min) 0.00 5 0.5 0.50 5 0.5 0.51 5 0.2 2.00 90 0.2 2.10 5 0.5 2.70 90 0.5 2.80 5 0.5 3.40 90 0.5 3.50 5 0.5 4.00 5 0.5 The mass spectrometry was performed on a Waters QTOF Synapt G2-S machine with electrospray ionization in positive mode (ES+). The mass spectra were deconvoluted with the Waters Biopharmalynx software. HRMS spectra were obtained for all tested examples and are depicted in Figures 20 to 25.
These results showed that all tested examples of the invention didn’t display the metabolization observed with reference ADC, ADC1 and ADC2.
It is ore apparent that the compouds of the invention have an anticancer ty and that the conjugates of the invention display an ed stability in mice plasma.
Accordingly, in another of its aspects, the invention also relates to the use of cryptophycin compounds of formula (I) or of the conjugates of formula (III) as anticancer agents.
The present invention, according to r of its aspects, also provides medicaments which comprise a ate of formula (III).
These ments are employed eutically, especially in the treatment of cancer.
According to another of its aspects, the present invention relates to pharmaceutical compositions comprising as active principle a conjugate of formula (III) according to the invention. These pharmaceutical compositions comprise an effective dose of at least one ate of formula (III) according to the invention and also at least one pharmaceutically acceptable excipient.
The said ents are selected, in accordance with the pharmaceutical form and method of administration desired, from the customary excipients, which are known to a person skilled in the The present invention, according to another of its aspects, also provides a method of treating the pathologies indicated above, which comprises administering to a patient an effective dose of a conjugate of a (III) according to the invention.

Claims (3)

1. Cryptophycin compound of formula (I): o O 1 m O p o O O HN R m R R 3 2 R 10 8 9 R N N O 7 R R R H 5 4 5 6 (I) wherein: ? R1 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- 10 C6)cycloalkyl group or a 6)heterocycloalkyl group; ? R4 and R5 represent, independently of each other, a hydrogen atom, a -(C1-C6)alkyl group, a - )alkyl-NH(R12) group, a -(C1-C6)alkyl-OH group, 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- 15 C6)cycloalkyl or a -(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, a -(C1-C6)alkyl group, a - (C1-C6)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- 20 C6)cycloalkyl group or a 6)heterocycloalkyl group; ? R9 represents one or more substituents of the phenyl s chosen, independently of each other, from: a hydrogen atom, -OH, a -(C1-C4)alkoxy group, a n atom, -NH2, a -NH(C1- C6)alkyl group, a -N(C1-C6)alkyl2 group, a -NH(C1-C6)cycloalkyl group or a (C3- C6)heterocycloalkyl group; 25 ? 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(R11), • (C1-C6)alkyl-OH, 30 • (C1-C6)alkyl-SH, • CO2H or C(=O)NH2; • (C1-C6)alkyl-CO2H or (C1-C6)alkyl-C(=O)NH2; or • )alkyl-N3. 20266725_1 (GHMatters) P44029NZ00 ? R11 and R12 represent, independently of each other, a hydrogen atom or a -(C1-C6)alkyl group.
2. Cryptophycin compound of formula (I) according to claim 1, having the following structure: o O 1 m O p o O O HN R m R 10 R 3 2 R 8 9 R N N O 7 R R R H 5 4 3. Cryptophycin compound of formula (I) according to any one of claim 1 or 2, characterized in that R1 represents a methyl group. 4. Cryptophycin compound of a (I) according to any one of claims 1 to 3, characterized in 10 that each of R2 and R3 represents a hydrogen atom. 5. Cryptophycin compound of formula (I) according any one of claims 1 to 3, characterized in that one of R2 and R3 represents a methyl group and the other one represents a hydrogen atom. 15 6. Cryptophycin compound of formula (I) according to any one of claims 1 to 3, terized in that R2 and R3 form together with the carbon atom to which they are attached a cyclopropyl group. 7. Cryptophycin compound of formula (I) according to any one of claims 1 to 6, characterized in 20 that each of R4 and R5 represents a methyl group. 8. Cryptophycin compound of formula (I) according to any one of claims 1 to 7, characterized in that R6 ents a hydrogen atom. 25 9. Cryptophycin nd of formula (I) according to any one of claims 1 to 8, characterized in that R7 and R8 represent ndently of each other a hydrogen atom or a (C1-C6)alkyl group. 10. Cryptophycin compound of formula (I) according to any one of claims 1 to 9, characterized in that R9 represents two tuents ndently selected from a methoxy group and a chlorine 30 atom. 11. Cryptophycin compound of formula (I) according to any one of claims 1 to 10, characterized in that R10 represents a hydrogen atom. 20266725_1 (GHMatters) P44029NZ00 12. Cryptophycin compound of formula (I) according to any one of claims 1 to 11, characterized in that: ? R1 represents a -(C1-C6)alkyl group; ? each of R2 and R3 represents a hydrogen atom; 5 ? R6 represents a en atom; ? R9 represents two substituents independently of each other selected from a methoxy group and a ne atom; and R10 ents a hydrogen atom. 10 13. Cryptophycin compound of formula (I) according to any one of claims 1 to 11, characterized in that: ? R1 represents a (C1-C6)alkyl group; ? R2 and R3 represents a (C1-C6)alkyl group, and the other one represents a hydrogen atom; 15 ? R6 represents a hydrogen atom; ? R9 represents two substituents ndently of each other selected from a methoxy group and a chlorine atom; and ? R10 represents a hydrogen atom. 20 14. Cryptophycin nd of formula (I) according to any one of claims 1 to 13, characterized in that W is selected from -(C1-C6)alkyl-NHR11, -(C1-C6)alkyl-OH, -(C1-C6)alkyl-SH and -(C1-C6)alkyl- CO2H. 15. Cryptophycin compound of formula (I) according to claim 14, characterized in that W 25 represents a -CH2-NH2 group or a -CH2-OH group. 16. Cryptophycin compounds of a (I) according to claim 1 which are selected from the following list: O O O O N O O HN Cl H N O O HN Cl 3 O 2 O N N O OMe N N O OMe H H H H O O O O HO O O HN Cl N O O HN Cl O 3 O N N O OMe N N O OMe H H H H 20266725_1 (GHMatters) P44029NZ00 O O O O H N O O HN Cl N O O HN Cl 2 O 3 O N N O OMe N N O OMe H H H H stereomer 1 O O O O H N O O HN Cl HO O O HN Cl 2 O O N N O OMe N N O OMe H H H H stereomer 1 stereomer 1 O O O O N O O HN Cl 3 H N O O HN Cl O 2 O N N O OMe N N O OMe H H H H stereomer 1 stereomer 1 O O O O HO O O HN Cl HO O O HN Cl O O N N O OMe N N O OMe H H H H O O O O HO O O HN Cl H N O O HN Cl O 2 O N N O OMe N N O OMe H H H H 5 stereomer 2 O O O O N O O HN Cl H O O HN Cl 3 N O 2 O N N O OMe N N O OMe H H H H O O O O H N O O HN Cl HO O O HN 2 Cl O O N N O OMe N N O OMe H H H H 20266725_1 ters) P44029NZ00 H N O O 2 HN Cl N N O OMe H H 17. Cryptophycin payload of a (II): o O 1 m O RCG -L-Y p o O O HN R m R R R 3 2 10 R 8 9 R N N O 7 R R 5 R H 6 (II) 5 wherein R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in claim 1 to 13, and ? Y isselected from a –(C1-C6)alkyl-NR11- group, a 6)alkyl-O- group, a -(C1-C6)alkyl-S- group , a -C(=O)O, C(=O)NH- group, a -(C1-C6)alkyl-C(=O)O- group, and a -(C1-C6)alkyl- C(=O)NH- group; 10 ? R11 represents a hydrogen atom or a -(C1-C6)alkyl group; ? L represents a linker; ? RCG1 represents a ve chemical group t at the end of the linker, L being of formula (IV): L2 (AA)w L1 15 in which: ? L1 represents ? a single bond or a -NR16(hetero)aryl-CR15R14-O-C(=O)- group if Y is -(C1-C6)alkyl- N(R11)-; ? a -NR18-(C2-C6)alkyl-NR17-C(=O)- group or a -NR16(hetero)aryl-CR15R14-O-C(=O)- 20 NR18-(C2-C6)alkyl-NR17-C(=O)- group if Y is -(C1-C6)alkyl- O- or -(C1-C6)alkyl- S-; ? a -NR16(hetero)aryl-CR15R14- group if Y is -C(=O)O-, -C(=O)NH-, (-C1-C6)alkyl- C(=O)O- or -(C1-C6)alkyl-C(=O)NH-; ? R14, R15, R16, R17 and R18 represent, ndently of each other, a hydrogen atom or a - (C1-C6)alkyl group; 25 ? (AA)w represents a sequence of w amino acids AA ted together via peptide bonds; ? w represents an integer ranging from 1 to 12; ? L2 represents a single bond , a -(C1-C6)alkyl- group, a –(C1-C6)alkyl-(OCH2CH2)i- group, a - (C1-C6)alkyl-(OCH2CH2)i-O(C1-C6)alkyl- group, a -(CH2CH2O)i(C1-C6)alkyl- group, a - 20266725_1 (GHMatters) P44029NZ00 CH(SO3H)-(C1-C6)alkyl- group, a 6)alkyl-CH(SO3H)- group, a 6)alkyl-cyclohexylgroup , a -NR19-(C1-C6)alkyl- group, a -NR20-(CH2CH2O)i(C1-C6)alkyl- group, a -NR21-arylgroup , a -NR21-heteroaryl- group, a -(C1-C6)alkyl-NR22C(=O)-(C1-C6)alkyl- group, or a -(C1- C6)alkyl-NR22C(=O)-(C1-C6)alkyl-(OCH2CH2)i- group; 5 ? R19,R20, R21 and R22 represent, independently of each other, a hydrogen atom or a -(C1- C6)alkyl group; ? i represents an integer n 1 and 50 ; AA denotes a natural or unnatural amino acid, of configuration D or L, chosen from: alanine (Ala), ?-alanine, ?-aminobutyric acid, 2-aminocyclohexylacetic acid, 2-aminophenylacetic acid, 10 arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), ?,?-dimethyl- ?-aminobutyric acid, ?,?-dimethyl-?-aminobutyric acid, ine (Gln), ic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), ?-acetyl-lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). 18. Cryptophycin payload of formula (II) according to claim 17, having the following structure: o O 1 m O RCG -L-Y 1 p o O O HN m O R R 10 R 3 2 R 8 9 R N N O 7 R R R H 5 4 19. Cryptophycin payload of formula (II) according to any one of claim 17 or 18, characterized in 20 that Y represents a -(C1-C6)alkyl-NR11- group. 20. Cryptophycin payload of formula (II) according to claim 19, characterized in that Y represents a --CH2-NH- group. 21. Cryptophycin payload of formula (II) ing to claim 17, characterized in that the sequence (AA)w has the formula: H O * ALK w * in which R23 represents the side chain of AA. 20266725_1 (GHMatters) P44029NZ00 22. Cryptophycin payload of formula (II) ing to any one of claim 17 or 21, characterized in that the sequence AA represents alanine (Ala), citrulline (Cit), glutamine (Gln), glycine (Gly), ?- acetyl-lysine (AcLys), or valine (Val). 5 23. Cryptophycin payload of formula (II) according to any one of claims 17 to 23, characterized in that RCG1 is chosen from: - -ZaRa group 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- 10 C10)heteroaryl or (C3-C7)heterocycloalkyl group or a succinimidyl group; - one of the following reactive groups: the maleimido O group; the haloacetamido Br or I O group with R13 representing a hydrogen atom or a (C1-C6)alkyl group; -Cl; -N3; - OH; -SH; -NH2; -C?CH, a group or a O-(C1-C6)alkyl hydroxylamine group. 24. phycin payload of formula (II) according to any one of claims 17 to 22, characterized in that L2 represents a -(C1-C6)alkyl- group, a -(C1-C6)alkyl-(OCH2CH2)i- group or a -CH(SO3H)- (C1-C6)alkyl- group 20 25. Cryptophycin ds of formula (II) according to claim 17, which are ed from the following list: O H O H O N N O O HN Cl N N O O O O O N N O OMe H H 20266725_1 (GHMatters) P44029NZ00 O O H H O N N O O HN Cl N N O O O O O N N O OMe H H O O H H O N N O O HN Cl N N O O O O O N N O OMe H H stereomer 1 O H O H O N N O O HN Cl N N O O O O O N N O OMe H H stereomer 1 O H O H O N N O O HN Cl N N O O O O O N N O OMe H H stereomer 2 O H O H O N N O O HN Cl N N O O H O O O N N O OMe H H O O O O H O H N N N O O O HN Cl O N N O O H O H O N N O OMe H H 25_1 (GHMatters) P44029NZ00 O NH O H O H O N N O N N H O H O O N O O HN Cl O O N N O OMe H H 26. ate of formula (III): o O 1 Ab-G-L-Y p o O O HN m O R R R R 3 2 R 10 8 9 R N N O 7 R R 5 R H 5 4 6 (III) wherein ? R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in claim 1 to 13; ? Y and L are as defined in claims 17 to 22; ? sents the product of reaction between RCG1, a reactive group present at the end of 10 the linker and RCG2, an onal reactive group present on Ab; ? Ab represents an antibody. 27. Conjugate of formula (III) according to claim 26 having the following structure: o O 1 Ab-G-L-Y p o O O HN m O R R R R 3 2 R 10 8 9 R N N O 7 R R 5 R H 28. Conjugate of formula (III) according to any one of claim 27 or 28, characterized in that RCG2 is ed from: • ?-amino groups borne by the side chains of lysine residues that are present at the surface of the antibody; 20 • a-amino groups of N-terminal amino acids of heavy and light chains of the antibody; • saccharide groups of the hinge region of the antibody; 20266725_1 (GHMatters) P44029NZ00 • thiols of cysteines generated by reducing intra-chain disulfide bonds of the antibody or the thiols of ered cysteines of the antibody; • amide groups borne by side chains of glutamine residues that are present at the surface of the antibody; 5 • aldehyde groups introduced into the antibody using formylglycine generating enzyme. 29. Conjugate of formula (III) according to any one of claim 26 or 27, characterized in that: • when RCG1 represents a N-hydroxysuccinimidyl ester, RCG2 represents a -NH2 group; • when RCG1 represents a maleimido or haloacetamido function or a -Cl group, RCG2 10 represents a -SH group; • when RCG1 represents a -N3 group, RCG2 ents a -C?CH group or an activated • when RCG1 represents a -OH or -NH2 group, RCG2 ents a carboxylic acid or amide on; 15 • when RCG1 represents a -SH group, RCG2 represents a maleimido or haloacetamido function; • when RCG1 represents a -C?CH function or an activated C?C, RCG2 represents a -N3 group; • when RCG1 represents a O-alkyl ylamine function or a Pictet-Spengler on 20 substrate, RCG2 represents an aldehyde or ketone function. 30. Conjugate of formula (III) ing to any one of claims 26 to 2930, characterized in that G is selected from: 31. Process for preparing the cryptophycin compound of formula (I) as defined in any one of claims 1 to 16 comprising the step: 20266725_1 (GHMatters) P44029NZ00 O OH R R O 1 1 PMBO PMBO R + R N O O O OH 7 R 6 R Sakurai alcohol D R NH 7 AD3 O R R H 3 2 O N HN O NH + O 2 R R R (i) R R O 9 3 2 R HO 9 5 4 O HO N O C B R R H 5 4 BC 1 R PMBO O PMBO O O O HN (ii) O O HN O R R O R R R 3 2 R R 3 2 R 8 9 N O 8 9 R N N N O 7 R R R H R 7 R R H 5 4 R 5 4 6 6 (iii, iv) 1 R O O O 1 O O HN (v) X O O HN O R R O R R R 3 2 R R 10 R 3 2 R 8 9 8 N N O X = N R N N O R 3 , P2 7 7 R R R H R R R 6 5 4 H 5 4 6 X = OH, P 4 H N O HN 2 O R O R R 10 R 3 2 R 8 N N O 9 P R 3 7 R R wherein: - Step (i) is a peptidic coupling between fragments AD3 and alternative BC in the presence of coupling reagents; 5 - Step (ii) is a macrocyclization by ring closing metathesis in the presence of a catalyst; - Step (iii) is a deprotection of the p-methoxybenzyl ether in acidic conditions; - Step (iv) is an oxidation of the alcohol using an oxidizing agent; - Step (v) is an uction of the epoxide by asymmetric Corey-Chaykovsky on using appropriately substituted isothiocineole-derived chiral sulfonium in the presence of a base; 10 - Step (vi) is a ion of the azido group. 32. Process for preparing a conjugate of formula ( III) as defined in any one of claims 26 to 30 comprising the steps of: 20266725_1 (GHMatters) P44029NZ00 (i) placing in contact and leaving to react: - an optionally buffered aqueous solution of an antibody, optionally ed by means of a modifying agent, 5 - a solution of a phycin payload of formula (II) as defined in one of claims 17 to 25, the chemical group RCG1 of the cryptophycin payloadof formula (II) being reactive towards the chemical groups RCG2 present on the antibody especially towards the amino groups present on antibodies, the said chemical groups RCG2 having been introduced, where appropriate, by the ing agent, 10 so as to attach the cryptophycin payload of formula (II) to the dy by formation of a covalent bond; (ii) and then optionally to te the conjugate formed in step (i) from the cryptophycin payload of a (II) and/or from the unreacted antibody and/or from any aggregates that may have formed. 33. Medicament, characterized in that it comprises a conjugate of formula (III) according to any one of claims 26 to 30. 34. Pharmaceutical composition, characterized in that it comprises a conjugate of formula (III) 20 according to any one of claims 26 to 30, and also at least one pharmaceutically acceptable excipient. 35. Use of the conjugate of formula (III) ing to any one of claims 26 to 30 in the manufacture of an anticancer agent. 36. Use of the conjugate of formula (III) according to any one of claims 26 to 30 in the manufacture of a medicament for treating cancer. 20266725_1 (GHMatters) P44029NZ00 “wmmE m :1 ooommr Qvum u ”nytrxixL ooowmr 3.5.03 .a.1%.1t ooowmr T ooommr on x _ ooommv m0 w ,d.LarurI~:M4w.Trrkl Goovmr 3m ,. , #0 M IXVELLL. 60525 ooommr MD WWm l he ooommw ‘,M.Smr?rrl». Esbumnm NO mw coo E _,_ mme w \“w“.WAWLWLix?ru oooomw c2533.. $u M _ _ m ” m E 2:9”. SUBSTITUTE SHEET (RULE 26) u ooowmr EB 000nm? ooommw 000mm ‘P ooovm 3m :J. ooommr .3525 no hf, ooommv ho 000 m gr oooom mmmE r 20:23.2 Doom: 959“. SUBSTITUTE SHEET (RULE 26) wme‘rr?ri Ev ooommv m ooowme ooommr m0 ooommr m0 ? .8525 3 ooommw Sam m0 *0 ooowmr 53.58% ooo mmmE oooomr 5:232 959". SUBSTITUTE SHEET (RULE 26) WNW? ooowmr 3 salxkxw u ,«au WmT.(SEQ?) coonmv mad . ND _/u
3. ooowme «., ,Imw on ,_ 1r}, x‘ X ooommw 8 i, W 4“
NZ741734A 2016-11-03 Novel cryptophycin compounds and conjugates, their preparation and their therapeutic use NZ741734B2 (en)

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EP15306751 2015-11-05
PCT/EP2016/076603 WO2017076998A1 (en) 2015-11-05 2016-11-03 Novel cryptophycin compounds and conjugates, their preparation and their therapeutic use

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