EA045708B1 - NEW PEPTIDE LINKERS AND CONJUGATES BASED ON CRYPTOPHICIN, THEIR PREPARATION AND THERAPEUTIC APPLICATION - Google Patents

Description

The present invention relates to new peptide linkers, new cryptophycin payloads, new cryptophycin-based conjugates, compositions containing them, and their therapeutic uses, such as use as anticancer agents. The present invention also relates to a method for preparing such conjugates.

These linkers are enzymatically cleaved in cell lysosomes by enzymes such as cathepsin B. Cathepsin B is a cysteine proteinase that belongs to the papain family and is one of the main lysosomal proteinases in mammals. It is involved in protein metabolism and in the maintenance of cellular metabolism, as well as several other physiological or pathological processes, such as the progression of tumors. Its overexpression, both at the genetic and protein levels, has been demonstrated in tumors, with increased protein also leading to increased enzymatic activity. This has been the basis for several approaches involving the use of prodrugs of chemotherapeutic agents (Int J Oncology 2013, 42, 373-383), as well as the concept of using cleavable linkers to produce antibody-drug conjugates (ADCs) (Bioconj Chem 2002, 13, 855869 ). With respect to ADCs, one common peptide linker consists of a valine citrulline (ValCit) sequence linked to a self-cleaving aminobenzyl alcohol moiety (J Org Chem 2002, 67, 1866-1872), as illustrated in Example 23 in WO2011/001052. The construct resulting from binding to a cytotoxic drug is quite hydrophobic, which can be difficult to achieve certain drug to antibody ratios (DAR), monomeric purity and ADC stability. In fact, despite significant optimization of conjugation, example 23 of WO 2011/00152 cannot be successfully conjugated to produce ADCs with satisfactory DAR and monomer purity, namely DAR > 2 and > 95% monomers.

Since drugs used for ADCs, such as tubulin or DNA binding agents, are essentially hydrophobic, there is a need for more hydrophilic peptide linkers to improve the solubility of the payload, where the payload is a compound containing a cytotoxic drug conjugated to the linker, and thus potentially its reactivity with respect to antibody conjugation and the stability of the resulting ADC. Increasing payload solubility should allow for increased DAR, monomeric purity, and ADC stability, especially in terms of aggregation propensity.

Brief description of the invention

The present invention relates to new peptide compounds selected from the compounds of formula (I):

Where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

P represents a hydrogen atom, -OH or activated O, where activated O is defined below.

L is a linker of formula (II):

О ^L2^(AA)w-L1^ _(πχ where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J _b J ₂ , J ₃ and J ₄ are selected independently from CA ₁ and N;

ALK represents a (C1- _C12 )alkylene group, for example (C1- _C6 )alkylene, for example in the form -( _CH2 ) _n- , wherein n is an integer ranging from 1 to 12, and , for example, ranging from 1 to 6;

- 1 045708

A1, A2, _A3 , _A4 , _A5 and _A6 independently represent a hydrogen atom or a ( _C1C6 )alkyl group, for example a hydrogen atom or a methyl group.

(AA)w is a sequence of w substituted AAs or unsubstituted amino acids AA _ns joined together by peptide bonds, wherein the substituted AA _s or unsubstituted AA _ns are defined below;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA _s , then L2 represents _a single bond, (C1- _C6 )alkyl group, ( _C1 - _C6 )alkyl-( _OCH2CH2 ) _i group, ( C1-C ₆ )alkyl(OSH ₂ CH ₂ ) _i -O(C ₁ -C ₆ )alkyl group, (CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, CH(SO ₃ H)- (C1C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, (C1-C ₆ )alkylcyclohexyl group, C(=O)-(CiC ₆ )alkyl group, C(= 0)-(C ₁ -C ₆ )alkyl-(oCH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) ₁ -0(c ₁ C ₆ ) alkyl group, C(=0)-(CH ₂ CH ₂ 0) _i (C ₁ -C ₆ )alkyl group, C(=O)-CH(SO ₃ H)-(C ₁ -C ₆ )alkyl group, group C(=O)-(C ₁ -C ₆ )alkyl-CH(SO ₃ H), C(=O)-(C ₁ -C ₆ )alkylcyclohexyl group, NA ₈ -(C1C ₆ )alkyl group, group NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i -O(C ₁ C ₆ )alkyl group, NA ₈ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C(=O)-NA ₈ - (C ₁ -C ₆ )alkyl group, C(=0)-NA ₈ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i group, C(=O)-NA ₈ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -O(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA group ₇ , NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ C ₆ )alkyl-NA ₇ C(=O) -(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i , (C ₁ - C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl( OCH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group (0CH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=0)-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ , C(=0)-NA ₇ -(CH ₂ CH ₂ 0) _i (C ₁ -C ₆ ) alkyl group, C(=O)-NA ₇ aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-( C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ (C1-C ₆ )alkyl group, C(=0)-(C ₁ -C ₆ )alkyl-C(= 0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=0)-(C ₁ -C ₆ )alkyl- (0CH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C1C ₆ )alkyl-NA ₇ C( =O)-(C ₁ -C ₆ )alkyl group, NA ₈ group -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA group ₇ (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C (=O)-(C ₁ -C ₆ )alkyl group, C(=0)-NA8-(C1-C6)alkyl-NA7C(=0)-(C1-C6)alkyl-(0CH2CH2)i group, C (=O)-NA8-(C1-C6)alkyl-C(=O)NA7-(C1C ₆ )alkyl group, C(=o)-NA ₈ -(C ₁ -C ₆ )alkyl-C(= 0)NA ₇ -(C ₁ -C ₆ )alkyl-(oСН ₂ CH ₂ ) _b C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group , group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(0CH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

if (AA)w is a sequence of w unsubstituted amino acids AA _ns , then L2 represents an NA ₇ -(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-NA ₇ group, NA ₇ -( CH ₂ CH ₂ O) _i (C ₁ C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -group, (C ₁ -C ₆ )alkyl-C (=O)NA ₇ (C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i - group, (C ₁ -C ₆ )alkyl-NA ₇ (C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -group, (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -group, C(=O)-NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C (=O)-NA ₇ -aryl group, C(=O)-NA ₇ heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkylNA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i -group, C(=O) -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C( =O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(oСН ₂ CH ₂ ) _i -group, C(=0)-(C ₁ -C ₆ )alkyl- (0CH ₂ CH ₂ ) _i NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -group, NA ₈ -(C ₁ -C ₆ )alkylC(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )αalkyl-C(=O) NA ₇ -(C ₁ -C ₆ )αalkyl-(OSH ₂ CH ₂ ) _i -group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ - (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i group, NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i - NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkylNA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i -group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(oСН ₂ CH ₂ ) _i -group, C(=O) -NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ (C1-C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -group or C(=O)NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

A7 is straight or branched, saturated or unsaturated, optional

- 2 045708 but a substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NHC(=O)-, -N(alkyl)C(=O)-, -C(=O)NH- , -C(=O)N(alkyl)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -CH(SO ₃ H)-, -O-, -C(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O)2-, -N(alkyl)S( =O) ₂ -, -S(=O) ₂ NH-, -SO ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP (=O)(OH)-,

-OP(=O)(OH)-O- or a heterocycloalkyl group, optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH- alkyl and -N(alkyl) ₂ ;

it should be understood that each A ₇ containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na);

A8 represents a hydrogen atom or a (C1- _C6 )alkyl group, such as a hydrogen atom or a methyl group;

i is an integer ranging from 1 to 50, such as 1 to 35.

The present invention further relates to cryptophycin payloads of formula (IV):

Where

R1 represents a (C1- _C6 )alkyl group;

R2 and R3 independently represent a hydrogen atom or a (C1- _C6 )alkyl group;

or alternatively, R ₂ and R ₃ form, together with the carbon atom to which they are attached, a (C ₃ -C ₆ )cycloalkyl or (C ₃ -C ₆ )heterocycloalkyl group;

R ₄ and R ₅ independently represent a hydrogen atom, or a (C1-C ₆ )alkyl group, or a (C ₁ -C ₆ )alkyl-NH(R ₁₂ ) group, or a (C1-C ₆ )alkyl group -OH, or a (C ₁ -C ₆ )alkyl-SH group, or a (C1-C ₆ )alkyl-CO ₂ H group;

or alternatively, R4 and _R5 form, together with the carbon atom to which they are attached, a (C ₃ -C ₆ )cycloalkyl or (C ₃ -C ₆ )heterocycloalkyl group;

X represents O or N(R ₆ );

R ₆ represents a hydrogen atom or a (C1-C ₆ )alkyl group;

_R7 and R8 independently represent a hydrogen atom, or a (C1- _C6 )alkyl group, or a (C1- _C6 )alkyl- _CO2H group, or a ( _C1 - _C6 )alkyl-N( C ₁ -C ₆ )alkyl;

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 at least one substituent on the phenyl ring independently selected from hydrogen, -OH, (C1- _C4 )alkoxy, halogen, -NH2, -NH( _C1 - _C6 )alkylα, -N (C ₁ C ₆ )alkyl ₂ , -NH(C ₁ -C ₆ )cycloalkyl or (C ₃ -C ₆ )heterocycloalkyl;

R10 represents at least one substituent on the phenyl ring selected from a hydrogen atom and a (C1- _C4 )alkyl group;

Y is -NR ₁₁ -(C ₁ -C ₆ )alkyl-, for example -NR ₁₁ -(CH2) _n -, such as

RCG^O-j

-O-(C ₁ -C6)alkyl-, for example -O-(CH ₂ ) _p -, such as

-S-(C ₁ -C ₆ )αalkyl-, for example -S-(CH ₂ )n-, such as where Y is located in the ortho-(o), meta-(m) or para-(p) position of the phenyl kernels;

R11 and R12 independently represent a hydrogen atom or a (C1- _C6 )alkyl, for example a hydrogen atom or a methyl group;

n is an integer ranging from 1 to 6;

- 3 045708

L is defined in formula (I) and is a linker of formula (II);

RCG1 is a reactive chemical group present at the end of a linker, wherein RCG1 is reactive with a chemical group present on a polypeptide such as an antibody.

The present invention further relates to conjugates of formula (V):

Where

R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and _Rn are defined in formula (IV);

X, Y and L are defined in formula (IV);

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

Ab is an antibody.

Each of the substituents R1-R12 may also assume one of the spatial configurations (eg, R or S, or alternatively Z or E) as described in the examples.

The compounds of formula (I), (II), (III), (IV) and (V) may contain at least one asymmetric carbon atom. Therefore, they can exist in the form of enantiomers or diastereoisomers. These enantiomers and diastereoisomers, as well as mixtures thereof, including racemic mixtures, form part of the present invention.

The compounds of formula (I), (II), (IV), including those illustrated, may exist in the form of bases or acid addition salts, for example pharmaceutically acceptable acids. For example, if these compounds contain the SO3H functional group, they may exist in the form of SO3- and alkali metal salts, such as SO3- and sodium (SO ₃ - ⁺ Na) salts.

Definitions.

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

Alkenyl Group: A hydrocarbon group obtained by removing one hydrogen atom from an alkene. The alkenyl group may be straight or branched. Examples that may be mentioned include ethenyl (-CH=CH ₂ , also called vinyl) and propenyl (-CH ₂ -CH=CH ₂ , also called allyl).

Alkoxy group: an -O-alkyl group in which the alkyl group is defined below.

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

Alkylene group: A saturated divalent group with the empirical formula -CnH2n-, obtained by removing two hydrogen atoms from an alkane. The alkylene group may be straight or branched. Examples that may be mentioned include methylene ( _-CH2- ), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-) and hexylene (-CH2CH2CH2CH2CH2CH2-) groups or the following branched groups

''' for example, an alkylene group represented by the formula -(CH2) _P- , where n is an integer; value ranges include limits (for example, a range of type n that is in the range from 1 to 6 or that is in the range from 1 to 6 includes limits 1 and 6).

Antibody: An antibody with affinity for a biological target, such as a monoclonal antibody. The function of an antibody is to direct a biologically active compound as a cytotoxic compound to a biological target. The antibody may be monoclonal, polyclonal or polyspecific; it may also be an antibody fragment; it may also be a murine, chimeric, humanized or human antibody. The antibody may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond (also called a full-length antibody). Terms are traditional

- 4 045708 new (or full-length) antibody refers to both the antibody containing the signal peptide (or propeptide, if any) and the mature form obtained by secretion and proteolytic processing of the chain(s). There are two types of light chains, lambda (l) and kappa (k). There are five main classes (or isotypes) of heavy chains that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains domains with a different sequence. The light chain contains two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, a variable domain (VH) and three constant domains (CH1, CH2 and _CH3 , collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and antigen specificity. The light (CL) and heavy (CH) chain constant region domains confer important biological properties such as antibody chain association, secretion, placental translocation, complement fixation, and FcR binding. The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of variable parts of one light chain and one heavy chain. Antibody specificity lies in the structural complementarity between the antibody paratope and the antigenic determinant. Antibody paratopes are formed by residues primarily from hypervariable or complementarity determining regions (CDRs). Residues from non-hypervariable or framework regions (FRs) sometimes influence the overall structure of the domain and hence the paratope. CDRs refer to amino acid sequences that together determine the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. Each of the immunoglobulin light and heavy chains has three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2H, CDR3-H, respectively. Therefore, the antigen binding region of a conventional antibody contains six CDRs, which include a set of CDRs from each of the heavy and light chain V regions.

As used herein, the term antibody refers to both conventional (full-length) antibodies and fragments thereof, as well as single-domain antibodies and fragments thereof, such as the heavy chain variable region of single-domain antibodies. (Conventional) antibody fragments typically contain a portion of the intact antibody, such as the antigen binding region or variable region of the intact antibody, and retain the biological function of the conventional antibody. Examples of such fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, nanobodies and diabodies.

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

Aryl group: A cyclic aromatic group containing 5 to 10 carbon atoms. Examples of aryl groups include phenyl, tolyl, xylyl, naphthyl.

Biological target: an antigen (or group of antigens), for example, located on the surface of cancer cells or stromal cells associated with a given tumor; such antigens may be, for example, a growth factor receptor, an oncogene product, or a mutated tumor suppressor gene product, an angiogenesis-related molecule, or an adhesion molecule.

Conjugate: Antibody-drug conjugate or ADC, i.e. a polypeptide, such as an antibody, to which at least one molecule of a cytotoxic compound is covalently attached via a linker.

Cycloalkyl group: A cyclic alkyl group containing 3 to 6 carbon atoms included in a cyclic structure. Examples that may be mentioned include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups.

DAR (Drug to Antibody Ratio): The average number of cytotoxic molecules attached via a linker to an antibody.

Halogen: Any of the four elements being fluorine, chlorine, bromine and iodine.

Heteroaryl group: An aryl group containing 2 to 10 carbon atoms and 1 to 5 heteroatoms, such as nitrogen, oxygen or sulfur, included in a ring and attached to the carbon atoms to form a ring. Examples of heteroaryl groups include pyridyl, pyrimidyl, thienyl, imidazolyl, triazolyl, indolyl, imidazopyridyl, pyrazolyl.

Heterocycloalkyl group: A cycloalkyl group containing 2 to 8 carbon atoms and 1 to 3 heteroatoms, such as nitrogen, oxygen or sulfur, included in a ring and attached to the carbon atoms to form a ring. Examples include aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, azetidinyl, oxetanyl and pyranyl.

Linker: A group of atoms or single bond that can covalently attach a cytotoxic compound to a polypeptide, such as an antibody, to form a conjugate.

Payload: A cytotoxic compound to which a linker is covalently attached.

Reactive Chemical Group: A group of atoms that can promote or participate in a chemical reaction.

In addition, as indicated throughout the present invention, the SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

- 5 045708

Abbreviations.

ADC: antibody-drug conjugate; ALK: (C1-C ₁₂ )alkylene group, for example (Cp C6)alkylene, for example, in the form -(CH ₂ ) _n -, wherein n is an integer ranging from 1 to 12, and, for example, ranging from 1 to 6; aq.: aquatic; Ar: argon; AUC: area under the curve; BCN: (1a,8a,9c)-bicyclo[6.1.0]non-4-yne-9-methanol; CHCl3: chloroform; CH3CN: acetonitrile; CO ₂ : carbon dioxide; CR: full answer; crypto stands for formula unit

where crypto means, for example, the cryptophycin compound of the example, one of the cryptophycin compounds D ₁ -D ₈ described in WO2011/001052, or one of the following cryptophycin compounds D1-D1 ₉ described in PCT/EP2016/076603:

- 6 045708

- 7 045708 where W represents (C ₁ -C ₆ )alkyl-NH(R ₁₁ ), for example (CH2) _n NHR11;

(C ₁ -C ₆ )alkyl-OH, for example (CH2) _n OH;

(C ₁ -C ₆ )alkyl-SH, for example (CH2) _n SH;

CO2H or C(=O)NH2;

(C1- _C6 )alkyl- _CO2H or ( _C1 - _C6 )alkyl-C(=O) _NH2 or ( _C1 - _C6 )alkyl- _N3 .

W is located in the ortho-(o), meta-(m) or para-(p) position of the phenyl ring;

R11 independently represents a hydrogen atom or a (C1- _C6 )alkyl group, for example a hydrogen atom or a methyl group;

n is an integer ranging from 1 to 6;

d: day; DAR: drug to antibody ratio (D1 refers to an ADC with a DAR of 1, D2 refers to an ADC with a DAR of 2, etc.); DBCO: dibenzylcyclooctyne; DCC: N,N'-dicyclohexylcarbodiimide; DCM: dichloromethane; DIEA: N,N-diisopropylethylamine; DMA: dimethylacetamide; DMAP: 4-(dimethylamino)pyridine; DMEM: Dulbecco's modified Eagle's medium; DMEM/F12: Dulbecco's modified medium and nutrient mixture F-12; DMF: dimethylformamide; DMSO: dimethyl sulfoxide; DPBS: Dulbecco's phosphate buffered saline; DSC: N, N'-disuccinimidyl carbonate; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; EDTA: ethylenediaminetetraacetic acid; EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; ELSD: Evaporative Light Scattering Detector; eq: equivalent; ES: electrospray; AtOAc: ethyl acetate; Et ₂ O: diethyl ether; note: example; FCS: fetal bovine serum; Fmoc: 9fluorenylmethoxycarbonyl; GI: electroinduction group; h: hour; H2O: water; Hal: halogen; HATU: 1[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate; HCl: hydrochloric acid; HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; HIC: hydrophobic interaction chromatography; HOAt: 1-hydroxy-7-azabenzotriazole; HOBt: 1hydroxybenzotriazole; HPLC: high performance liquid chromatography; HRMS: High Resolution Mass Spectrometry; IC ₅₀ : average inhibitory concentration; i.e.: id est, which means that is; IEC: ion exchange chromatography; iPrOH: 2-propanol; iPr ₂ O: diisopropyl ether; iv: intravenously; MeCN: acetonitrile; MeOH: methanol; MeTHF: 2-methyltetrahydrofuran; MFCO: 1-fluoro-2-cyclooctyne-1-carboxylic acid; MgSO ₄ : magnesium sulfate; min: minute; MsCl: methanesulfonyl chloride; MTBE: methyl tert-butyl ether; MTD: maximum tolerated dose; NaH: sodium hydride; NaCl: sodium chloride; NaHCO ₃ : sodium hydrogen carbonate; n.a.: not defined; NHS: Nhydroxysuccinimide; NMP: 1-methyl-2-pyrrolidone; NMR: nuclear magnetic resonance; PABA: para-aminobenzyl alcohol; PBS: phosphate buffered saline; PEG: polyethylene glycol; PNGase F: peptide N-glycosidase F; ppm: parts per million; PR: partial response; QS: quantum satis, which means quantity required; Q-Tof: quadrupole time-of-flight; quant: quantitative yield; RCG: reactive chemical group; RT: room temperature; saturated: saturated; sc: subcutaneous; SCID: severe combined immunodeficiency; SEC: size exclusion chromatography; T3P: propylphosphonic anhydride; TBAF: tetrabutylammonium fluoride; TFA: trifluoroacetic acid; TFS: tumor free survivor; THF: tetrahydrofuran; TLC: thin layer chromatography; t _1/2 : half-life; t _R : retention time; UPLC: ultra performance liquid chromatography; UV: ultraviolet radiation.

Figures

Fig. 1 - in vivo effectiveness approx. 6 for the MDA-MB-231 xenograft in SCID mice.

Fig. 2 - in vivo effectiveness approx. 16, approx. 19, approx. 23 and approx. 32 against the MDA-MB-231 xenograft in SCID mice at 2.5 mg/kg.

Fig. 3 - in vivo effectiveness approx. 16, approx. 19, approx. 23 and approx. 32 against the MDA-MB-231 xenograft in SCID mice at 1.25 mg/kg.

Fig. 4 - in vivo effectiveness approx. 26 for the MDA-MB-231 xenograft in SCID mice.

Fig. 5 - in vivo effectiveness approx. 29 for the MDA-MB-231 xenograft in SCID mice.

Fig. 6 - in vivo effectiveness approx. 35 for the MDA-MB-231 xenograft in SCID mice.

Fig. 7 - in vivo effectiveness approx. 41 for the MDA-MB-231 xenograft in SCID mice.

- 8 045708

Detailed Description of the Invention

The present invention relates to new peptide compounds selected from the compounds of formula (I):

Where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

L is a linker of formula (II) as defined below;

P represents a hydrogen atom, -OH or activated O.

Examples of activated O that may be mentioned include

where the cation is, for example, sodium, potassium or cesium;

or

group

wherein GI represents at least one electroinductive group such as NO2 or a halogen atom (-Hal), for example a fluorine atom (-F). They may be, for example, the following groups:

or

Linker L, which is present in formula (I), is represented by formula (II):

ABOUT

L2 (AA)W LI ί _(P) where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J1, _J2 , J3 and _J4 are independently selected from CA1 and N;

ALK represents a (C ₁ -C ₁₂ )alkylene group, for example (C1 -C6)alkylene, for example in the form -(CH ₂ ) _n -, wherein n is an integer ranging from 1 to 12, and , for example, ranging from 1 to 6;

A1, _A2 , A3, _A4 , _A5 and _A6 independently represent a hydrogen atom or a ( _C1C6 )alkyl group, for example a hydrogen atom or a methyl group.

- 9 045708 (AA)w is a sequence of w substituted AAAs or unsubstituted amino acids AA _ns joined together by peptide bonds, wherein the substituted AAAs or unsubstituted AA _ns are defined below;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AAs, _then L2 represents a single bond, (C1- _C6 )alkyl group, ( _C1 - _C6 )alkyl-( _OCH2CH2 ) _i group, (C1 -C ₆ )alkyl(OSH ₂ CH ₂ ) _i -O(C ₁ -C ₆ )alkyl group, (CН ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, CH(SO ₃ H)-( C ₁ C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, (C1-C ₆ )alkylcyclohexyl group, C(=O)-(CiC ₆ )alkyl group, C( =0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i -0(c ₁ C ₆ )alkyl group, C(=0)-(CH ₂ CH ₂ 0) _i (C ₁ -C ₆ )alkyl group, C(=O)-CH(SO ₃ H)-(C ₁ -C ₆ )alkyl group , C(=O)-(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C(=O)-(C ₁ -C ₆ )alkylcyclohexyl group, NA ₈ -(C1C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i group, NA ₈ -(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i -O(C ₁ C ₆ )alkyl group , NA ₈ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), C(=O)-NA ₈ group -(C ₁ -C ₆ )alkyl group, C(=0)-NA ₈ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i group, C(=O)-NA ₈ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -O(C ₁ -C ₆ )alkyl group, C(=0)-NA ₈ -(CH ₂ CH ₂ 0) _i (C ₁ -C ₆ )alkyl group , group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), NA ₇ -(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl- NA ₇ , NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ C ₆ )alkyl-NA ₇ C(=O )-(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group (OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ ) group alkyl(OCH ₂ CH ₂ ) _b (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=0)-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ ) alkyl group, C(=O)-NA ₇ aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-( C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ (C1-C ₆ )alkyl group, C(=0)-(C ₁ -C ₆ )alkyl-C(= 0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=0)-(C ₁ -C ₆ )alkyl- (0CH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C1C ₆ )alkyl-NA ₇ C( =O)-(C ₁ -C ₆ )alkyl group, NA ₈ group -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA group ₇ (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C (=O)-(C ₁ -C ₆ )alkyl group, C(=0)-NA8-(C1-C6)alkyl-NA7C(=0)-(C1-C6)alkyl-(0CH2CH2)i group, C (=O)-NA8-(C1-C6)alkyl-C(=O)NA7-(C1C ₆ )alkyl group, C(=o)-NA ₈ -(C ₁ -C ₆ )alkyl-C(= 0)NA ₇ -(C ₁ -C ₆ )alkyl-(oСН ₂ CH ₂ ) _b C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group , group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(0CH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

if (AA)w is a sequence of w unsubstituted amino acids AA _ns , then L2 is

NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ group, NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₇ - aryl group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ C( =0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, group ( C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA group ₇ , C(=O)NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ ) alkyl group, C(=O)-NA ₇ -aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl group -NA ₇ C(=0)(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-(CiC ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O )-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ ) group alkyl-(0CH ₂ CH ₂ ) _i , C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ - group (C ₁ -C ₆ )alkyl-NA ₇ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -) group C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ - (C ₁ C ₆ )alkyl group, NA ₈ group -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ - (C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH _{2) group} ) _i , NA ₈ -(C1C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ ) group alkyl-NA ₇ , C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ - group (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl -C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C) group ₆ )alkyl-(oСН ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)- group NA8-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl(0cH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

A7 is a straight or branched, saturated or unsaturated, optionally substituted C ₁ -C ₁₆ hydrocarbon chain, optionally with at least one methylene unit

- 10 045708 but independently replaced by -NHCO-, -N(alkyl)C(=O)-, -C(=O)NH-, -C(=O)N(alkyl)-, -OC(=O)- , -C(=O)O-, -OC(=O)O-, -CH(OH)-, -CH(SO3H)-, -O-, -C(=O)-, -S(=O )-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O)2NH-, -S(=O)2N (alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O - or a heterocycloalkyl group, optionally substituted by at least one substituent, identical or different, selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -N(alkyl) ₂ ; it should be understood that each A ₇ containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO3 ^-+ Na).

A8 represents a hydrogen atom or a (C1-C6)alkyl group, for example a hydrogen atom or a methyl group;

i is an integer ranging from 1 to 50, such as 1 to 35.

In accordance with a specific embodiment, the present invention relates to compounds of formula (I):

RCG1-LP _(I) where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

P represents a hydrogen atom, -OH or activated O;

L is a linker of formula (II):

O d L2 (AA)w L1 _(πχ where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J _b J ₂ , J3 and J ₄ are selected independently from CA1 and N;

ALK represents a (C1- _C12 )alkylene group, for example (C1-C6)alkylene, for example in the form -( _CH2 ) _n- , wherein n is an integer ranging from 1 to 12, and, for example, ranging from 1 to 6;

A1, _A2 , A3, _A4 , _A5 and _A6 independently represent a hydrogen atom or a (C1C6)alkyl group, for example a hydrogen atom or a methyl group.

(AA)w is a sequence of w substituted AA _s or unsubstituted amino acids AA _ns joined together by peptide bonds;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA _s , then L2 represents _a single bond, (C1- _C6 )alkyl group, ( _C1 - _C6 )alkyl-( _OCH2CH2 ) _i group, ( C1-C ₆ )alkyl(OSH ₂ CH ₂ ) _i -O(C ₁ -C ₆ )alkyl group, (CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, CH(SO ₃ H)- (C1C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, (C1-C ₆ )alkylcyclohexyl group, C(=O)-(C1C ₆ )alkyl group, C(= O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -O(C ₁ C ₆ ) alkyl group, C(=O)-(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-CH(SO ₃ H)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C(=O)-(C ₁ -C ₆ )alkylcyclohexyl group, NA8-(C1C ₆ )alkyl group, NA group ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -O(C ₁ C ₆ )alkyl group, NA ₈ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), C(=O)-NA8-(C1 -C6)alkyl group, C(=O)-NA8-(C1-C6)alkyl-(OCH2CH2)i group, C(=O)-NA8-(CiC ₆ )alkyl-(OCH ₂ CH ₂ ) _i -O (C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA _{7 group, NA 7 -} (CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C1C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl group-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl(OCH ₂ CH ₂ )i, (C ₁ -C ₆ )alkyl -NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl group-NA ₇ -(C ₁ -C ₆ )alkyl

- 11 045708 (OSH ₂ CH ₂ ) _b (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -( C|-C ₆ )αalkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ group, C(=0)-NA ₇ -(CH ₂ CH ₂ 0) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O) -(C ₁ -C ₆ )alkyl group, C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) group ₁ , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ (C1-C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C group (=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group , group C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=0)-(C ₁ -C ₆ ) alkyl-(0CH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C _{6 )} alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA 7 group, NA ₈ -(C ₁ C ₆ )alkyl- NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl group-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C( =O)NA ₇ (C ₁ -Cb)alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ - group (C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ - (C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl- NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-NA8-(C1-C6)αalkyl-NA7C(=O)-(C1-C6)alkyl-(OCH2CH2) group i, C(=O)-NA8-(C1-C6)alkyl-C(=O)NA7-(C1C ₆ )alkyl group, C(=o)-NA ₈ -(C ₁ -C ₆ )alkyl group C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(oCH ₂ CH ₂ ) ₁ , C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(oCH ₂ CH ₂ ) ₁ or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group;

if (AA)w is a sequence of w unsubstituted amino acids AA _ns , then L2 represents an NA ₇ -(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-NA ₇ group, NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ ) alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(0CН ₂ CH ₂ ) _group , (C ₁ -C ₆ )alkyl-C(= O)NA ₇ (C1-C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _{1 group} , (C ₁ -C ₆ )alkyl-NA ₇ (C1-C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group, (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(= O)-(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -aryl group, C(=O)-NA ₇ heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C group (=O)-(C ₁ -C ₆ )αalkylNA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CН ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ ) αlkyl-C(=O)NA ₇ -(C ₁ -C ₆ )αalkyl group, C(=O)(C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ ) group alkyl-(0CH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C) group ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , c(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ - (C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ - C ₆ )alkyl group, NA ₈ -(C ₁ C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group, NA ₈ -(C ₁ - C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ ) group )alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA group ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)- (C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(oCН ₂ CH) group ₂ ) ₁ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ C ₆ )alkyl group, C(=O)-NA ₈ -( group C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) ₁ , C(=O)-NA ₈ -(C ₁ C ₆ )alkyl- NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group;

A ₇ is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NHC(=O)-, -N(alkyl)C(=O)- , -C(=O)NH-, -C(=O)N(alkyl)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH (OH)-, -CH(SO ₃ H)-, -O-, -C(=O)-, -S(=O)-, -S(=O)2-, -NHS(=O)2 -, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, - P(=O)(OH)O-, -O-P(=O)(OH)-, -O-P(=O)(OH)-O- or a heterocycloalkyl group optionally substituted by at least one substituent, identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -M(alkyl) ₂ , it being understood that each _A7 containing a SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na);

A ₈ represents a hydrogen atom or a (C1-C ₆ )alkyl group, for example a hydrogen atom or a methyl group;

I represents an integer ranging from 1 to 50, such as from 1 to 35;

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

According to another specific embodiment, the present invention relates to

- 12 045708 to compounds of formula (I):

Where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

P represents a hydrogen atom, -OH or activated O;

L is a linker of formula (II):

О ^L2^(AA)W-L1+ _(πχ where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J1, J ₂ , J3 and J ₄ are selected independently from CA ₁ and N;

ALK represents a (C1- _C12 )alkylene group, for example (C1-C6)alkylene, for example in the form -( _CH2 ) _P- , wherein n is an integer ranging from 1 to 12, and, for example, ranging from 1 to 6;

A1, _A2 , A3, _A4 , _A5 and _A6 independently represent a hydrogen atom or a ( _C1C6 )alkyl group, such as a hydrogen atom or a methyl group.

(AA)w is a sequence of w substituted AA _s or unsubstituted amino acids AA _ns joined together by peptide bonds;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA _s , _then L2 represents a single bond, (C1- _C6 )alkyl group, ( _C1 - _C6 )alkyl-( _OCH2CH2 ) ₁ group, ( C1-C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ -O(C ₁ -C ₆ )alkyl group, (CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, CH(SO ₃ H)- (C ₁ C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, (C1-C ₆ )alkylcyclohexyl group, C(=O)-(C1C ₆ )alkyl group, C group (=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O(C ₁ C ₆ )alkyl group, C(=O)-( _CH2CH2O ) ₁ ( _{C1 - C6} )alkyl group, C(=O)-CH( _8O3H )-( _C1 - _C6 )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C(=O)-(C1-C ₆ )alkylcyclohexyl group, NA ₈ -(C ₁ C ₆ )alkyl group group, group NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O(C ₁ C ₆ ) alkyl group, NA ₈ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), C(=O)- group NA ₈ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ group, C(=O)-NA ₈ -( C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ ) alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, NA7-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl group -NA7, NA7-(CH ₂ CH ₂ O) ₁ (-C ₁ C ₆ )alkyl group, NA7-aryl group, NA7-heteroaryl group, (C1C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ group, (C ₁ -C ₆ )alkyl-C(= O)NA7-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA7-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _{1 group} , (C ₁ -C ₆ )alkyl-NA7-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA7-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ group, (C ₁ - C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA7-(C ₁ -C ₆ )alkyl group, C(=O)-NA7-(C ₁ -C ₆ )alkyl group, C(=O)- group (C ₁ -C ₆ )alkyl-NA77, C(=O)-NA7-(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-NA7aryl group, C(=O )-NA7-heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-(C1-C _b ) group )alk _I l-NA7C(=O)-(C1-C _b )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-(C1-C6)alkyl-C(=O)NA7(C1-C ₆ )alkyl group, group C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA7-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O) -(C1C ₆ )alkyl-NA7-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA7-(C ₁ -C ₆ )alkyl-(OSH ₂ CH) group ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA7-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ ) group alkyl-NA7, NA ₈ -(C ₁ C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA7C(=O)- group (C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA7-(C ₁ -C ₆ )alkyl group, NA ₈ -( group C ₁ -C ₆ )alkyl-C(=O)NA7(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-NA7-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkylNA7-(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _{1 group} , NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA7-(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA7 group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl group, group

- 13 045708

C(=O)-NA ₈ -(C ₁ -C ₆ )αlkyl-NA ₇ C(=O)-(C ₁ -C ₆ )αlkyl-(OSH ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ C ₆ )alkyl group, C(=O)-NA group ₈ -(C ₁ -C ₆ )alkyl-C(= O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(OCH ₂ CH ₂ )i or C(=O)-NA ₈ -( C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group;

if (AA)w is a sequence of w unsubstituted amino acids AAns, then L2 represents an NA ₇ -(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-NA ₇ group, NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) ₁ , (C ₁ -C ₆ )alkyl-C(=O )NA ₇ (C1-C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _{1 group} , (C ₁ -C ₆ )alkyl-NA ₇ (C1-C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group, (C1- C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O) group -(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -aryl group, C(=O)-NA ₇ heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(= O)-(C ₁ C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl -C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-(C ₁ -C ₆ )αлkul-C(=O)NA ₇ -(C ₁ -C ₆ ) αlkyl-(OSH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)(C1-C ₆ )alkyl group, group NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ C ₆ )alkyl -C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl group-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl(OSN ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, group C(= O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkylNA7C(=O)-(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA8-(C1C ₆ )alkyl-C(=0)NA _{7 group} -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) ₁ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-8-NA(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group;

A ₇ is a straight or branched, saturated or unsaturated, optionally substituted C ₁ -C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NHC(=O)-, -N(αalkyl)C(=O) -, -C(=O)NH-, -C(=O)N(alkyl)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, - CH(OH)-, -CH(SO ₃ H)-, -O-, -C(=O)-, -S(=O)-, -S(=O)2-, -NHS(=O) 2-, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group optionally substituted by at least one substituent identical or different , selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -N(alkyl) ₂ , it should be understood that each A ₇ containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na);

A8 represents a hydrogen atom or a (C1- _C6 )alkyl group, for example a hydrogen atom or a methyl group;

is an integer ranging from 1 to 50, such as from 1 to 35, wherein said substituted amino acids AAs are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent (C1-C ₈ )alkyl group, preferably T represents

or

group U represents a single bond, -NHC(=O)-, -N(alkyl)C(=O)-, -C(=O)NH-,

- 14 045708

-C(=O)N(αalkyl)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, -OC( =O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C(=O)-, -OP(=O)-, -S(=O )-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, ^(=OZhalkyl)- , -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)- or -OP(=O)(OH)-O, for example, group U represents -NH-C(=O)- or -C(=O)NH-;

A9 is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(αalkyl)C(=O) -, -C(=O)NH-, -C(=O)N(alkyl)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)-, - C(=O)O-, -OC(=O)O-, -CH(OH)-, -CH(SO3H)-, -CH(O-alkyl)-, -CHF-, -CF2-, -S -, -Se-, -O-, -NH-, -N(alkyl)-, -N ⁺ H(alkyl)-, -N ⁺ (alkyl) ₂ -, -C(=O)-, -OP( =O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH) , -OP(=O)(OH)-O- or a heterocycloalkyl group, optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH- alkyl and -N(alkyl) ₂ , for example, A9

ΗΟ^γ^ΟΗ it represents -[(CH ₂ ) ₂ -O]4-CH ₃ , -[(CH2)2-O]24-CH3, '

O Λ ° ⁿ he o γ, ^{OH He} , ^He or -[(CH2)2-O)]4-(CH2)2-C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A9 containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

According to another specific embodiment, the present invention provides compounds of formula (I):

RCG1-LP _(I) where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

P represents a hydrogen atom, -OH or activated O;

L is a linker of formula (II):

ABOUT

L2 ^Н (AA)w _(πχ where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J1, _J2 , J3 and _J4 are independently selected from CA1 and N;

ALK represents a (C1- _C12 )alkylene group, for example (C1- _C6 )alkylene, for example in the form -( _CH2 ) _n- , wherein n is an integer ranging from 1 to 12, and , for example, ranging from 1 to 6;

A _b A ₂ , A ₃ , A ₄ , A ₅ and A ₆ independently represent a hydrogen atom or a (C1C ₆ )alkyl group, for example a hydrogen atom or a methyl group.

(AA)w is a sequence of w substituted AA _s or unsubstituted amino

- 15 045708 AA _ns acids linked together by peptide bonds;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AAs, then L2 represents _a single bond, (C1- _C6 )alkyl group, ( _C1 - _C6 )alkyl-( _OCH2CH2 ) _i group, (C1 -C ₆ )alkyl(OSH ₂ CH ₂ ) _i -O(C ₁ -C ₆ )alkyl group, (CН ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, CH(SO ₃ H)-( C1C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, (C1-C ₆ )alkylcyclohexyl group, C(=O)-(CiC ₆ )alkyl group, C(=0) group )-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i -0(c ₁ C ₆ )alkyl group, C(=0)-(CH ₂ CH ₂ 0) _i (C ₁ -C ₆ )alkyl group, C(=O)-CH(SO ₃ H)-(C ₁ -C ₆ )alkyl group, group C(=O)-(C ₁ -C ₆ )alkyl-CH(SO ₃ H), C(=O)-(C ₁ -C ₆ )alkylcyclohexyl group, NA ₈ -(C1C ₆ )alkyl group, NA group ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i -O(C ₁ C ₆ )alkyl group, NA ₈ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), C(=O)-NA ₈ -( C ₁ -C ₆ )alkyl group, C(=0)-NA ₈ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i group, C(=O)-NA ₈ -(C1C ₆ )alkyl -(OSH ₂ CH ₂ ) _i -O(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C( group =O)-NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA7 group, NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (Cp C ₆ )alkyl-NA ₇ C(=O)-(C ₁ - C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C( =O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(0CH ₂ CH ₂ ) _{group i} , (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=0)-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA7, C(=0)-NA ₇ -(CH ₂ CH ₂ 0) _i (C ₁ -C ₆ )alkyl group, C(=O) -NA ₇ aryl group, C(=O)-NA ₇ heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group , group C(=0)-(C ₁ -C ₆ )alkyl-NA7C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ - C ₆ )alkyl-C(=O)NA ₇ (C1-C ₆ )alkyl group, C(=0)-(C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C) group ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(CiC ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i -NA ₇ -( C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C ₁ C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, group NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ (C ₁ -C ₆ )alkyl group (OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, group C( =O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=0)-NA8-(C1-C6)alkyl-NA7C(=0)-(C1-C6)alkyl-(0CH2CH2)i group, C(=O)-NA8-(C1- C6)alkyl-C(=O)NA7-(C1C ₆ )alkyl group, C(=o)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C) group ₆ )alkyl-(oСН ₂ CH ₂ ) _i , (=OC)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(0CH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

if (AA)w is a sequence of w unsubstituted amino acids AAns, then L2 represents an NA ₇ -(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-NA ₇ group, NA ₇ -(CH ₂ CH ₂ O)i(C ₁ C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(0СН ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O )NA ₇ (C1-C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _{i group} , (C ₁ -C ₆ )alkyl-NA ₇ (C1-C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O) group )-(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ - aryl group, C(=O)-NA ₇ heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C( group =O)-(C1C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=o)-(C ₁ -C ₆ )alkyl- C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group -(oСН ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ ) alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)(C1-C ₆ )alkyl group , group NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ C ₆ )alkyl- C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl(OSH _{2) group} CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C) group ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(oCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O )-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ C ₆ )alkyl-NA ₇ C(=O)-(c ₁ -C ₆ )alkyl-(oСН ₂ CH ₂ ) _i , C(=0) -NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl group C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(oСН ₂ CH ₂ ) _i , c(=O)-NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

A7 is a straight or branched, saturated or unsaturated, optionally substituted C ₁ -C ₁₆ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NHC(=O)-, -N(alkyl)C(=O)- , -C(=O)NH-, -C(=O)N(alkyl)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH (OH)-, -CHCSOjH)-, -о-, -C(=O)-, -S(=O)-, -S(=O)2-, -NHS(=O)2-, -N (alkyl)S(=O)2-, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O) )(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O- or hetero- 16 045708 cycloalkyl group, optionally substituted by at least one substituent, identical or different, selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -N(alkyl) ₂ , it being understood that each A ₇ containing the SO3H functional group may be in the forms salts such as alkali metal salts, for example sodium salts (SO3 ^-+ Na);

A8 represents a hydrogen atom or a (C1-C6)alkyl group, for example a hydrogen atom or a methyl group;

i represents an integer ranging from 1 to 50, such as from 1 to 35, wherein said substituted amino acids AAs are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent (C1-C ₈ )alkyl group, preferably T represents “ ' or ' group U represents a single bond, -NHC(=O)-, -N(αalkyl)C( =O)-, -C(=O)NH-C(=O)N(alkyl)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C(=O)-, - OR(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O)2-, -N(alkyl)S(=O) ₂ -, -S(= O)2NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH) - or -OP(=O)(OH)-O, for example the group U is -NH-C(=O)- or -C(=O)NH-;

A9 is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(alkyl)C(=O) -, -C(=O)NH-, -C(=O)N(alkyl)-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, - OC(=O)O-, -CH(OH)-, -CH(SO ₃ H)-, -CH(O-alkyl)-, -CHF-, -CF2-, -S-, -Se-, - O-, -NH-, -N(αalkyl)-, -N ⁺ H(alkyl)-, -N ⁺ (alkyl) ₂ -, -C(=O)-, -OP(=O)-, -S (=O)-, -S(=O) ₂ -, -NHS(=O)2-, N(alkyl)S(=O)2-, -S(=O)2NH-, -S(=O) )2N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH) -O- or heterocycloalkyl group, optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom, NH2, -NH-alkyl and

-N(alkyl) ₂ , for example, A9 is -[(CH ₂ ) ₂ -O]4-CH ₃ , -[(CH ₂ ) ₂ -O] ₂₄ -CH ₃ , ' k θ θ Jk OH t T OH OH

HO^y^OH lu^on he he he or -[(CH ₂ ) ₂ -O)] ₄ -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H; ' it should be understood that each A ₉ containing the SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

According to another specific embodiment, the present invention provides compounds of formula (I):

RCG1-LP _(I) where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

P represents a hydrogen atom, -OH or activated O;

L is a linker of formula (II):

O (M)w LI

- 17 045708 where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J1, J2, J3 and J ₄ are independently selected from CA ₁ and N;

ALK represents a (C1- _C12 )alkylene group, for example (C1-C6)alkylene, for example in the form -( _CH2 ) _n- , wherein n is an integer ranging from 1 to 12, and, for example, ranging from 1 to 6;

A ₁ , A ₂ , A3 , A ₄ , A5 and A ₆ independently represent a hydrogen atom or a (C ₁ C ₆ )alkyl group, for example a hydrogen atom or a methyl group.

(AA)w is a sequence of w substituted AAs or unsubstituted amino acids AA _ns joined together by peptide bonds;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AAs, then L2 represents a single bond, a (C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group, ( C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ -O(C ₁ -C ₆ )alkyl group, (CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, CH(SO ₃ H) -(C ₁ C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, (C ₁ -C ₆ )alkylcyclohexyl group, C(=O)-(C ₁ C ₆ )alkyl group, group C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O (C ₁ C ₆ )alkyl group, C(=O)-(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-CH(SO ₃ H)-(C ₁ - C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C(=O)-(C ₁ -C ₆ )alkylcyclohexyl group, NA ₈ -(C ₁ C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ group, NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O( C ₁ C ₆ )alkyl group, NA ₈ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ ) alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C (=O)-NA ₈ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ group, C(=O) -NA ₈ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ , NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ C ₆ )alkyl -NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) group ₁ , (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(OCH ₂ CH ₂ ) ₁ , (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)- NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA7 group, C(=O)-NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C( =O)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH _{2) group} CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ (C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ ) group )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA 7 group, NA _{8 -} (C ₁ C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ ) group alkyl(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ ) group alkyl-C(=O)NA ₇ (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group , group NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ - C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )αalkyl-NA ₇ C(=O)- group (C ₁ -C ₆ )αalkyl-(OSH ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O )-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ ) an alkyl group;

if (AA)w is a sequence of w unsubstituted amino acids AA _ns , then L2 represents an NA ₇ -(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-NA7 group, NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , (C ₁ -C ₆ )alkyl-C(=O )NA ₇ (C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _{1 group} , (C ₁ -C ₆ )alkyl-NA ₇ (C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group, ( C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C( group =O)-(C ₁ -C ₆ )alkyl-NA ₇ ,

- 18 045708

C(=O)-NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ aryl group, C(=O)-NA ₇ heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ C ₆ )alkyl-NA ₇ group C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ - C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) ₁ , C(= O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C _{6 )} group )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) ₁ NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ - group (C ₁ -C ₆ )alkyl-NA ₇ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, NA ₈ -(C ₁ -C) group ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , NA ₈ -(C ₁ -C ₆ )alkylC(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _{1 group} , NA8(C ₁ - C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-NA group ₈ -(C ₁ -C ₆ )alkylNA ₇ C(=O)-(C ₁ -C ₆ )αalkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-NA ₈ -(C ₁ -C ₆ ) alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA8-(C1-C6)alkyl-C(=O)NA7-(C1-C6)alkyl- group (OCH2CH2)1, C(=O)-NA8-(C1-C6)alkyl-NA7-(C1C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ or C(=O)-NA8(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group;

A ₇ is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NHC(=O)-, -N(alkyl)C(=O)- , -C(=O)NH-, -C(=O)N(alkyl)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH (OH)-, -CH(SO ₃ H)-, -O-, -C(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, - P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group, optionally substituted by at least one substituent, identical or different, selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -N(alkyl) ₂ , it being understood that each A ₇ containing the SO3H functional group may be in the forms salts such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na);

A8 represents a hydrogen atom or a (C1- _C6 )alkyl group, such as a hydrogen atom or a methyl group;

I represents an integer ranging from 1 to 50, such as from 1 to 35, wherein said substituted amino acids AAs are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent (C1-C ₈ )alkyl group, preferably T represents or ' group U represents a single bond, -NHC(=O)-, -N(alkyl)C(=O )-, -C(=O)NH-C(=O)N(αlkyl)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)-, -C (=O)O-, -OC(=O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C(=O)-, -OP( =O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)- or -OP(=O)(OH)-O, for example the group U is -NH-C(=O)- or -C(=O)NH-;

A ₉ represents a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(alkyl)C(=O )-, -C(=O)NH-, -C(=O)N(alkyl)-, -NHC(=NH)NH-OC(=O)-, -C(=O)O-, -OC (=O)O-, -CH(OH)-, -CH(SO ₃ H)-, -CH(O-alkyl)-, -CHF-, -CF2-, -S-, -Se-, -O -, -N(alkyl)-, -N ⁺ H(alkyl)-, -N ⁺ (alkyl) ₂ -, -OP(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -S(=O)2NH-, -S(=O)2N(alkyl)-, -P(=O)( OH)-, -P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group optionally substituted by at least one substituent , identical or different, selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -N(alkyl) ₂ , for example, A ₉ is -[(CH ₂ ) ₂ - O] ₄ -CH ₃ , -[(CH ₂ ) ₂ -O] ₂₄ -CH ₃ ,

- 19 045708

or -[(CH2)2-O)]4-(CH2)2-C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A9 containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

According to another specific embodiment, the present invention provides compounds of formula (I):

Where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

P represents a hydrogen atom, -OH or activated O;

L is a linker of formula (II):

ABOUT

L2 ^Н (AA)w L1 _(πχ where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J1, _J2 , J3 and _J4 are independently selected from CA1 and N;

ALK represents a (C1- _C12 )alkylene group, for example (C1-C6)alkylene, for example in the form -( _CH2 ) _n- , wherein n is an integer ranging from 1 to 12, and, for example, ranging from 1 to 6;

A _b A ₂ , A ₃ , A ₄ , A5 and A ₆ independently represent a hydrogen atom or a (C ₁ C6)alkyl group, for example a hydrogen atom or a methyl group.

(AA)w is a sequence of w substituted AAs or unsubstituted amino acids AA _ns joined together by peptide bonds;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA _s , then L2 represents a single bond, a (C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group, (C1-C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ -O(C ₁ -C ₆ )alkyl group, (CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, CH(SO ₃ H) -(C1C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, (C1-C ₆ )alkylcyclohexyl group, C(=O)-(C1C ₆ )alkyl group, C( group =O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O(C ₁ C ₆ )alkyl group, C(=O)-(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-CH(SO ₃ H)-(C ₁ -C ₆ )alkyl group , C(=O)-(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C(=O)-(C ₁ -C ₆ )alkylcyclohexyl group, NA ₈ -(C1C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _{1 group} , NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -O(C ₁ C ₆ )alkyl group , NA ₈ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H) group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ group -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-NA8-(C1C ₆ ) alkyl-(OSH ₂ CH ₂ ) ₁ -O(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C group (=O)-NA ₈ -(C ₁ -C ₆ )alkyl-CH(SO ₃ H), NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ group, NA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C1C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ group, (C ₁ -C ₆ )alkyl-C (=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) ₁ group , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) group ₁ , (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl

- 20 045708 group, C(=O)-(C ₁ -C ₆ )alkyl-NA7 group, C(=O)-NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-NA7aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O) -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ (C1-C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ group - (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0) -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C ₁ C ₆ )alkyl-NA ₇ C(=O) -(C ₁ -C ₆ )alkyl group, NA ₈ group -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl(OCH ₂ CH ₂ )i,NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ ) group )alkylNA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O )-(C ₁ -C ₆ )alkyl group, C(=O)-NA8-(C1-C6)alkyl-NA7C(=O)-(C1-C6)alkyl-(OCH2CH2)i group, C(=O )-NA8-(C1-C6)alkyl-C(=O)NA7-(C1C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA group ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i ,C(=O)-NA ₈ (C ₁ -C6)alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C (=O)-NA ₈ -(C ₁ -C6)alkyl-NA ₇ -(C ₁ -C ₆ )alkyl(OCH ₂ CH ₂ )i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

if (AA)w is a sequence of w unsubstituted amino acids AA _ns , then L2 represents an NA ₇ -(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-NA7 group, NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ -C6)alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group , group (C ₁ -C ₆ )alkyl-NA ₇ C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O) NA ₇ (C1- _C6 )alkyl group, group (C1 _- C6)alkyl-C(=0)NA ₇ -(C1 _- C6)alkyl-( _{0CH2CH2 ) i} , (C1 _- C6) alkyl-NA ₇ (C1-C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , (C ₁ -C ₆ ) alkyl-(OSH ₂ CH ₂ ) _i NA ₇ -(C ₁ -C6)alkyl group, C(=O)-NA ₇ -(C ₁ -C6)alkyl group, C(=O)-(C ₁ - C6)alkyl-NA ₇ , C(=O)-NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -aryl group, C(=O )-NA7heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ C ₆ ) group )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C1-C6)alkyl-C(=O)NA7-(C1-C6)alkyl-(OCH2CH2)i group, C(=O)- (C1-C6)alkyl-NA7-(C1C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) group _i , C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ group -(C ₁ -C ₆ )alkyl- NA ₇ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)(C1-C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O) group )-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ C6)alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ group -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl(OCH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -( C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C ₆ )alkyl-(OCH ₂ CH ₂ ) _group , NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ group, C(=O) -NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ C6)alkyl-NA7C( =O)-(C1-C6)alkyl-(OCH2CH2)i, C(=O)-NA8-(CrC6)alkyl-C(=O)NA7-(C _r C6)alkyl group, C(=O) group -NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C6)alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C6)alkyl-(OCH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group;

A ₇ is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NHC(=O)-, -N(αalkyl)C(=O)- , -C(=O)NH-, -C(=O)N(alkyl)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH (OH)-, -CH(SO ₃ H)-, -O-, -C(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, S(=O)2NH-, -S(=O)2N(alkyl)-, -P(=O)(OH)-, -P(= O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group, optionally substituted by at least one substituent, identical or different, selected from - OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and N(alkyl) ₂ , it should be understood that each A7 containing the SO ₃ H functional group can be in salt forms such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na);

A ₈ represents a hydrogen atom or a (C1-C ₆ )alkyl group, for example a hydrogen atom or a methyl group;

i represents an integer ranging from 1 to 50, such as from 1 to 35, wherein said substituted amino acids AAs are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent (C1-C ₈ )alkyl group, preferably T represents

or

group U represents a single bond, -NHC(=O)-, -N(alkyl)C(=O)-, -C(=O)NH-, -C(=O)N(αalkyl)-, -NHC (=O)NH-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C(=O)-, -OP(=O)-, -S(=O)-, -S(=O)2-, -NHS (=O)2-, -N(alkyl)S(=O)2-, -S(=O)2NH-, -S(=O)2N(alkyl)-, -P(=O)(OH) -, -P(=O)(OH)O-, -O-P(=O)(OH)- or -O-P(=O)(OH)-O, for example the group U is -NH-C(=O )- or -C(=O)NH-;

A ₉ represents a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(alkyl)C(=O )-, -C(=O)NH-, -C(=O)N(alkyl)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, - CH(SO3H)-, -CH(O-alkyl)-, -CHF-, -CF2-, -Se-, -O-, -N(αalkyl)-, -N ⁺ H(alkyl)-, -N ⁺ (alkyl)2-, -OP(=O)-, -S(=O)-, -S(=O)2-, -NHS(=O)2-, -N(alkyl)S(=O) 2--S(=O)2NH-, -S(=O)2N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(= O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group, optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom , -NH2, -NH-alkyl and -N(alkyl) ₂ , for example, A9 is pre-

is -[(CH ₂ ) ₂ -O] ₄ -CH ₃ , -[(CH ₂ ) ₂ -O] ₂₄ -CH ₃ ,

or -[(CH2)2-O)]4-(CH2)2-C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A9 containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

According to another specific embodiment, the present invention provides compounds of formula (I):

RCG1-LP _(I) where

RCG1 is a reactive chemical group that is reactive with a chemical group present on a polypeptide such as an antibody;

P represents a hydrogen atom, -OH or activated O;

L is a linker of formula (II):

ABOUT

-L2 (AA)wL1~ (P), where

L1 is represented by formula (III):

where if P represents a hydrogen atom, then x=0 or 1, and y=1, and z=0;

if P is -OH, then x=y=z=0;

if P is activated O, then x=1, and y=z=0, or x=y=z=1;

J1, _J2 , J3 and _J4 are independently selected from CA1 and N;

ALK is a (C1- _C12 )alkylene group, for example (C1-C6)alkylene, for example in the form

- 22 045708

-(CH2) _P -, wherein n is an integer ranging from 1 to 12, and, for example, ranging from 1 to 6;

A1, _A2 , A3, _A4 , _A5 and _A6 independently represent a hydrogen atom or a ( _CpC6 )alkyl group, for example a hydrogen atom or a methyl group.

(AA)w is a sequence of w substituted AA _s or unsubstituted amino acids AA _ns joined together by peptide bonds;

w is an integer ranging from 1 to 12, such as 1 to 6, such as 2 or 3;

(AA)w contains at least one substituted amino acid AA _s and/or w unsubstituted amino acids AA _ns , and L2 is a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group;

A7 is defined above.

In accordance with at least one embodiment, (AA)w contains at least one substituted amino acid AA _s and L2 is a (C1- _C6 )alkyl group, for example a ( _CH2 ) ₃ group;

C(=O)-(C ₁ -C ₆ )alkyl group, for example the C(=O)-(CH ₂ ) ₃ group; or a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, for example a (CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ group, in which A7 is as defined above.

In accordance with at least one embodiment, (AA)w contains at least one substituted amino acid AA _s and L2 is a (C1- _C6 )alkyl group, for example a ( _CH2 ) ₃ group;

C(=O)-(C ₁ -C ₆ )alkyl group, for example the C(=O)-(CH ₂ ) ₃ group; or a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, for example a -(CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ - group, in which A7 represents a -C group (=O)-(CH ₂ ) ₂ -C(=O)-NH-(Ch ₂ ) ₂ -SO ₃ H;

it should be understood that each A7 containing the SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

In another embodiment, (AA)w contains w unsubstituted amino acids AA _ns , and L2 is a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, such as a (CH ₂ ) ₂ group -NA ₇ -(CH ₂ ) ₂ in which A7 is defined above.

In another embodiment, (AA)w contains w unsubstituted amino acids AA _ns , and L2 is a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, such as a (CH ₂ ) ₂ group -NA ₇ -(CH ₂ ) ₂ in which A7 represents the group C(=O)-[(CH ₂ ) ₂ -O] _and -CH ₃ where a is an integer ranging from 1 to 50 , for example, ranging from 1 to 24, such as 4, 7 and 24, for example, A7 represents a C(=O)-[(CH ₂ ) ₂ -O] ₄ -CH ₃ group, a C(=O) group )-[(CH ₂ ) ₂ _O] ₇ -CH ₃ or group C(=O)-[(CH ₂ ) ₂ -O] ₂₄ -CH ₃ ;

group -C(=O)-(Ch ₂ ) ₂ -C(=O)-NH-(Ch ₂ ) ₂ _SO ₃ H or group C(=O)-(CH ₂ ) ₂ -C(=O)- NH-[(CH ₂ ) ₂ -O] _a -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H, where a is an integer ranging from 1 to 50, in a range such as from 1 to 24, for example 4, for example, A7 represents the group -C(=O)-(CH ₂ ) ₂ -C(=O)-NH-[(CH ₂ ) ₂ .O] ₄ -(CH ₂ ) ₂ .C(=O)-NH-(CH ₂ ) ₂ SO3H;

it should be understood that each A ₇ containing a SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na);

In accordance with at least one embodiment, (AA)w contains at least one substituted amino acid AA _s and/or w unsubstituted amino acids AA _ns , and L2 represents an NA ₇ -(C ₁ -C ₆ )alkyl group, a group ( C ₁ -C ₆ )alkyl-NA ₇ , NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NA ₇ -aryl group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA7C(=O)-(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -( C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O) -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -aryl group, C(=O)-NA ₇ heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-(CiC ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OCH ₂ CH) group ₂ ) _i , C(=o)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ ) group )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(oСН ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group _, NA ₈ -(C ₁ -C ₆ )alkyl-NA 7 group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)(C1-C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group (OSH ₂ CH ₂ ) _i , NA ₈ -(C1C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C( group =O)NA ₇ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ group -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-(oCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl -NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C1- 23 045708

C6)alkyl-NA ₇ C(=O)-(Cl-C6)alkyl-(OSH ₂ CH ₂ )i, C(=O)-NA ₈ -(C ₁ -C6)alkyl-C(=O)NA ₇ -(C ₁ -C6)alkyl group, C(=O)-NA group ₈ -(Cl-C6)alkyl-C(=O)NA ₇ -(Cl-C6)alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-ML ₈ -(C1- _C6 )alkylCL ₇ -(C1- _C6 )alkyl group, C(=O)-NA ₈ -( _C1 - _C6 )alkyl-NA ₇ group - (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ - C ₆ )alkyl group; where i, A7 and _A8 are defined above.

In another embodiment, (LL)w contains at least one substituted amino acid LL, and/or w unsubstituted amino acids LL _P8 , and L2 is an NA ₇ -(C ₁ C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-NA ₇ , NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, NΛ ₇ aryl group, CL ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ group C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , (C ₁ -C ₆ )-Calkyl(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ )i, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)- NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-CL ₇ (CH ₂ CH ₂ O) _i (C ₁ -C6) alkyl group, C(=O)-CL ₇ -aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C( =O)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl-(OCH ₂ CH) group ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C1C ₆ )alkyl group C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ ) alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ group, NA ₈ -(C ₁ -C ₆ ) group alkyl-NA ₇ , NA ₈ -(C ₁ -C ₆ )alkylNA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O) NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkylNA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group or C(=O)-Na ₈ -(C ₁ -C ₆ )alkyl-NA ₇ - (C ₁ -C ₆ )alkyl group; where i, A ₇ and A ₈ are defined above.

According to another embodiment, (ΛΛ)w contains at least one substituted amino acid LL, and/or w unsubstituted amino acids LL _|K , and L2 is an NA ₇ -(C ₁ C ₆ )alkyl group, a (C ₁ - C ₆ )alkyl-NA ₇ , NA ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, (C1-C ₆ )alkylNA ₇ C(=O)-(C ₁ -C ₆ ) alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C1C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ cH ₂ ) _i , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i group, ( C1C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O) group )-(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₇ -(CН ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-(C1- C ₆ )alkylNA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, c(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=OHCl-C ₆ )alkyl-( OCH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ );, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C1-C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ group, NA ₈ -(C ₁ -C) group ₆ )alkyl-NA ₇ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C( =O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ (C1-C ₆ )alkyl group, C(=O)-NA ₈ -( C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, C(=O )-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group or C(=O)-NA ₈ -(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group; where i, A ₇ and A ₈ are defined above.

According to another embodiment, (ΛΛ)w contains at least one substituted amino acid LL, and/or w unsubstituted amino acids LL _|K , and L2 is an NA ₇ -(C ₁ C ₆ )alkyl group, a (C ₁ - C ₆ )alkyl-NA ₇ , (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C1C ₆ )alkyl-C(=O)NA ₇ -( C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-CL ₇ (C1-C ₆ )alkyl group, C group (=O)-(C ₁ -C ₆ )alkyl-NA ₇ , C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, C (=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C1-C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ (C1-C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(= O)-(C ₁ C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group or C( =O)-NA ₈ -(C ₁ C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group; where A ₇ and L ₈ are defined above.

In accordance with at least another embodiment (LL^ contains at least one substituted amino acid LL, and/or w unsubstituted amino acids LL _P ,, and L2 contains A ₇ representing the group C(=O)-[( _CH2 ) ₂ -O] ₄ -CH ₃ ; group C(=O)-[(CH ₂ ) ₂ -O] ₇ -CH ₃ ; group C(=O)[(CH2)2-O]24-CH ₃ ; group C(=O)-(CH2)2-C(=O)-NH-(CH2)2-SO ₃ H; group C(=O)-(CH2)2-C(=O)-IN-[ (CH2)2O]4-(CH2)2-C(=O)-NH-(CH2)2-SO ₃ H;

it should be understood that each A ₇ containing a SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ⁺ Na).

- 24 045708

In accordance with at least another embodiment, (AA)w contains at least one substituted amino acid AAAs and/or w unsubstituted amino acids _AAns , and L2 is

L1 may be one of the following (III1-5):

R L1 Examples —NAg-ALKNA,— (NI) n j^A 9 1 ,D ^J 1 ^ O NyALK ΝΑ,, (|||2) NA^jf ¹ ' He (1113) ι 2 XA ^J 1 Io ^ (PI) -NA/Jp activated O AA ^J i ON^ ALK NA ₅ (1115)

- 25 045708 where

J1, _J2 , J3 and _J4 are independently selected from CA1 and N;

ALK represents a (C1- _C12 )alkylene group, for example (C1- _C6 )alkylene, for example in the form -( _CH2 ) _n- , wherein n is an integer ranging from 1 to 12, and , for example, ranging from 1 to 6;

A1, _A2 , A3, _A4 , _A5 and _A6 independently represent a hydrogen atom or _a ( _C1C6 )alkyl group, for example a hydrogen atom or a methyl group.

AA stands for amino acid. The amino acid is a compound of the formula NH2CHA10-COOH, where A ₁₀ represents the side chain of AA. AA may be a substituted AAAs or an unsubstituted AA _ns amino acid.

Unsubstituted amino acid AA _ns refers to a natural or unnatural amino acid of D- or L-configuration identical to alanine (Ala), β-alanine, γ-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine (Arg ), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), α,α-dimethyl-γ-aminobutyric acid, β,β-dimethyl-γ-aminobutyric acid, glutamine (Gln), glutamic acid (Glu), glycine (Gly), homocysteine, selenocysteine, homoselenocysteine, histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), ε-acetyl lysine (AcLys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val) or a derivative thereof.

For example, AA _ns is alanine (Ala), citrulline (Cit), glycine (Gly), isoleucine (Ile), leucine (Leu), lysine (Lys), s-acetyllysine (AcLys), phenylalanine (Phe), tryptophan ( Trp) and valine (Val).

Substituted amino acids AA _s are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent (C1-C ₈ )alkyl group, for example, T represents or ' group U represents a single bond, -NHC(=O)-, -N(alkyl)C(= O)-, -C(=O)NH-, -C(=O)N(αalkyl)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)- , -C(=O)O-, -OC(=O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C(=O)-, -OR(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S( =O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)( OH)- or -OP(=O)(OH)-O, for example, the group U is -NH-C(=O)- or -C(=O)NH-;

A9 is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(alkyl)C(=O) -, -C(=O)NH-, -C(=O)N(alkyl)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)-, - C(=O)O-, -OC(=O)O-, -CH(OH)-, -CH(SO ₃ H)-, -CH(O-alkyl)-, -CHF-, -CF2-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -N ⁺ H(alkyl)-, -N ⁺ (alkyl) ₂ -, -C(=O)-, - OP(=O)-, -S(=O)-, -S(=O) ₂ -, NHS(=O)2-, -N(alkyl)S(=O)2-, -S(=O )2NH-, -S(=O)2N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group, optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH- alkyl and -N(alkyl) ₂ , for example, A9 represents [(CH ₂ ) ₂ -O] ₄ -CH ₃ , [(CH ₂ ) ₂ -O] ₂₄ -CH ₃ ,

or [(CH2)2-O)]4-(CH2)2-C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A ₉ containing a SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

- 26 045708

According to a specific embodiment, the substituted amino acids AAs are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent (C1-C ₈ )alkyl group, for example, T represents or ^; group U represents a single bond, -NHC(=O)-, -N(alkyl)C(=O)-, -C(=O)NH-, -C(=O)N(αalkyl)-, -NHC (=O)NH-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C(=O)-, -OP(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS (=O)2-, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)( OH)-, -P(=O)(OH)O-, -OP(=O)(OH)- or -OP(=O)(OH)-O, for example the group U is -NH-C( =O)- or -C(=O)NH-;

A9 is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(alkyl)C(=O) -, -C(=O)NH-, -C(=O)N(alkyl)-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, - OC(=O)O-, -CH(OH)-, -CH(SO ₃ H)-, -CH(C-alkyl)-, -CHF-, -CF2-, -S-, -Se-, - O-, -NH-, -N(αalkyl)-, -N+H(alkyl)-, -N+(alkyl) ₂ -, -C(=O)-, -OP(=O)-, -S( =O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O)2NH-, -S(=O) ) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH) )-O- or heterocycloalkyl group, optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -N(alkyl) ₂ , for example, A9 represents [(CH ₂ ) ₂ -O] ₄ -CH ₃ , [(CH ₂ ) ₂ -O] ₂₄ -CH ₃ ,

or [(CH2)2-O)]4-(CH2)2-C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A9 containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

According to another specific embodiment, the substituted amino acids AA _s are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent+1 (C1-C ₈ )alkyl group, for example, T represents “T· or ' group U represents a single bond, -NHC(=O)-, -N (alkyl)C(=O)-, -C(=O)NH-, -C(=O)N(αalkyl)-, -NHC(=O)NH-, -NHC(=NH)NH-, - OC(=O)-, -C(=O)O-, -OC(=O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C (=O)-, -OR(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, - OP(=O)(OH)- or -OP(=O)(OH)-O, for example, the group U is -NH-C(=O)- or -C(=O)NH-;

A9 is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(alkyl)C(=O) -, -C(=O)NH-, -C(=O)N(alkyl)-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, - OC(=O)O-, -CH(OH)-, -CH(SO ₃ H)-, -CH(O-alkyl)-, -CHF-, -CF2-, -S-, -Se-, - ABOUT-,

- 27 045708

-N(alkyl)-, -N+H(alkyl)-, -K+(alkyl) ₂ -, -OP(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS(=O) ₂ -, -N(alkyl)S(=O) ₂ -, -S(=O) ₂ NH-, -S(=O) ₂ N(alkyl)-, -P(=O )(OH)-, -P(=O)(OH)O-, -OP(=O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group optionally substituted by at least one substituent, identical or different, selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and N(alkyl) ₂ , for example, A9 is [(CH ₂ ) ₂ -O ] ₄ -CH3, [(CH ₂ ) ₂ -O] ₂₄ -CH3,

or [(CH ₂ ) ₂ -O)] ₄ -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO3H;

it should be understood that each A9 containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

According to another specific embodiment, the substituted amino acids AA _s are characterized by formula (VI):

Where

T represents a saturated or unsaturated, linear or branched trivalent (C1- _C8 )alkyl group, for example, T represents or

group U represents a single bond, -NHC(=O)-, -N(αlkyl)C(=O)-, -C(=O)NH-, -C(=O)N(alkyl)-, -NHC (=O)NH-, -NHC(=NH)NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -S-, -Se-, -O-, -NH-, -N(alkyl)-, -C(=O)-, -OP(=O)-, -S(=O)-, -S(=O) ₂ -, -NHS (=O) ₂ -, -N(alkyl)S(=O)2-, -S(=O) ₂ NH-, -S(=O)2N(alkyl)-, -P(=O)(OH )-, -P(=O)(OH)O-, -OP(=O)(OH)- or -OP(=O)(OH)-O, for example the group U is -NH-C(= O)- or -C(=O)NH-;

A ₉ represents a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -NH-C(=O)-, -N(alkyl)C(=O )-, -C(=O)NH-, -C(=O)N(alkyl)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, - CH(SO ₃ H)-, -CH(C-alkyl)-, -CHF-, -CF2-, -Se-, -O-, -N(αlkyl)-, -N ⁺ H(alkyl)-, - N+(αalkyl)2-, -OP(=O)-, -S(=O)-, -S(=O)2-, -NHS(=O)2-, -N(alkyl)S(=O )2-, -S(=O)2NH-, -S(=O)2N(alkyl)-, -P(=O)(OH)-, -P(=O)(OH)O-, -OP (=O)(OH)-, -OP(=O)(OH)-O- or a heterocycloalkyl group, optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, -alkyl, halogen atom, -NH2, -NH-alkyl and -N(alkyl) ₂ , for example, A9 represents [(CH2)2-O]4-CH3, [(CH2)2-O]24-CH3,

or [(CH2)2-O)]4-(CH2)2-C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A ₉ containing a SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

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Examples of Ts that may be mentioned include

In accordance with at least one embodiment, the substituted amino acids AA _s are characterized by formula (VI), wherein T represents a saturated or unsaturated, linear or branched trivalent (C1- _C8 )alkyl group, such as

or

the group U is -NH-C(=O)- or -C(=O)NH-;

A ₉ is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, where optionally at least one methylene unit is independently replaced by -C(=O)NH-, -CH(OH)-, -CH( SO ₃ H)-, -O-, -NH-, or -C(=O)-, or a heterocycloalkyl group optionally substituted by at least one substituent identical or different selected from -OH, -O-alkyl, - alkyl, for example, A9 is [(CH ₂ ) ₂ -O] ₄ CH3, [(CH2)2-O)]24-CH3,

or [(CH2)2-O]4-(CH2)2-C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A9 containing the SO3H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

According to another embodiment, the substituted amino acids AA _s are characterized by formula (VI) wherein T is

or the group U is -NH-C(=O)- or -C(=O)NH-; A ₉ represents [(CH ₂ ) ₂ -O] ₄ -CH ₃ , [(CH2)2-O]24-CH3,

or [(CH ₂ ) ₂ -O] ₄ -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H;

it should be understood that each A ₉ containing a SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

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According to yet another embodiment, the substituted amino acids AAs are characterized by formula (VI), wherein or

T represents

the group U is -NH-C(=O)- or -C(=O)NH-;

A9 represents -[(CH2)2-O]b- _CH3 , where b is an integer ranging from 1 to 50, for example, ranging from 1 to 24, such as 4, 7 and 24, for example 4 or 24.

The sequence (AA) _W is characterized by the formula:

wherein A10 is a side chain of one of the substituted AAs or unsubstituted AAns amino acids described above.

Examples of unsubstituted amino acid (AAns)w sequences are: GlyGly, Phe-Lys, Val-Lys, Val-AcLys, Val-Cit, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Ala, Phe-Cit, Phe-Gly, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Phe, Gly-Gly-Gly, Gly-Ala-Phe, Gly- Phe-Gly, GlyVal-Cit, Gly-Phe-Leu-Cit (SEQ ID No. 3), Gly-Phe-Leu-Gly (SEQ ID No. 4), Ala-Leu-Ala-Leu (SEQ ID No. 5).

In accordance with at least one embodiment, the sequence (AA _ns )w of unsubstituted amino acids AAns is selected from the following list: Gly-Gly, Phe-Lys, Val-Lys, Val-AcLys, Val-Cit, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Ala, Phe-Cit, Phe-Gly, Leu-Cit, Ile-Cit, TrpCit, Phe-Ala, Ala-Phe, Gly- Gly-Gly, Gly-Ala-Phe, Gly-Phe-Gly, Gly-Val-Cit, Gly-Phe-Leu-Cit (SEQ ID No. 3), Gly-Phe-Leu-Gly (SEQ ID No. 4 ), and Ala-Leu-Ala-Leu (SEQ ID No. 5), for example, Val-Ala and Val-Cit, for example, Val-Ala.

In accordance with at least another embodiment, the sequence (AAs)w containing at least one substituted amino acid AA _s is selected from the list:

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- 31 045708

Among the linkers L of formula (II), which are the object of the present invention, the following compounds may be mentioned, for example:

- 32 045708

- 33 045708

- 34 045708

- 35 045708

Examples of RCG1 that may be mentioned include:

(i) a reactive group R _a Z _a -C(=O)-, in which

Z _a represents a single bond, -O- or -NH, for example -O-, and

R _a represents a hydrogen atom, a (C ₁ -C 6 )alkyl, (C 3 -C ₇ )cycloalkyl, alkenyl, aryl, heteroaryl or (C 3 -C ₇ )heterocycloalkyl group. The aryl group, heteroaryl group and/or (C ₃ C ₇ )heterocycloalkyl group may be substituted with 1 to 5 atoms/groups selected from a halogen atom such as a fluorine atom, an alkyl group, an alkoxy group, a hydroxyl group, an oxo group, a nitro group and a cyano group;

ABOUT

[Γ^Ν^

.. ⁰ · (ii) one of the following reactive groups: maleimido group of haloacetami- ^21

Cl, Br or I Π subgroup θ ’, with R21 representing a hydrogen atom or a (C1-C6)alkyl group, for example Me; Cl-; N3-; BUT-; HS-; activated disulfide such as

- 36 045708

N.H.

O-alkylhydroxylamine or Pictet-Spengler reaction substrate such as described in Agarwal P., et al., Bioconjugate Chem 2013, 24, 846-851.

According to a specific embodiment, RCG1 may be:

(i) a reactive group R _a Z _a -C(=O)-, in which

Z _a represents a single bond, -O- or -NH, for example -O-, and

R _a represents a hydrogen atom, a (C ₁ -C ₆ )alkyl, (C ₃ -C ₇ )cycloalkyl, alkenyl, aryl, heteroaryl or (C ₃ -C ₇ )heterocycloalkyl group. The aryl group, heteroaryl group and/or (C ₃ C ₇ )heterocycloalkyl group may be substituted with 1 to 5 atoms/groups selected from a halogen atom such as a fluorine atom, an alkyl group, an alkoxy group, a hydroxyl group, an oxo group, a nitro group and a cyano group;

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

C1, Br or I y g ⁰ 'wherein R21 represents a hydrogen atom or a (C ₁ -C ₆ )alkyl group,

for example Me; Cl-; N3-; BUT-; HS-; activated disulfide such as 'H2N-;

HC^C- or activated C=C, for example, cyclooctyne fragment, for example DBCO-amine

’ or BCN ’ or MFCO

O-alkylhydroxylamine or Pictet-Spengler reaction substrate such as

Chem 2013, 24, 846-851.

NH/

described in Agarwal P., et al., Bioconjugate

For example, R _a Z _a - can represent HO-, CH3O-, CH ₂ =CH-CH ₂ O-,

M=H or (O-NHS),

O SO ₃ M

Wherein the cation is, for example, sodium, potassium or cesium, or

GI group

wherein GI represents at least one electroinductive group such as -NO2 or a halogen atom, for example a fluorine (F) atom. They may be, for example, the following groups:

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For example, RCG1 can be selected from one of those described in the examples, that is, selected from the following groups:

COOH

The present invention further relates to cryptophycin payloads of formula (IV):

Where

R1 represents a (C1-C6)alkyl group,

R2 and _R3 independently represent a hydrogen atom or a (C1-C6)alkyl group;

or alternatively, R2 and _R3 form, together with the carbon atom to which they are attached, a ( _C3 -C6)cycloalkyl or ( _C3 -C6)heterocycloalkyl group;

R4 and _R5 independently represent a hydrogen atom, or a (C1-C6)alkyl group, or a ( _C1 - _C6 )alkyl-NH( _R12 ) group, or a (C1- _C6 )alkyl-OH group , or a (C ₁ -C ₆ )alkyl-SH group, or a (C ₁ -C ₆ )alkyl-C(=O) ₂ H group;

or alternatively, R ₄ and R ₅ form, together with the carbon atom to which they are attached, a (C ₃ -C ₆ )cycloalkyl or (C ₃ -C ₆ )heterocycloalkyl group;

X represents O or N(R ₆ );

R6 represents a hydrogen atom or a (C1- _C6 )alkyl group;

R ₇ and R8 independently represent a hydrogen atom, or a (C1- _C6 )alkyl group, or a ( _C1 - _C6 )alkyl-C(=O) _2H group, or a ( _C1 - _C6 ) group )alkyl-N(C ₁ -C ₆ )alkyl ₂ ;

or alternatively, R ₇ and R ₈ form, together with the carbon atom to which they are attached, a (C ₃ -C ₆ )cycloalkyl group or a (C ₃ -C ₆ )heterocycloalkyl group;

R9 represents at least one substituent on the phenyl ring independently selected from hydrogen, -OH, (C1- _C4 )alkoxy, halogen, -NH2, -NH( _{C1- C6} )alkyl, or N (C ₁ -C ₆ )alkyl ₂ , or -NH(C ₁ -C ₆ )cycloalkyl, or (C ₃ -C ₆ )heterocycloalkyl group;

R ₁₀ represents at least one substituent on the phenyl ring selected from a hydrogen atom and a (C1- _C4 )alkyl group;

Y represents

-NR ₁₁ -(C ₁ -C ₆ )alkyl-, for example -NR ₁₁ -(CH2) _n -, such as

RCG^LNR^^CH^n

Yu

-O-(C1-C ₆ )alkyl-, for example -O-(CH2) _P -, such as

RCG^-O-fCH^n

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-8-(C ₁ -C ₆ )alkyl-, for example -S-(CH ₂ )n-, such as

wherein Y is located in the ortho-(o), meta-(m) or para-(p) position of the phenyl ring;

R11 and R12 independently represent a hydrogen atom or a (C1-C6)alkyl, for example a hydrogen atom or a methyl group;

n is an integer ranging from 1 to 6;

L is defined in formula (I) and is a linker of formula (II) as defined in the present invention;

RCG1 is a reactive chemical group present at the end of linker L, wherein RCG1 is reactive with a chemical group present on a polypeptide such as an antibody. RCG1 is defined above.

Each of the substituents R1-R12 may also assume one of the spatial configurations (eg, R or S, or alternatively Z or E) as described in the examples. The compounds of formula (IV) may contain at least one asymmetric carbon atom. Therefore, they can exist in the form of enantiomers or diastereoisomers. These enantiomers and diastereoisomers, as well as mixtures thereof, including racemic mixtures, form part of the present invention.

As an example, the cryptophycin compound of formula (IV) may be one of the cryptophycin compounds of formula (I) described in WO2011/001052 (eg, one of D1-D8) or in PCT/EP2016/076603 (eg, one of D1- D ₁₉ ), as mentioned above.

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

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which R1 represents a (C1- _C6 )alkyl, for example a methyl group.

Among the compounds of formula (IV) which are the object of the present invention, the group of compounds consists of compounds in which each of R2 and _R3 represents a hydrogen atom.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which one of R2 and _R3 represents a (C1- _C6 )alkyl, for example a methyl group, and the other represents a hydrogen atom.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which R2 and _R3 form, together with the carbon atom to which they are attached, a (C ₃ -C ₆ )cycloalkyl group, for example a cyclopropyl group.

Among the compounds of formula (IV) which are the object of the present invention, the group of compounds consists of compounds in which R4 and _R5 each represent a (C1- _C6 )alkyl, for example a methyl group.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which X represents an oxygen atom, that is, O.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which X represents NH.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which R ₇ and R 8 independently represent a hydrogen atom or a (C1-C ₆ )alkyl group, for example an isobutyl group or a neopentyl group.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which R ₉ represents two substituents selected from a methoxy group and a chlorine atom, for example, the two R ₉ substituents are 3-Cl and 4-methoxy . For example, the phenyl ring contains two substituents at positions 3 and 4 of the phenyl ring.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which R ₁₀ represents a hydrogen atom.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which Y is located in the para position of the phenyl ring.

Among the compounds of formula (IV) which are the object of the present invention, the group of compounds consists of compounds in which Y represents NR ₁₁ -(C ₁ -C ₆ )alkyl, such as NR11(C1-C ₃ )alkyl, for example NH- CH2.

Among the compounds of formula (IV) which are the object of the present invention, the group of compounds consists of compounds in which L of formula (II) contains at least one substituted amino acid AA in the sequence of w amino acids (AA)w, L1 and L2 are defined in the formula (II).

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which L of formula (II) contains a sequence of w unsubstituted amino acids AA _ns , L1 and L2 as defined in formula (II).

- 39 045708

Among the compounds of formula (IV) which are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA _s , and L2 is (C1-C ₆ )alkyl group, for example a (CH ₂ ) ₃ group;

C(=O)-(C1-C ₆ )alkyl group, for example the C(=O)-(CH ₂ ) ₃ group; or a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, for example a (CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ group, in which A7 is as defined above.

Among the compounds of formula (IV) that are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA _s , and L2 is (C1-Cb) an alkyl group, for example a (CH ₂ ) ₃ group;

a C(=O)-(C ₁ -C6)alkyl group, for example a C(=O)-(CH ₂ ) ₃ group; or a (C ₁ -C6)alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, for example a -(CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ - group, in which A7 represents a -C( =O)-(CH ₂ ) ₂ -C(=O)-NH-(Ch ₂ ) ₂ -SO ₃ H;

it should be understood that each A7 containing the SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which (AA)w in L of formula (II) contains w unsubstituted amino acids AA _ns and L2 is (C ₁ -C ₆ )alkyl -NA ₇ -(C ₁ -C ₆ )alkyl group, for example the (CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ group, in which A7 is defined above.

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds in which (AA)w in L of formula (II) contains w unsubstituted amino acids AA _ns and L2 is (C ₁ -C6)alkyl- NA ₇ -(C ₁ -C ₆ )alkyl group, for example a (CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ group, in which A7 represents a C(=O)-[(CH ₂ ) ₂ -O group ] _a -CH ₃ where a is an integer ranging from 1 to 50, for example ranging from 1 to 24, such as 4, 7 and 24, for example A7 is a C(=O) group -[(CH ₂ ) ₂ -O] ₄ -CH ₃ , group C(=O)-[(CH ₂ ) ₂ -O] ₇ -CH ₃ or group C(=O)-[(CH ₂ ) ₂ - O] ₂₄ -CH ₃ ;

group -C(=O)-(Ch ₂ ) ₂ -C(=O)-NH-(Ch ₂ ) ₂ -sO ₃ H or group C(=O)-(CH ₂ ) ₂ -C(=O) -NH-[(CH ₂ ) ₂ -O] _a -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H, where a is an integer ranging from 1 to 50, in a range such as from 1 to 24, for example 4, for example, A7 represents the group -C(=O)-(CH ₂ ) ₂ -C(=O)-NH-[(CH ₂ ) ₂ .O ] ₄ -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H;

it should be understood that each A7 containing the SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na);

Among the compounds of formula (IV) which are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA _s and/or w unsubstituted amino acids AA _ns , and L2 represents an NA7(C1- _C6 )alkyl group, a ( _C1 - _{C6 )alkyl-NA7 group, NA7 -(CH2CH2O)i (C1-C6 ) alkyl group , NA7aryl group} , NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O) group -(C ₁ -C ₆ )alkyl-(OcH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ - C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA _{7 group} , C(=O)-NA ₇ (CH ₂ CH ₂ OTS1-Sb) alkyl group, C(=O)-NA ₇ -aryl group, C(=O)-NA ₇ -heteroaryl group, C(=O)- (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ C(=0) group -(C ₁ C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-(CiC ₆ )alkyl-C(=0)NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=0)-(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=0)-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl group -NA ₇ , NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C group (=O)-(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ C ₆ )alkyl group , group NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl group, group NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C (=O)-NA ₈ (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-Na ₈ -(C ₁ -C ₆₎ group )alkyl-NA ₇ C(=O)-(C ₁ C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O) NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(oСН) group ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i or C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C6)alkyl group; where i, A7 and A ₈ are defined above.

Among the compounds of formula (IV) that are the object of the present invention, a preferred group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AAAs and/or w unsubstituted amino acids AA _ns , and L2 represents

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CL ₇ -(C1-C ₆ )alkyl group, (C1-C ₆ )alkyl-ML ₇ group, NL ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, CL ₇ -aryl group , CL ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-CL ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NΛ ₇ C(=0) group )-(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)kL ₇ -(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-C(=O)NΛ ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ ) alkyl group, (C ₁ -C ₆ )alkyl-NΛ ₇ -(C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _{i group} , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i - NΛ ₇ -(C ₁ C ₆ )alkyl group, C(=O)-CL ₇ -(C1-C ₆ )alkyl group, C(=O)-(C1-C ₆ )alkyl-CL ₇ group, C( =O)NL ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-CL ₇ -aryl group, C(=O)-CL ₇ -heteroaryl group, C( =O)-(C ₁ -C ₆ )alkyl-CL ₇ C(=O)-(C ₁ -C ₆ )alkyl group, group C(=O)-(C1-C ₆ )alkyl-ML ₇ C( =O)(C ₁ -C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ ) alkyl group, group C(=O)-(C1C ₆ )alkyl-C(=O)NΛ ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NΛ ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂₎ group CH ₂ ) _i , group CL ₈ -(C1-C ₆ )alkyl-CL ₇ , NA ₈ -(C|-C ₆ )αalkylCL ₇ C(=O)-(C1-C ₆ )alkyl group, CL ₈ - (C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ C6)alkyl-NA7-(C ₁ -C6)alkyl group, group C (=O)-CL ₈ -(C1-C ₆ )alkyl-CL ₇ , C(=O)-ML ₈ -(C1-C ₆ )alkylCL ₇ C(=O)-(C1-C ₆ )alkyl group , C(=O)-NA ₈ -(C ₁ -C6)alkyl-C(=O)NA7-(C ₁ -C6)alkyl group or C(=O)-ML ₈ -(C ₁ -C ₆ ) alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group; where i, A7 and A ₈ are defined above.

Among the compounds of formula (IV) that are the object of the present invention, a more preferred group of compounds consists of compounds in which (LL)w in L of formula (II) contains at least one substituted amino acid LA _s and/or w unsubstituted amino acids LL _P8 , and L2 represents

CL ₇ -(C1-C ₆ )alkyl group, (C1-C ₆ )alkyl-ML ₇ group, NL ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CL ₇ C(=O)-(C ₁ -C ₆ )alkyl group, group (C ₁ -C ₆ )alkyl-CL ₇ C(=O)-(C ₁ -C ₆ )alkyl(0CH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)CL ₇ group -(C ₁ C ₆ )alkyl-(OSN ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CL group ₇ -(C ₁ C ₆ )alkyl-(0CH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NΛ ₇ -(C ₁ -C ₆ )alkyl group, C( =O)-NA7-(C ₁ C ₆ )alkyl group, C(=O)-(C1-C ₆ )alkyl-CL ₇ group, C(=O)-NL ₇ -(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C6)alkyl-NA7C(=O)-(C ₁ -C6)alkyl group, C(=O)-(C ₁ -) group C ₆ )alkyl-CL ₇ C(=OHC ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-(C1C ₆ )alkyl-C(=O)NL ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O )-(C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NL ₇ -(C ₁ -C ₆ ) group alkyl-(OSH ₂ CH ₂ ) _i , CL ₈ -(C1-C ₆ )alkyl-CL ₇ group, NA ₈ -(C|-C ₆ )αalkylCL ₇ C(=O)-(C1-C ₆ )alkyl group, CL ₈ -(C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ C6)alkyl-NA7-(C ₁ -C6) alkyl group, C(=O)-CL ₈ -(C1-C ₆ )alkyl-CL ₇ group, C(=O)-ML ₈ -(C1-C ₆ )alkylCL7C(=O)-(C1-C ₆ )alkyl group, C(=O)-CL ₈ -(C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group or C(=O)-ML ₈ -( C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group; where i, A7 and A ₈ are defined above.

Among the compounds of formula (IV) that are the object of the present invention, an even more preferred group of compounds consists of compounds in which (LL)w in L of formula (II) contains at least one substituted amino acid LL ₈ and/or w unsubstituted amino acids AA _l , _s , and L2 represents a CL ₇ -(C1-C ₆ )alkyl group, a (C1-C ₆ )alkyl-ML ₇ group, (C1-C ₆ )alkyl-CL ₇ C(=O)(C1- C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ C ₆ )alkyl group, C(=O)-ML ₇ -(C1-C ₆ )alkyl group, C(=O)-(C1-C ₆ )alkyl-CL ₇ group, C(=O)-(C1C ₆ ) alkyl-ML ₇ C(=O)-(C1-C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group, CL ₈ -(C1-C ₆ )alkyl-CL ₇ group, NA ₈ -(C |-C ₆ )αalkylCL ₇ C(=O)-(C1-C ₆ )alkyl group, CL ₈ -(C ₁ -C ₆ )alkyl-C(=O)CL ₇ -(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ C6)alkyl-NA7-(C ₁ -C6)alkyl group, group C(=O)-CL ₈ -(C1-C ₆ )alkyl-CL ₇ , C(=O)- ML ₈ -(C1- _C6 )alkylCL ₇ C(=O)-(C1- _C6 )alkyl group, C(=O)-NA ₈ -(C1 _- C6)alkyl-C(=O)NA7- (C ₁ -C6)alkyl group or C(=O)-ML ₈ -(C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group; where A7 and A ₈ are defined above.

Among the compounds of formula (IV) that are the object of the present invention, the most preferred group of compounds consists of compounds in which (LL)w in L of formula (II) contains at least one substituted amino acid LL ₈ and/or w unsubstituted amino acids LL _P8 , and L2 represents a (C ₁ -C ₆ )alkyl-CL ₇ -(C ₁ -C ₆ )alkyl group; where A7 is defined above.

Among the compounds of formula (IV) that are the object of the present invention, the group of compounds consists of compounds in which (LL)w in L of formula (II) contains at least one substituted amino acid LL ₈ and/or w unsubstituted amino acids LL _P8 , and L2 contains A7, which is a group C(=O)-[(CH ₂ ) ₂ -O] ₄ -CH ₃ ; group C(=O)-[(CH ₂ ) ₂ -O] ₇ -CH ₃ ; group C(=O)-[(CH ₂ ) ₂ -O] ₂₄ CH ₃ ; group C(=O)-(CH2)2-C(=O)-NH-(CH2)2-SO3H; group C(=O)-(CH2)2-C(=O)-KH-[(CH2)2-O]4-(CH2)2C(=O)-NH-(CH2)2-SO3H;

it should be understood that each A7 containing the SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ' ⁺ Na).

Among the compounds of formula (IV) that are the object of the present invention, the group of compounds consists of compounds in which (LL)w in L of formula (II) contains at least one substituted amino acid LL ₈ and/or w unsubstituted amino acids LL _P8 , and L2 represents

- 41 045708

Among the compounds of formula (IV), which are the object of the present invention, a group of compounds consists of compounds containing in L the sequence (AAs)w containing at least one substituted amino acid AA _s , wherein (AA _s )w is selected from the list:

- 42 045708

or

Among the compounds of formula (IV) which are the object of the present invention, a group of compounds consists of compounds with the following structure (beta-epoxy configuration):

Where

R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, n, RCG1 X, Y and L are defined in formula (IV).

All of these subgroups, taken alone or in combination, are part of the present invention.

- 43 045708

In accordance with at least one embodiment, the present invention relates to compounds of formula (IV), wherein

RCG1 is COOH,

L is one of the following compounds of formula (II):

- 44 045708

- 45 045708

- 46 045708

- 47 045708

Y represents NR ₁₁ -(C ₁ -C ₆ )alkyl, in which R11 represents a hydrogen atom, for example, Y represents NH-CH2;

R ₁ represents a (C ₁ -C ₆ )alkyl group, for example a methyl group;

R2 and _R3 independently represent a hydrogen atom or _a ( _CrC6 )alkyl group, for example a methyl group;

R4 and R5 independently represent a (C1- _C6 )alkyl group, for example a methyl group;

R ₇ and R ₈ independently represent a hydrogen atom or a (C ₁ -C ₆ )alkyl group, for example an isobutyl group or a neopentyl group, for example, one of R ₇ and R ₈ represents a ( _{C 6} )alkyl group , for example an isobutyl group or a neopentyl group, and the other of R ₇ and R ₈ represents a hydrogen atom;

X represents an oxygen atom or NH;

R9 represents two substituents selected from (CrC ₄ )alkoxy group, for example methoxy group, and a halogen atom, for example chlorine atom, for example R9 represents 3-Cl and 4-methoxy; And

R ₁₀ represents a hydrogen atom.

- 48 045708

Among the compounds of formula (IV) which are the object of the present invention, mention may be made in particular of the following compounds:

- 49 045708

- 50 045708

- 51 045708

- 52 045708

- 53 045708

- 54 045708

The present invention further relates to conjugates of formula (V):

Where

R1, R2, R3, R4, R5, _R6 , R7, _R8 , R9, _R10 , R11 and R12 are defined in formula (IV);

X, Y and L are defined in formula (IV);

- 55 045708

G represents the reaction product between RCG1, a reactive chemical group present at the end of the linker L of formula (II), and RCG2, an orthogonal reactive chemical group present on a polypeptide such as an antibody (Ab);

Ab is an antibody.

Attachment of a cryptophycin payload of formula (IV) to an antibody to produce a conjugate of formula (V) is achieved through a reaction between a reactive chemical group RCG1 present on the payload as defined above, which is reactive with a reactive chemical group RCG2 present on a polypeptide such like an antibody. The reaction between RCG1 and RCG2 allows the compound of formula (IV) to attach to the antibody through the formation of a covalent bond. In the conjugate of formula (V), the RCG1 and RCG2 portions may remain to provide linker attachment to the antibody.

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

(i) ε-amino groups of lysines located on the side chains of lysine residues that are present on the surface of the antibody;

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

(iii) hinge region saccharide groups;

(iv) cysteine thiols formed by reduction of intrachain disulfide bonds, or engineered cysteine thiols;

(v) amide groups located on the side chains of certain glutamine residues that are present on the surface of the antibody;

(vi) aldehyde groups introduced using formylglycine-forming enzyme.

Recently, other approaches to conjugation have been considered, for example, the introduction of cysteines through mutation (Junutula J.R., et al., Nature Biotechnology 2008, 26, 925-932), the introduction of unnatural amino acids, which allows the use of other types of chemical structures (Axup J.Y., et al. ., PNAS 2012, 109, 40, 16101-16106), OR conjugation using antibody glycans (Zhou Q., et al., Bioconjugate Chem. 2014, 25, 510-520). The use of cysteine-bridged dibromomaleimides (Behrens C.R., et al., Mol. Pharmaceutics 2015 3986-3998) and bis-sulfone reagents (Bryant P., et al., Mol. Pharmaceutics 2015 1872-1879) for cross-linking antibodies has also been described. , and it can be used in the present invention.

Another approach to site-specific modification of antibodies is based on enzymatic conjugation 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-forming enzyme (Hudak J.E., et al., Angew. Chem. Int. Ed. 2012, 51, 4161-4165). For a review of site-specific conjugation strategies, see Agarwal P. and Bertozzi C.R., Bioconjugate Chem 2015, 26, 176-192. These conjugation technologies can also be applied to the cryptophycin payloads described in the present invention.

A polypeptide, such as an antibody, can also be chemically modified to introduce new RCG2 reactive chemical groups. Thus, it is well known to those skilled in the art how to modify an antibody by introducing a modifying agent, for example, an activated disulfide, thiol, meleimilo-, haloacetamido-, azido-, alkyne or cycloalkyne groups (see in particular WO2005/ 077090, page 14 and WO2011/001052). The modification allows for improved conjugation reactions and a wider variety of RCG1 groups.

For example, if RCG1 is of type (ii) above, the antibody can be chemically modified using an appropriate modifying agent or one or more unnatural amino acids introduced into it so as to introduce the appropriate functional groups of RCG2. For example:

if RCG1 is an N-hydroxysuccinimidyl ester, RCG2 is a -NH2 group;

if RCG1 is a maleimido functional group, a haloacetamido functional group, a chlorine atom or an activated disulfide, RCG2 may be an SH group;

if RCG1 is an N3 group, RCG2 may be a C^CH functional group or an activated CAC functional group, such as a cyclooctyne moiety;

if RCG1 represents an OH group or an NH ₂ group, RCG2 may represent a carboxylic acid functional group or an amide functional group;

if RCG1 is an SH group, RCG2 may be a maleimido functional group, a haloacetamido functional group, or an activated disulfide functional group;

if RCG1 is a C^CH functional group or an activated CAC functional group, RCG2 may be an N3 group;

- 56 045708 if RCG1 is an O-alkylhydroxylamine functional group or a Pictet-Spengler reaction substrate, RCG2 may be an aldehyde or ketone functional group.

Examples of G that may be mentioned include

For example, G represents the following groups:

or for example

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which R ₁ represents (C ₁ -C6)alkyl, for example a methyl group.

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which each of R ₂ and R ₃ represents a hydrogen atom.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which one of R2 and _R3 represents a (C1-C6)alkyl group, for example a methyl group, and the other represents a hydrogen atom.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which R2 and _R3 form, together with the carbon atom to which they are attached, a (C ₃ -C6)cycloalkyl group, for example a cyclopropyl group.

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which R4 and R5 each represent a (C1-C6)alkyl group, in particular a methyl group.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which X represents an oxygen atom, that is, O.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which X represents NH.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which R ₇ and R 8 independently represent a hydrogen atom or a (C1-C6)alkyl group, for example an isobutyl group or a neopentyl group.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which R ₉ represents two substituents selected from a methoxy group and a chlorine atom. For example, the phenyl ring contains two substituents at positions 3 and 4 of the phenyl ring. For example, 3-Cl and 4-methoxy.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which R ₁₀ represents a hydrogen atom.

Among the compounds of formula (v) which are the object of the present invention, a group of compounds consists of compounds in which Y is located in the para position of the phenyl ring.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which Y represents NR ₁₁ -(C ₁ -C ₆ )alkyl, such as NR ₁₁ -(C ₁ -C ₃ )alkyl, for example NH-CH2.

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which L of formula (II) contains at least one substituted

- 57 045708 amino acid AAs in the sequence of w amino acids (AA)w, L1 and L2 are defined in formula (II).

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which L of formula (II) contains a sequence of w unsubstituted amino acids AA _ns , L1 and L2 as defined in formula (II).

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA _s , and L2 is (C1-Cb) an alkyl group, for example a (CH ₂ ) ₃ group;

a C(=O)-(C ₁ -C6)alkyl group, for example a C(=O)-(CH ₂ ) ₃ group; or a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, for example a (CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ group, in which A7 is as defined above.

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA _s , and L2 is (C1-C ₆ )alkyl group, for example a (CH ₂ ) ₃ group;

C(=O)-(C ₁ -C ₆ )alkyl group, for example the C(=O)-(CH ₂ ) ₃ group; or a (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, for example a -(CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ - group, in which A7 represents a -C group (=O)-(CH ₂ ) ₂ -C(=O)-NH-(Ch ₂ ) ₂ -SO ₃ H;

it should be understood that each A7 containing the SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which (AA)w in L of formula (II) contains w unsubstituted amino acids AAns, and L2 is (C ₁ -C ₆ )alkyl- An NA ₇ -(C ₁ -C ₆ )alkyl group, for example a (CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ group, in which A7 is defined above.

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which (AA)w in L of formula (II) contains w unsubstituted amino acids AA _ns and L2 is (C ₁ -C ₆ )alkyl -NA ₇ -(C ₁ -C ₆ )alkyl group, for example a (CH ₂ ) ₂ -NA ₇ -(CH ₂ ) ₂ group, in which A7 represents a C(=O)-[(CH ₂ ) ₂ - group O] _a -CH ₃ where a is an integer ranging from 1 to 50, for example, ranging from 1 to 24, such as 4, 7 and 24, for example, A7 represents the group C(=O )-[(CH ₂ ) ₂ -O] ₄ -CH ₃ , group C(=O)-[(CH ₂ ) ₂ -O] ₇ -CH ₃ or group C(=O) ^[(CH 2 ⁾ 2 ^{- O]} 24 ^-CH 3 ^; group -C(=O)-(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H or group C(=O)-(CH ₂ ) ₂ -C(=O) -NH-[(CH ₂ ) ₂ -O] _a -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H, where a is an integer ranging from 1 to 50, in a range such as from 1 to 24, for example 4, for example, A7 represents the group -C(=O)-(CH ₂ ) ₂ -C(=O)-NH-[(CH ₂ ) ₂ -O ] ₄ -(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ SO3H;

it should be understood that each A ₇ containing a SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AAAs and/or w unsubstituted amino acids AA _ns , and L2 represents an NA7(C1- _C6 )alkyl group, a ( _C1 - _{C6 )alkyl-NA7 group, NA7 -(CH2CH2O)i (C1-C6 ) alkyl group , a NA7aryl} group, NA ₇ -heteroaryl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-NA ₇ C(=O)- group (C ₁ -C ₆ )alkyl-(0cH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, (C ₁ -C) group ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group , group (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ group, C (=O)-NA7(CH ₂ CH ₂ O) _i (C ₁ -C ₆ )alkyl group, C(=O)-NA ₇ -aryl group, C(=O)-NA ₇ -heteroaryl group, C( =O)-(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-NA ₇ C group (=O)-(C ₁ C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, group c(=o)-(c ₁ C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , C(=O)- (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C1-C6)alkyl-NA7-(C1-C6)alkyl-(OCH2CH2)i group, C(=O)-(C1-C6)alkyl-(OCH2CH2)i-NA7-(C1C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, NA ₈ -(C ₁ - C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ ) group )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ C ₆ )alkyl group, NA ₈ -(C ₁ -C ₆ ) group )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ -C ₆ )alkylNA ₇ -(C ₁ -C ₆ )alkyl group , group NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , NA ₈ -(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) _i -NA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-NA ₇ group, C(=O)-NA ₈ (C ₁ - C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-Na ₈ -(C ₁ -C ₆ )alkyl-NA ₇ C(=O)- group (C ₁ C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , C(=O)-NA ₈ -(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈

- 58 045708 (Cl-C6)alkyl-C(=O)NL ₇ -(Cl-C6)alkyl-(OSH ₂ CH ₂ ) _i , C(=O)-KA ₈ -(C ₁ -C ₆ )alkyl -MA ₇ -(C ₁ -C ₆ )alkyl group, group C(=O)-NA ₈ -(Cl-C6)alkyl-NΛ ₇ -(Cl-C6)alkyl-(OSH ₂ CH ₂ ) _i or C (=O)-IA ₈ -(C ₁ -Cb)alkyl(OSH ₂ CH ₂ ) ₁ -MA ₇ -(C ₁ -C ₆ )alkyl group; where i, A7 and A ₈ are defined above.

Among the compounds of formula (V) that are the object of the present invention, a preferred group of compounds consists of compounds in which (ΛΛ)w in L of formula (II) contains at least one substituted amino acid LL _s and/or w unsubstituted amino acids AA _P8 . and L2 represents a KA7-(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )alkyl-HA ₇ group, NL ₇ -(CH ₂ CH ₂ O) _i (ClC ₆ )alkyl group, KA7-aryl group, KA7-heteroaryl group, (C ₁ -C ₆ )alkyl-HA ₇ C(=O)-(C ₁ C ₆ )alkyl group, (C1-Cb)alkyl-MA ₇ C(=O)-( C1-Cb)alkyl-(OCH _{2 CH2} ) _b ( _C1 - _C6 )alkyl-C(=O)KA7(C1- _C6 )alkyl group, (C1-Cb)alkyl-C(=O) group KA ₇ -(C1-Cb)alkyl-(OSH ₂ CH ₂ y (C1-Cb)alkyl-MA7(C1-C ₆ )alkyl group, (Cl-C ₆ )alkyl-NA ₇ -(Cl-C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , (C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ KA7-(C ₁ -C ₆ )alkyl group, C(=O)-KA7-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-KA ₇ group, C(=O)-NA ₇ -(CH ₂ CH ₂ O) _i (Cl-C ₆ )alkyl group, C(=O)-KA ₇ -aryl group, C(=O)-MA7heteroaryl group, C(=O)-(C ₁ -C ₆ )alkyl-KA7C(=O)-(C ₁ -C ₆ )alkyl group, group C(=O)-(C1C ₆ )alkyl-NA ₇ C(=O)-(C ₁ -C ₆ )alkyl-(OCH ₂ CH ₂ ) _i , C(=o)-(C ₁ -C ₆ )alkyl-C(=O)KA7-(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C(=O)NA ₇ -(C) group ₁ -C ₆ )alkyl-(oCH ₂ CH ₂ ) _i , C(=O)-(C ₁ -C ₆ )alkyl-KA ₇ -(C ₁ C ₆ )alkyl group, C(=O)-( group C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) _i , group NΛ ₈ -(C1C ₆ )alkyl-MA7, KA ₈ -(C ₁ -C ₆ ) alkyl-MA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, KA ₈ -(C ₁ -C ₆ )alkyl-C(=O)KA ₇ -(C ₁ C ₆ )alkyl group, NL ₈ -(Cl-C6)alkyl-NL ₇ -(Cl-C6)alkyl group, C(=O)-KA group ₈ -( _{C1- C6} )alkylKA7, C(=O)-KA ₈ -(C ₁ -C ₆ )alkyl-KA ₇ C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-KA ₈ -(C ₁ -C ₆ )alkyl-C(=O)KA ₇ (C1-C ₆ )alkyl group or C(=O)-KA ₈ -(C ₁ -C ₆ )alkyl-KA ₇ -(C ₁ -C ₆ )alkyl group; where i, A7 and A ₈ are defined above.

Among the compounds of formula (V) which are the object of the present invention, a more preferred group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA ₈ and/or w unsubstituted amino acids AA _P8 , and L2 represents

KA7-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-KA7 group, KA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, (Cl-C6) alkyl-NΛ ₇ C(=O)-(Cl-C6)alkyl group, (C ₁ -C ₆ )alkyl-KA7C(=O)-(C ₁ -C ₆ )alkyl(OCH ₂ CH ₂ ) _{1 group} , (C ₁ -C ₆ )alkyl-C(=O)KA7-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-C(=O)KA7-(C ₁ C ₆ )alkyl group -(OSH ₂ CH ₂ ) ₁ , (C1-C6)alkyl-NΛ ₇ -(Cl-C6)alkyl group, group (C ₁ -C ₆ )alkyl-KA7-(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , (Cl-C6)alkyl-(OSH ₂ CH ₂ ) _i -NΛ ₇ -(Cl-C6)alkyl group, C(=O)-NΛ ₇ -(C1C ₆ )alkyl group, C( group =O)-(C ₁ -C ₆ )alkyl-KA7, C(=O)-MA ₇ -(CH ₂ CH ₂ O) ₁ (C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-MA7C(=O)-(C ₁ -C ₆ )alkyl group, group C(=O)-(C ₁ -C ₆ )alkyl-KA ₇ C(=O)-(C ₁ C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C1-C ₆ )alkyl-C(=O)KA7-(C ₁ -C ₆ )alkyl group, c(=o) group -(Cr C ₆ )alkyl-C(=O)KA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ , C(=O)-(C ₁ -C ₆ )alkyl-KA7- (C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-MA ₇ -(C ₁ -C ₆ )alkyl-(OSH ₂ CH ₂ ) ₁ group, KA ₈ - group (C ₁ -C ₆ )alkyl-KA ₇ , KA ₈ -(C1-C ₆ )alkylKA7C(=O)-(C ₁ -C ₆ )alkyl group, KA ₈ -(C ₁ -C ₆ )alkyl-C (=O)KA ₇ -(C ₁ -C ₆ )alkyl group, NL ₈ -(C1C6)alkyl-NL ₇ -(Cl-C6)alkyl group, C(=O)-KA ₈ -(C ₁ -) group C ₆ )alkyl-KA ₇ , C(=O)-KA ₈ -(C ₁ -C ₆ )alkylKA7C(=O)-(C ₁ -C ₆ )alkyl group, C(=O)-NΛ ₈ -( Cl-C6)alkyl-C(=O)NΛ ₇ -(Cl-C6)alkyl group or C(=O)-MA ₈ -(C ₁ -C ₆ )alkyl-KA ₇ -(C ₁ -C ₆ ) an alkyl group; where ί, A7 and A ₈ are defined above.

Among the compounds of formula (V) which are the object of the present invention, an even more preferred group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA ₈ and/or w unsubstituted amino acids ΛΛ _l , _s , and L2 represents

KA7-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-KA7 group, (C ₁ -C ₆ )alkyl-KA ₇ C(=O)-(C ₁ -C ₆ )alkyl group , (C ₁ -C ₆ )alkyl-C(=O)KA7-(C ₁ -C ₆ )alkyl group, (C ₁ -C ₆ )alkyl-KA7-(C ₁ -C ₆ )alkyl group, C( =O)-KA ₇ -(C ₁ -C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-MA ₇ group, C(=O)-(C ₁ -C ₆ )alkyl -KA7C(=O)(C1-C ₆ )alkyl group, C(=O)-(C ₁ -C ₆ )alkyl-C(=O)KA7-(C ₁ -C ₆ )alkyl group, C(= O)-(C1- _C6 )alkylKA7-( _C1 - _{C6 )} alkyl group, _KA8- ( _C1 - _C6 )alkyl-KA7 group, _KA8- ( _C1 - _C6 )alkyl-MA ₇ C(=O)-( _{C1C6 )} alkyl group, KA ₈ -( _C1 - _C6 )alkyl-C(=O)KA ₇ -( _C1 - _C6 )alkyl group, KA ₈ -( C ₁ -C ₆ )alkyl-MA ₇ (C1-C ₆ )alkyl group, C(=O)-KA ₈ -(C ₁ -C ₆ )alkyl-KA ₇ group, C(=O)-MA ₈ - (C ₁ -C ₆ )alkyl-KA ₇ C(=O)-(C ₁ C ₆ )alkyl group, C(=O)-KA ₈ -(C ₁ -C ₆ )alkyl-C(=O)KA ₇ -(C ₁ -C ₆ )alkyl group or C(=O)-KA ₈ -(C1C ₆ )alkyl-KA ₇ -(C ₁ -C ₆ )alkyl group; where A7 and A ₈ are defined above.

Among the compounds of formula (V) which are the object of the present invention, the most preferred group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA ₈ and/or w unsubstituted amino acids LL _l , _s , and L2 represents a (C ₁ -C ₆ )alkyl-KA7-(C ₁ -C ₆ )alkyl group; where A7 is defined above.

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AA ₈ and/or w unsubstituted amino acids LL _l , _s , and L2 contains A ₇ representing the group C(=O)-[(CH2)2-O]4-CH ₃ ; group C(=O)-[(CH2)2-Ob-CH ₃ ; group C(=O)-[(CH2)2-O]24-CH ₃ ;

- 59 045708 group C(=O)-(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H; group C(=O)-(CH ₂ ) ₂ -C(=O)-NH-[(CH ₂ ) ₂ -O] ₄ -(CH ₂ ) ₂ -C(=O)NH-(CH2)2- SO3H;

it should be understood that each A ₇ containing a SO ₃ H functional group may be in salt forms, such as alkali metal salts, for example sodium salts (SO ₃ ^-+ Na).

Among the compounds of formula (V) which are the object of the present invention, the group of compounds consists of compounds in which (AA)w in L of formula (II) contains at least one substituted amino acid AAAs and/or w unsubstituted amino acids AAns, and L2 represents yourself

- 60 045708

Among the compounds of formula (V), which are the object of the present invention, a group of compounds consists of compounds containing in L the sequence (AA _s )w containing at least one substituted amino acid AAs, wherein (AA _s )w is selected from the list:

- 61 045708

O HE or

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds in which the Ab is an antibody to Epha2, for example the antibody hu2H11_R35R74 presented under SEQ ID NO: 1 (light chain of the antibody hu2H11_R35R74) and under SEQ ID NO : 2 (antibody heavy chain hu2H11_R35R74), which correspond to SEQ ID NO: 16 and SEQ ID NO:18, respectively, presented in WO2011039724 A1.

In accordance with at least one embodiment, the present invention relates to compounds of formula (V), wherein

Ab is an antibody;

G represents

or .

Y represents an NR ₁₁ -(C ₁ -6)alkyl group in which _RB represents a hydrogen atom, for example, Y represents an NH-CH ₂ group;

- 62 045708

L represents

- 63 045708

R1 represents a (C1- _C6 )alkyl group, for example a methyl group;

R2 and _R3 independently represent a hydrogen atom or a (C1- _C6 )alkyl group, for example a methyl group;

R ₄ and R 5 independently represent a (C1-C ₆ )alkyl group, for example a methyl group;

R ₇ and R ₈ independently represent a hydrogen atom or a (C1-C ₆ )alkyl group, for example an isobutyl group or a neopentyl group, for example one of R ₇ and R8 represents a (C1-C ₆ )alkyl group, for example an isobutyl group or a neopentyl group, and the other of R ₇ and R 8 represents a hydrogen atom;

X represents an oxygen atom or NH;

R9 represents two substituents selected from a (C1- _C4 )alkoxy group, for example a methoxy group, and a halogen atom, for example a chlorine atom, for example, R9 represents 3-Cl and 4-methoxy; And

R ₁₀ represents a hydrogen atom.

- 64 045708

Among the compounds of formula (V) which are the object of the present invention, a group of compounds consists of compounds with the following structure (beta-epoxy configuration):

Where

R1, R2, R3, R4, R5, R6, R7, _R8 , R9, _R10 , R11, R12, n, RCG1, X, Y and L are defined in formula (IV).

All of these subgroups, taken alone or in combination, are part of the present invention.

Among the compounds of formula (V) which are the subject of the present invention, mention may be made of the following compounds:

- 65 045708

-66045708

where Ab is defined in formula (V) of the present invention.

In accordance with the present invention, compounds of general formula (I), (II), (III), (IV) and (V) can be prepared using the following methods.

- 67 045708

Preparation of linkers of formula (II).

Preparation of spacers with improved hydrophilicity.

Scheme 1

Step (i): introducing a carboxylic acid protecting group using an allyl bromide and a base such as, for example, cesium carbonate to produce an allyl ester.

Step (ii): Removal of the Boc protecting group from the amine using an HCl solution (eg dioxane solution) or TFA.

Step (iii): addition of the amine to the tert-butyl acrylate in the presence of a base such as, for example, DIEA.

Step (iv): coupling of the amine with an activated carboxylic acid, such as an NHS ester or acyl chloride, in the presence of a base such as, for example, DIEA.

Step (v): deprotection of the tert-butyl ester using an HCl solution (eg dioxane solution) or TFA.

Step (vi): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA.

Stage (vii): combination with succinic anhydride.

Step (viii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA and coupling with an amine.

Scheme 1 shows the synthesis starting from Boc-β-Ala-OH (CAS number [3303-84-2]), however other Boc-protected alkylamino acids that are commercially available can also be used if n is in the range of 3 to 10. It depicts a synthesis using succinic anhydride, but glutaric anhydride or alkyl diacids, which are commercially available, can also be used when n is in the range of 3 to 10.

Combination of spacers with improved hydrophilicity and classic dipeptides.

- 68 045708

Scheme 2

linker 2

Step (i): peptide coupling using coupling reagents such as, for example, EDC and HOBt, and deprotection of the Fmoc group from the amine in the presence of a base, such as, for example, pi peridine.

Step (ii): for spacer 3, activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA, and coupling of spacers 3 and 4 with the dipeptide.

Step (iii): deprotection of the tert-butyl ester using an HCl solution (eg dioxane solution) or TFA.

Step (iv): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA and coupling with an amine.

Step (v): Removal of the Boc protecting group from the amine using an HCl solution (eg dioxane solution) or TFA.

Scheme 2 depicts the synthesis of linkers using the dipeptide Val-Ala, but other dipeptides can also be used; it shows synthesis using spacers 3 or 4, but spacers 1 or 2 can also be used; It depicts a synthesis using Boc-mono-protected ethylenediamine, but other Boc-mono-protected diamines that are commercially available can also be used when n is in the range of 3 to 10.

Preparation of dipeptides with improved hydrophilicity.

Preparation of dipeptides with improved hydrophilicity from amino acids containing a carboxylic acid group on their side chain

Scheme 3

dipeptide 1

- 69 045708

Scheme 4

Step (i): peptide coupling between a carboxylic acid and an amine using coupling reagents such as, for example, EDC and HOBt.

Step (ii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA.

Step (iii): peptide coupling between an NHS ester and an amine in the presence of a base such as, for example, sodium bicarbonate or DIEA.

Step (iv): deprotection of the tert-butyl ester using an HCl solution (eg dioxane solution) or TFA.

Step (v): peptide coupling of the dipeptide with Boc-mono-protected ethylenediamine using a coupling reagent such as, for example, T3P.

Step (vi): Removal of the Boc protecting group from the amine using an HCl solution (eg dioxane solution) or TFA.

Step (vii): peptide coupling of the dipeptide with p-aminobenzyl alcohol using coupling reagents such as, for example, EEDQ.

Step (viii): activation of benzyl alcohol as p-nitrophenyl carbonate by treatment with p-nitrophenyl chloroformate in the presence of a base such as eg DIEA.

Step (ix): formation of a carbamate between the activated alcohol and the amine in the presence of a base such as for example DIEA.

Scheme 3 shows the synthesis of dipeptides starting from Val, however, other amino acids listed above can be used; it shows a synthesis using L-Glu tert-butyl ester (CAS number [45120-30-7]), but other amino acids containing a carboxylic acid group on their side chain, such as tert-butyl ester, can also be used L-Asp ester (CAS number [4125-93-3]), D-Asp tert-butyl ester (CAS number [148823-36-3]), D-Glu mpem-butyl ester (CAS number [25456- 76-2]), 2-aminohexanedioic acid 1-tert-butyl ester (CAS number [1245806-58-9]) or 2-aminoheptanedioic acid 1-mpem-butyl ester (CAS number [1888498-03-0]). Scheme 4 shows the synthesis of dipeptides using p-aminobenzyl alcohol (CAS number [623-04-1]), however other aminobenzyl alcohol compounds that are commercially available can also be used, such as 4-(1-hydroxyethyl) -aniline (racemic (CAS number [14572-89-5]) or enantiomerically pure (R) (CAS number [210754-25-9]) or (S) (CAS number [500229-84-5])), 4 -amino-a,a-dimethylbenzenemethanol (CAS number [23243-04-1]), 4-amino-a-methoxy-a-methylbenzenemethanol (CAS number [1379318-81-6]), 4-amino-a-methyl-a -trifluoromethylbenzenemethanol (CAS number [851652-56-7]), 2-aminobenzenemethanol (but

- 70 045708 CAS measures [5344-90-1]), 2-amino-a-methylbenzenemethanol (racemic (CAS number [10517-50-7]) or enantiomerically pure (R) (CAS number [3205-21-8] ) or (S) (CAS number [3205-21-8])), 6-amino-3-pyridinemethanol (CAS number [113293-71-3]), 6-amino-a-methyl-3-pyridinemethanol (CAS number [ 133505483-5]), 6-amino-a-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]), 6-amino-a,a-dimethyl-3-pyridinemethanol (CAS number [843646-03-8] ), 5-amino-3-pyridinemethanol (CAS number [873651-92-4]), 2-amino-3-pyridinemethanol (CAS number [23612-57-9]), 2-amino-a-methyl-3-pyridinemethanol ( racemic (CAS number [869567-91-9]) or enantiomerically pure (R) (CAS number [936718-01-3]) or (S) (CAS number [936718-00-2])), 2-amino- a-ethyl-3-pyridinemethanol (CAS number [914223-90-8]), 2-amino-a,a-dimethyl-3pyridinemethanol (CAS number [213666-96-7]), 3-amino-4-pyridinemethanol ( CAS No. [152398-05-5]), 3-amino-a-methyl-4-pyridinemethanol (CAS No. [1242470-88-7]), 3-amino-a,a-methyl-4-pyridinemethanol (CAS No. [13357] -81-8]), 4-amino-3-pyridinemethanol (CAS number [138116-34-4]), 4-amino-a-methyl-3pyridinemethanol (CAS number [741223-49-4]), 4-amino -a,a-methyl-3-pyridinemethanol (CAS number [1339013-26-1]), 3-amino-2-pyridinemethanol (CAS number [52378-63-9]), 3-amino-a-methyl-2pyridinemethanol (CAS number [954240-54-1]), 3-amino-a,a-methyl-2-pyridinemethanol (CAS number [89943857-4]); It depicts a synthesis using Boc-mono-protected ethylenediamine, but other Boc-mono-protected diamines that are commercially available can also be used when n is in the range of 3 to 10.

Preparation of dipeptides with improved hydrophilicity based on amino acids containing an amino group on their side chain.

Scheme 5

Or, alternatively, dipeptide 5 can be prepared according to the synthesis described in Scheme 6.

- 71 045708

Scheme 7

Scheme 6

NHBoc

NH ₂

CIH

ABOUT

Step (i): peptide coupling between a carboxylic acid and an amine using coupling reagents such as, for example, EDC and HOBt.

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Step (ii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA.

Step (iii): peptide coupling between an NHS ester and an amine in the presence of a base such as, for example, sodium bicarbonate or DIEA.

Step (iv): Removal of the Boc protecting group from the amine using an HCl solution (eg dioxane solution) or TFA.

Step (v): peptide coupling of the dipeptide with Boc-mono-protected ethylenediamine using a coupling reagent such as, for example, T3P.

Step (vi): peptide coupling of the dipeptide with p-aminobenzyl alcohol using coupling reagents such as, for example, EEDQ.

Step (vii): activation of benzyl alcohol as p-nitrophenyl carbonate by treatment with p-nitrophenyl chloroformate in the presence of a base such as eg DIEA.

Step (viii): formation of a carbamate between the activated alcohol and the amine in the presence of a base such as DIEA.

Schemes 5 and 6 depict the synthesis of dipeptides starting from Val, however, other amino acids listed above can be used. Scheme 5 shows a synthesis using Boc-protected side chain L-Lys (CAS number [2418-95-3]), but other amino acids containing an amino group on their side chain butoxycarbonylamino)propanoic acid butoxycarbonylamino)propanoic acid butoxycarbonylamino can also be used )butanoic acid (S)-3-amino-2-(tert(S)-3-amino-2-(tert(S)-4-amino-2-(tert(R)-4-amino-2-( tertiary chains such as, for example, (CAS No. [73259-81-1]), (CAS No. [76387-70-7]), (CAS No. [25691-37-6]), butoxycarbonylamino)butanoic acid (CAS No. [80445-78-9]), Boc side chain protected L-ornithine (CAS number [21887-64-9]), Boc side chain protected D-ornithine (CAS number [159877-12-0]) , Boc-protected on the D-Lys side chain (CAS number [106719-44-2]), (2S)-2-amino-7[[(1,1-dimethylethoxy)carbonyl]amino]heptanoic acid (CAS number [ 1142814-17-2]) or (2R)-2-amino7-[[(1,1-dimethylethoxy)carbonyl]amino]heptanoic acid (CAS number [117833-90-6]). Scheme 7 shows the synthesis of dipeptides with using p-aminobenzyl alcohol (CAS number [623-04-1]), however other aminobenzyl alcohol compounds that are commercially available can also be used, such as, for example, 4-(1-hydroxyethyl)-aniline (racemic (CAS number [14572-89-5]) or enantiomerically pure (R) (CAS number [210754-25-9]) or (S) (CAS number [500229-84-5])), 4-aminoα,α-dimethylbenzenemethanol ( CAS No. [23243-04-1]), 4-amino-a-methoxy-a-methylbenzenemethanol (CAS No. [1379318-81-6]), 4-amino-a-methyl-a-trifluoromethylbenzenemethanol (CAS No. [851652] -56-7]), 2-aminobenzenemethanol (CAS number [5344-90-1]), 2-amino-a-methylbenzenemethanol (racemic (CAS number [10517-50-7]) or enantiomerically pure (R) (CAS number [ 3205-21-8]) or (S) (CAS number [3205-218])), 6-amino-3-pyridinemethanol (CAS number [113293-71-3]), 6-amino-a-methyl-3 -pyridinemethanol (CAS number [1335054-83-5]), 6-amino-a-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]), 6-amino-a,adimethyl-3-pyridinemethanol (CAS number CAS [843646-03-8]), 5-amino-3-pyridinemethanol (CAS no.

[873651-92-4]), 2-amino-3-pyridinemethanol (CAS number [23612-57-9]), 2-amino-a-methyl-3pyridinemethanol (racemic (CAS number [869567-91-9]) or enantiomerically pure (R) (CAS No. [936718-01-3]) or (S) (CAS No. [936718-00-2])), 2-amino-a-ethyl-3-pyridinemethanol (CAS No. [914223] -90-8]), 2-amino-a,a-dimethyl-3-pyridinemethanol (CAS number [213666-96-7]), 3-amino-4pyridinemethanol (CAS number [152398-05-5]), 3 -amino-a-methyl-4-pyridinemethanol (CAS number [124247088-7]), 3-amino-a,a-methyl-4-pyridinemethanol (CAS number [13357-81-8]), 4-amino-3 -pyridinemethanol (CAS number [138116-34-4]), 4-amino-a-methyl-3-pyridinemethanol (CAS number [741223-49-4]), 4-aminoa,a-methyl-3-pyridinemethanol (CAS number CAS [1339013-26-1]), 3-amino-2-pyridinemethanol (CAS number [52378-63-9]), 3-amino-a-methyl-2-pyridinemethanol (CAS number [954240-54-1] ), 3-amino-a,a-methyl-2pyridinemethanol (CAS number [899438-57-4]); It depicts a synthesis using Boc-mono-protected ethylenediamine, but other Boc-mono-protected diamines that are commercially available can also be used when n is in the range of 3 to 10.

Preparation of cryptophycin compounds.

Cryptophycin compounds can be prepared as described in WO2011/001052 for cases where X=O and PCT/EP2016/076603 for cases where X=NH.

Preparation of new payloads with cryptophycin of formula (IV) for cases where Y=NH-CH ₂ .

Generation of new cryptophycin payloads containing a spacer with improved hydrophilicity.

Without PABA fragment.

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Scheme 8

Step (i): peptide coupling between a cryptophycin derivative and a dipeptide using coupling reagents such as, for example, EDC and HOBt.

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

Step (iii): peptide coupling with spacer 1 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 2 in the presence of a base, such as, for example, DIEA.

Step (iv): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium.

Step (v): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA.

Alternatively, novel cryptophycin payloads containing a spacer with improved hydrophilicity can also be prepared as depicted in Scheme 9.

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Scheme 9

Step (i): activation of the carboxylic acid of linker 1 as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA and coupling with cryptophycinamine.

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

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

Schemes 8 and 9 depict the synthesis of payloads using C52 p-benzylamine, but other cryptophycin compounds can also be used; they depict synthesis using Val-Ala dipeptides, but other dipeptides can also be used; They depict synthesis using spacers 1 or 2, but spacers 3 or 4 can also be used.

With a RABA fragment.

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Scheme 10

Step (i): peptide coupling between Fmoc-Val-Ala-OH and 4-aminobenzyl alcohol in the presence of a coupling reagent such as, for example, EEDQ.

Step (ii): activation of benzyl alcohol as p-nitrophenyl carbonate by treatment with p-nitrophenyl chloroformate in the presence of a base such as eg DIEA.

Step (iii): formation of a carbamate between the activated alcohol and cryptophycinamine in the presence of a base such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (iv): peptide coupling with spacer 1 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 2 in the presence of a base, such as, for example, DIEA.

Step (v): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium.

Step (vi): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA.

Scheme 10 depicts the synthesis of payloads using Val-Ala dipeptides, but other dipeptides can also be used; it depicts a synthesis using p-aminobenzyl alcohol (CAS number [623-04-1]), but other aminobenzyl alcohol compounds that are commercially available, such as 4-(1-hydroxyethyl)aniline, can also be used (racemic (CAS number [14572-89-5]) or enantiomerically pure (R) (CAS number [210754-25-9]) or (S) (CAS number [500229-84-5])), 4-amino -a,a-dimethylbenzenemethanol (CAS number [23243-04-1]), 4-amino-amethoxy-a-methylbenzenemethanol (CAS number [1379318-81-6]), 4-amino-a-methyl-a-trifluoromethylbenzenemethanol (CAS No. [851652-56-7]), 2-aminobenzenemethanol (CAS No. [5344-90-1]), 2-amino-amethylbenzenemethanol (racemic (CAS No. [10517-50-7]) or enantiomerically pure (R ) (CAS No. [3205-21-8]) or (S) (CAS No. [3205-21-8])), 6-amino-3-pyridinemethanol (CAS No. [113293-71-3]), 6-amino- a-methyl-3-pyridinemethanol (CAS number [1335054-83-5]), 6-amino-a-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]), 6-amino-a,a- dimethyl-3-pyridinemethanol (CAS No. [843646-03-8]), 5-amino3-pyridinemethanol (CAS No. [873651-92-4]), 2-amino-3-pyridinemethanol (CAS No. [23612-57-9] ]), 2amino-a-methyl-3-pyridinemethanol (racemic (CAS number [869567-91-9]) or enantiomerically pure

- 76 045708 (R) (CAS number [936718-01-3]) or (S) (CAS number [936718-00-2])), 2-amino-a-ethyl-3-pyridinemethanol (CAS number [914223] -90-8]), 2-amino-a,a-dimethyl-3-pyridinemethanol (CAS number [213666-96-7]), 3-amino-4-pyridinemethanol (CAS number [152398-05-5]), 3 -amino-a-methyl-4-pyridinemethanol (CAS number [1242470-88-7]), 3-amino-a,a-methyl-4-pyridinemethanol (CAS number [13357-81-8]), 4-amino -3-pyridinemethanol (CAS number [138116-34-4]), 4-amino-a-methyl-3-pyridinemethanol (CAS number [74122349-4]), 4-amino-a,a-methyl-3-pyridinemethanol (CAS number CAS [1339013-26-1]), 3-amino-2-pyridinemethanol (CAS number [52378-63-9]), 3-amino-a-methyl-2-pyridinemethanol (CAS number [954240-54-1] ), 3-amino-a,amethyl-2-pyridinemethanol (CAS number [899438-57-4]); it depicts a synthesis using pbenzylamine C52, but other cryptophycin compounds can also be used; It shows synthesis using spacers 1 or 2, but spacers 3 or 4 can also be used.

Preparation of new payloads with cryptophycin of formula (IV) containing dipeptides with improved hydrophilicity.

Without PABA fragment.

Scheme 11

Step (i): peptide coupling between a cryptophycin derivative and a dipeptide using coupling reagents such as, for example, EDC and HOBt, and deprotection of the Fmoc group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): combination with glutaric anhydride; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iii).

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

Scheme 11 depicts the synthesis of payloads using C52 p-benzylamine, but other cryptophycin compounds can also be used; it depicts a synthesis using dipeptide 1, but dipeptide 5 can also be used; It depicts a synthesis using glutaric anhydride, but succinic anhydride or alkyl diacids, which are commercially available, can also be used when n is in the range of 3 to 10.

With a RABA fragment.

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Scheme 12

Step (i): formation of a carbamate between the activated alcohol and cryptophycinamine in the presence of a base, such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): combination with glutaric anhydride; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iii).

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

Scheme 12 depicts the synthesis of payloads using C52 p-benzylamine, but other cryptophycin compounds can also be used; it depicts a synthesis using dipeptide 6, but dipeptide 2 can also be used.

Generation of new cryptophycin payloads of formula (IV) containing both a spencer and a dipeptide with improved hydrophilicity.

Without PABA fragment.

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Scheme 13

Step (i): peptide coupling between a cryptophycin derivative and a dipeptide using coupling reagents such as, for example, EDC and HOBt, and deprotection of the Fmoc group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): peptide coupling with spacer 3 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 4 in the presence of a base, such as, for example, DIEA.

Step (iii): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iv).

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

Scheme 13 depicts the synthesis of payloads using C52 p-benzylamine, but other cryptophycin compounds can also be used; it depicts a synthesis using dipeptide 5, but dipeptide 1 can also be used; It shows synthesis using spacers 3 or 4, but spacers 1 or 2 can also be used.

With a RABA fragment.

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Scheme 14

Step (i): formation of a carbamate between the activated alcohol and cryptophycinamine in the presence of a base, such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): peptide coupling with spacer 3 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 4 in the presence of a base, such as, for example, DIEA.

Step (iii): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iv).

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

Scheme 14 depicts the synthesis of payloads using C52 p-benzylamine, but other cryptophycin compounds can also be used; it shows a synthesis using dipeptide 6, but dipeptide 2 can also be used; It shows synthesis using spacers 3 or 4, but spacers 1 or 2 can also be used.

Preparation of new payloads with cryptophycin of formula (IV) for cases where Y=O-CH ₂ or S-CH2.

Generation of new cryptophycin payloads containing a spacer with improved hydrophilicity.

Without PABA fragment.

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Scheme 15

Step (i): formation of a carbamate between the activated alcohol cryptophycin and the amine in the presence of a base such as for example DIEA.

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

Step (iii): peptide coupling with spacer 1 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 2 in the presence of a base, such as, for example, DIEA.

Step (iv): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium.

Step (v): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA.

Scheme 15 depicts the synthesis of payloads using C52 activated p-benzyl alcohol, but other cryptophycin compounds can also be used; it depicts a synthesis using Val-Ala dipeptides, but other dipeptides can also be used; it depicts a synthesis using Boc-mono-protected ethylenediamine, however other Boc-mono-protected diamines that are commercially available can also be used when n is in the range from 3 to 10; It shows synthesis using spacers 1 or 2, but spacers 3 or 4 can also be used.

Alternatively, novel cryptophycin payloads of formula (IV) containing a spacer with improved hydrophilicity can also be prepared as depicted in Scheme 16.

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Scheme 16

Step (i): formation of a carbamate between the activated alcohol cryptophycin and linker 2 in the presence of a base such as eg DIEA.

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

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

Scheme 16 depicts the synthesis of payloads using activated C52 p-benzyl alcohol, but other cryptophycin compounds can also be used.

With a RABA fragment.

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Scheme 17

Step (i): peptide coupling between Fmoc-Val-Ala-OH and 4-aminobenzyl alcohol in the presence of a coupling reagent such as, for example, EEDQ.

Step (ii): activation of benzyl alcohol as p-nitrophenyl carbonate by treatment with p-nitrophenyl chloroformate in the presence of a base such as eg DIEA.

Step (iii): formation of a carbamate between the activated alcohol and Boc-mono-protected ethylenediamine in the presence of a base such as eg DIEA.

Step (iv): Removal of the Boc protecting group from the amine using an HCl solution (eg dioxane solution) or TFA.

Step (v): formation of a carbamate between the activated alcohol cryptophycin and the amine in the presence of a base, such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (vi): peptide coupling with spacer 1 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 2 in the presence of a base, such as, for example, DIEA.

Step (vii): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium.

Step (viii): activation of the carboxylic acid as an NHS ester by treatment with DSC in the presence of a base such as eg DIEA.

Scheme 17 depicts the synthesis of payloads using Val-Ala dipeptides, however other dipeptides can also be used; it depicts a synthesis using p-aminobenzyl alcohol (CAS number [623-04-1]), but other aminobenzyl alcohol compounds that are commercially available, such as 4-(1-hydroxyethyl)aniline, can also be used (racemic (CAS number [14572-89-5]) or enantiomerically pure (R) (CAS number [210754-25-9]) or (S) (CAS number [500229-84-5])), 4-amino -a,a-dimethylbenzenemethanol (CAS number [23243-04-1]), 4-amino-amethoxy-a-methylbenzenemethanol (CAS number [1379318-81-6]), 4-amino-a-methyl-a-trifluoromethyl

- 83 045708 benzenemethanol (CAS number [851652-56-7]), 2-aminobenzenemethanol (CAS number [5344-90-1]), 2-amino-amylbenzenemethanol (racemic (CAS number [10517-50-7]) or enantiomerically pure (R) (CAS No. [3205-21-8]) or (S) (CAS No. [3205-21-8])), 6-amino-3-pyridinemethanol (CAS No. [113293-71-3] ), 6-amino-a-methyl-3-pyridinemethanol (CAS number [1335054-83-5]), 6-amino-a-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]), 6-amino- a,a-dimethyl-3-pyridinemethanol (CAS number [843646-03-8]), 5-amino3-pyridinemethanol (CAS number [873651-92-4]), 2-amino-3-pyridinemethanol (CAS number [23612] -57-9]), 2amino-a-methyl-3-pyridinemethanol (racemic (CAS number [869567-91-9]) or enantiomerically pure (R) (CAS number [936718-01-3]) or (S) (CAS number [936718-00-2])), 2-amino-a-ethyl-3-pyridinemethanol (CAS number [914223-90-8]), 2-amino-a,a-dimethyl-3-pyridinemethanol ( CAS No. [213666-96-7]), 3-amino-4-pyridinemethanol (CAS No. [152398-05-5]), 3-amino-a-methyl-4-pyridinemethanol (CAS No. [1242470-88-7]) , 3-amino-a,a-methyl-4-pyridinemethanol (CAS number [13357-81-8]), 4-amino-3pyridinemethanol (CAS number [138116-34-4]), 4-amino-a-methyl -3-pyridinemethanol (CAS number [74122349-4]), 4-amino-a,a-methyl-3-pyridinemethanol (CAS number [1339013-26-1]), 3-amino-2-pyridinemethanol (CAS number [ 52378-63-9]), 3-amino-a-methyl-2-pyridinemethanol (CAS number [954240-54-1]), 3-amino-a,amethyl-2-pyridinemethanol (CAS number [899438-57- 4]); it depicts a synthesis using Boc-mono-protected ethylenediamine, but other Boc-mono-protected diamines that are commercially available can also be used when n is in the range from 3 to 10; it depicts a synthesis using activated p-benzyl alcohol C52, but other cryptophycin compounds can also be used; It shows synthesis using spacers 1 or 2, but spacers 3 or 4 can also be used.

Preparation of new payloads with cryptophycin of formula (IV) containing dipeptides with improved hydrophilicity.

Without PABA fragment.

Scheme 18

Step (i): formation of a carbamate between the activated alcohol cryptophycin and the dipeptide in the presence of a base, such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): combination with glutaric anhydride; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iii).

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

Scheme 18 shows the synthesis of payloads using activated p-benzyl

- 84 045708 C52 alcohol, however, other cryptophycin compounds can also be used; it depicts a synthesis using dipeptide 7, but dipeptide 3 can also be used; it shows a synthesis using glutaric anhydride, but succinic anhydride or alkyl diacids, which are commercially available, can also be used if n is in the range of 3 to 10. With PABA moiety

Scheme 19

dipeptide 8 I®

HNyR ₂₆ o

Step (i): formation of a carbamate between the activated alcohol cryptophycin and the dipeptide in the presence of a base, such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): combination with glutaric anhydride; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iii).

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

Scheme 19 depicts the synthesis of payloads using activated C52 p-benzyl alcohol, but other cryptophycin compounds can also be used; it shows a synthesis using dipeptide 8, but dipeptide 4 can also be used; It depicts a synthesis using glutaric anhydride, but succinic anhydride or alkyl diacids, which are commercially available, can also be used when n is in the range of 3 to 10.

Generation of new cryptophycin payloads of formula (IV) containing both a spencer and a dipeptide with improved hydrophilicity.

Without PABA fragment.

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Scheme 20

Step (i): formation of a carbamate between the activated alcohol cryptophycin and the dipeptide in the presence of a base, such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): peptide coupling with spacer 3 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 4 in the presence of a base, such as, for example, DIEA.

Step (iii): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iv).

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

Scheme 20 depicts the synthesis of payloads using C52 p-benzylamine, but other cryptophycin compounds can also be used; it depicts a synthesis using dipeptide 7, but dipeptide 3 can also be used; It shows synthesis using spacers 3 or 4, but spacers 1 or 2 can also be used.

With a RABA fragment.

- 86 045708

Scheme 21

Step (i): formation of a carbamate between the activated alcohol cryptophycin and the dipeptide in the presence of a base, such as, for example, DIEA, and removal of the Fmoc protecting group from the amine in the presence of a base, such as, for example, piperidine.

Step (ii): peptide coupling with spacer 3 using coupling reagents such as, for example, EDC and HOBt, or coupling with spacer 4 in the presence of a base, such as, for example, DIEA.

Step (iii): deprotection of the allylic ester in the presence of a catalyst such as, for example, tetrakis(triphenylphosphine)palladium; one additional step of removing the protecting group from the side chain of the substituted amino acid may be necessary before step (iv).

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

Scheme 21 depicts the synthesis of payloads using C52 p-benzylamine, but other cryptophycin compounds can also be used; it shows a synthesis using dipeptide 8, but dipeptide 4 can also be used; It shows synthesis using spacers 3 or 4, but spacers 1 or 2 can also be used.

Preparation of conjugates of formula (V).

The conjugates of formula (V) of the present invention can be prepared by a process comprising at least the steps of:

(i) bringing into contact and causing the reaction:

an optionally buffered aqueous solution of an antibody, optionally modified with a modifying agent, and a solution of a cryptophycin payload of formula (IV) as defined herein, wherein the chemical group RCG1 in the cryptophycin payload of formula (IV) is reactive with the chemical group RCG2 present on a polypeptide such as an antibody, especially with respect to amino groups present on antibodies, wherein said chemical groups of RCG2 have been introduced, if necessary, by means of a modifying agent, so as to attach the cryptophycin payload of formula (IV) to the antibody through

- 87 045708 formation of covalent bonds;

(ii) and then optionally separating the conjugate of formula (V) formed in step (i) from the cryptophycin payload of formula (IV), and/or from the unreacted antibody, and/or from any aggregates that may have formed.

In one embodiment, for example, in step (ii), the conjugate of formula (V) from step (i) is separated only from the unreacted cryptophycin payload of formula (IV) and any aggregates formed, and any unreacted antibody remains in solution.

The purpose of contacting is to cause a reaction between the chemical groups of RCG1 and RCG2 to cause the cryptophycin payload of formula (IV) to attach to a polypeptide, such as an antibody, by forming a covalent bond; for example, if RCG1 is RaZa-C(=O), then the reaction preferentially occurs on functional amino groups of the antibody, such as the ε-amino groups found on the side chains of the lysine (Lys) residues of the antibody, and the α-amino groups of the N-terminal amino acids of heavy and antibody light chains. In this case, a conjugate is obtained with the following formula: mAb-[NH-C(=O)-L*-Y-Crypto] _d , wherein L* is a fragment of linker L containing the RaZa-C(=O) group as RCG1 , and d represents the drug-to-antibody ratio, or DAR;

if RCG1 is a chlorine atom or a maleimido or haloacetamido group, the antibody may contain thiol chemical groups;

if RCG1 is an azido group, the antibody may contain a C^CH moiety or an activated triple bond, such as a cyclooctyne group;

if RCG1 is an NH ₂ group, the reaction can occur on the amide functional group of the antibody using enzymatic catalysis, such as amide groups found on the side chains of glutamine (Gln) residues of the antibody. In this case, a conjugate is obtained with the following formula: mAb-[C(=O)-NH-L*-Crypto] _d , wherein L* is a fragment of the linker L, which contains the NH2 group as RCG1, and thus L represents the L*NH ₂ group, and d represents the drug-antibody ratio, or DAR;

if RCG1 is a C^CH group or an activated C^C group such as a cyclooctyne moiety, the antibody may contain azido groups.

The term aggregates refers to associates that can form between two or more antibodies, where the antibodies may have been modified by conjugation. Aggregates can form under the influence of a wide variety of parameters, such as high concentration of antibody in solution, pH of solution, high shear forces, amount of grafted drugs and their hydrophobic nature, temperature (see references given in the introduction J. Membrane Sci. 2008, 318 , 311-316), however, the influence of some of them has not been clearly established. In the case of proteins or antibodies, reference may be made to AAPS Journal, Protein Aggregation and Bioprocessing 2006, 8(3), E572-E579. The aggregate content can be determined using known techniques such as SEC (see in this regard Analytical Biochemistry 1993, 212 (2), 469-480).

The aqueous antibody solution can be buffered with buffers, such as potassium phosphate, or HEPES, or a mixture of buffers, such as buffer A, described below. The buffer depends on the nature of the antibody. The cryptophycin payload of formula (IV) is dissolved in a polar organic solvent such as DMSO or DMA.

The reaction occurs at a temperature that is generally in the range from 20°C to 40°C. The reaction time may range from 1 to 24 hours. The reaction between the antibody and the cryptophycin payload of formula (IV) can be monitored by SEC using a refractometric and/or ultraviolet detector and/or HRMS to determine its extent. If the degree of substitution is insufficient, the reaction can be prolonged and/or a cryptophycin compound can be added. For more information regarding specific terms, please refer to the Examples section. Specific embodiments are described in Examples 3, 6, 16,19, 23, 26, 29, 32, 35 and 41.

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

After step (i) or (ii), the conjugate solution can be subjected to step (iii) ultrafiltration and/or diafiltration. Thus, after these steps, the conjugate is obtained in aqueous solution.

Antibody.

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

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In one embodiment, the antibody is a monospecific antibody, i.e. the antibody specifically binds to only one target. Alternatively, it may be a multispecific antibody.

In one embodiment, the antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody.

The antibody in accordance with the present invention specifically binds to a target, thereby directing the biologically active compound as a cytotoxic compound towards said target. As used herein, the expressions specifically binds, or specifically binds to, or binds to, or the like. mean that the antibody or antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (KD) of at least about 1x10 ^-8 M or less (eg, a lower KD value indicates stronger binding). Methods for determining the presence of specific binding between two molecules are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been characterized, for example, by their specific binding to a target and/or target antigen using surface plasmon resonance, for example BIACORE™.

The target typically corresponds to a protein that is expressed on the surface of cells, for example, a protein that is expressed on the surface of tumor cells.

In one embodiment, the target is an EphA2 receptor. The EphA2 receptor is an ephrin receptor and is also called Eph receptor A2 or epithelial cell receptor tyrosine protein kinase. An antibody that specifically binds to the EphA2 receptor, for example, may correspond to one of the antibodies described in WO2008/010101 or WO2011/039724.

The antibody may optionally be modified with a modifying agent to facilitate the attachment of the payload to cryptophycin, as previously described. In particular, the antibody may 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 EphA2 receptor antagonist antibody. The sequence of hu2H11R3574 is provided under SEQ ID NO: 1 (antibody light chain hu2H11_R35R74) and SEQ ID NO:2 (antibody heavy chain hu2H11_R35R74), which correspond to SEQ ID NO: 16 and SEQ ID NO:18, respectively, presented in WO2011039724 A1.

Conjugate.

The conjugate typically contains from about 1 to 10 cryptophycin compounds covalently attached to the antibody (this represents the degree of grafting, or drug-to-antibody ratio, or DAR). This number varies depending on the nature of the antibody and cryptophycin compound, as well as the operating conditions used in the conjugation process (eg, number of cryptophycin compound equivalents relative to the antibody, reaction time, nature of the solvent, and any cosolvent). Bringing the antibody and cryptophycin compound into contact produces a mixture containing several conjugates that characteristically differ from each other by different DAR values; optionally unreacted antibody; optionally aggregates. The DAR value that is determined in the final solution thus corresponds to the average DAR value. The DAR value can be calculated based on the deconvolution of the SEC-HRMS spectrum of the conjugate. The DAR (HRMS) value, for example, is greater than 0.5, for example, is in the range of 1 to 10, such as a range of 2 to 7.

The conjugate can be used as an anticancer agent. Thanks to the presence of antibodies, a conjugate is obtained that is highly selective for tumor cells rather than healthy cells. This makes it possible to direct the cryptophycin compound into an environment similar to them or directly to them. It is possible to treat solid cancers or hematological malignancies. The conjugate can 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 typically ranging from 1 to 10 mg/ml. This solution can be administered as a perfusion on its own or can be re-diluted to form a perfusion solution.

Examples

The following examples describe the preparation of certain compounds in accordance with the present invention. Such examples are not limiting and merely illustrate the present invention.

Analytical methods used.

High pressure liquid chromatography - mass spectrometry (LCMS).

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Method A.

Spectra were acquired on a Waters UPLC-SQD system in positive and/or negative electrospray mode (ES+/-). The chromatographic conditions were as follows:

column: ACQUITY VEIN C ₁₈ -1.7 µm - 2.1x50 mm; solvents: A: H ₂ O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 50°C; flow rate: 0.8 ml/min; gradient (2.5 min.): from 5 to 100% B in 1.8 min.; 2.4 min: 100% B; 2.45 min: from 100 to 5% B in 0.05 min.

Method B.

Spectra were acquired on a Waters UPLC-SQD system in positive and/or negative electrospray mode (ES+/-). The chromatographic conditions were as follows:

column: ACQUITY VEIN C ₁₈ -1.7 µm - 2.1x50 mm; solvents: A: H ₂ O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 50°C; flow rate: 0.6 ml/min; gradient (2 min.): from 5 to 50% B in 1 min.; from 50 to 100% B in 0.3 minutes; 100% B for 0.15 min; from 100 to 5% B in 0.3 min and 5% B in 0.25 min.

Method C.

Spectra were acquired on a Waters UPLC-SQD system in positive and/or negative electrospray mode (ES+/-). The chromatographic conditions were as follows:

column: ACQUITY VEIN C ₁₈ -1.7 µm - 2.1x50 mm; solvents: A: H ₂ O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 50°C; flow rate: 0.8 ml/min; gradient (5 min.): from 5 to 100% B in 4.2 min.; 4.6 min: 100% B; 4.8 min: 5% B.

Method D

Spectra were acquired on a Waters XeVo - Qtof system in positive electrospray mode (ES+). The chromatographic conditions were as follows:

column: ACQUITY VEIN C ₁₈ -1.7 µm - 2.1x100 mm; solvents: A: H ₂ O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 45°C; flow rate: 0.6 ml/min; gradient (5.3 min): 5% B from 0 to 0.3 min, 4 min: 100% B; 4.6 min: 100% B; 5.3 min: 5% B.

Method E

Spectra were acquired on a Waters UPLC-SQD system in positive and/or negative electrospray mode (ES+/-). The chromatographic conditions were as follows:

column: ACQUITY VEIN C ₁₈ -1.7 µm - 2.1x50 mm; solvents: A: H ₂ O (0.1% formic acid), B: CH3CN (0.1% formic acid); column temperature: 45°C; flow rate: 0.8 ml/min; gradient (10 min.): from 5 to 100% B in 8.6 min.; 9.6 min: 100% B; 9.8 min: 5% B.

1H nuclear magnetic resonance (NMR).

1H NMR spectra were obtained on a Bruker Avance spectrometer, or on a model DRX-300, DRX-400 or DRX-500. Chemical shifts (δ) are given in ppm.

Size exclusion chromatography-high resolution mass spectrometry (SEC-HRMS).

Chromatographic analysis was performed on an Agilent HP1100 instrument and a Waters BEN SEC 200 1.7 μm (2.1x150 mm) column at 30°C with a flow rate of 0.5 ml/min, and isocratic elution (A) 25 mM ammonium formate + 1% formic acid/(B) CH ₃ CN+0.1% formic acid 70/30 for 15 min. Mass spectrometry was performed on a Waters QTOF-II instrument with electrospray ionization in positive mode (ES+). Mass spectra were deconvoluted using Waters MaxEntl software.

Analytical size exclusion chromatography (SEC).

Analysis was performed on a Waters Alliance HPLC system or a Hitachi Lachrom HPLC system equipped with a photodiode array detector and a Tosoh Bioscience TSKgel G3000 SWXL 5 µm (7.8 x 300 mm) column with a flow rate of 0.5 ml/min, and isocratic elution for 30 min buffer with pH 7 containing 0.2 M KCl, 0.052 M KH ₂ PO ₄ , 0.107 M K2HPO4 and 20% by volume isopropanol.

Buffers.

Buffer A (pH 6.5): NaCl (50 mM), potassium phosphate buffer (50 mM), EDTA (2 mM).

Buffer B (pH 6.5): NaCl (140 mM), potassium sodium phosphate (9.6 mM).

DPBS (pH 7.2): KCl (2.7 mM), NaCl (137 mM), KH2PO4 (1.47 mM), Na ₂ HPO ₄ (8.10 mM).

PBS (pH 7.4): KH2PO4 (1.06 mM), NaCl (155.17 mM), Na ₂ HPO ₄ -7H ₂ O (2.97 mM).

General method used to produce an antibody-drug conjugate (ADC).

A solution of the antibody in an aqueous buffer consisting of a mixture of buffer A and 1N. HEPES at a ratio of 96:4 was treated with an excess (5-10 equiv) of a solution of approximately 10 mM cryptophycin payload in DMA such that the final antibody concentration was 3 mg/ml and the percentage of DMA in the aqueous buffer was 20%. After stirring for 1-4 hours, the mixture was analyzed using SECHRMS to determine the DAR in the monomeric antibody population. If the DAR was found to be insufficient (<3.5-4), the mixture was treated with an additional excess (1-5 eq.) of cryptophycin solution in DMA for 2-4 additional hours at RT. while stirring. The mixture was purified by gel filtration using a Superdex 200 pg matrix (HiLoad 16/60 or 26/60 desalting column, GEHealthcare) pre-equilibrated in a pH 6.5 aqueous buffer (buffer B or DPBS) containing

- 90 045708 compressed with 10-20% NMP, or Sephadex™ G25 matrix (Hiprep 26/10 desalting column, GEHealthcare), pre-equilibrated in an aqueous pH 6.5 buffer (buffer B or DPBS) containing 5-10% NMP. Fractions containing monomeric conjugated antibody were pooled and concentrated on Amicon Ultra-15 (Ultracel 10k or 50k membrane, Millipore) to a concentration of 2 to 5 mg/ml. Buffer exchange or dilution was then performed in a suitable buffer to formulate the conjugate in the final buffer. In the case of buffer exchange, this was accomplished by gel filtration using a Sephadex G25 matrix (NAP-5, NAP-10, NAP-25/PD-10 or Hiprep 26/10 desalting columns, GEHealthcare) pre-equilibrated with the final aqueous buffer, composition and whose pH corresponds to each conjugate. Finally, the conjugate was filtered through a Steriflip® filter block (0.22 μm Durapore® PVDF membrane, Millipore). The final conjugate was analyzed by UV spectrometry or SEC-HPLC to measure conjugate concentration, by SEC-HPLC to determine monomeric purity, and by SEC-HRMS to determine DAR based on deconvolution of the conjugate mass spectrum.

Synthesis of Examples 1-3: PEG4-Val-Ala-C52-benzylamine, PEG4-Val-Ala-C52-benzylamine NHS ester and corresponding ADC

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Compound 1: Allyl-3-((tert-butoxycarbonyl)amino)propanoate.

To a solution of Boc-in-Ala-OH (CAS number [3303-84-2], 2 g, 10.04 mmol) in DMF (50 ml) was added cesium carbonate (6.88 g, 21.09 mmol) and allyl bromide (965.52 µl, 11.05 mmol). The reaction medium was stirred at room temperature. during the night. At the same time, the reaction medium was concentrated in vacuo, then diluted with toluene and concentrated in vacuo several times. The crude product was diluted with water (100 ml) and extracted with AtOAc (3x300 ml). The combined organic phases were washed with brine, dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 90 g silica gel (DCM/MeOH gradient elution) to give 1.5 g of compound 1 (65%).

^1H NMR (400 MHz, δ in ppm, DMSO-d6): 1.3V (s, 9H); 2.45 (t, J=6.9 Hz, 2H); 3.17 (q, J=6.9 Hz, 2H); 4.54 (m, 2H); 5.20 (qd, J=1.5 and 10.6 Hz, 1H); 5.29 (qd, J=1.9 and 17.3 Hz, 1H); 5.91(m, 1H); 6.85 (wide t, J=6.9 Hz, 1H).

Compound 2: Allyl 3-aminopropanoate-2,2,2-trifluoroacetate.

To a solution of compound 1 (1.5 g, 6.54 mmol) in DCM (30 ml) was added TFA (5.09 ml, 65.42 mmol). The reaction medium was stirred at room temperature. during the night. At the same time, the reaction medium was concentrated in vacuo, then diluted with toluene and concentrated in vacuo twice to obtain 1.6 g of compound 2 (quantitative).

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Compound 3: allyl-3-((3-(tert-butoxy)-3-oxopropyl)amino)propanoate.

To a solution of compound 3 (1.6 g, 6.58 mmol) in DMSO (15 ml) was added DIEA (2.30 ml, 13.16 mmol) and tert-butyl acrylate (973.49 μl, 6.58 mmol). The reaction medium was heated for 1 hour at 60°C using microwaves. At the same time, the reaction medium was diluted with H ₂ O (10 ml) and extracted with AtOAc. The combined organic phases were washed with brine, dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 80 g silica gel (DCM/MeOH gradient elution) to give 678 mg of compound 3 (40%).

1H NMR (400 MHz, δ in ppm, DMSO-d6): 1.39 (s, 9H); 1.73 (wide m, 1H); 2.29 (t, J=6.9 Hz, 2H); 2.44 (t, J=6.9 Hz, 2H); 2.68 (t, J=6.9 Hz, 2H); 2.73 (t, J=6.9 Hz, 2H); 4.53 (m, 2H); 5.20 (qd, J=1.5 and 10.6 Hz, 1H); 5.30 (qd, J=1.9 and 17.3 Hz, 1H); 5.91 (m, 1H).

Compound 4: tert-butyl-2,5,8,11-tetraoxatetradecane-14-oate.

Under argon, a mixture of triethylene glycol monomethyl ether (CAS number [112-35-6], 3 g, 17.36 mmol) and sodium (3.99 mg, 173.57 μmol) in THF (9 ml) was stirred for 2 h at 50°C, then overnight at room temperature. At the same time, tert-butyl acrylate (3.08 ml, 20.83 mmol) was added, stirred for 2 hours at 40°C, then overnight at room temperature. The reaction medium was then concentrated in vacuo, then diluted with H2O and extracted with EtOAc. The combined organic phases were washed with brine, dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 90 g silica gel (heptane/EtOAc gradient elution) to give 3.3 g of 4 (65%).

Compound 5: 2,5,8,11-tetraoxatetradecane-14-oic acid.

To a solution of compound 4 (2.5 g, 8.55 mmol) in DCM (30 ml) was added TFA (6.65 ml, 85.51 mmol). The reaction medium was stirred overnight at room temperature. At the same time, the reaction medium was concentrated in vacuo, then diluted with toluene and concentrated in vacuo three times to obtain 3 g of compound 5 (quantitative).

Compound 6: 2,5,8,11-tetraoxatetradecane-14-oil chloride.

Thionyl chloride (3.68 mL, 50.79 mmol) was added to compound 5 (800 mg, 3.39 mmol). The reaction medium was heated for 3 hours at 60°C. After cooling, the reaction medium was concentrated in vacuo, diluted with DCM and concentrated in vacuo twice to obtain 870 mg of compound 6 (quantitative).

Compound 7: allyl-15-(3-(tert-butoxy)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15azaoctadecane-18-oate.

At 0°C under argon, a solution of compound 6 (738.21 mg, 2 .90 mmol) in DCM (3 ml). After 2 hours, the reaction medium was washed with H2O (10 ml), then with brine, dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 40 g silica gel (DCM/MeOH gradient elution) to give 410 mg of compound 7 (33%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 50/50; 1.39 (s, 4.5 H); 1.40 (s, 4.5 H); 2.39 (t, J=7.2 Hz, 1H); 2.47 to 2.60 (partially hidden m, 4H); 2.66 (t, J=7.2 Hz, 1H); 3.23(s, 1.5H); 3.24 (s, 1.5 H); 3.38 to 3.65 (m, 18 H); 4.54 (m, 2H); 5.19 to 5.34 (m, 2H); 5.85 to 5.97 (m, 1H).

Compound 8: 15-(3-(allyloxy)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecane-18-oic acid.

A solution of compound 7 (150 mg, 315.41 µmol) in DCM (3 ml) and TFA (236.66 µl, 3.15 mmol) was stirred at RT. during the night. At the same time, the reaction medium was concentrated in vacuo, diluted with toluene and concentrated in vacuo three times to obtain 130 mg of compound 8 (98%).

Compound 9: Allyl-15-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-14-oxo-2,5,8,11tetraoxa-15-azaoctadecane-18-oate.

To a solution of compound 8 (130 mg, 309.92 µmol) in THF (5 ml) was added DIEA (104.53 µl, 619.83 µmol) and DSC (97.21 mg, 371.90 µmol). The reaction medium was stirred at room temperature. during the night. At this time, the reaction medium was concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/iPrOH gradient elution) to give 107 mg of compound 9 (67%).

Compound 10: (9H-fluoren-9-yl)methyl-((2S)-1 -(((2S)-1 -((4-((2R,3 S)-3-(1 -((3 S ,10R,16S,E)-10-(3 chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexade13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate.

To a solution of (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-( 3chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 150 mg, 214.82 µmol) HOBt (31.93 mg, 236.31 µmol), EDC (41.83 µL, 236.31 µmol) and Fmoc-Val-Ala-OH (CAS number [ 150114-97-9], 79.36 mg, 193.34 µmol). The reaction medium was stirred for 3 hours at room temperature. H ₂ O (20 ml) was then added and the mixture was extracted with DCM (2x15 ml). The combined organic phases were washed with brine, dried over MgSO4, filtered, concentrated in vacuo and purified by flash chromatography on 5 g silica gel (DCM/MeOH gradient elution) with

- 93 045708 obtaining 200 mg of compound 10 (85%).

Compound 11: (2S)-2-αmino-N-((2S)-1-((4-((2R)-3-((S)-1-((3S,10R,16S,E)-10 -(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en16-yl)ethyl)oxirane -2-yl)benzyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide.

A mixture of compound 10 (200 mg, 183.37 µmol) in DCM (10 ml) and piperidine (183.39 µl, 1.83 mmol) was stirred for 2 h at RT. The reaction medium was concentrated in vacuo, solubilized in DCM before adding Et ₂ O. The precipitate was filtered, washed with Et ₂ O and dried to obtain 180 mg of crude material, which was purified by flash chromatography on 10 g aminopropyl modified silica gel (gradient elution DCM/ MeOH) to obtain 136 mg of compound 11 (85%).

Compound 12: αlyl-15-(3-(((2S)-1-(((2S)-1-((4-((2R,3S)-3-(1-((3S,10R,16S, E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en16-yl )ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3oxopropyl)-14-oxo -2.5 ,8,11-tetraoxa-15-azaoctadecane-18-oate.

To a solution of compound 11 (60 mg, 69.09 μmol) in THF (5 ml) was added compound 9 (42.82 mg, 82.91 μmol) and DIEA (17.48 μl, 103.63 μmol). The reaction medium was stirred for 3 hours at room temperature. At this time, it was concentrated in vacuo and purified by flash chromatography on 10 g silica gel (DCM/MeOH gradient elution) to give 60 mg of compound 12 (68%).

1H NMR (MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60/40; 0.78 (d, J=7.0 Hz, 6H); 0.80 to 0.87 (m, 6H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.12(s, 3H); 1.24 (divided d, J=7.0 Hz, 3H); 1.30 (m, 1H); 1.51 to 1.63 (m, 2H); 1.75 to 1.83 (m, 1H); 1.95 (m, 1H); 2.26 (m, 1H); 2.30 to 2.73 (m, 8 H); 2.95 to 3.05 (m, 3H); 3.23(s, 3H); 3.31 (hidden m, 1H); 3.40 to 3.64 (m, 18H); 3.81(s, 3H); 3.87 (d, J=2.1 Hz, 1H); 4.12 to 4.33 (m, 5H); 4.53 (m, 1.2H); 4.55 (m, 0.8H); 4.91(m, 1H); 5.11(m, 1H); 5.21(m, 1H); 5.28 (qd, J=1.9 and 17.3 Hz, 0.6 H); 5.31 (qd, J=1.9 and 17.3 Hz, 0.4H); 5.78 (divided d, J=15.7 Hz, 1H); 5.85 to 5.95 (m, 1H); 6.47 (ddd, J=4.2, 11.5 and 15.7 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.17 (dd, J=2.3 and 8.7 Hz, 1H); 7.19 to 7.26 (m, 5 H); 7.28 (d, J=2.3 Hz, 1H); 7.94 (d, J=9.2 Hz, 0.4H); 8.04 (m, 1H); 8.10 (d, J=7.6 Hz, 0.6 H); 8.32 (m, 1H); 8.35 (d, J=8.2 Hz, 1H).

Example 1: 15-(3-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R, 16S,E)-10-(3chloro-4-methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-ene- 16-yl)ethyl)oxiran2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo2, 5,8,11-tetraoxa-15-azaoctadecane-18-oic acid.

Under argon, tetrakis(triphenylphosphine)palladium(0) (2.76 mg, 2.36 μmol) and 1,3-dimethylbarbituric acid ( 22.58 mg, 141.74 µmol). The reaction medium was stirred for 1 hour at room temperature. and concentrated in vacuo. The crude product was purified by flash chromatography on 4 g silica gel (DCM/MeOH gradient elution) to give 31 mg of the compound of Example 1 (53%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.79 (d, J=7.0 Hz, 6H); 0.80 to 0.88 (m, 6H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.12(s, 3H); 1.23 (m, 3H); 1.31(m, 1H); 1.51 to 1.62 (m, 2H); 1.80 (m, 1H); 1.95 (m, 1H); 2.27 (m, 1H); 2.35 to 2.60 (partially hidden m, 6H); 2.62 to 2.73 (m, 2H); 2.93 to 3.06 (m, 3H); 3.23(s, 3H); 3.28 to 3.35 (hidden m, 1H); 3.36 to 3.65 (m, 18 H); 3.81(s, 3H); 3.87 (d, J=2.0 Hz, 1H); 4.11 to 4.34 (m, 5H); 4.91(m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.5 Hz, 1H); 6.48 (ddd, J=3.8, 11.3 and 15.5 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.17 (dd, J=2.0 and 8.7 Hz, 1H); 7.24 (m, 5 H); 7.29 (d, J=2.0 Hz, 1H); 7.95 to 8.18 (m, 2H); 8.30 to 8.46 (m, 2H); 12.27 (wide m, 1H). LCMS (A): ES mass/charge=615 [M+2H] ² +; mass/charge=1227 [MN] ^- ; mass/charge=1229 [M+H]+; Rt=1.26 min.

Example 2: 2,5-dioxopyrrolidin-1-yl-15-(3-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S )-1-((3S,10R,16S,E)-10(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4- dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2yl) amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecane-18-oate.

To a solution of the compound of Example 1 (30 mg, 24.39 µmol) in THF (5 ml) was added DSC (7.65 mg, 29.27 µmol) and DIEA (9.88 µl, 58.54 µmol). The reaction medium was stirred at room temperature. overnight, then concentrated in vacuo and purified by flash chromatography on 5 g diol-modified silica gel (DCM/iPrOH gradient elution) to give 17 mg of Example 2 (53%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 70/30; 0.79 (d, J=7.0 Hz, 6H); 0.80 to 0.87 (m, 6H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.12(s, 3H); 1.21 to 1.34 (m, 4H); 1.51 to 1.62 (m, 2H); 1.80 (m, 1h); 1.93 (m, 1h); 2.27 (m, 1H); 2.35 to 2.72 (partially hidden m, 6H); 2.81 (wide s, 4h); 2.88 to 3.06 (m, 5H); 3.23(s, 3H); 3.28 to 3.37 (hidden m, 1H); 3.40 to 3.79 (m, 18H); 3.81(s, 3H); 3.88(s, 1H); 4.13 to 4.33 (m, 5H); 4.90 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.5 Hz, 1H); 6.46 (ddd, J=3.8,11.5 and 15.5 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.17 (wide s, 1H); 7.24 (m, 5 H); 7.29 (wide s, 1H); 7.94 (d, J=8.8 Hz, 0.3 H); 8.06 (m, 1H); 8.12 (d, J=8.1 Hz, 0.7 H); 8.32 (m, 1H); 8.36 (d, J=8.1 Hz, 1H). LCMS (A): ES mass/charge=663.5 [M+2H] ²⁺ ; mass/charge=1324 [M-N] ^- ; mass/charge=1326 [M+H]+; mass/charge=1374 [M-H+HCO2H] ^- ; Rt=1.3 min.

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Example 3: hu2Hl l_R35-74-Ex2.

The general method described previously was used to prepare the compound of Example 3. 15 mg of hu2Hl l_R35-74 was reacted with 55.2 μl of a 9.1 mM solution of the compound of Example 2 in DMA (10 eq.) for 3 hours 30 minutes . After purification on Superdex 200 pg in buffer B pH 6.5 + 10% NMP, concentration on Amicon Ultra-15, buffer exchange with Nap-10 in buffer B pH 6.5 + 5% NMP and filtration on a PVDF filter with size 0.22 μm pores yielded 3.36 mg of the compound from Example 3 as a colorless clear solution at a concentration of 2.24 mg/ml with a DAR of 3.4 (HRMS), monomer purity of 99.8% and an overall yield of 22%.

SEC-HRMS: mass/charge=149354 (unconjugated mAb); mass/charge=150563 (D1); mass/charge=151774 (D2); mass/charge=152966 (D3); mass/charge=154169 (D4); mass/charge=155410 (D5); mass/charge= 156623 (D6); mass/charge= 157835 (D7); mass/charge= 159051 (D8).

Synthesis of Examples 4-6: Sulfo-Va1-A1a-C52-benzylamine, NHS-sulfo-Va1-A1a-C52-benzylamine ester and corresponding ADC

Compound 13: 4-((3-(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4oxobutanoic acid.

A solution of compound 3 (958 mg, 3.72 mmol) in DCM (5 ml) and succinic anhydride (752.65 mg, 7.45 mmol) was stirred for 3 h at room temperature. The reaction medium was concentrated in vacuo and purified

-95045708 flash chromatography on 24 g silica gel (DCM/MeOH gradient elution) to give 1.3 g of 13 (98%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 50/50; 1.39 (s, 4.5 H); 1.40 (s, 4.5 H); 2.33 to 2.43 (m, 3H); 2.45 to 2.55 (partially hidden m, 4H); 2.69 (t, J=7.5 Hz, 1H); 3.40 (t, J=7.5 Hz, 1h); 3.45 (t, J=7.5 Hz, 1H); 3.53 (t, J=7.5 Hz, 1H); 3.59 (t, J=7.5 Hz, 1H); 4.53 (m, 1H); 4.59 (m, 1H); 5.19 to 5.35 (m, 2H); 5.91(m, 1H); 12.06 (wide m, 1H).

Compound 14: 2-(4-((3-(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4oxobutanamido)ethanesulfonic acid.

A mixture of compound 13 (1.3 g, 3.64 mmol) in THF (12 ml) and DSC (1.14 g, 4.36 mmol) and DIEA (1.84 ml, 10.91 mmol) was stirred for 5 h at room temperature After this time, a solution of taurine (718.75 mg, 5.46 mmol) in water (9 ml) was added and the medium was stirred at room temperature. during the night. At the same time, DSC (1.14 g, 4.36 mmol) and DIEA (1.84 ml, 10.91 mmol) were added and the medium was stirred at room temperature. during the night. At the same time, taurine (2.4 g, 18.2 mmol) in water (5 ml) was added and the reaction mixture was stirred for 1 h at room temperature, then concentrated and purified by flash chromatography on 70 g C ₁₈ -grafted silica gel (gradient elution CH ₃ CN/H ₂ O) to obtain 1.28 g of compound 14 (76%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 50/50; 1.39 (s, 4.5 H); 1.40 (s, 4.5 H); 2.26 (t, J=7.2 Hz, 2H); 2.38 (t, J=7.5 Hz, 1H); 2.47 to 2.55 (partially hidden t, 6H); 2.69 (t, J=7.5 Hz, 1h); 3.28(m, 2H); 3.40 (t, J=7.5 Hz, 1H); 3.44 (t, J=7.5 Hz, 1H); 3.52 (t, J=7.5 Hz, 1H); 3.58 (t, J=7.5 Hz, 1H); 4.53 (m, 1h); 4.58 (m, 1H); 5.19 to 5.35 (m, 2H); 5.91(m, 1H); 7.70 (wide t, J=6.5 Hz, 1H).

Compound 15: 3-(N-(3-(allyloxy)-3-oxopropyl)-4-oxo-4-((2-sulfoethyl)amino)butanamido)propanoic acid.

A solution of compound 14 (300 mg, 645.81 µmol) in DCM (5 ml) and TFA (484.57 µl, 6.46 mmol) was stirred at RT. during the night. At the same time, TFA (250 μl) was added. The reaction medium was stirred for several hours and concentrated in vacuo, then diluted with toluene and concentrated in vacuo twice to obtain 260 mg of compound 15 (99%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 50/50; 2.27 (t, J=7.2 Hz, 2H); 2.40 (t, J=7.6 Hz, 1H); 2.48 to 2.56 (partially hidden m, 6H); 2.68 (t, J=7.6 Hz, 1H); 3.28(m, 2H); 3.40 (t, J=7.6 Hz, 1H); 3.46 (t, J=7.6 Hz, 1H); 3.50 (t, J=7.6 Hz, 1H); 3.59 (t, J=7.6 Hz, 1H); 4.54 (m, 1H); 4.57 (m, 1H); 5.18 to 5.35 (m, 2H); 5.91(m, 1H); 7.65 (wide t, J=6.5 Hz, 1H). LCMS (D): ES mass/charge=409 [M+H]+; Rt=1.49 min.

Compound 16: (4S,7S)-12-(3-(αlyloxy)-3-oxononyl)-1-(4-((2R,3R)-3-((S)-l-((3S,10R, 16S,E)-10(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13- en-16-yl)ethyl)oxiran-2-yl)phenyl)-7-isopropyl-4-methyl-3,6,9,13,16-pentaoxo-2,5,8,12,17-pentaazanonedecane-19 -sulfonic acid.

Under argon, DIEA (23.30 μL, 138.18 μmol), HOBt (9.72 mg, 69.09 μmol) was added to a solution of compound 15 (28.22 mg, 69.09 μmol) in DMF (3 ml). , EDC (12.49 µl, 69.09 µmol) and a solution of compound 11 (60 mg, 69.09 µmol) in DMF (2 ml). The reaction medium was stirred at room temperature. during the night. Compound 15 (28.22 mg, 69.09 µmol), DIEA (23.30 µl, 138.18 µmol), HOBt (9.72 mg, 69.09 µmol), EDC (12.49 µl) was added to complete the reaction , 69.09 µmol) and the reaction medium was stirred at room temperature. within 1 day. At the same time, the reaction medium was concentrated in vacuo and purified by flash chromatography on 70 g C ₁₈ -grafted silica gel (CH ₃ CN/H ₂ O gradient elution) to obtain 24 mg of compound 16 (24%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60/40; 0.79 (d, J=7.0 Hz, 6H); 0.80 to 0.87 (m, 6H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.23 (d, J=7.0 Hz, 3H); 1.30 (m, 1H); 1.52 to 1.63 (m, 2H); 1.80 (m, 1H); 1.95 (m, 1H); 2.27 (m, 3H); 2.34 to 2.58 (partially hidden m, 6H); 2.63 to 2.73 (m, 2h); 2.93 to 3.03 (m, 3H); 3.25 to 3.59 (partially hidden m, 9H); 3.81(s, 3H); 3.88 (d, J=1.4 Hz, 1H); 4.10 to 4.33 (m, 5 H); 4.53 (m, 1.2H); 4.56 (m, 0.8 H); 4.91(m, 1H); 5.10 (m, 1H); 5.20 (m, 1H); 5.29 (qd, J=1.8 and 17.3 Hz, 0.6 H); 5.31 (qd, J=1.8 and 17.3 Hz, 0.4H); 5.79 (dd, J=1.8 and 15.4 Hz, 1H); 5.90 (m, 1H); 6.47 (ddd, J=3.8, 11.8 and 15.4 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.18 (dd, J=2.4 and 8.7 Hz, 1H); 7.24 (m, 5 H); 7.29 (d, J=2.4 Hz, 1H); 7.69 (t, J=5.8 Hz, 1H); 7.97 (d, J=8.8 Hz, 0.4H); 8.04 (d, J=7.7 Hz, 0.4H); 8.07 (d, J=8.8 Hz, 0.6 H); 8.11 (d, J=7.7 Hz, 0.6 H); 8.31 (t, J=6.3 Hz, 1H); 8.38 (d, J=8.3 Hz, 1H); 11.83 (wide m, 1H). LCMS (D): ES mass/charge=1256 [M-N] ^- ; Rt=2.58 min.

Example 4: (4S,7S)-1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4- methoxybenzyl)-3-isobutyl6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)phenyl )-7-isopropyl-4-methyl-3,6,9-trioxo-12-(4-oxo-4-((2-sulfoethyl)amino)butanoyl)-2,5,8,12-tetraazapentadecane-15- ovalic acid.

Under argon, DMF (0.5 mL), 1,3-dimethylbarbituric acid (9.11 mg, 57.19 μmol) was added to a solution of compound 16 (24 mg, 19.06 µmol) in CH ₃ CN (2 ml). and tetrakis(triphenylphosphine)palladium(0) (1.11 mg, 0.953 µmol). The reaction medium was stirred for 1 h at room temperature, then concentrated in vacuo and purified by flash chromatography on 10 g

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C ₁₈ -grafted silica gel (gradient elution CH ₃ CN/H2O) to obtain 16 mg of the compound from example 4 (70%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): conformer mixture 60/40; 0.79 (d, J=7.0 Hz, 6H); 0.80 to 0.86 (m, 6H); 0.99(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.12(s, 3H); 1.24 (d, J=7.0 Hz, 3H); 1.31(m, 1H); 1.52 to 1.62 (m, 2h); 1.80 (m, 1H); 1.95 (m, 1H); 2.21 to 2.31 (m, 3H); 2.34 to 2.59 (partially hidden m, 8 H); 2.62 to 2.75 (m, 2H); 2.94 to 3.05 (m, 3H);

3.25 to 3.58 (partially hidden m, 7H); 3.81(s, 3H); 3.88 (d, J=1.9 Hz, 1H); 4.09 to 4.33 (m, 5 H); 4.90 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.5 Hz, 1H); 6.47 (ddd, J=4.0, 11.4 and 15.5 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.17 (dd, J=2.2 and 8.7 Hz, 1H); 7.21 to 7.26 (m, 5 H); 7.29 (d, J=2.2 Hz, 1H); 7.69 (m, 1H); 7.97 (d, J=9.0 Hz, 0.4H); 8.04 (d, J=7.7 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6 H); 8.12 (d, J=7.7 Hz, 0.6 H); 8.31 (t, J=6.5 Hz, 1H); 8.38 (d, J=8.2 Hz, 1H); 12.2 (wide m, 1H). LCMS (A): ES mass/charge=609.5 [M+2H] ² +; mass/charge=1216 [M-N] ^- ; mass/charge=1218 [M+H]+; Rt=1.72 min.

Example 5: (4S,7S)-1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4- methoxybenzyl)-3-isobutyl6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)phenyl )-12-(3 -((2,5-dioxopyrrolidin-1 -yl)oxy)-3-oxopropyl)-7-isopropyl-4-methyl-3,6,9,13,16-pentaoxo-2,5 ,8,12,17-pentaazanonedecane-19-sulfonic acid.

Under argon, a mixture of the compound of Example 4 (16 mg, 13.13 µmol) in DMF (1 ml), DSC (4.12 mg, 15.75 µmol) and DIEA (2.66 µl, 15.75 µmol) was stirred at k.t. during the night. At this time, the reaction medium was concentrated in vacuo and purified by flash chromatography on 5 g C ₁₈ grafted silica gel (CH ₃ CN/H ₂ O gradient elution) to obtain 12.5 mg of the compound of Example 5 (73%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60/40; 0.79 (d, J=7.0 Hz, 6H); 0.80 to 0.86 (m, 6H); 0.99(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.23 (d, J=7.0 Hz, 3H); 1.31(m, 1H); 1.51 to 1.62 (m, 2H); 1.80 (m, 1H); 1.93 (m, 1H); 2.21 to 2.31 (m, 3H); 2.35 to 2.59 (partially hidden m, 6H); 2.62 to 2.74 (m, 2H); 2.81 (wide s, 4H); 2.85 to 3.05 (m, 4H); 3.20 to 3.68 (partially hidden m, 8H); 3.81(s, 3H); 3.88 (d, J=1.9 Hz, 1H); 4.10 to 4.33 (m, 5H); 4.90 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.5 Hz, 1H); 6.46 (ddd, J=4.0, 11.4 and 15.5 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.18 (dd, J=2.2 and 8.7 Hz, 1H); 7.21 to 7.26 (m, 5 H); 7.29 (d, J=2.2 Hz, 1H); 7.69 (m, 1H); 7.97 (d, J=9.0 Hz, 0.4H); 8.05 (d, J=7.7 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6 H); 8.12 (d, J=7.7 Hz, 0.6 H); 8.31 (t, J=6.5 Hz, 1H); 8.38 (d, J=8.2 Hz, 1H). LCMS (A): ES mass/charge=658 [M+2H] ²⁺ ; mass/charge=1313 [M-N] ^- ; mass/charge=1315 [M+H]+; Rt=1.82 min.

Example 6: hu2H11_R35-74-Ex5.

The general method described previously was used to prepare the compound of Example 6. 15 mg of hu2H11_R35-74 was reacted with 42 μl of a 10 mM solution of the compound of Example 5 in DMA (4 eq.) for 1 h 20 min, then 42 were added sequentially µl of a 10 mM solution of the compound from example 5 in DMA (4 equiv.) for 1 hour 20 min, 53 µl of a 10 mM solution of the compound from example 5 in DMA (5 equiv.) for 1 hour 30 min and finally 42 µl of 10 mM a solution of the compound from example 5 in DMA (4 eq.) for 1 hour. After purification on Superdex 200 pg in buffer B with pH 6.5 + 20% NMP, concentration on Amicon Ultra-15, replacement of the buffer with PD-10 in buffer B with pH 6.5 + 5% NMP and filtration on a PVDF filter with a pore size of 0.22 μm, 3.33 mg of the compound from example 6 was obtained in the form of a colorless transparent solution at a concentration of 1.33 mg/ml with DAR 4, 05 (HRMS), monomer purity 99% and overall yield 22%.

SEC-HRMS: mass/charge=150568 (D1); mass/charge=151768 (D2); mass/charge=152971 (D3); mass/charge=154172 (D4); mass/charge=155375 (D5); mass/charge=156574 (D6); mass/charge= 157944 (D7); mass/charge=158984 (D8).

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Synthesis of Example 7: Sulfo-Va1-A1a-azacrintobenzylamine

Compound 17: tert-butyl-L-valyl-L-alaninate.

To a solution of Fmoc-Val-OH (CAS number [68858-20-8], 3 g, 8.66 mmol) in DCM (60 ml) under Ar atmosphere was added EDC (2.02 g, 12.99 mmol), HOBt (1.46 g, 10.40 mmol) and L-Alanine tert-butyl ester hydrochloride (CAS number [13404-22-3], 1.61 g, 8.66 mmol). The reaction medium was stirred for 1 day at RT, quenched with H2O (30 ml) and extracted with DCM (3x30 ml). The combined organic phases were washed with brine, dried over MgSO ₄ , filtered, concentrated in vacuo to obtain 5 g of Fmoc-protected dipeptide as a white solid (qty.). To a solution of this intermediate (5 g, 8.66 mmol) in DCM (50 ml) was added piperidine (8.64 ml, 86.63 mmol). The reaction medium was stirred for 1 hour at room temperature. and concentrated in vacuo. The crude product was diluted with Et ₂ O and filtered to give 1.77 g of a solid, which was further purified by two successive flash chromatography runs on 120 g and 50 g of silica gel (DCM/MeOH gradient elution) to give 500 mg of compound 17 in as a white solid (24%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.79 (d, J=6.9 Hz, 3H); 0.89 (d, J=6.9 Hz, 3H); 1.24 (d, J=7.3 Hz, 3H); 1.40(s,9H); 1.62 (wide s, 2H); 1.86(m, 1H); 2.95 (d, J=5.2 Hz, 1H); 4.15 (m, 1H); 8.09 (wide d, J=7.3 Hz, 1H).

Compound 18: (5S,8S)-13-(3-(allyloxy)-3-oxopropyl)-8-isopropyl-2,2,5-trimethyl-4,7,10,14,17pentaoxo-3-oxa-6 ,9,13,18-tetraazaicosane-20-sulfonic acid.

To a solution of compound 15 (250 mg, 612.1 µmol) in DMF (6 ml) under Ar atmosphere was added DIEA (309.7 µl, 1.84 mmol) and DSC (163.34 mg, 612.1 µmol). The reaction medium was stirred for 1 hour at room temperature, then a solution of compound 17 (149.56 mg, 612.1 μl) in DMF (1 ml) was added. The reaction medium was stirred for 2 hours at room temperature. and purified by reverse phase chromatography on a C ₁₈ 5 μm, 30 x 100 mm column (gradient elution MeCN+0.07% TFA/H ₂ O+0.07% TFA) to give 215 mg of compound 18 as a white foam (55 %).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): conformer mixture 60/40; 0.84 (d, J=6.9 Hz 3 H); 0.87 (d, J=6.9 Hz, 3H); 1.12 (d, J=7.3 Hz, 3H); 1.39(s,9H); 1.92 (m, 1H); 2.20 to 2.70 (partially hidden m, 10H); 3.28(m, 2H); 3.34 to 3.60 (m, 4H); 4.10 (m, 1H); 4.20 (m, 1H); 4.53 (td, J=1.6 and 5.5 Hz, 1.2H); 4.57 (td, J=1.6 and 5.5 Hz, 0.8 H); 5.20 (m, 1H); 5.30 (m, 1H); 5.90 (m, 1H); 7.69 (wide t, J=6.4 Hz, 1H); 7.91 (d, J=9.6 Hz, 0.4H); 8.04 (d, J=9.6 Hz, 0.6 H); 8.23 (d, J=7.2 Hz, 0.4H); 8.30 (d, J=7.2 Hz, 0.6 H).

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Compound 19: (3-(N-(3-(allyloxy)-3-oxopropyl)-4-oxo-4-((2-sulfoethyl)amino)butanamido)propanoyl)-L-valyl-L-alanine.

To a solution of compound 18 (215 mg, 338.72 µmol) in DCM (5 ml) was added TFA (508.3 µl, 6.77 mmol). The reaction medium was stirred for 1 day at RT, concentrated in vacuo and co-evaporated with toluene (3x) to obtain 200 mg of compound 19 as a white foam (quantitative).

Compound 20: (4S,7S)-12-(3-(αlyloxy)-3-oxopropyl)-1-(4-((2R,3R)-3-((S)-1-((3S,7S, 10R,16S,E)-10-(3chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexade -13-en16-yl)ethyl)oxiran-2-yl)phenyl)-7-isopropyl-4-methyl-3,6,9,13,16-pentaoxo-2,5,8,12,17-pentaazanonedecane- 19sulfonic acid.

To a solution of compound 19 (200 mg, 345.6 µmol) in DMF (9 ml) was added DSC (108 mg, 413.2 µmol) and DIEA (200 µl, 1.185 mmol). The reaction medium was stirred for 6 hours at room temperature, then DSC (108 mg, 413.2 µmol) and DIEA (200 µl, 1.185 mmol) were added. The reaction medium was stirred at room temperature. overnight, then DSC (50 mg, 191.3 µmol) and DIEA (100 µl, 592.5 µmol) were added and stirred for 1 h at room temperature. (3S,7S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2yl) was added to 3 ml of the activated ester solution. ethyl)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec13-ene-2,5,9,12-tetraone (which can be synthesized as described in PCT/EP2016/076603 starting from methyl (3S)-3-amino-2,2-dimethylbutanoate, [MFCD09256689], 70 mg, 96.51 µmol), EDC (17 µl, 77 .8 µmol) and HOBt (19 mg, 124 µmol). The reaction medium was stirred for 1 h at RT, concentrated in vacuo and purified by flash chromatography on 10 g _C18 -modified silica gel (MeCN/ _H2O gradient elution) to obtain 30 mg of compound 20 as a white varnish (24%).

1H NMR (600 MHz, δ in ppm, DMSO-d ₆ ): 0.79 to 0.90 (m, 15 H); 0.98 (m, 3H); 1.00(s, 3H); 1.03 (d, J=7.3 Hz, 3H); 1.19(s, 3H); 1.24 (divided d, J=7.0 Hz, 3H); 1.34 (d, J=14.1 Hz, 1H); 1.81(m, 1H); 1.91(m, 1H); 1.98 (dd, J=9.3 and 14.1 Hz, 1H); 2.25 (m, 3H); 2.38 (m, 1H); 2.50 (partially hidden m, 5H); 2.60 (m, 1H); 2.68 (m, 1H); 2.91(m, 1H); 2.96(m, 2H); 3.25 to 3.58 (partially hidden m, 9H); 3.80(s, 3H); 3.91(m, 1H); 4.00 to 4.35 (m, 6H); 4.53 (m, 2H); 5.03 (m, 1H); 5.20 (m, 1H); 5.30 (m, 1H); 5.89 (d, J=16.1 Hz, 1H); 5.91(m, 1H); 6.44 (ddd, J=5.0, 10.5 and 16.1 Hz, 1H); 7.03 (d, J=8.7 Hz, 1H); 7.19 to 7.28 (m, 5 H); 7.31(s, 1H); 7.69 (m, 1H); 7.86 (d, J=8.2 Hz, 1H); 7.91 (m, 1H); 7.95 to 8.17 (m, 2H); 8.32 (m, 1H); 8.39 (m, 1H). LCMS (A): ES mass/charge=1283 [M-N] ^- ; mass/charge=1285 [M+H]+; mass/charge=1307 [M+Na]+; Rt=1.93 min.

Example 7: (4S,7S)-1-(4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro- 4-methoxybenzyl)-6,6,7trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran2-yl )phenyl)-7-isopropyl-4-methyl-3,6,9-trioxo-12-(4-oxo-4-((2-sulfoethyl)amino)butanoyl)-2,5,8,12tetraazapentadecane-15- ovalic acid.

Tetrakis(triphenylphosphine)palladium(0) (1.38 mg, 1.17 μmol) was added to a solution of compound 20 (30 mg, 23.33 µmol) in DCM (2 ml) and DMF (0.3 ml) under Ar atmosphere. and 1,3-dimethylbarbituric acid (11.15 mg, 69.99 µmol). The reaction medium was stirred for 1 hour at room temperature. and purified by reverse phase chromatography on 1 g _C18 -modified silica gel (MeCN/H2O gradient elution) to give 15 mg of a white powder, which was further purified on silica gel (DCM/MeOH/H2O gradient elution) to give 7 mg of the compound from example 7 in the form of white varnish (24%).

- 99 045708

Synthesis of Examples 8 and 9: PEG4-Val-Ala-PABA-C52-benzylamine and PEG4-Val-Ala-PABA-C52-benzylamine NHS ester

Compound 21: (9H-fluoren-9-yl)methyl-(^)-1 -(((S)-1 -((4-(hydroxymethyl)phenyl)amino)-1 oxopropan-2-yl)amino)- 3-methyl-1-oxobutan-2-yl)carbamate.

4-Aminobenzyl alcohol (612 mg, 4 .87 mmol) and EEDQ (1.22 g, 4.87 mmol). The reaction medium was stirred at room temperature. for 24 hours, then additional 4-aminobenzyl alcohol (612 mg, 4.87 mmol) and EEDQ (1.22 g, 4.87 mmol) were added and stirred for 2 hours. The reaction medium was then concentrated in vacuo; Et ₂ O was added to the crude product, the mixture was stirred and filtered to obtain a brown solid, which was diluted in Et ₂ O and the mixture was stirred at room temperature. during the night. The mixture was then filtered and the precipitate was dried to obtain 1.3 g of compound 21 as a brown solid (qty).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d, J=7.0 Hz, 3H); 1.30 (d, J=7.0 Hz, 3H); 2.00 (m, 1H); 3.91(m, 1H); 4.19 to 4.34 (m, 4H); 4.43 (d, J=5.7 Hz, 2H); 5.08 (t, J=5.7 Hz, 1H); 7.23 (d, J=7.9 Hz, 2H); 7.31 (t, J=7.9 Hz, 2H); 7.40 (m, 3H); 7.53 (d, J=9.1 Hz, 2H); 7.73 (t, J=8.6 Hz, 2H); 7.89 (d, J=7.9 Hz, 2H); 8.16 (d, J=7.3 Hz, 1H); 9.91(s, 1H). LCMS (A): ES mass/charge=292; mass/charge=393; mass/charge=516 [M+N]+; Rt=1.21 min.

Compound 22: (9H-fluoren-9-yl)methyl-((S)-3-methyl-1 -(((S)-1 -((4-((((4-nitrophenoxy)carbonyl)oxy)methyl )phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate.

To a solution of compound 21 (1.3 g, 2.52 mmol) in DMF (10 ml) under Ar atmosphere was added DIEA (2.55 ml, 15.13 mmol), the solution was cooled at 0°C before adding 4-nitrophenylchloroformate ( 1.57 g, 7.56 mmol). The reaction medium was stirred for 2 hours at room temperature. 20 ml H2O was added and the medium was extracted twice with EtOAc (10 ml). The combined organic phases were washed with brine, dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 120 g silica gel (DCM/MeOH gradient elution) to give 600 mg

- 100 045708 compound 22 in the form of a solid (35%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d, J=7.0 Hz, 3H); 1.31 (d, J=7.0 Hz, 3H); 1.99 (m, 1H); 3.91(m, 1H); 4.18 to 4.35 (m, 3H); 4.43 (m, 1H); 5.24(s, 2H); 7.31 (t, J=7.9 Hz, 2H); 7.35 to 7.44 (m, 5 H); 7.56 (d, J=9.1 Hz, 2H); 7.63 (d, J=8.6 Hz, 2H); 7.74 (m, 2H); 7.88 (d, J=7.9 Hz, 2H); 8.19 (d, J=7.3 Hz, 1H); 8.31 (d, J=9.1 Hz, 2H); 10.07(s, 1H).

Compound 23: 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl-4-((2R,3R)-3-((S)-1((3S, 10R, 16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa8,11-diazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzylcarbamate/

To a solution of (3S,10R,16S,E)-16-((S)-1-(2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-(3chloro -4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77 , 100 mg, 143.2 µmol) in THF (5 ml) under argon was added compound 22 (117 mg, 171.9 µmol) and DIEA (60.4 µl, 358 µmol). The reaction medium was stirred at room temperature. in an Ar atmosphere overnight. Piperidine (142.9 μL, 1.43 mmol) was then added and the reaction mixture was stirred for 1 hour at room temperature. After concentration in vacuo, the crude medium was purified by flash chromatography on 5 g silica gel (DCM/MeOH gradient elution) to obtain 100 mg of compound 23 as a white solid (73%).

Compound 24: allyl-15-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)- 1-((3S,10R,16S,E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8 ,11-diazacyclohexadec-13-en16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)-methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1 - oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecane-18-oate.

To a solution of compound 23 (100 mg, 98.3 μmol) in THF (5 ml) under Ar atmosphere was added compound 9 (50.8 mg, 98.3 μmol) and DIEA (24.9 μl, 147.4 μmol). The reaction medium was stirred for 1 h at RT, concentrated in vacuo and purified by flash chromatography on 5 g silica gel (DCM/MeOH gradient elution) to obtain 128 mg of compound 24 as a white solid (91%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60:40; 0.78 (d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8 H); 0.84 (d, J=7.0 Hz, 1.2H); 0.86 (d, J=7.0 Hz, 1.8 H); 0.88 (d, J=7.0 Hz, 1.2H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.30 (m, 4H); 1.57 (m, 2H); 1.80 (m, 1H); 1.95 (m, 1H); 2.26 (m, 1H); 2.40 (m, 2H); 2.52 to 2.72 (m, 6H); 2.95 to 3.04 (m, 3H); 3.22(s, 3H); 3.30 (m, 1H); 3.38 to 3.63 (m, 18 H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.15 to 4.28 (m, 4H); 4.40 (m, 1H); 4.51 to 4.57 (m, 2H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.98(s, 2H); 5.10 (m, 1H); 5.20 (m, 1H); 5.30 (m, 1H); 5.80 (d, J=16.0 Hz, 1H); 5.91(m, 1H); 6.47 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.29 to 7.32 (m, 8 H); 7.58 (m, 2H); 7.76 (t, J=6.5 Hz, 1H); 7.92 (d, J=9.0 Hz, 0.4H); 8.04 (d, J=9.0 Hz, 0.6 H); 8.15 (d, J=7.0 Hz, 0.4H); 8.21 (d, J=7.0 Hz, 0.6 H); 8.32 (d, J=8.0 Hz, 1H); 9.88(s, 0.4H); 9.93 (s, 0.6 H). LCMS (A): ES mass/charge=677; mass/charge=1418 [M+H]+; mass/charge=1462 [M-H+HCO ₂ N]'; Rt=1.21 min.

Example 8: 15-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1- ((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutane -2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecane-18-oic acid.

To a solution of compound 24 (58 mg, 40.9 µmol) in DCM (20 ml) under Ar atmosphere was added 1,3dimethylpyrimidine-2,4,6(1H, 3H, 5H)-trione (19.5 mg, 122.6 µmol) and tetrakis(triphenylphosphine)palladium(0) (2.4 mg, 2.0 µmol). The reaction medium was stirred for 3 hours at RT, concentrated in vacuo and purified by flash chromatography on 5 g silica gel (DCM/MeOH gradient elution) to give 53 mg of the compound of Example 8 as a white solid (94% ).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers; 0.78 (d, J=6.8 Hz, 6H); 0.86 (m, 6H); 0.99(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.30 (m, 1H); 1.38(m, 3H); 1.57 (m, 2H); 1.79 (m, 1H); 2.04 (m, 1h); 2.10 to 2.59 (partially hidden m, 7H); 2.69 (m, 2H); 2.93 to 3.03 (m, 3H); 3.22(s, 3H); 3.30 (hidden m, 1H); 3.40 to 3.64 (m, 18 H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.05 to 4.27 (m, 4H); 4.36 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97(s, 2H); 5.10 (m, 1H); 5.80 (d, J=16.0 Hz, 1H); 5.90 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 6.46 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 N); 7.68 (m, 2H); 7.82 (m, 1H); 8.40 to 8.65 (m, 2H); 9.21 (m, 1H); 10.20 (s, 1H). LCMS (A): ES mass/charge=637; mass/charge=1376 [M-N] ^- ; mass/charge=1378 [M+H]+; Rt=1.3 min.

Example 9: 2,5-dioxopyrrolidin-1-yl-15-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R) -3-((S)-1-((3 S, 10R, 16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9, 12-tetraoxo-1,4-dioxa-8,11diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)amino)-1 oxopropan-2-yl) amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15azaoctadecane-18-oate

To a solution of the compound of Example 8 (49 mg, 35.5 µmol) in DMF (2 ml) under Ar atmosphere was added DSC (13.9 mg, 53.3 µmol) and DIEA (9.0 µl, 53.3 µmol) . The reaction medium was stirred at room temperature. V

- 101 045708 during the night. Additional DSC (13.9 mg, 53.3 µmol) and DIEA (9.0 µl, 53.3 µmol) were added and stirred for 8 hours. The reaction medium was concentrated in vacuo and purified by flash chromatography on 10 g diol-modified silica gel (DCM/iPrOH gradient elution) to give 20 mg of Example 9 as a white solid (38%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 70:30 mixture of conformers; 0.78 (d, J=6.8 Hz, 6H); 0..82 (d, J=7.0 Hz, 2.1H); 0.84 (d, J=7.0 Hz, 0.9H); 0.86 (d, J=7.0 Hz, 2.1H); 0.88 (d, J=7.0 Hz, 0.9H); 1.00(s, 3H); 1.05 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.27 (m, 1H); 1.30 (m, 3H); 1.57 (m, 2H); 1.80 (m, 1H); 1.95 (m, 1H); 2.25 (m, 1H); 2.40 (m, 1H); 2.52 to 2.75 (m, 7 H); 2.80(s, 4H); 2.87 to 3.08 (m, 3H); 3.22(s, 3H); 3.32 (hidden m, 1H); 3.40 to 3.68 (m, 18 H); 3.80(s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.16 to 4.28 (m, 4H); 4.38 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97(s, 2H); 5.10 (m, 1H); 5.79 (d, J=16.0 Hz, 1H); 6.46 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to 7.31 (m, 8 H); 7.58 (m, 2H); 7.78 (t, J=6.0 Hz, 1H); 7.94 (d, J=9.0 Hz, 0.3 H); 8.08 (d, J=9.0 Hz, 0.7 H); 8.19 (d, J=7.0 Hz, 0.3 H); 8.25 (d, J=7.0 Hz, 0.7 H); 8.35 (d, J=8.0 Hz, 1H); 9.90(s, 0.3H); 9.95 (s, 0.7 H). LCMS (A): ES mass/charge=698; mass/charge=1475 [M+H]+; Rt=1.35 min.

Synthesis of Example 10: PEC7-Val-Ala-PABA-C52-benzylamine

Compound 25: tert-butyl-2,5,8,11,14,17,20-heptaoxatricosane-23-oate.

Na (0.7 mg, 32.4 μmol) was added to a solution of hexaethylene glycol monomethyl ether (926.0 µl, 3.24 mmol) in THF (3 ml) under Ar atmosphere. The reaction medium was stirred for 1 hour at room temperature. until Na dissolution was complete, then tert-butyl acrylate (570 μL, 3.89 mmol) was added and stirred at room temperature. during the night. The reaction medium was then concentrated in vacuo, diluted with H2O and extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over MgSO ₄ , filtered and concentrated in vacuo to give 1.04 g of compound 25 as a colorless oil (76%).

- 102 045708 ¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 1.40 (s, 9H); 2.40 (t, J=6.4 Hz, 2H); 3.23(s, 3H); 3.43 (m, 2H); 3.47 to 3.54 (m, 22H); 3;59 (t, J=6.4 Hz, 2H).

Compound 26: 2,5,8,11,14,17,20-heptaoxatricosan-23-oic acid.

To a solution of compound 25 (1.04 g, 2.45 mmol) in DCM (15 ml) was added TFA (1.84 ml, 24.5 mmol). The reaction medium was stirred at room temperature. overnight, concentrated in vacuo, diluted with DCM and co-evaporated with toluene (3x) to give 1 g of compound 26 as a colorless oil (qt).

Compound 27: 2,5,8,11,14,17,20-heptaoxatricosan-23-oil chloride.

To compound 26 (400 mg, 1.09 mmol) was added MsCl (1.27 ml, 16.3 mmol); the reaction medium was heated for 3 hours at 60°C, concentrated in vacuo, diluted with DCM and concentrated in vacuo (3x) to obtain 420 mg of compound 27 as a colorless oil (quantitative).

Compound 28: allyl-24-(3-(tert-butoxy)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24azaheptacosane-27-oate.

To a solution of compound 3 (200 mg, 777.2 µmol) in DCM (5 ml) was added DIEA (262 µl, 1.55 mmol). The reaction medium was cooled at 0°C and a solution of compound 27 (360.8 mg, 932.7 μmol) in DCM (2 ml) was added dropwise at 0°C. The reaction medium was stirred for 1 hour at room temperature. and concentrated in vacuo. The crude product was purified by two sequential flash chromatography runs on 12 g and 10 g silica gel (DCM/MeOH gradient elution) to give 72 mg of compound 28 (15%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 50:50; 1.39 (s, 4.5 H); 1.40 (s, 4.5 H); 2.22 to 2.75 (partially hidden m, 6H); 3.23(s, 3H); 3.38 to 3.63 (m, 30H); 4.55 (m, 2H); 5.19 to 5.36 (m, 2H); 5.90 (m, 1H).

Compound 29: 24-(3-(allyloxy)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24azaheptacosane-27-oic acid.

To a solution of compound 28 (72 mg, 118.5 μmol) in DCM (3 ml) was added TFA (100 μl, 1.33 mmol); the reaction medium was stirred at room temperature. overnight, then additional TFA (100 μL, 1.33 mmol) was added and stirred for 1 day. The reaction medium was concentrated in vacuo, diluted with DCM and co-evaporated with toluene (3x) to give 66 mg of compound 29 (qt).

Compound 30: allyl-24-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-23-oxo2,5,8,11,14,17,20-heptaoxa-24- Azaheptacosane-27-oate.

To a solution of compound 29 (66 mg, 119.7 μmol) in THF (2 ml) under Ar atmosphere was added DIEA (20.2 μl, 119.9 μmol) and DSC (34.4 mg, 131.6 μmol); the reaction medium was stirred for 4 days at RT, concentrated in vacuo and purified by flash chromatography on 900 mg diol-modified silica gel (DCM/iPrOH gradient elution) to give 35 mg of compound 30 (45%).

Compound 31: allyl-24-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)- 1-((3S,10R,16S,E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8 ,11-diazacyclohexadec-13-en16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)-methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1 - oxobutan-2-yl)amino)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24-azaheptacosane-27-oate.

To a solution of compound 23 (45 mg, 44.2 μmol) in THF (5 ml) was added compound 30 (28.7 mg, 44.2 μmol) and DIEA (11.2 μl, 66.3 μmol). The reaction medium was stirred at room temperature. overnight, diluted with H2O (10 ml) and extracted with AtOAc (3x 10 ml). The combined organic phases were washed with brine, dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 5 g silica gel (DCM/MeOH gradient elution) to give 35 mg of compound 31 as a white solid (51%) .

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60:40; 0.80 (divided d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8 H); 0.84 (d, J=7.0 Hz, 1.2H); 0.86 (d, J=7.0 Hz, 1.8 H); 0.88 (d, J=7.0 Hz, 1;2H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.28(m, 1H); 1.30 (m, 3H); 1.56 (m, 2H); 1.79 (m, 1H); 1.95 (m, 1H); 2.27 (m, 1H); 2.35 to 2.60 (partially hidden m, 6H); 2.62 to 2.73 (m, 2H); 2.93 to 3.05 (m, 3H); 3.23(s, 3H); 3.33 (hidden m, 1H); 3.38 to 3.64 (m, 30H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.15 to 4.27 (m, 4H); 4.38 (m, 1H); 4.52 to 4.58 (m, 2H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97(s, 2H); 5.10 (m, 1H); 5.50 (m, 1H); 5.30 (m, 1H); 5.80 (d, J=16.0 Hz, 1H); 5.90 (m, 1H); 6.47 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.15 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.59 (m, 2H); 7.78 (t, J=6.5 Hz, 1H); 7.95 (d, J=9.0 Hz, 0.4H); 8.07 (d, J=9.0 Hz, 0.6 H); 8.18 (d, J=7.0 Hz, 0.4H); 8.27 (d, J=7.0 Hz, 0.6 H); 8.36 (d, J=8.0 Hz, 1H); 9.90(s, 0.4H); 9.95 (s, 0.6 H). LCMS (A): ES mass/charge=698; mass/charge=1552 [M+H]+; mass/charge=1595 [M-H+HCO2H] ^- ; Rt=1.39 min.

- 103 045708

Example 10: 24-(3-(((S)-1 -(((S)-1 -((4-((((4-((2R,3R)-3-((S)-1 - ((3S, 10R, 16S,E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2 -yl)amino)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24-azaheptacosane-27 oic acid.

To a solution of compound 31 (30 mg, 19.3 μmol) in DCM (5 ml) under Ar atmosphere was added dimethylbarbituric acid (9.24 mg, 58.0 μmol) and tetrakis(triphenylphosphine)palladium(0) (1.13 mg , 0.97 µmol). The reaction medium was stirred for 3 hours at RT, then concentrated in vacuo and purified by two sequential flash chromatography runs on 5 g and 0.9 g silica gel (DCM/MeOH gradient elution) to obtain 14 mg of the compound of the example 10 as a white solid (50%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 50:50 mixture of conformers; 0.78 (divided d, J=6.8 Hz, 6H); 0.81 to 0.89 (m, 6H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.12(s, 3H); 1.28(m, 1H); 1.30 (d, J=7.0 Hz, 1.5 H); 1.32 (d, J=7.0 Hz, 1.5 H); 1.51 to 1.62 (m, 2H); 1.79 (m, 1H); 1.96 (m, 1H); 2.26 (m, 1H); 2.33 to 2.60 (partially hidden m, 6H); 2.61 to 2.73 (m, 2H); 2.93 to 3.03 (m, 3H); 3.22(s, 3H); 3.30 (hidden m, 1H); 3.38 to 3.62 (m, 30H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.12 to 4.29 (m, 4H); 4.39 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.96(s, 2H); 5.10 (m, 1H); 5.78 (d, J=16.0 Hz, 1H); 6.46 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.59 (m, 2H); 7.79 (m, 1H); 8.04 (m, 0.5 H); 8.10 (d, J=9.0 Hz, 0.5 H); 8.25 to 8.41 (m, 2H); 9.95(s, 0.5H); 9.98 (s, 0.5 H); 12.20 (m, 1H). LCMS (D): ES mass/charge=769; mass/charge=1511 [M+H]+; Rt=3.16 min.

Synthesis of Examples 11 and 12: PEG24-Val-Ala-PABA-C52-benzylamine and PEG24-ValAla-PABA-C52-benzylamine NHS ester

- 104 045708

Compound 32: allyl-75-(3-(tert-butoxy)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35, 38 ,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontane-78-oate.

To a solution of compound 3 (45 mg, 174.9 µmol) in THF (5 ml) under Ar atmosphere were added MEODPEG(24)-NHS (212.4 mg, 174.9 µmol) and DIEA (44.2 µl, 262, 3 µmol). The reaction medium was stirred at room temperature. overnight, then concentrated in vacuo and purified by flash chromatography on 20 g silica gel (DMC/MeOH gradient elution) to give 125 mg of a mixture of compound 32 (60%) and MEO-DPEG(24)-NHS (40%). This mixture was diluted in THF (3 ml), 15 mg of compound 3 and 20 μl of DIEA were added; the reaction medium was stirred at room temperature. overnight, then concentrated in vacuo and purified by flash chromatography on 12 g silica gel to give 105 mg of compound 32 (44%).

Ή NMR (400 MHz, δ in ppm, DMSO-d6): 1.40 (s, 9H); 2.25 to 2.78 (m, 6H); 3.22(s, 3H); 3.32 to 3.70 (m, 98 H); 4.52 (m, 2H); 5.20 (m, 1H); 5.30 (m, 1H); 5.90 (m, 1H). LCMS (A): ES mass/charge=679 [M+2H] ²⁺ ; mass/charge=1356 [M+N] ⁺ ; mass/charge=1400 [M-H+HCO2H]-; Rt=1.15 min.

Compound 33: 75-(3-(allyloxy)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,

44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontane-78-oic acid.

To a solution of compound 32 (105 mg, 77.4 µmol) in DCM (2 ml) was added TFA (116.2 µl, 1.55 mmol); the reaction medium was stirred at room temperature. overnight, then 150 μl of TFA was added and the reaction was carried out for another day. The reaction medium was then concentrated in vacuo, diluted with DCM, co-evaporated with toluene (3x) to give 110 mg of crude product, which was purified by RP-HPLC on a C ₁₈ SunFire 5 µm column (Waters, 30x100 mm, MeCN+ gradient elution 0.07% TFA/H ₂ O+0.07% TFA) to obtain 60 mg of compound 33 (60%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 50:50; 2.37 to 2.60 (partially hidden m, 6H); 3.23(s, 3H); 3.32 to 3.44 (partially hidden m, 38 H); 3.47 to 3.52 (m, 58 H); 3.60 (m, 2H); 4.54(m,

- 105 045708

2H); 5.22 (dm, J=10.5 Hz, 1H); 5.30 (dm, J=17.3 Hz, 1H); 5.84 to 5.98 (m, 1H); 12.32 (wide m, 1H).

Compound 34: allyl-75-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)- 1-((3S,10R,16S,E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8 ,11-diazacyclohexadec-13-en16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)-methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1 -oxobutan-2-yl)amino)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47 ,50,53, 56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontane-78-oate.

To a solution of compound 33 (30 mg, 23.1 µmol) in THF (5 ml) under Ar atmosphere were added DIEA (11.7 µl, 69.2 µmol) and DSC (6.03 mg, 23.1 µmol). The reaction medium was stirred for 3 hours at room temperature, then compound 23 (23.5 mg, 23.1 µmol) was added and stirred for 1 hour. The reaction medium was concentrated in vacuo and purified by flash chromatography on 5 g silica gel to give 23 mg of compound 34 (40%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): conformer mixture 60:40: 0.80 (d separated, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8 H); 0.84 (d, J=7.0 Hz, 1.2H); 0.86 (d, J=7.0 Hz, 1.8 H); 0.88 (d, J=7.0 Hz, 1.2H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.28 (m, 1 H); 1.30 (d, J=7.0 Hz, 3H); 1.51 to 1.62 (m, 2H); 1.80 (m, 1H); 1.96 (m, 1H); 2.26 (m, 1H); 2.38 (m, 1H); 2.45 to 2.61 (partially hidden m, 5 H); 2.61 to 2.72 (m, 2H); 2.93 to 3.05 (m, 3H); 3.23(s, 3H); 3.32 (hidden m, 1H); 3.40 to 3.77 (m, 98 H); 3.80 (s. 3H); 3.88 (d, J=2.3 Hz, 1H; 4.14 to 4.27 (m, 4H); 4.39 (m, 1H); 4.51 to 4.60 (m, 2H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97 (s, 2H); 5.10 (m, 1H); 5.18 to 5.33 (m, 2H) ; 5.78 (d, J=16.0 Hz, 1H); 5.90 (m, 1H); 6.45 (m, 1H); 7.05 (d, J=8.6 Hz, 1H) ; 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.59 (m, 2H); 7.77 (t, J=6.5 Hz, 1H); 7.95 (d, J=9.0 Hz, 0.4H); 8.07 (d, J=9.0 Hz, 0.6 H); 8.19 (d, J=7.0 Hz, 0.4H) 8.25 (d, J=7.0 Hz, 0.6 H) 8.36 (d, J=8.0 Hz, 1H); 9.90 (s, 0.4H); 9.96 (s, 0.6H).

Example 11: 75-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1- ((3S,10R,16S,E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2 -yl)amino)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53 , 56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontane-78-oic acid.

To a solution of compound 34 (37 mg, 161 µmol) in DCM under Ar atmosphere was added 1,3dimethylbarbituric acid (5.13 mg, 32.2 µmol) and tetrakis(triphenylphosphine)palladium(0) (0.94 mg, 0.80 µmol); the reaction medium was stirred at room temperature. for 2 hours, then concentrated in vacuo and purified by flash chromatography on 5 g diol-modified silica gel (DCM/MeOH gradient elution) to give 16 mg of Example 11 (44%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d6): 0.77 (d, J=7.0 Hz, 6H); 0.80 to 0.90 (m, 6H); 0.99(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.28(m, 1H); 1.30 (d, J=7.0 Hz, 1.5 H); 1.32 (d, J=7.0 Hz, 1.5 H); 1.52 to 1.61 (m, 2H); 1.79 (m, 1H); 1.98(m, 1H); 2.22 to 2.58 (partially hidden m, 7 H); 2.68(m, 2H); 2.92 to 3.04 (m, 3H); 3.21(s, 3H); 3.30 (partially hidden m, 1H); 3.37 to 3.67 (m, 98 H); 3.81(s, 3H); 3.88(s, 1H); 4.08 to 4.28 (m, 4H); 4.39 (m, 1H); 4.90 (m, 1H); 4.98(s, 2H); 5.10 (m, 1H); 5.80 (d, J=16.0 Hz, 1H); 6.48 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.04 (d, J=8.6 Hz, 1H); 7.16 (wide d, J=8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.57 to 7.67 (m, 2H); 7.80 (m, 1H); 8.20 (m, 1H); 8.35 to 8.70 (m, 2H); 10.08 (m, 1H); 12.30 (wide m, 1H). LCMS (D): ES mass/charge=698; mass/charge=2259 [M+H]+; Rt=3.14 min.

Example 12: 2,5-dioxopyrrolidin-1-yl-75-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R) -3-((S)-1((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12- tetraoxo-1,4-dioxa-8,11diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl) amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26, 29,32,35 ,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontane-78-oate.

To a solution of the compound of Example 11 (16 mg, 7.1 µmol) in DCM was added DIEA (1.2 µl, 7.1 µmol) and DSC (1.8 mg, 7.1 µmol). The reaction medium was stirred for 3 hours at RT, then concentrated in vacuo and purified by flash chromatography on 1.3 g diol-modified silica gel (DCM/iPrOH gradient elution) to give 5 mg of a mixture of the compound of Example 12 ( 30%) and the compound from example 11 (70%).

LCMS (D): ES mass/charge=698; mass/charge=2356 [M+H]+; t _R =3.2 min.

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Synthesis of Example 13: Sulfo-PEG4-Val-Ala-PABA-C52-Benzylamine

Compound 3 5: 4-((3 -(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4oxobutanoic acid.

To a solution of compound 3 (100 mg, 388.6 µmol) in DCM (5 ml) was added succinic anhydride (78.6 mg, 777.2 µmol). The reaction medium was stirred at room temperature. overnight, then concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/MeOH gradient elution) to give 102 mg of compound 35 as a colorless oil (73%).

Compound 36: 1-ammonio-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecane-18-sulfonate.

To a solution of 15-(Boc-amino)-4,7,10,13-tetraoxapentadecanoic acid (500 mg, 1.3 mmol) in THF (5 mL) under Ar atmosphere was added DIEA (438.4 μL, 2.6 mmol ) and DSC (381.6 mg, 1.43 mmol). The reaction medium was stirred for 6 hours at room temperature, then 50 mg of DSC was added and stirring was carried out

- 107 045708 at k.t. during the night. At this time, taurine (821.6 mg, 6.5 mmol) and H ₂ O (1 ml) were added to the reaction medium. The reaction medium was stirred at room temperature. overnight, concentrated in vacuo and purified by preparative LCMS on a C ₁₈ SunFire 5 µm column (Waters, 30x100 mm, gradient elution MeCN+0.07% TFA/H ₂ O+0.07% TFA) to give 341 mg compounds 36 (70%).

1H NMR (400 MHz, δ in ppm, DMSO-d6): 2.28 (t, J=6.5 Hz, 2H); 2.60 (m, 2H); 2.99 (m, 2H); 3.32 (m, 2H); 3.45 to 3.65 (m, 16 H); 7.77 (broad m, 4H). IR spectrum in the case of a tablet with KBr; main absorption bands in reciprocal centimeters: 3000; 1780; 1648; 1556; 1200-1170; 1200; 1100; 1040.

Compound 37: 24-(3-(tert-butoxy)-3-oxopropyl)-4,20,23,27-tetraoxo-7,10,13,16,28-pentaoxa3,19,24-triazagentriacont-30-ene -1-sulfonate.

DIEA (141.6 μL, 839.4 μmol) and DSC (73.1 mg, 279.8 μmol) were added to a solution of compound 35 (100 mg, 279.8 μmol) in THF. The reaction mixture was stirred for 2 hours at room temperature, then a solution of compound 36 (104.2 mg, 279.8 µmol) in DMF and DIEA (141.6 µl, 839.4 µmol) was added. The reaction medium was stirred for 2 hours at RT, concentrated in vacuo and purified using preparative LCMS on a C ₁₈ SunFire 5 µm column (Waters, 30x100 mm, gradient elution MeCN+0.07% TFA/H ₂ O+0 .07% TFA) to obtain 118 mg of compound 37 (60%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 50:50 mixture of conformers; 1.39 (s, 4.5 H); 1.40 (s, 4.5 H); 2.27 (t, J=6.5 Hz, 2H); 2.31 (t, J=7.0 Hz, 2H); 2.38 (t, J=7.0 Hz, 1H); 2.45 to 2.56 (hidden m, 6H); 2.68 (m, 1H); 3.18 (q, J=6.0 Hz, 2H); 3.29 (m, 2H); 3.36 to 3.60 (m, 20H); 4.51 to 4.59 (m, 2H); 5.17 to 5.25 (m, 2H); 5.85 to 5.95 (m, 1H); 7.75 (t, J=6.0 Hz, 1H); 7.88 (t, J=6.0 Hz, 1H).

Compound 3 8: 24-(2-carboxyethyl)-4,20,23,27-tetraoxo-7,10,13,16,28-pentaoxa-3,19,24triazagentriacont-30-ene-1-sulfonate.

To a solution of compound 37 (118 mg, 166.0 µmol) in DCM (5 ml) was added TFA (249 µl, 3.32 mmol). The reaction medium was stirred at room temperature. overnight, concentrated in vacuo, diluted with DCM and co-evaporated with toluene (3x) to give 100 mg of compound 38 as a colorless oil (92%).

Compound 39: (2S,5S)-10-(3-(allyloxy)-3-oxopropyl)-1 -((4-((((4-((2R,3R)-3-((S)-1 -((3S, 10R,

16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,11,14,30-hexaoxo- 18,21,24,27-tetraoxa-3,6,10,15,31-pentaazatriacontane-33-sulfonic acid

To a solution of compound 38 (100 mg, 152.7 μmol) in THF (3 ml) and DMF (1 ml) was added DIEA (77 μl, 458.2 μmol) and DSC (39.9 mg, 152.7 μmol). The reaction medium was stirred for 2 hours at room temperature. before adding a solution of compound 23 (124 mg, 121.9 µmol) in THF (1 ml) and DMF (0.5 ml) and DIEA (100 µl). The reaction medium was stirred for 2 h at RT, concentrated in vacuo and purified by reverse phase flash chromatography on 75 g _C18 -modified silica gel ( _H2O /MeCN gradient elution) to give 60 mg of compound 39 in as a white solid (24%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60:40; 0.78 (d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8 H); 0.84 (d, J=7.0 Hz, 1.2H); 0.86 (d, J=7.0 Hz, 1.8 H); 0.88 (d, J=7.0 Hz, 1.2H); 0.99(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.28(m, 1H); 1.30 (d, J=7.0 Hz, 3H); 1.50 to 1.62 (m, 2H); 1.80 (m, 1H); 1.96(m, 1H); 2.20 to 2.55 (partially hidden m, 11H); 2.61 to 2.75 (m, 2H); 2.94 to 3.05 (m, 3H); 3.10 to 3.65 (partially hidden, 27 H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.13 to 4.28 (m, 4H); 4.38 (m, 1H); 4.53 (m, 1.2H); 4.56 (m, 0.8 H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97(s, 2H); 5.10 (m, 1H); 5.18 to 5.33 (m, 2H); 5.80 (d, J=16.0 Hz, 1H); 5.90 (m, 1H); 6.47 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.59 (m, 2H); 7.72 (t, J=6.5 Hz, 1H); 7.79 (t, J=6.0 Hz, 1H); 7.89 (t, J=6.5 Hz, 1H); 7.95 (d, J=9.0 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6 H); 8.19 (d, J=7.0 Hz, 0.4H); 8.25 (d, J=7.0 Hz, 0.6 H); 8.36 (d, J=8.0 Hz, 1H); 9.90(s, 0.4H); 9.95 (s, 0.6 H). LCMS (D): ES mass/charge=913; mass/charge=1654 [M+N] ⁺ ; Rt=3.87 min.

Example 13: 24-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1- ((3S,10R,16S,E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2 -yl)amino)-3-oxopropyl)-4,20,23-trioxo-1-sulfo-7,10,13,16-tetraoxa-3,19,24-triazaheptacosane-27-oic acid

To a solution of compound 39 (60 mg, 36.3 μmol) in DMF (5 ml) was added 1,3-dimethylbarbituric acid (17.3 mg, 108.8 μmol) and tetrakis(triphenylphosphine)palladium(0) (2, 1 mg, 1.8 µmol). The reaction medium was stirred at room temperature. overnight, concentrated in vacuo and purified by two flash chromatography runs on 30 g and 20 g C ₁₈ -modified silica gel to obtain two 6 mg batches of the compound of Example 13 as a white solid (20% overall yield).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60:40; 0.78 (d, J=6.8 Hz, 6H); 0.81 to 0.89 (m, 6H); 1.00(s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11(s, 3H); 1.28(m, 1H); 1.30 (d, J=7.0 Hz, 3H); 1.58 (m, 2H); 1.80 (m, 1H); 1.96 (m, 1H); 2.21 to 2.58 (partially hidden m, 11H); 2.69 (m, 2H); 2.93 to 3.68 (partially

- 108 045708 hidden m, 28 H); 3.80(s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.11 to 4.28 (m, 4H); 4.38 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.98(s, 2H); 5.10 (m, 1H); 5.79 (d, J=16.0 Hz, 1H); 6.47 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.59 (m, 2H); 7.72 (t, J=6.5 Hz, 1h); 7.79 (t, J=6.0 Hz, 1H); 7.89 (t, J=6.5 Hz, 1H); 7.97 (d, J=9.0 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6 H); 8.19 (d, J=7.0 Hz, 0.4H); 8.25 (d, J=7.0 Hz, 0.6 H); 8.36 (d, J=8.0 Hz, 1H); 9.90(s, 0.4H); 9.94 (s, 0.6 H); 12.15 (wide m, 1H). LCMS (A): ES mass/charge=807.5 [M+2H]2+; mass/charge=1612 [M-N] ^- ; mass/charge=1614 [M+N] ⁺ ; Rt=1.72 min.

Synthesis of Examples 14-16: Glutaryl-Val-PEG4Gln-C52-benzylamine, Glutaryl-ValPEG4Gln-C52-benzylamine NHS ester and corresponding ADC

example 16

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Compound 40: (S)-4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5(tert-butoxy)-5-oxopentane acid.

To a solution of H-Glu-OtBu (CAS number [45120-30-7], 959 mg, 4.7 mmol) and NaHCO ₃ (400 mg, 4.77 mmol) in H ₂ O (80 ml) with stirring using magnetic stirrer, a solution of (S)-2,5dioxopyrrolidin-1-yl-2-((((9H-fluoren-9-yl)methoxy)-carbonyl)amino)-3-methylbutanoate (2 g, 4.6 mmol) in THF (80 ml). The reaction medium was stirred at room temperature. during the night. The medium was concentrated in vacuo, then saturated aqueous NaHCO ₃ (1 L) was added, followed by extraction with Et ₂ O (2x250 ml), the organic phases were excluded. The aqueous phase was acidified with QS 1N. aqueous HCl solution until pH 2 and extracted with DCM (3x250 ml), the combined organic phases were dried over MgSO ₄ , filtered and concentrated in vacuo to give 2.05 g of compound 40 (85%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.87 (d, J=6.9 Hz, 3H); 0.90 (d, J=6.9 Hz, 3H); 1.38(s,9H); 1.73 to 2.02 (m, 3H); 2.24 to 2.30 (m, 2H); 3.90 (dd, J=7.3 and 9.0 Hz, 1H); 4.14 (m, 1H); 4.19 to 4.32 (m, 3H); 7.27 to 7.45 (m, 5H); 7.75 (t, J=7.6 Hz, 2H); 7.89 (d, J=7.6 Hz, 2H); 8.22 (wide d, J=7.0 Hz, 1H); 12.05 (wide s, 1H).

Compound 41: (S)-mpem-butyl-18-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3 methylbutanamido)-15-oxo-2,5 ,8,11-tetraoxa-14-azanonedecane-19-oate.

To a solution of compound 40 (200 mg, 381 µmol) in DCM (6 ml), PEG4-NH2 (CAS number [85030-56-4], 115 mg, 554.9 µmol), HOBt (82 mg, 606.8 µmol) and EDC (100 µl, 565 µmol). The reaction medium was stirred at room temperature. during the night. The crude medium was purified twice by flash chromatography on silica gel (DCM/MeOH gradient elution) to obtain 190 mg of compound 41 (70%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d6): 0.87 (d, J=6.8 Hz, 3H); 0.90 (d, J=6.8 Hz, 3H); 1.38(s,9H); 1.72 to 1.93 (m, 2H); 1.99 (m, 1H); 2.14 (t, J=7.8 Hz, 2H); 3.18(m, 2H); 3.23(s, 3H); 3.37 (t, J=6.0 Hz, 2H); 3.41(m, 2H); 3.46 to 3.53 (m, 10H); 3.92 (dd, J=7.l and 8.8 Hz, 1H); 4.09 (m, 1H); 4.17 to 4.34 (m, 3H); 7.32 (m, 3H); 7.41 (t, J=7.6 Hz, 2H); 7.70 to 7.81 (m, 3H); 7.88 (d, J=7.6 Hz, 2H); 8.18 (d, J=7.3 Hz, 1H). LCMS (A): ES mass/charge=714 [M+H]+; mass/charge=758 [M-H+HCO2H]-; Rt=1.41 min.

Compound 42: (S)-18-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)15-oxo-2,5,8,11 -tetraoxa-14-azanonedecane-19-oic acid.

A 4 M solution of HCl in 1,4-dioxane (5 ml) was added to compound 41 (190 mg, 266 µmol) with magnetic stirring. The reaction medium was stirred at room temperature. for 4 hours. At the same time, the reaction medium was concentrated in vacuo and purified by flash chromatography on 15 g of silica gel (gradient elution DCM/MeOH/NH ₄ OH) to obtain 70 mg of compound 42 (40%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d6): 0.84 (d, J=6.6 Hz, 3H); 0.86 (d, J=6.6 Hz, 3H); 1.70 to 1.90 (m, 2H); 2.01 to 2.15 (m, 3H); 3.15 (m, 2H); 3.23(s, 3H); 3.36 (t, J=6.4 Hz, 2H); 3.42(m, 2H); 3.45 to 3.53 (m, 10H); 3.80 (wide m, 1H); 3.83 (dd, J=6.8 and 8.7 Hz, 1H); 4.19 to 4.34 (m, 3H); 7.33 (m, 2H); 7.41 (t, J=7.6 Hz, 2H); 7.59 (wide m, 2H); 7.73 (d, J=7.6 Hz, 1H); 7.76 (d, J=7.6 Hz, 1H); 7.89 (d, J=7.6 Hz, 2H); 8.00 (wide m, 1H). LCMS (A): ES mass/charge=656 [M-N] ^- ; mass/charge=658 [M+H]+; RT=1.19 min.

Compound 43: (S)-2-((S)-2-amino-3-methylbutanamido)-N 1 -(4-((2R,3R)-3-((S)-1 -((3S,10R , 16S,E)-10(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)-N5-(2,5,8,11 -tetraoxatridecan-13 -yl)pentanediamide.

To a solution of (E)-(3 S, 10R, 16S)-16-(S)-1 -[(2R,3R)-3 -(4-aminomethylphenyl)oxiranyl]-ethyl}-10-(3-chloro- 4methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 58 mg , 83 µmol) in DMF (5 ml) with magnetic stirring, compound 42 (70 mg, 106 µmol), HOBt (20 mg, 148 µmol) and EDC (23 µl, 130 µmol) were added. The reaction medium was stirred at room temperature. for 24 hours. At the same time, piperidine (80 μl, 810 μmol) was added to the medium and stirring was maintained for 1 hour. The reaction medium was then concentrated in vacuo and purified by flash chromatography on 20 g of silica gel (gradient elution DCM/ MeOH) to obtain 60 mg of compound 43 (65%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.77 (d, J=6.9 Hz, 3H); 0.79 (d, J=6.3 Hz, 6H); 0.87 (d, J=6.9 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=6.4 Hz, 3H); 1.12(s, 3H); 1.31(m, 1H); 1.52 to 1.63 (m, 2H); 1.72 to 1.98 (m, 4H); 2.05 to 2.15 (m, 2H); 2.26 (m, 1H); 2.62 to 2.73 (m, 2H); 2.94 to 3.04 (m, 4H); 3.18 (q, J=5.9 Hz, 2H); 3.23(s, 3H); 3.33 (partially hidden m, 1H); 3.39 (t, J=5.9 Hz, 2H); 3.42(m, 2H); 3.47 to 3.52 (m, 10H); 3.81(s, 3H); 3.87 (d, J=1.4 Hz, 1H); 4.21 to 4.36 (m, 4H); 4.91 (dd, J=3.6 and 9.6 Hz, 1H); 5.10 (dd, J=5.5 and 11.8 Hz, 1H); 5.79 (d, J=15.3 Hz, 1H); 6.46 (ddd, J=3.8, 11.6 and 15.3 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17 (dd, J=1.9 and 8.5 Hz, 1H); 7.21 to 7.25 (m, 5 H); 7.28 (d, J=1.9 Hz, 1H); 7.89 (t, J=5.5 Hz, 1H); 8.07 (wide m, 1H); 8.37 (d, J=8.0 Hz, 1H); 8.45 (t, J=5.9 Hz, 1H). LCMS (A): ES mass/charge=558 [M+2H] ²⁺ ; mass/charge=1115 [M+H]+; mass/charge=1159 [M-H+HCO2H]-; Rt=0.96 min.

Example 14: (18S,21S)-18-((4-((2R,3R)-3-((S)-1 -((3 S,10R,16S,E)-10-(3 -chloro- 4-methoxybenzyl)-3 isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxirane

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2-yl)benzyl)carbamoyl)-21-isopropyl-15,20,23-trioxo-2,5,8,11-tetraoxa-14,19,22-triazaheptacosane27-oic acid.

Glutaric anhydride (9 mg, 79 µmol) was added to a solution of compound 43 (55 mg, 49.3 µmol) in DMF (3 ml), and the reaction medium was stirred at room temperature. for 2 hours. At the same time, the medium was concentrated in vacuo and purified by flash chromatography on 10 g of silica gel (DCM/MeOH gradient elution) to obtain 38 mg of the compound from example 14 (62%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d6): 0.81 (divided d, J=6.6 Hz, 6H); 0.85 (d, J=6.3 Hz, 3H); 0.87 (d, J=6.3 Hz, 3H); 0.98(s, 3H); 1.03 (d, J=6.6 Hz, 3H); 1.11(s, 3H); 1.35 (m, 1H); 1.52 to 2.34 (m, 15 H); 2.71 (m, 2H); 2.89 to 3.02 (m, 3H); 3.09 to 3.21 (m, 2h); 3.23(s, 3H); 3.30 to 3.40 (partially hidden m, 3H); 3.42(m, 2H); 3.46 to 3.51 (m, 10H); 3.81(s, 3H); 3.87(s, 1H); 4.03 to 4.24 (m, 4H); 4.37 (m, 1H); 4.96 (m, 1H); 5.11(m, 1H); 5.81 (d, J=15.3 Hz, 1 H); 6.46 (ddd, J=3.3, 11.4 and 15.3 Hz, 1 H); 7.04 (d, J=8.5 Hz, 1 H); 7.19 (dd, J=1.9 and 8.5 Hz, 1H); 7.22 (d, J=8.3 Hz, 2H); 7.27 (d, J=8.3 Hz, 2H); 7.30 (d, J=1.9 Hz, 1H); 7.32 (m, 1H); 7.85 to 8.99 (wide m, 5 H); 12.12 (wide m, 1H). LCMS (A): ES mass/charge=615 [M+2H] ² +; mass/charge=1227 [M-N] ^- ; mass/charge=1229 [M+H]+; Rt=1.26 min.

Example 15: (18S,21S)-2,5-dioxonirrolidin-1-yl-18-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )-10-(3chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec13-en-16-yl) ethyl)oxiran-2-yl)benzyl)carbamoyl)-21-isopropyl-15,20,23-trioxo-2,5,8,11-tetraoxa14,19,22-triazaheptacosane-27-oate.

To a solution of the compound of Example 14 (32 mg, 26 µmol) in DMF (3 ml) DSC (8 mg, 31.2 µmol) and DIEA (8.6 µl, 52 µmol) were added with magnetic stirring. The reaction medium was stirred at room temperature. during the night. Then 4 mg DSC was added and stirred at room temperature. maintained for 1 hour. At the same time, the reaction medium was diluted with MeTHF (10 ml), washed with H ₂ O (5 ml). The aqueous phase was extracted with MeTHF (3x5 ml). The combined organic phases were dried over MgSO ₄ , filtered and concentrated in vacuo. The medium was purified by flash chromatography on 4 g silica gel (DCM/iPrOH gradient elution) to give 28 mg of Example 15 (81%).

% NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.78 (d, J=6.6 Hz, 6H); 0.83 (d, J=6.9 Hz, 3H); 0.85 (d, J=6.9 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=6.4 Hz, 3H); 1.12(s, 3H); 1.30 (m, 1H); 1.52 to 1.64 (m, 2H); 1.75 to 1.93 (m, 5 H); 1.98(m, 1H); 2.12 (m, 2H); 2.22 to 2.34 (m, 3H); 2.62 to 2.72 (m, 4H); 2.81(s,4H); 2.93 to 3.04 (m, 3H); 3.18 (q, J=6.0 Hz, 2H); 3.23(s, 3H); 3.33 (partially hidden m, 1H); 3.39 (t, J=6.0 Hz, 2H); 3.42(m, 2H); 3.47 to 3.52 (m, 10H); 3.81(s, 3H); 3.87 (d, J=1.6 Hz, 1H); 4.15 (dd, J=6.6 and 8.2 Hz, 1H); 4.19 to 4.32 (m, 4H); 4.91 (dd, J=3.6 and 9.6 Hz, 1H); 5.10 (m, 1H); 5.79 (d, J=15.3 Hz, 1H); 6.47 (ddd, J=3.7, 11.3 and 15.3 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17 (dd, J=2.2 and 8.5 Hz, 1H); 7.21 to 7.26 (m, 5 H); 7.28 (d, J=2.2 Hz, 1H); 7.85 (t, J=6.0 Hz, 1H); 7.92 (d, J=8.2 Hz, 1H); 8.08 (d, J=7.7 Hz, 1H); 8.32 (t, J=6.0 Hz, 1H); 8.37 (d, J=8.0 Hz, 1H). LCMS (A): ES mass/charge=1326 [M+H]+; mass/charge=1348 [M+Na]+; mass/charge=1370 [M-H+HCO2H]-; Rt=1.31 min.

Example 16: hu2H11_R35-74-Ex15.

The general method described previously was used to prepare the compound of Example 16. 28.8 mg of hu2H11_R35-74 was reacted with 115.2 μl of a 10.05 mM solution of the compound of Example 15 in DMA (6 eq.) for 2 h 30 min. After purification on Superdex 200 pg in buffer B pH 6.5 + 10% NMP, concentration on Amicon Ultra-15, dilution in buffer B pH 6.5 to a final concentration of NMP at 5% and filtration on a PVDF filter with size 0.22 μm pore yielded 16.12 mg of ADC, which contained 2.2% residual compound from Example 14. This batch was concentrated on Amicon Ultra-15 and purified on Sephadex G25 to yield 13.16 mg of compound from Example 16 as a colorless clear solution at a concentration of 1.4 mg/ml with a DAR of 2.8 (HRMS), monomeric purity of 99.7% and an overall yield of 43%.

SEC-HRMS: wt/charge=149398 (unconjugated mAb); mass/charge=150610 (D1); mass/charge=151822 (D2); mass/charge=153035 (D3); mass/charge=154248 (D4); mass/charge=155458 (D5); mass/charge= 156672 (D6); mass/charge=1578881 (D7).

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Synthesis of Examples 17-19: Glutaryl-Val-glucose-Gln-C52-benzylamine, NHS-glutaryl-Val-glucose-Gln-C52-benzylamine ester and corresponding ADC

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Compound 44: (2S,3S,4S,5R,6R)-2-(iodomethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol.

To a solution of methyl β-D-glucopyranoside hemihydrate (CAS number [7000-27-3], 1 g, 4.67 mmol), imidazole (642 mg, 9.34 mmol) and triphenylphosphine (2.47 g, 9. 33 mmol) in THF (50 mg), which was stirred under reflux, a solution of iodine (2.37 g, 9.33 mmol) in THF (10 ml) was added dropwise. Reflux was maintained for 3 hours. After cooling at 0°C, the reaction medium was filtered, and the filtrate was concentrated in vacuo. The mixture was purified by flash chromatography on 150 g silica gel (DCM/MeOH gradient) to give 1 g of compound 44 (63%).

Compound 45: (2R,3S,4S,5R,6R)-2-(azidomethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol.

NaN ₃ (322 mg, 4.93 mmol) was added to a solution of compound 44 (1 g, 3.29 mmol) in DMF (10 mg) with magnetic stirring. The reaction medium was heated at 50°C for 16 hours. At the same time, after cooling at room temperature. the medium was concentrated in vacuo and purified by flash chromatography on 80 g silica gel (DCM/MeOH gradient elution) to give 550 mg of compound 45 (76%).

Compound 46: triol hydrochloride.

(2R,3S,4S,5R,6R)-2-(aminomethyl)-6-methoxytetrahydro-2H-pyran-3,4,5 Solution of compound 45 (550 mg, 2.51 mmol) and triphenylphosphine (1.33 g, 5.02 mmol) in 1,4-dioxane (12 ml) and MeOH (3 ml) were stirred at room temperature. within 30 min. At the same time, water (1.5 ml) was added to the reaction medium and stirred at room temperature. maintained overnight. Unpurified environment

- 113 045708 was centered in vacuo, then diluted with DCM (10 ml) and 1N. aqueous HCl solution (5 ml), the aqueous phase was extracted with DCM (2x10 ml). The aqueous phase was concentrated in vacuo, then diluted with MeOH and concentrated in vacuo to give 560 mg of compound 46 (97%).

Compound 47: (S)-1-tert-butyl-5-(2,5-dioxopyrrolidin-1-yl)-2-((S)-2-((((9H-fluoren-9yl)methoxy)carbonyl) amino)-3-methylbutanamido)pentanedioate.

To a solution of compound 40 (2 g, 3.81 mmol) in THF (100 ml), DIEA (1.29 ml, 7.62 mmol), DSC (1.95 g, 7.62 mmol) and DMF (20 ml). The reaction medium was stirred at room temperature. in an Ar atmosphere overnight. After concentration in vacuo, the mixture was purified by flash chromatography on 150 g silica gel (eluting with DCM/EtOAc) to give 1.67 g of compound 47 (71%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.89 (d, J=6.8 Hz, 3H); 0.91 (d, J=6.8 Hz, 3H); 1.39(s,9H); 1.84 to 2.10 (m, 3H); 2.65 to 2.77 (m, 2H); 2.80(s, 4H); 3.88 (dd, J=7.2 and 8.7 Hz, 1H); 4.18 to 4.31 (m, 4H); 7.32 (m, 2H); 7.41 (m, 3H); 7.73 (d, J=7.6 Hz, 1H); 7.75 (d, J=7.6 Hz, 1H); 7.88 (d, J=7.6 Hz, 2H); 8.31 (wide d, J=7.1 Hz, 1H). LCMS (A): ES mass/charge=566; mass/charge=622 [M+N]+; mass/charge=666 [M-H+HCO ₂ N]'; Rt=1.49 min.

Compound 48: (S)-tert-butyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3methylbutanamido)-5-oxo-5-(( ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2yl)methyl)amino)pentanoate.

To a solution of compound 47 (500 mg, 804 μmol) in DMF (10 ml), compound 46 (185 mg, 805 μmol) and DIEA (165 μl, 978 μmol) were added with magnetic stirring. The reaction medium was stirred at room temperature. for 4 hours. At the same time, 37 mg of compound 46 and 35 μl of DIEA were added to the reaction medium, stirring was maintained for 1 hour. The medium was then concentrated in vacuo and purified by flash chromatography on 50 g of silica gel (gradient elution DCM/ MeOH) to obtain 480 mg of compound 48 (85%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.87 (d, J=6.7 Hz, 3H); 0.89 (d, J=6.7 Hz, 3H); 1.38(s,9H); 1.70 to 2.04 (m, 3H); 2.19 (t, J=7.9 Hz, 2H); 2.86 to 2.98 (m, 2H); 3.01 to 3.16 (m, 3H); 3.35(s, 3H); 3.52 (m, 1H); 3.92 (dd, J=7.2 and 8.4 Hz, 1H); 3.99 (d, J=7.6 Hz, 1h); 4.09 (m, 1H); 4.16 to 4.36 (m, 3H); 4.95 (wide d, J=4.6 Hz, 1H); 4.99 (d, J=5.1 Hz, 1H); 5.03 (d, J=4.9 Hz, 1H); 7.27 to 7.37 (m, 3H); 7.41 (t, J=7.6 Hz, 2H); 7.75 (divided d, J=7.0 Hz, 2H); 7.85 (wide m, 1H); 7.89 (d, J=7.6 Hz, 2H); 8.23 (wide d, J=7.1 Hz, 1H). LCMS (A): ES mass/charge=700 [M+H]+; mass/charge=744 [M-H+HCO2H]-; Rt=1.22 min.

Compound 49: (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-oxo-5((((2R ,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)amino)pentanoic acid.

A 4 M solution of HCl in 1,4-dioxane (30 ml) was added to compound 48 (480 mg, 686 μmol) with magnetic stirring. The reaction medium was stirred at room temperature. for 4 hours. At the same time, the medium was concentrated in vacuo and purified by flash chromatography on 30 g of silica gel (eluting with DCM/MeOH/H ₂ O) to obtain 260 mg of compound 49 (59%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.83 (d, J=6.6 Hz, 3H); 0.85 (d, J=6.6 Hz, 3H); 1.69 to 1.90 (m, 2H); 2.01 to 2.15 (m, 3H); 2.86 to 2.97 (m, 2H); 2.99 to 3.16 (m, 3H); 3.35(s, 3H); 3.51 (m, 1H); 3.82(m, 2H); 3.99 (d, J=7.8 Hz, 1H); 4.20 to 4.33 (m, 3H); 4.95 (wide d, J=3.7 Hz, 1H); 5.03 (d, J=4.9 Hz, 1H); 5.06 (wide d, J=4.6 Hz, 1H); 7.33 (m, 2H); 7.40 (t, J=7.6 Hz, 2H); 7.52 (wide d, J=6.4 Hz, 1H); 7.61 (wide d, J=9.3 Hz, 1H); 7.73 (d, J=7.6 Hz, 1H); 7.76 (d, J=7.6 Hz, 1H); 7.89 (d, J=7.6 Hz, 2H); 8.10 (wide m, 1H). LCMS (A): ES mass/charge=642 [M-N] ^- ; mass/charge=644 [M+N]+; Rt=1.01 min.

Compound 50: (S)-2-((S)-2-amino-3-methylbutanamido)-N 1 -(4-((2R,3R)-3-((S)-1 -((3S,10R , 16S,E)-10(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)-N5-(((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran- 2-yl)methyl)pentanediamide.

To a solution of (E)-(3S,10R,16S)-16-(S)-1-[(2R,3R)-3-(4-aminomethylphenyl)oxiranyl]-ethyl}-10-(3-chloro4-methoxybenzyl )-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 110 mg, 157 µmol) in DMF (6 ml) was added with compound 49 (122 mg, 189 µmol), HOBt (34 mg, 252 µmol) and EDC (43 µl, 236 µmol) with magnetic stirring. The reaction medium was stirred at room temperature. during the night. Then, 25 mg of compound 49 and 25 μl of EDC were added to the medium, and stirring was maintained for 1 hour. At the same time, piperidine (160 μl, 1.58 mmol) was added to the medium. After 2 hours, the medium was concentrated in vacuo and purified by flash chromatography on 20 g silica gel (DCM/MeOH gradient elution) to give 78 mg of compound 50 (45%).

1H NMR (500 MHz, δ in ppm, DMSO-d6): 0.76 (d, J=6.9 Hz, 3H); 0.79 (d, J=6.3 Hz, 6H); 0.87 (d, J=6.9 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=6.6 Hz, 3H); 1.12(s, 3H); 1.30 (m, 1H); 1.50 to 1.98 (m, 8 H); 2.12 (m, 2H); 2.26 (m, 1H); 2.68(m, 2H); 2.86 to 3.14 (m, 9H); 3.32 (partially hidden m, 1H); 3.38(s, 3H); 3.52 (m, 1H); 3.81(s, 3H); 3.87(s, 1H); 4.01 (d, J=7.4 Hz, 1H); 4.20 to 4.36 (m, 4h); 4.90 (m, 1H); 4.98 (wide d, J=3.6 Hz, 1H); 5.02 (d, J=4.9 Hz, 1H); 5.06 (d, J=4.7 Hz, 1H); 5.10 (m, 1H); 5.79 (d, J=15.6 Hz, 1H); 6.47 (m, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17 (wide d, J=8.5 Hz, 1H); 7.21 to 7.26 (m, 5 H); 7.29 (wide s, 1H); 7.90 (m, 1H);

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8.08 (wide m, 1H); 8.37 (d, J=8.0 Hz, 1H); 8.48 (m, 1H). LCMS (A): ES mass/charge=551 [M+2H]2+; mass/charge=1099 [M-N] ^- ; mass/charge=1101 [M+H]+; mass/charge=1145 [M-H+HCO ₂ N]-; Rt=0.9 min.

Example 17: 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )-10-(3-chloro-4-methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-ene-16- yl)ethyl)oxiran2-yl)benzyl)amino)-1,5-dioxo-5-((((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H- pyran2-yl)methyl)amino)pentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid.

Glutaric anhydride (14 mg, 125.8 μmol) was added to a solution of compound 50 (77 mg, 69.9 μmol) in DMF (4 ml) with magnetic stirring. The reaction medium was stirred at room temperature. for 2 hours. The reaction medium was then concentrated in vacuo and purified by flash chromatography on 10 g silica gel (DCM/MeOH gradient elution) to give 54 mg of Example 17 (64%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.80 (d, J=6.0 Hz, 6H); 0.86 (d, J=6.6 Hz, 3H); 0.88 (d, J=6.6 Hz, 3H); 0.99(s, 3H); 1.03 (d, J=6.6 Hz, 3H); 1.12(s, 3H); 1.34 (m, 1H); 1.53 to 1.85 (m, 6H); 1.90 to 2.33 (m, 9h); 2.65 to 2.76 (m, 2H); 2.87 to 3.14 (m, 8 H); 3.32 (partially hidden m, 1H); 3.35(s, 3H); 3.53 (m, 1H); 3.81(s, 3H); 3.87 (d, J=1.9 Hz, 1H); 4.00 (d, J=7.9 Hz, 1H); 4.03 to 4.26 (m, 4H); 4.33 (m, 1H); 4.93(m, 1H); 5.02 (wide m, 3H); 5.10 (m, 1H); 5.80 (d, J=15.3 Hz, 1H); 6.46 (ddd, J=3.5, 11.0 and 15.3 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.18 (dd, J=2.2 and 8.5 Hz, 1H); 7.22 (d, J=8.2 Hz, 2H); 7.26 (d, J=8.2 Hz, 2H); 7.30 (m, 2H); 7.98 to 8.65 (wide m, 5 H). LCMS (A): ES mass/charge=608 [M+2H] ²⁺ ; mass/charge=1213 [M-N] ^- ; mass/charge=1215 [M+H]+; Rt= 1.15 min.

Example 18: 2,5-dioxopyrrolidin-1-yl-5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1 -((3S,10R,16S,E)-10-(3chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8, 11-diazacyclohexadec13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1,5-dioxo-5-((((2R,3S,4S,5R,6R)-3,4, 5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)amino)pentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5oxopentanoate.

Example 17 (45 mg, 37 µmol) was diluted in toluene and concentrated in vacuo. DSC (11.4 mg, 44.4 µmol) and DIEA (19 µl, 112.6 µmol). The reaction medium was stirred at room temperature. in an Ar atmosphere for 3 hours. DMF and DSC were then added to the medium until the reaction was completed. After 3 h, the medium was diluted with water (5 ml) and extracted with MeTHF (3x10 ml). The combined organic phases were dried over MgSO4, filtered and concentrated in vacuo. The mixture was purified by flash chromatography on 4 g silica gel (DCM/iPrOH gradient elution) to give 21 mg of Example 18 (43%).

1H NMR (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.0 Hz, 6H); 0.83 (d, J=6.9 Hz, 3H); 0.85 (d, J=6.9 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=6.3 Hz, 3H); 1.12(s, 3H); 1.30 (m, 1H); 1.52 to 1.63 (m, 2H); 1.75 to 2.02 (m, 6H); 2.09 to 2.34 (m, 5 H); 2.68 (m, 4H); 2.81(s,4H); 2.88 to 3.18 (m, 7 H); 3.32 (partially hidden m, 1H); 3.38(s, 3H); 3.51 (m, 1H); 3.81(s, 3H); 3.88(s, 1H); 4.01 (d, J=7.7 Hz, 1H); 4.16 (dd, J=7.1 and 7.7 Hz, 1H); 4.19 to 4.35 (m, 5 H); 4.90 (m, 1H); 4.97 (d, J=4.l Hz, 1H); 5.01 (d, J=4.9 Hz, 1H); 5.06 (d, J=4.9 Hz, 1H); 5.10 (m, 1H); 5.79 (d, J=15.3 Hz, 1H); 6.47 (m, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17 (wide d, J=8.5 Hz, 1H); 7.20 to 7.26 (m, 5 H); 7.28 (wide s, 1H); 7.86 (wide t, J=5.6 Hz, 1H); 7.91 (d, J=7.9 Hz, 1H); 8.08 (d, J=7.4 Hz, 1H); 8.33 (t, J=5.8 Hz, 1H); 8.38 (d, J=8.2 Hz, 1H). LCMS (A): ES mass/charge=1310 [MH]-; mass/charge=1356 [M-H+HCO2H]-; mass/charge=1312 [M+H]+; mass/charge=656.5 [M+2N] ²⁺ ; Rt=1.2 min.

Example 19: hu2H11_R35-74-Ex18.

The general method described previously was used to prepare the compound of Example 19. 29.02 mg of hu2H11_R35-74 was reacted with 116.6 μl of a 10 mM solution of the compound of Example 18 in DMA (6 eq.) for 2 hours 30 minutes. After purification on Superdex 200 pg in buffer B pH 6.5 + 10% NMP, concentration on Amicon Ultra-15, dilution in buffer B pH 6.5 to a final concentration of NMP at 5% and filtration on a PVDF filter with size 0.22 μm pores yielded 23.1 mg of the compound from Example 19 as a colorless clear solution at a concentration of 2.2 mg/ml with a DAR of 2.8 (HRMS), monomeric purity of 99.9% and overall yield of 78%. Non-protein bound drug levels exceeded the 1% threshold: ADC was concentrated on Amicon Ultra-15, purified on Sephadex G25 in pH 6.5+5% NMP buffer B and filtered on a pore size PVDF filter 0.22 µM to obtain 17.25 mg of the compound of Example 19 as a colorless clear solution at a concentration of 1.50 mg/ml with a DAR of 2.55 (HRMS), monomeric purity of 99.7% and an overall yield of 59%.

SEC-HRMS: wt/charge=149405 (unconjugated mAb); mass/charge=150602 (D1); mass/charge=151800 (D2); mass/charge=152997 (D3); mass/charge=154196 (D4);

mass/charge=155393 (D5); mass/charge=156589 (D6); mass/charge=157791 (D7). Synthesis of Example 20: Glutaryl-Val-glucamine-Gln-C52-benzylamine

- 115 045708

HE is he

Compound 51: (S)-2,5-dioxopyrrolidin-1-yl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3methylbutanoate.

To a solution of Fmoc-Val-OH (CAS number [68858-20-8], 5 g, 14.44 mmol) in THF (40 ml) was added NHS (1.71 g, 14.44 mmol) and DCC (2, 98 g, 14.44 mmol). The reaction medium was stirred for 5 hours at RT, then 0.2 equivalents of NHS and DCC were added and stirring was carried out for 4 hours at RT.

- 116 045708

The medium was filtered, the solid was washed twice with THF (2x25 ml) and the filtrate was concentrated in vacuo to give 6.5 g of compound 51 as a white meringue (qt).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 1.03 (d, J=6.9 Hz, 6H); 2.20 (m, 1H); 2.81(s,4H); 4.18 to 4.39 (m, 4H); 7.32 (m, 2H); 7.42 (m, 2H); 7.74 (m, 2H); 7.90 (d, J=7.6 Hz, 2H); 8.12(d,J=8.5Hz, 1H).

Compound 52: (S)-4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5(tert-butoxy)-5-oxopentane acid.

To a solution of H-Glu-OtBu (CAS number [45120-30-7], 3.14 g, 15.16 mmol) and sodium bicarbonate (1.40 g, 16.46 mmol) in H ₂ O (60 ml) add a solution of compound 51 (6.3 g, 14.43 mmol) in THF (240 ml). The reaction medium was stirred for 16 hours at room temperature. and concentrated in vacuo. The crude product was diluted in H2O (700 ml) to form a gel, which was stirred for 15 minutes, then extracted twice with Et ₂ O (200 ml). The aqueous suspension was acidified to pH 3 with 5 N. HCl and extracted with DCM (4x200 ml). The combined organic phases were dried over MgSO ₄ , filtered and concentrated in vacuo to give 7.98 g of compound 52 as a white meringue (qty).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.87 (d, J=6.9 Hz, 3H); 0.90 (d, J=6.9 Hz, 3H); 1.38(s,9H); 1.77 (m, 1H); 1.91(m, 1H); 1.98(m, 1H); 2.28 (m, 2H); 3.90 (dd, J=7.1 and 9.1 Hz, 1H); 4.10 to 4.37 (m, 4H); 7.32 (m, 2H); 7.38 (d, J=9.1 Hz, 1H); 7.41 (m, 2H); 7.73 (m, 2H); 7. 89 (d, J=7.6 Hz, 2H); 8.20 (d, J=7.5 Hz, 1H); 12.21 (wide m, 1H).

Compound 53: (S)-1-tert-butyl-5-(2,5-dioxopyrrolidin-1-yl)-2-((S)-2-((((9H-fluoren-9yl)methoxy)carbonyl) amino)-3-methylbutanamido)pentanedioate.

To a solution of compound 52 (500 mg, 953.1 µmol) in THF (30 ml) was added DIEA (320 µl, 1.9 mmol), DSC (498.3 mg, 1.91 mmol) and DMF (6 ml). The reaction medium was stirred at room temperature. overnight, then 30 mg of DSC was added to the medium and stirring was carried out for 24 hours. The reaction medium was concentrated in vacuo, co-evaporated twice with toluene and purified by flash chromatography on 50 g of silica gel (DCM/iPrOH gradient elution) to obtain 506 mg compound 53 as a white oil (85%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.89 (d, J=6.9 Hz, 3H); 0.91 (d, J=6.9 Hz, 3H); 1.39(s,9H); 1.75 to 2.11 (m, 3H); 2.73 (m, 2H); 2.80(s, 4H); 3.89 (dd, J=7.3 and 9.1 Hz, 1H); 4.20 to 4.38 (m, 4H); 7.32 (m, 2H); 7.42 (m, 3H); 7.75 (m, 2H); 7.80 (m, 2H); 8.31 (d, J=7.5 Hz, 1H).

Compound 54: (2S,3R,4R,5R)-1-amino-6-((tert-butyldiphenylsilyl)oxy)hexane-2,3,4,5-tetraol

A suspension of D-glucamine (CAS number [488-43-7], 200 mg, 1.10 mmol) in DMF (2 ml) was cooled at 0°C, then imidazole (82.0 mg, 1.19 mmol) was added and tert-butylchlorodiphenylsilane (334.4 mg, 1.19 mmol). The reaction medium was stirred for 1 hour at 0°C, quenched with H ₂ O (20 ml) and stirred for 10 minutes. The medium was extracted with AtOAc (3x50 ml), the combined organic phases were dried over MgSO4, filtered, concentrated in vacuo, co-evaporated twice with toluene and purified by reverse phase flash chromatography on 31 g _C18 -modified silica gel (gradient elution with H ₂ O/MeCN) to obtain 218 mg of compound 54 as a white foam (47%).

1H NMR (400 MHz, δ in ppm, DMSO-d6): 1.00 (s, 9H); 2.62 (dd, J=6.3 and 12.7 Hz, 1H); 2.74 (dd, J=4.5 and 12.7 Hz, 1H); 3.40 (partially hidden m, 1H); 3.49 (m, 1H); 3.55 to 3.72 (m, 3H); 3.88(m, 1H); 4.48 (wide m, 4H); 7.42 (m, 6H); 7.69 (m, 4H).

Compound 55: (S)-tert-butyl-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3 methylbutanamido)-5-(((2S, 3R,4R,5R)-6-((tert-butyldiphenylsilyl)oxy)-2,3,4,5-tetrahydroxyhexyl)amino)-5oxopentanoate.

To a solution of compound 53 (300 mg, 482.6 μmol) in DMF (2 ml) was added a solution of compound 54 (214.6 mg, 511.5 μmol) in DMF (3 ml). The reaction medium was stirred for 1 h at RT, concentrated in vacuo, co-evaporated with toluene (3x) and purified by flash chromatography on 25 g silica gel (gradient elution DCM/MeOH/H2O) to obtain 270 mg of compound 55 in the form of white foam (60%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.87 (d, J=6.9 Hz, 3H); 0.90 (d, J=6.9 Hz, 3H); 0.99(s,9H); 1.38(s,9H); 1.80 (m, 1H); 1.90 (m, 1H); 1.98(m, 1H); 2.17 (t, J=7.7 Hz, 2H); 3.03 (m, 1H); 3.26(m, 1H); 3.49 (m, 1H); 3.52 to 3.60 (m, 4H); 3.83 (m, 1H); 3.92 (dd, J=7.3 and 9.2 Hz, 1H); 4.08 (m, 1H); 4.18 to 4.31 (m, 4H); 4.33 (d, J=6.4 Hz, 1H); 4.63 (d, J=5.8 Hz, 1H); 4.78 (d, J=4.7 Hz, 1H); 7.31 (m, 2H); 7.36 (d, J=9.2 Hz, 1H); 7.41 (m, 8 H); 7.67 (m, 4H); 7.69 to 7.76 (m, 3H); 7.89 (m, 2H); 8.23 (d, J=7.5 Hz, 1H).

Compound 56: (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5(((2S,3R,4R, 5R)-6-((tert-butyldiphenylsilyl)oxy)-2,3,4,5-tetrahydroxyhexyl)amino)-5-oxopentanoic acid.

Compound 55 was diluted in DCM to form a gel, which was cooled at 0°C, then a 1:1 mixture of DCM/TFA was added slowly at 0°C. The reaction medium was stirred for 30 minutes. at 0°C, then for 5 hours at RT, concentrated in vacuo and co-evaporated with toluene (3x) to obtain a white solid, which was ground in iPr ₂ O, filtered, washed twice with

- 117 045708 with Et2O (5 ml), dried and finally purified by flash chromatography on 15 g silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 149 mg of compound 56 as a white solid (59% ).

¹ H NMR (400 MHz, δ in ppm, DMSO-d6): 0.84 (d, J=6.8 Hz, 3H); 0.86 (d, J=6.8 Hz, 3H); 0.99(s,9H); 1.82(m, 1H); 1.90 (m, 1H); 2.03 to 2.14 (m, 3H); 3.00 (m, 1H); 3.25 (m, 1H); 3.48 (m, 1H); 3.65 (m, 4H); 3.82 to 3.92 (m, 3H); 4.20 to 4.37 (m, 3H); 4.40 (d, J=6.0 Hz, 1H); 4.45 (d, J=6.0 Hz, 1H); 4.80 (m, 1H); 5.12 (m, 1H); 7.32 (m, 2H); 7.41 (m, 8 H); 7.55 (m, 2H); 7.65 to 7.82 (m, 7H); 7.89 (d, J=8.0 Hz, 2H). LCMS (A): mass/charge=868 [M-N] ^- ; mass/charge=870 [M+N]+; Rt=1.55 min.

Compound 57: (S)-2-((S)-2-amino-3-methylbutanamido)-N5-((2S,3R,4R,5R)-6-((tert-butyldiphenylsilyl)oxy)-2,3,4 ,5-tetrahydroxyhexyl)-N1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3chloro-4-methoxybenzyl)-3- isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec13-en-16-yl)ethyl)oxiran-2-yl)benzyl)pentanediamide.

To a solution of compound 56 (67.3 mg, 77.3 µmol) and (3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4(aminomethyl)phenyl )oxiran-2-yl)ethyl)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2, 5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 45 mg, 64.5 µmol) in DMF and DCM (10 ml) was added HOBt (11.5 mg, 85.1 µmol) and EDC (11 .4 mg, 70.9 µmol). The reaction medium was stirred for 3 hours at room temperature, then quenched with H2O (15 ml) and stirred for 10 minutes. The aqueous phase was extracted with DCM (3x20 ml), the combined organic phases were dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 5 g silica gel (DCM/MeOH gradient elution) to give 64 mg of Fmoc-protected intermediate compound dipeptide cryptophycin as a white solid (64%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d6): 0.76 (d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 3H); 0.86 (d, J=7.0 Hz, 3H); 0.99(s, 12H); 1.04 (d, J=7.1 Hz, 3H); 1.10(s, 3H); 1.30 (m, 1H); 1.55 (m, 2H); 1.80 (m, 2H); 1.90 (m, 1H); 2.00 (m, 1H); 2.14 (m, 2H); 2.26 (m, 1H); 2.68(m, 2H); 2.92 to 3.08 (m, 4H); 3.22 to 3.38 (partially hidden m, 2H); 3.49 (m, 1H); 3.58 to 3.69 (m, 4H); 3.81(s, 3H); 3.82(m, 1H); 3.87 (d, J=2.0 Hz, 1H); 3.91(m, 1H); 4.18 to 4.38 (m, 9H); 4.68(m, 1H); 4.82 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m, 1H); 5.79 (wide d, J=16.0 Hz, 1H); 6.47 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.0 Hz, ¹ H); 7.17 (dd, J=2.5 and 8.0 Hz, 1H); 7.20 to 7.27 (m, 5 H); 7.29 (d, J=2.3 Hz, 1H); 7.31 (t, J=7.8 Hz, 2H); 7.34 to 7.48 (m, 7H); 7.65 to 7.79 (m, 9H); 7.90 (d, J=7.8 Hz, 2H); 8.10 (d, J=8.3 Hz, 1H); 8.38 (d, J=8.0 Hz, 1H); 8.41 (t, J=6.5 Hz, 1H). LCMS (A): ES mass/charge=775 [M+2H] ²⁺ ; mass/charge=1549 [M+H]+; mass/charge=1549 [M+N] ₂ N] ^- ; Rt=1.78 min.

To a solution of this intermediate (62 mg, 40.0 μmol) in DCM (8 ml) was added piperidine (59.8 μl, 600.0 μmol). The reaction medium was stirred for 4 h at RT, concentrated in vacuo and purified by flash chromatography on 5 g silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 49 mg of compound 57 as a white varnish (92 %).

Compound 58: (6R,7R,8R,9S,15S,18S)-15-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)- 10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en16-yl)ethyl) oxiran-2-yl)benzyl)carbamoyl)-6,7,8,9-tetrahydroxy-18-isopropyl-2,2-dimethyl-12,17,20trioxo-3,3-diphenyl-4-oxa-11,16 ,19-triaza-3-silatetracosane-24-oic acid.

To a solution of compound 57 (23 mg, 17.3 μmol) in DCM (3 ml) was added a solution of glutaric anhydride (2.22 mg, 19.1 μmol) in DCM (2 ml). The reaction medium was stirred for 3 h at room temperature, partially concentrated in vacuo (to 2 ml) and purified by flash chromatography on 5 g silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 17 mg of compound 58 in the form of white varnish (68%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.80 (divided d, J=6.8 Hz, 6H); 0.6 (divided d, J=7.0 Hz, 6H); 0.99(s, 12H); 1.04 (d, J=7.1 Hz, 3H); 1.11(s, 3H); 1.32(m, 1H); 1.52 to 1.60 (m, 2H); 1.72 to 2.00 (m, 7H); 2.08 (m, 1H); 2.13 (m, 1H); 2.20 to 2.37 (m, 3 H); 2.69 (m, 2H); 2.92 to 3.07 (m, 4H); 3.20 (m, 1H); 3.30 (hidden m, 1H); 3.45 (m, 1H); 3.55 to 3.70 (m, 4H); 3.80(s, 3H); 3.83 (m, 1H); 3.87 (d, J=2.3 Hz, 1H); 4.07 to 5.00 (m, 11H); 5.10 (m, 1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.47 (m, 1H); 7.04 (d, J=8.6 Hz, ¹ H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.28 (m, 5 H); 7.29 (d, J=2.3 Hz, 1H); 7.41 (m, 6H); 7.68 (m.4H); 8.05 (d, J=7.9 Hz, 1H; 8.23 (t, J=6.5 Hz, 1H); 8.30 to 8.60 (m, 3H). LCMS (A): ES mass/charge=721 [M+2N] ²⁺ ; mass/charge=1439 [M-N] ^- ; mass/charge=1441 [M+N]+; Rt=1.52 min.

Example 20: 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )-10-(3-chloro-4-methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-ene-16- yl)ethyl)oxiran2-yl)benzyl)amino)-1,5-dioxo-5-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)pentane-2 -yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid.

A solution of compound 57 (46 mg, 31.9 µmol) in THF (3 ml) was cooled at 0°C, then a 1 M solution of TBAF in THF (35.1 µl, 35.1 µmol) was added dropwise at 0°C. The reaction medium was stirred for 2 h at 0°C, quenched with H2O (100 µl), stirred for 10 min and extracted with DCM (5 ml). The organic phase was dried over MgSO ₄ , filtered and concentrated in vacuo: LCMS analysis indicated the presence of residual starting material. Unrefined product

- 118 045708 was made in DCM (3 ml), the solution was cooled at 0°C, then a 1 M solution of TBAF in THF (35.1 μl, 35.1 μmol) was added dropwise at 0°C. The reaction medium was stirred for 3 h at 0°C, quenched with H2O (100 µl), stirred for 10 min and extracted with DCM (20 ml). The organic phase was dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 5 g silica gel (gradient elution DCM/MeOH/H ₂ O to MeOH), then by reverse phase flash chromatography on 3 g C ₁₈ -modified silica gel (gradient elution H ₂ O/MeCN) to obtain 17 mg of the compound from example 20 as a white varnish (44%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d6): 0.80 (divided d, J=6.8 Hz, 6H); 0.6 (divided d, J=7.0 Hz, 6H); 0.99(s, 12H); 1.04 (d, J=7.1 Hz, 3H); 1.11(s, 3H); 1.32(m, 1H); 1.52 to 1.60 (m, 2H); 1.72 to 2.00 (m, 7H); 2.08 (m, 1H); 2.13 (m, 1H); 2.20 to 2.37 (m, 3H); 2.69 (m, 2H); 2.92 to 3.07 (m, 4H); 3.20 (m, 1H); 3.30 (hidden m, 1H); 3.45 (m, 1H); 3.55 to 3.70 (m, 4H); 3.80(s, 3H); 3.83 (m, 1H); 3.87 (d, J=2.3 Hz, 1H); 4.07 to 5.00 (m, 11H); 5.10 (m, 1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.47 (m, 1H); 7.04 (d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.28 (m, 5 H); 7.29 (d, J=2.3 Hz, 1H); 7.41 (m, 6H); 7.68 (m, 4H); 8.05 (d, J=7.9 Hz, 1H); 8.23 (t, J=6.5 Hz, 1H); 8.30 to 8.60 (m, 3H). LCMS (A): ES mass/charge=602 [M+2H]2+; mass/charge=1201 [M-N]-; mass/charge=1203 [M+N] ⁺ ; Rt=1.1 min.

Synthesis of Examples 21-23: Glutaryl-Val-PEG4Lys-C52-benzylamine, NHS-glutaryl-ValPEG4Lys-C52-benzylamine ester and corresponding ADC

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Compound 59: (S)-20-((tert-butoxycarbonyl)amino)-14-oxo-2,5,8,11-tetraoxa-15-azagenicosan21-oic acid.

To a solution of (S)-6-amino-2-((tert-butoxycarbonyl)amino)hexanoic acid (CAS number [1373428-6], 73.9 mg, 300 µmol) in THF (300 µl) with magnetic stirring 2,5-dioxopyrrolidin-1-yl-2,5,8,11-tetraoxatetradecane-14-oate (CAS number [622405-78-1], 100 mg, 300 µmol), DIEA (53.3 µl, 300 µmol) and H2O (100 µl). The reaction medium was stirred at room temperature. overnight, then concentrated in vacuo. The crude medium was purified by flash chromatography on 5 g _C18 -grafted silica gel (H2O/CH3CN gradient elution) to give 81 mg of compound 59 (58%). LCMS (V): ES mass/charge=463 [M-N]-; mass/charge=465 [M+N]+; tR=0.86 min.

Compound 60: (S)-20-amino-14-oxo-2,5,8,11-tetraoxa-15-azagenicosan-21-oic acid hydrochloride.

A 4 M solution of HCl in 1,4-dioxane (1 mL) was added to compound 59 (150.6 mg, 324.2 μmol) with magnetic stirring. The reaction medium was stirred at room temperature. overnight, then concentrated in vacuo. AtOAc was added and the mixture was concentrated in vacuo to obtain 156 mg of compound 60 (quantitative).

LCMS (V): ES mass/charge=508 [M-N]-; mass/charge=510 [M+N] ⁺ ; tR=l,23 min.

Compound 61: (S)-20-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)14-oxo-2,5,8,11 -tetraoxa-15-azagenicosan-21-oic acid.

To a solution of compound 60 (130 mg, 275 µmol) in water (0.5 ml) was added NaHCO ₃ (55.6 mg, 661 µmol) with magnetic stirring, then (S)-2,5-dioxopyrrolidine- 1yl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoate (CAS number [130878-68-1], 144.4

- 120 045708 mg, 330.8 µmol) in THF (1.5 ml). The reaction medium was stirred at room temperature. during the night. Then NaHCO ₃ (30 mg), THF (500 µl), H2O (200 µl) and (S)-2,5-dioxopyrrolidin1-yl-2-((((9H-fluoren-9-yl) methoxy)carbonyl)amino)-3-methylbutanoate (28 mg). After 3 h, the medium was concentrated in vacuo, then diluted with H2O (20 ml) and ~2-3 ml of aqueous saturated Na2CO3. The aqueous phase was extracted twice with EtOAc (20 ml). The combined organic phases were washed with brine (20 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. The mixture was purified by flash chromatography on 15 g silica gel (elution EtOAc/MeOH/H ₂ O 85:1:0.5 v/v). After concentration, the crude product was diluted with toluene and concentrated in vacuo to give 75.6 mg of compound 61 (40%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.85 (d, J=7.1 Hz, 3H); 0.87 (d, J=7.1 Hz, 3H); 1.21 to 1.40 (m, 4H); 1.50 to 1.73 (m, 2H); 2.01 (m, 1H); 2.27 (t, J=6.6 Hz, 2H); 2.98(m, 2H); 3.23(s, 3H); 3.41(m, 2H); 3.43 to 3.52 (m, 10H); 3.57 (t, J=6.6 Hz, 2H); 3.88(m, 1H); 4.01(m, 1H); 4.19 to 4.34 (m, 3H); 7.31 (m, 2H); 7.38 to 7.46 (m, 3H); 7.70 to 7.91 (m, 6H); 12.51 (m, 1H). LCMS (A): ES mass/charge=684 [M-N] ^- ; mass/charge=686 [M+N]+; Rt=1.2 min.

Compound 62: N-((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R)-3-((S)-1-(( 3S,10R,16S,E)10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11- diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-6-oxohexyl)-2,5,8,11-tetraoxatetradecane-14amide.

A solution of (E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R) was added to compound 61 (70 mg, 102.1 μmol) with magnetic stirring and an Ar atmosphere. )-3-(4-aminomethylphenyl)oxiranyl]ethyl} -10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13 - ene2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 76.2 mg, 109.1 µmol) in THF (3 ml) followed by the addition of HOBt (17.7 mg, 131 µmol) and EDC (23 µl, 131 µmol). The reaction medium was stirred at room temperature. during the night. Piperidine (32.8 µl, 327.4 µmol) was then added to the medium and stirring was maintained for 4 hours 30 minutes. At this time, the medium was concentrated in vacuo, diluted with DMA and purified by flash chromatography on 15 g C ₁₈ grafted silica gel (H2O/CH3CN gradient elution) to give 66 mg of compound 62 (53%).

LCMS (V): ES mass/charge=572; mass/charge=1187 [M-H+HCO2H]-; t _R =1.18 min.

Example 21: (20S,23 S)-20-((4-((2R,3R)-3-((S)-1 -((3 S,10R,16S,E)-10-(3-chloro -4-methoxybenzyl)-3 isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran2-yl )benzyl)carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8,11-tetraoxa-15,21,24-triazanonacozan-29oic acid.

Glutaric anhydride (11.85 mg, 103.8 μmol) was added to a solution of compound 62 (66 mg, 57.7 µmol) in DMF (4 ml) with magnetic stirring. The reaction medium was stirred at room temperature. for 2 hours. At the same time, the medium was concentrated in vacuo and purified by flash chromatography on 10 g of silica gel (DCM/MeOH gradient elution) to obtain 48.6 mg of the compound of Example 21 (80%).

1H NMR (500 MHz, δ in ppm, DMSO- _d6 ): 0.81 (m, 6H); 0.85 (d, J=7.5 Hz, 3H); 0.87 (d, J=7.5 Hz, 3H); 0.98(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.10(s, 3H); 1.16 to 1.44 (m, 5 H); 1.55 to 1.79 (m, 7H); 1.90 to 2.10 (m, 3H); 2.14 to 2.29 (m, 3H); 2.31 (t, J=6.7 Hz, 2H); 2.66 to 2.78 (m, 2H); 2.85 to 2.99 (m, 4H); 3.05 (m, 1H); 3.23(s, 3H); 3.32 (partially hidden m, 1H); 3.42(m, 2H); 3.44 to 3.51 (m, 10H); 3.57 (m, 2H); 3.81(s, 3H); 3.88 (d, J=1.6 Hz, 1H); 4.04 to 4.25 (m, 4H); 4.35 (m, 1H); 4.94 (m, 1H); 5.10 (m, 1H); 5.80 (d, J=15.0 Hz, 1H); 6.45 (ddd, J=4.0, 11.2 and 15.0 Hz, 1H); 7.04 (d, J=8.6 Hz, 1H); 7.19 (dd, J=1.7 and 8.6 Hz, 1H); 7.22 (d, J=8.4 Hz, 2H); 7.28 (d, J=8.4 Hz, 2H); 7.30 (d, J=1.7 Hz, 1H); 7.32 (wide m, 1H); 7.75 to 9.42 (wide m, 5 H); 12.06 (wide m, 1H). LCMS (A): ES mass/charge=629 [M+2H] ²⁺ ; mass/charge=1255 [M-N] ^- ; mass/charge=1257 [M+H]+; Rt=1.26 min.

Example 22: (20S,23S)-2,5-dioxopyrrolidin-1-yl-20-((4-(2R,3R)-3-((S)-1-((3S,10R,16S,E) -10-(3chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec13-en-16-yl)ethyl )oxiran-2-yl)benzyl)carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8,11-tetraoxa-15,21,24-triazanonacosane-29-oate.

Toluene (10 ml) was added to the compound of Example 21 (46 mg, 36.6 µmol) and concentrated in vacuo followed by dilution with THF (5 ml), DCM (2 ml) and DMF (200 µl). DSC (11.5 mg, 43.9 μmol) was then added followed by DIEA (18.5 μL, 109.7 μmol). The reaction medium was stirred at room temperature. for 4 hours. At the same time MeTHF (7 ml) and H ₂ O (3 ml) were added and the aqueous phase was extracted twice with MeTHF (5 ml). The combined organic phases were washed with H ₂ O (3 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. The crude medium was purified by flash chromatography on 4 g silica gel (DCM/iPrOH gradient elution) to give 33.3 mg of Example 22 (67%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.78 (d, J=6.6 Hz, 6H); 0.83 (d, J=7.1 Hz, 3H); 0.84 (d, J=7.1 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=6.9 Hz, 3H); 1.12(s, 3H); 1.20 to 1.43 (m, 5 H); 1.50 to 1.69 (m, 4H); 1.76 to 1.87 (m, 3H); 1.98(m, 1H); 2.21 to 2.35 (m, 5 H); 2.62 to 2.73 (m, 4H); 2.81(s,4H); 2.94 to 3.04 (m, 5 H); 3.23(s,

- 121 045708

3H); 3.32 (partially hidden m, 1H); 3.42(m, 2H); 3.44 to 3.51 (m, 10H); 3.58 (t, J=6.4 Hz, 2H); 3.81(s, 3H); 3.87 (d, J=1.1 Hz, 1H); 4.17 (dd, J=7.3 and 7.8 Hz, 1H); 4.21 to 4.31 (m, 4H); 4.90 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.1 Hz, 1H); 6.47 (ddd, J=3.5, 11.5 and 15.1 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17 (dd, J=1.6 and 8.5 Hz, 1H); 7.20 to 7.25 (m, 5 H); 7.28 (d, J=1.6 Hz, 1H); 7.79 (t, J=5.7 Hz, 1H); 7.91 (d, J=8.5 Hz, 1H); 7.95 (d, J=8.3 Hz, 1H); 8.37 (m, 2H). LCMS (C): ES mass/charge=677.5 [M+2H] ²⁺ ; mass/charge=1354 [M+N] ⁺ ; mass/charge=1399 [M-H+HCO2H]-; Rt=4.41 min.

Example 23: hu2H11_R35-74-Ex22.

The general method described previously was used to prepare the compound of Example 23. 44.64 mg of hu2H11_R35-74 was reacted with 209.2 μl of a 10 mM solution of the compound of Example 22 in DMA (7 eq.) for 3 hours 30 minutes. Purification on Sephadex G25 in buffer B pH 6.5 + 5% NMP and concentration on Amicon Ultra-15 gave 36.6 mg of the compound from Example 23 as a colorless clear solution at a concentration of 1.83 mg/ml with DAR 3 .6 (HRMS), monomeric purity of 98.8% and overall yield of 82%. Non-protein bound drug levels exceeded the 1% threshold: ADC was concentrated on Amicon Ultra-15, purified on Sephadex G25 in pH 6.5+5% NMP buffer B and filtered on a pore size PVDF filter 0.22 µM to obtain 32.6 mg of the compound of Example 23 as a colorless clear solution at a concentration of 1.63 mg/ml with a DAR of 3.3 (HRMS), monomeric purity of 98.3% and overall yield of 73%.

SEC-HRMS: wt/charge=149411 (unconjugated mAb); mass/charge=150649 (D1); mass/charge=151889 (D2); mass/charge=153129 (D3); mass/charge=154369 (D4); mass/charge=155608 (D5); mass/charge=156850 (D6); mass/charge=158091 (D7); mass/charge=159331 (D8); mass/charge=160583 (D9).

Synthesis of Examples 24-26: Glutaryl-Val-PEG24Lys-C52-benzylamine, NHS-glutaryl-Val-PEG24Lys-C52-benzylamine ester and corresponding ADC

- 122 045708

Compound 63: (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6((tert-butoxycarbonyl)amino)hexane acid.

To a solution of H-Lys(Boc)-OH (CAS number [2418-95-3], 310 mg, 1.26 mmol) and NaHCO ₃ (105 mg, 1.25 mmol) in water (5 ml) and THF ( 5 ml) while stirring with a magnetic stirrer, a solution of (S)-2,5-dioxopyrrolidin-1-yl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3methylbutanoate ( CAS number [68858-20-8], 310 mg, 1.15 mmol) in THF (15 ml). The reaction medium was stirred at room temperature. for 3 hours. At the same time, the reaction medium was concentrated in vacuo, then diluted with water (200 ml), acidified with QS 5 N. aqueous HCl solution and extracted with DCM (2x200 ml). The combined organic phases were dried over MgSO ₄ , filtered and concentrated in vacuo to give 510 mg of compound 63 (53%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.85 (d, J=6.8 Hz, 3H); 0.87 (d J=6.8 Hz, 3H); 1.20 to 1.39 (m, 4H); 1.34(s,9H); 1.48 to 1.73 (m, 2H); 1.99 (m, 1H); 2.86(m, 2H); 3.89 (dd, J=7.7 and 8.4 Hz, 1H); 4.08 (m, 1H); 4.17 to 4.32 (m, 3H); 6.72 (wide m, 1H); 7.32 (m, 2H); 7.38 to 7.46 (m, 3H); 7.74 (m, 2H); 7.89 (d, J=7.6 Hz, 2H); 8.00 (wide m, 1H); 12.54 (wide s, 1H). LCMS (A): ES mass/charge=344; mas

- 123 045708 sa/charge=468; mass/charge=566 [M-N] ^- ; mass/charge=568 [M+H]+; Rt=1.41 min.

Compound 64: (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3methylbutanamido)-6-aminohexanoic acid.

A solution of 4 M HCl in 1,4-dioxane was added to compound 63 (340 mg, 599 µmol), and the reaction medium was stirred at room temperature. for 1 hour. At the same time, the reaction medium was concentrated in vacuo, followed by the addition of Et ₂ O and filtration. The resulting white solid was purified by flash chromatography on 15 g silica gel (elution DCM/MeOH/NH ₄ OH 12:3:0.5 v/v/v) to give 190 mg of compound 64 (63%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.83 (d, J=6.6 Hz, 3H); 0.85 (d, J=6.6 Hz, 3H); 1.21 to 1.70 (m, 6H); 2.04 (m, 1H); 2.71 (t, J=7.1 Hz, 2H); 3.84 (dd, J=6.7 and 8.7 Hz, 1H); 3.91(m, 1H); 4.18 to 4.35 (m, 3H); 7.22 (wide m, 3H); 7.33 (m, 2H); 7.42 (t, J=7.6 Hz, 2H); 7.54 (d, J=9.0 Hz, 1H); 7.69 (wide m, 1H); 7.74 (m, 2H); 7.89 (d, J=7.6 Hz, 2H). LCMS (A): ES mass/charge=244; mass/charge=466 [M-N] ^- ; mass/charge=468 [M+N]+; Rt=0.81 min.

Compound 65: (S)-80-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)74-oxo-2,5,8,11 ,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azagenoctacontane-81-ova acid.

To a solution of compound 64 (130 mg, 258.0 µmol) in THF (20 ml) was added a solution of sodium bicarbonate (50 mg, 589.2 µmol) in H ₂ O (8 ml) and MEO-DPeG(24)-NHS ( CAS number [756525-94-7], 40θ mg, 329.4 µmol). The reaction medium was stirred for 20 h at RT, partially concentrated in vacuo, then diluted with H2O (10 ml), acidified to pH 3 using Amberlite IR-120 (H) ion exchange resin (CAS number [78922-04 -0]), filtered, concentrated in vacuo and purified by three sequential flash chromatography runs on 40g, 25g and 12g silica gel (gradient elution DCM/MeOH/H ₂ O) to give 214 mg of compound 65 as white varnish (53%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d, J=7.0 Hz, 3H); 1.22 to 1.40 (m, 4H); 1.58 (m, 1H); 1.67 (m, 1H); 1.99 (m, 1H); 2.28 (t, J=6.6 Hz, 2H); 2.99 (m, 2H); 3.23(s, 3H); 3.40 to 3.70 (m, 92H); 3.56 (t, J=6.6 Hz, 2H); 3.90 (m, 1H); 4.08 (wide m, 1H); 4.19 to 4.31 (m, 3H); 7.31 (wide t, J=7.8 Hz, 2H); 7.41 (wide t, J=7.8 Hz, 3H); 7.73 (t, J=7.8 Hz, 2H); 7.76 (m, 1H); 7.89 (d, J=7.8 Hz, 2H); 8.00 (m, 1H); 12.53 (m, 1H). LCMS (D): ES mass/charge=794 [M+H+Na] ²⁺ ; mass/charge=1567 [M+H]+; Rt=2.9 min.

Compound 66: N-((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R)-3-((S)-1-(( 3S, 10R, 16S,E)10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11- diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-6-oxohexyl)-2,5,8,11,14,17,20,23,26,29,32, 35,38,41, 44,47,50,53,56,59,62,65,68,71-tetracosaoxatetraheptacontan-74-amide.

To a solution of (E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethylphenyl)oxiranyl]-ethyl}-10-(3chloro-4- methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 71 mg , 101.7 µmol) in DMF (5 ml) was added a solution of compound 65 (214 mg, 136.6 µmol) in DMF (5 ml), HOBt (22 mg, 156.3 µmol) and EDC (27 µl, 152, 5 µmol). The reaction medium was stirred for 3 hours at room temperature. before adding piperidine (91 µl, 915.3 µmol). Stirring was carried out for 1 hour 30 minutes. at RT, the reaction mixture was concentrated in vacuo and purified by two sequential flash chromatography runs on 25 g and 12 g silica gel (DCM/MeOH gradient elution) to give 58 mg of compound 66 as a clear varnish (28%) .

1H NMR (500 MHz, δ in ppm, DMSO-d6): 0.76 (d, J=7.0 Hz, 3H); 0.78 (d, J=6.8 Hz, 6H); 0.88 (d, J=7.0 Hz, 3H); 0.99(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.25 to 1.41 (m, 5 H); 1.51 to 1.70 (m, 4H); 1.80 (m, 1H); 1.93 (m, 1H); 2.25 (m, 1H); 2.28 (t, J=6.7 Hz, 2H); 2.55 (wide m, 2H); 2.68(m, 2H); 2.99 (m, 5 H); 3.23(s, 3H); 3.32 (hidden m, 1H); 3.40 to 3.55 (m, 92H); 3.58 (t, J=6.7 Hz, 2H); 3.80(s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.20 to 4.35 (m, 4H); 4.91 (dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m, 1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.46 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 and 8.6 Hz, 1H); 7.22 (m, 5 H); 7.29 (d, J=2.4 Hz, 1H); 7.79 (t, J=6.7 Hz, 1H); 8.00 (d, J=8.8 Hz, 1H); 8.36 (d, J=8.0 Hz, 1H); 8.47 (t, J=6.2 Hz, 1H). LCMS (D): ES mass/charge=1013 [M+2H] ²⁺ ; mass/charge=2025 [M+N]+; Rt=2.64/2.62 min (mixture of isomers 85/15).

Example 24: (80S,83S)-80-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4 -methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran2-yl)benzyl) carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50, 53 ,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanoneoctacontane-89-oic acid.

To a solution of compound 66 (58 mg, 28.7 µmol) in DCM (7 ml) was added glutaric anhydride (5.85 mg, 48.7 µmol). The reaction medium was stirred for 2 hours at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (gradient elution DCM/MeOH/H2O) to obtain 35 mg of the compound of Example 24 as a white varnish (57 %).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.78 (d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 3H); 0.84 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.11(s, 3H); 1.18 to 1.41 (m, 5 H); 1.50 to 1.75 (m, 6H); 1.80(m,

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1H); 1.98(m, 1H); 2.15 to 2.27 (m, 5 H); 2.30 (t, J=6.7 Hz, 2H); 2.69 (m, 2H); 2.98 (m, 5 H); 3.23(s, 3H); 3.32 (hidden m, 1h); 3.40 to 3.65 (m, 94H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.14 (m, 1H); 4.20 to 4.33 (m, 4H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m, 1H); 5.79 (d, J=15.8 Hz, 1H); 6.47 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.24 (m, 5 H); 7.29 (d, J=2.4 Hz, 1H); 7.80 (t, J=6.0 Hz, 1H); 7.86 (d, J=8.8 Hz, 1H); 7.92 (d, J=8.0 Hz, 1H); 8.37 (m, 2H); 12.10 (wide m, 1H). LCMS (D): ES mass/charge=1069.5 [M+2H] ² +; mass/charge=2138 [M+H]+; Rt=2.96/2.93 min (mixture of isomers 85/15).

Example 25: (80S,83S)-2,5-dioxopyrrolidin-1-yl-80-((4-((2R,3R)-3-((S)-1 -((3 S,10R, 16S, E)-10-(3chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec13-en-16-yl )ethyl)oxiran-2-yl)benzyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23, 26,29,32,35, 38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanoneoctacontane-89-oate.

To a solution of the compound of Example 24 (35 mg, 16.4 µmol) in THF (3 ml) was added DSC (4.28 mg, 16.4 µmol) and DIEA (2.7 µl, 16.4 µmol). The reaction medium was stirred for 20 hours at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/iPrOH gradient elution) to give 12.0 mg of the compound from Example 25 as a white varnish (33 %).

¹ H NMR (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 3H); 0.84 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.15 to 1.40 (m, 5H); 1.50 to 1.71 (m, 4H); 1.75 to 1.86 (m, 3H); 1.97 (m, 1H); 2.18 to 2.32 (m, 5H); 2.67 (m, 4H); 2.80(s, 4H); 2.98 (m, 5 H); 3.22(s, 3H); 3.32 (hidden m, 1H); 3.40 to 3.77 (m, 94H); 3.80(s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.16(m, 1H); 4.20 to 4.32 (m, 4H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m, 1H); 5.78 (dd, J=2.0 and 15.8 Hz, 1H); 6.47 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.24 (m, 5 H); 7.29 (d, J=2.4 Hz, 1H); 7.79 (t, J=6.0 Hz, 1H); 7.90 (d, J=8.8 Hz, 1H); 7.94 (d, J=8.0 Hz, 1H); 8.36 (m, 2H). LCMS (D): ES mass/charge=746; mass/charge=2235 [M+H]+; Rt=3.08/3.06 min (mixture of isomers 89/11).

Example 26: hu2H11_R35-74-Ex25.

The general method described previously was used to prepare the compound of Example 26. 55.8 mg of hu2H11_R35-74 was reacted with 261.6 μl of a 10 mM solution of the compound of Example 25 in DMA (7 eq.) for 3 h 15 min. then 131 μl of a 10 mM solution of the compound of Example 25 in DMA (3.5 eq.) was added over 5 hours 30 minutes. After overnight storage at 4°C, purification on Superdex 200 pg in buffer B pH 6.5 + 20% NMP, concentration on Amicon Ultra-15, buffer exchange with Sephadex G25 in buffer B pH 6.5 + 5 % NMP, concentration on Amicon Ultra-15 and filtration on a PVDF filter with a pore size of 0.22 μm gave 33.35 mg of the compound from example 26 as a colorless transparent solution at a concentration of 2.9 mg/ml with a DAR of 3.9 ( HRMS), monomer purity 100% and overall yield 60%.

SEC-HRMS: mass/charge=151487 (D1); mass/charge=153615 (D2); mass/charge=155740 (D3); mass/charge=157863 (D4); mass/charge=159981 (D5); mass/charge=162112 (D6); mass/charge= 164240 (D7); mass/charge=166356 (D8).

- 125 045708

Synthesis of Examples 27-29: Glutaryl-Val-PEG24Lys-azacryptobenzylamine, Glutaryl-Val-PEG24Lys-azacryptobenzylamine NHS ester and corresponding ADC

Compound 67: N-((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R)-3-((S)-1-(( 3S,7S, 10R, 16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4 ,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-6-oxohexyl)-2,5,8,11,14,17,20,23,26 , 29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatetraheptacontan-74-amide.

To a solution of (3 S,7S,10R, 16S,E)-16-((S)-1 -((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)- 10-(3chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12tetraone (which can be synthesized as described in PCT/EP2016/076603, starting from methyl (3S)-3amino-2,2-dimethylbutanoate, [MFCD09256689], 75 mg, 103.4 µmol) in DMF (5 ml), a solution of compound 65 (211 mg) was added , 134.7 µmol) in DMF (5 ml), HOBt (21 mg, 155.4 µmol) and EDC (20 µl, 123.0 µmol). The reaction medium was stirred for 4 hours at room temperature. before adding piperidine (95 µl, 961.9 µmol); Stirring was carried out for 2 h at RT, then the reaction medium was concentrated in vacuo and purified by flash chromatography on 25 g silica gel (DCM/MeOH gradient elution) to obtain 132 mg of compound 67 as a colorless oil (62%) .

- 126 045708 ¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.76 (d, J=7.0 Hz, 3H); 0.87 (m, 15 H); 1.00(s, 3H); 1.03 (d, J=7.1 Hz, 3H); 1.15 to 1.40 (m, 5 H); 1.18(s, 3H); 1.46 to 2.00 (m, 8H); 2.25 (m, 1H); 2.28 (t, J=6.7 Hz, 2H); 2.60 (m, 1H); 2.69 (dd, J=11.2 and 14.5 Hz, 1H); 2.91 (dd, J=2.3 and 7.5 Hz, 1H); 2.92 to 3.04 (m, 4H); 3.23(s, 3H); 3.34 to 3.77 (m, 95 H); 3.80(s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.08 (m, 2H); 4.20 to 4.34 (m, 3H); 5.04 (m, 1H); 5.78 (dd, J=2.0 and 15.8 Hz, 1H); 6.44 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.02 (d, J=8.6 Hz, 1H); 7.17 to 7.25 (m, 5 H); 7.32 (d, J=2.4 Hz, 1H); 7.78 (t, J=6.0 Hz, 1H); 7.85 (d, J=8.0 Hz, 1H); 7.92 (d, J=7.0 Hz, 1H); 8.00 (d, J=8.8 Hz, 1H); 8.40 (t, J=7.5 Hz, 1H); 8.46 (t, J=6.0 Hz, 1H). LCMS (D): ES mass/charge=2051 [M+H]+; Rt=2.57/2.55 min (mixture of isomers 95/5).

Example 27: (80S,83S)-80-((4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro -4-methoxybenzyl)6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxy ran-2-yl)benzyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41 ,44,47, 50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanoneoctacontane-89-oic acid.

To a solution of compound 67 (132 mg, 64.3 µmol) in DCM (10 ml) was added glutaric anhydride (9 mg, 78.9 µmol). The reaction medium was stirred for 2 hours at RT, concentrated in vacuo and purified by two successive flash chromatography runs on 4 g and 12 g silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 71 mg of compound from example 27 in the form of white varnish (51%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d6): 0.80 to 0.90 (m, 18 H); 1.00(s, 3H); 1.03 (d, J=7.1 Hz, 3H); 1.15 to 1.42 (m, 5 H); 1.18(s, 3H); 1.51 to 1.74 (m, 4H); 1.80 (m, 1H); 1.98 (m, 2H); 2.15 to 2.32 (m, 7H); 2.60 (m, 1H); 2.70 (dd, J=11.2 and 14.5 Hz, 1H); 2.91 (dd, J=2.3 and 7.5 Hz, 1H); 2.95 (dd, J=3.4 and 14.5 Hz, 1H); 3.00 (m, 2H); 3.24(s, 3H); 3.40 to 3.67 (m, 95 H); 3.80(s, 3H); 3.90 (d, J=2.3 Hz, 1H); 4.00 to 4.34 (m, 6H); 5.04 (m, 1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.44 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.02 (d, J=8.6 Hz, 1H); 7.17 to 7.25 (m, 5 H); 7.32 (d, J=2.4 Hz, 1H); 7.80 (t, J=6.0 Hz, 1H); 7.86 (d, J=8.6 Hz, 2H); 7.92 (t, J=6.5 Hz, 2H); 8.37 (t, J=6.0 Hz, 1H); 8.41 (t, J=7.5 Hz, 1H); 12.00 (m, 1H). LCMS (D): ES mass/charge=722; mass/charge=2165 [M+H]+; Rt=2.95/2.92 min (mixture of isomers 95/5).

Example 28: 2,5-dioxopyrrolidin-1-yl-(80S,83S)-80-((4-((2R,3R)-3-((S)-1-((3S,7S, 10R, 16S ,E)-10(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17, 20,23,26, 29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanoneoctacontane-89oate.

To a solution of the compound of Example 27 (33 mg, 15.2 µmol) in THF (3 ml) was added DSC (4 mg, 15.6 µmol) and DIEA (2.55 µl, 15.4 µmol). The reaction medium was stirred for 20 h at room temperature, then DSC (1 mg, 3.9 µmol) and DIEA (1 µl, 6.0 µmol) were added. The reaction medium was stirred for 2 h at RT, concentrated in vacuo and purified by flash chromatography on 4 g of silica gel (gradient elution DCM/iPrOH/MeCN) to obtain 11 mg of the compound from example 28 as a white varnish (32 %).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.80 to 0.90 (m, 18 H); 1.00(s, 3H); 1.03 (d, J=7.1 Hz, 3H); 1.12 to 1.42 (m, 5H); 1.19(s, 3H); 1.55 (m, 1H); 1.65 (m, 1H); 1.82 (m, 3H); 1.96 (m, 2H); 2.28(m, 3H); 2.67 (m, 4H); 2.80(s, 4H); 2.91 (dd, J=2.3 and Hz, 1H); 2.95 (dd, J=3.4 and 14.5 Hz, 1H); 3.00 (m, 2H); 3.23(s, 3H); 3.35 to 3.67 (m, 95 H); 3.80(s, 3H); 3.90 (d, J=2.3 Hz, 1H); 4.03 to 4.32 (m, 6H); 5.03 (m, 1H); 5.90 (dd, J=2.0 and 15.8 Hz, 1H); 6.44 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.03 (d, J=8.6 Hz, 1H); 7.17 to 7.25 (m, 5 H); 7.32 (d, J=2.4 Hz, 1H); 7.79 (t, J=6.0 Hz, 1H); 7.85 (d, J=8.7 Hz, 1H); 7.90 (d, J=8.7 Hz, 1H); 7.93 (m, 2H); 8.35 (t, J=6.5 Hz, 1H); 8.40 (d, J=7.1 Hz, 1H).

Example 29: hu2H11_R35-74-Ex28.

The general method described previously was used to prepare the compound of Example 29. 44.64 mg of hu2H11_R35-74 was reacted with 210 μl of a 10 mM solution of the compound of Example 28 in DMA (7 eq.) for 2 hours, then 210 was added sequentially µl of a 10 mM solution of the compound of Example 28 in DMA (7 equiv.) for 2 hours 30 minutes and 90 µl of a 10 mM solution of the compound of Example 28 in DMA (3 equiv.) for 2 hours. After storing overnight at 4 °C, purification with Superdex 200 pg in buffer B with pH 6.5+20% NMP, concentration with Amicon Ultra-15, buffer replacement with Sephadex G25 in buffer B with pH 6.5+5% NMP and filtration with PVDF- filter with a pore size of 0.22 μm, 30.1 mg of the compound of Example 29 were obtained as a colorless clear solution at a concentration of 2.51 mg/ml with DAR 4 (HRMS), monomer purity of 98.4% and overall yield of 67%.

SEC-HRMS: mass/charge=151502 (D1); mass/charge=153649 (D2); mass/charge=155798 (D3); mass/charge=157939 (D4); mass/charge=160100 (D5); mass/charge=162262 (D6).

- 127 045708

Synthesis of Examples 30-32: Glutaryl-Va1-glucuronic Lys-C52-benzylamine, Glutaryl-Va1-glucuronic Lys-C52-benzylamine NHS ester and corresponding ADC

Compound 68: (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6((2S,3S,4S,5R ,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-carboxamido)hexanoic acid.

Sodium solution was introduced into the round-bottomed flask while stirring with a magnetic stirrer.

- 128 045708 1-O-methyl-in-O-glucuronic acid salt (CAS number [58189-74-5], 100 mg, 434 µmol) followed by water (5 ml) and QS Amberlite (CAS number [9037- 24-5]) until pH2 was reached, the medium was filtered.

In a round bottom flask, while stirring with a magnetic stirrer, NaHCO ₃ (43 mg, 511 µmol) in water (5 ml) was added to a solution of compound 64 (50 mg, 99 µmol) in THF (4 ml); the reaction medium was stirred at room temperature. within 5 min. Then, the glucuronic acid solution described above was added to the reaction medium, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2yl)-4-methylmorpholinium chloride N-hydrate (CAS number [3945-69- 5], 67 mg, 242 µmol). The reaction medium was stirred at room temperature. overnight, then concentrated in vacuo. The crude medium was purified by flash chromatography on 5 g silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 23 mg of compound 68 (35%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.84 (d, J=6.7 Hz, 3H); 0.86 (d, J=6.7 Hz, 3H); 1.13 to 1.42 (m, 4H); 1.52 to 1.72 (m, 2H); 2.05 (m, 1H); 2.96 to 3.08 (m, 2H); 3.14 (m, 1H); 3.30 (partially hidden m, 1H); 3.38(s, 3H); 3.53 (m, 1H); 3.78 to 3.97 (m, 2H); 4.09 (dd, J=2.3 and 7.8 Hz, 1H); 4.19 to 4.34 (m, 3H); 5.01 to 5.08 (wide m, 1H); 5.13 (wide m, 1H); 5.27 (m, 1H); 7.33 (m, 2H); 7.42 (t, J=7.6 Hz, 2H); 7.52 to 7.62 (wide m, 2H); 7.75 (m, 2H); 7.89 (d, J=7.6 Hz, 2H); 7.93 (wide m, 1H); 12.00 (m, 1H). LCMS (A): ES mass/charge=656 [M-N] ^- ; mass/charge=658 [M+H]+; Rt=1.05 min.

Compound 69: (2S,3S,4S,5R,6R)-N-((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R) -3-((S)-1((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12- tetraoxo-1,4-dioxa-8,11diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-6-oxohexyl)-3,4,5-trihydroxy-6methoxytetrahydro-2H- pyran-2-carboxamide.

To a solution of (E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethylphenyl)oxiranyl]-ethyl}-10-(3chloro-4- methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 90 mg , 129 µmol) with magnetic stirring, a solution of compound 68 (135 mg, 205 µmol) in DMF (5 ml) was added, followed by the addition of HOBt (30 mg, 222 µmol) and EDC (36 µl, 203 µmol). The reaction medium was stirred at room temperature. for 1 hour. At the same time, DMA (1 ml) was added and stirring was maintained overnight. EDC (20 μl) was then added and the reaction medium was stirred for another 2 hours. Piperidine (12.9 μl, 129 μmol) was then added to the medium. After 1 hour 30 minutes. After stirring, the reaction medium was concentrated in vacuo and purified using 3 sequential flash chromatography operations on silica gel (isocratic elution DCM/MeOH/H ₂ O 40:5:0.5 v/v/v and DCM/MeOH/NH ₄ OH 12 :3:0.5 v/v/v) to obtain 47 mg of compound 69 (33%).

^1H NMR (500 MHz, δ in ppm, DMSO-d6): 0.77 to 0.81 (m, 9H); 0.88 (d, J=6.6 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=6.6 Hz, 3H); 1.12(s, 3H); 1.20 to 1.71 (m, 9H); 1.80 (m, 1H); 1.93 (m, 1H); 2.28 (m, 1H); 2.62 to 2.73 (m, 2H); 2.93 to 3.14 (m, 9H); 3.28 to 3.36 (partially hidden m, 2H); 3.39(s, 3H); 3.53 (d, J=9.5 Hz, 1H); 3.81(s, 3H); 3.87 (d, J=1.7 Hz, 1H); 4.09 (m, 1H); 4.19 to 4.36 (m, 4H); 4.91(m, 1H); 5.00 to 5.15 (m, 4H); 5.79 (d, J=15.9 Hz, 1H); 6.48 (m, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.1 and 8.6 Hz, 1H); 7.20 to 7.25 (m, 5 H); 7.28 (d, J=2.1 Hz, 1H); 7.92 (wide t, J=5.9 Hz, 1H); 8.02 (wide m, 1H); 8.35 (d, J=7.8 Hz, 1H); 8.46 (wide t, J=6.2 Hz, 1H). LCMS (A): ES mass/charge=558 [M+2H] ²⁺ ; mass/charge=1113 [M-N] ^- ; mass/charge=1115 [M+H]+; mass/charge=1159 [M-H+HCO2H]-; Rt=0.9 min.

Example 30: 5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E )-10-(3-chloro-4-methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-ene-16- yl)ethyl)oxiran2-yl)benzyl)amino)-1-oxo-6-((2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2- carboxamido)hexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid.

DMF (4 mL) was added to compound 69 (47 mg, 42.1 μmol) and glutaric anhydride (8.5 mg, 74.5 μmol) was added with magnetic stirring; the reaction medium was stirred at room temperature. for 2 hours. At the same time, the crude medium was concentrated in vacuo and purified by 2 sequential flash chromatography runs on silica gel (gradient elution DCM/MeOH/H ₂ O and DCMMeOH/NH ₄ OH) to obtain 17 mg of the compound of the example 30 (33%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.81 (divided d, J=6.4 Hz, 6H); 0.84 (d, J=6.6 Hz, 3H); 0.86 (d, J=6.6 Hz, 3H); 0.98(s, 3H); 1.04 (d, J=6.6 Hz, 3H); 1.11(s, 3H); 1.17 to 2.32 (wide m, 18 H); 2.66 to 2.74 (m, 2H); 2.91 to 3.03 (m, 5 H); 3.10 (wide m, 1H); 3.18 (t, J=8.8 Hz, 1H); 3.28 to 3.36 (partially hidden m, 2H); 3.37(s, 3H); 3.60 (m, 1H); 3.81(s, 3H); 3.87 (d, J=1.6 Hz, 1H); 4.07 (m, 2H); 4.14 to 4.26 (m, 3H); 4.31(m, 1H); 4.92(m, 1H); 5.06 to 5.13 (m, 2H); 5.35 (m, 2H); 5.80 (d, J=15.3 Hz, 1H); 6.45 (ddd, J=3.6, 11.1 and 15.3 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.18 (dd, J=2.1 and 8.5 Hz, 1H); 7.22 (d, J=8.4 Hz, 2H); 7.25 (d, J=8.4 Hz, 2H); 7.29 (d, J=2.1 Hz, 1H); 7.31 (m, 1H); 7.77 to 8.81 (m, 5 H). LCMS (A): ES mass/charge=1227 [M-N] ^- ; mass/charge=1229 [M+H]+; mass/charge=615; Rt=1.16 min.

- 129 045708

Example 31: 2,5-dioxopyrrolidin-1-yl-5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1 -((3S,10R,16S,E)-10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8, 11 -diazacyclohexadec- 13-en-16ul)ethyl)oxupan-2-ul)benzyl)amino)-1-oxo-6-((2S,3S,4S,5R,6R)-3,4,5-trihydroxy- 6-methoxytetrahydro-2H-pyran-2-carboxamido)hexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate.

DMF (3 mL), DSC (4.2 mg, 15.6 μmol) and DIEA (6 μL, 34.5 μmol) were added to the compound of Example 30 (14 mg, 11.4 μmol) with magnetic stirring. . The reaction medium was stirred at room temperature. for 4 hours. At the same time, the medium was diluted with MeTHF (10 ml) and washed with H ₂ O (5 ml). The aqueous phase was extracted with MeTHF (10 ml), the combined organic phases were dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/iPrOH gradient elution) to give 3.5 mg of compound from examples 31 (23%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.78 (d, J=6.6 Hz, 6H); 0.82 (d, J=7.1 Hz, 3H); 0.84 (d, J=7.1 Hz, 3H); 0.99(s, 3H); 1.04 (d, J=6.6 Hz, 3H); 1.12(s, 3H); 1.17 to 1.70 (m, 9H); 1.77 to 1.87 (m, 3H); 1.98(m, 1H); 2.23 to 2.32 (m, 3H); 2.65 to 2.70 (m, 4H); 2.81(s,4H); 2.93 to 3.07 (m, 6H); 3.15 (m, 1H); 3.28 to 3.38 (partially hidden m, 2H); 3.40(s, 3h); 3.53 (d, J=9.6 Hz, 1H); 3.81(s, 3H); 3.87 (d, J=1.4 Hz, 1H); 4.09 (d, J=7.7 Hz, 1H); 4.17 (dd, J=6.9 and 8.2 Hz, 1H); 4.20 to 4.33 (m, 4H); 4.90 (dd, J=3.4 and 9.5 Hz, 1H); 5.04 (m, 2H); 5.10 (m, 1H); 5.14 (d, J=5.2 Hz, 1H); 5.79 (dd, J=0.8 and 15.3 Hz, 1H); 6.47 (m, 1H); 7.05 (d, J=8.8 Hz, 1H); 7.17 (dd, J=1.6 and 8.8 Hz, 1H); 7.20 to 7.25 (m, 5 H); 7.28 (d, J=1.6 Hz, 1H); 7.86 to 7.99 (m, 3H); 8.37 (m, 2H). LCMS (A): ES mass/charge=1370 [M-H+HCO2H]-; ES mass/charge=1326 [M+H]+; mass/charge=663.5; Rt =1.21 min.

Example 32: hu2H11_R35-74-Ex31.

The general method described previously was used to prepare the compound of Example 32. 27.9 mg of hu2H11_R35-74 was reacted with 107 μl of a 10.5 mM solution of the compound of Example 31 in DMA (6 eq.) for 2 h 30 min. then 107 μl of a 10.5 mM solution of the compound of Example 31 in DMA (6 eq.) was added over 4 hours 30 minutes. After purification on Superdex 200 pg in buffer B pH 6.5 + 10% NMP, concentration on Amicon Ultra-15, dilution in buffer B pH 6.5 to a final concentration of 5% NMP and filtration on a pore size PVDF filter 0.22 µM yielded 16.7 mg of the compound of Example 32 as a colorless clear solution at a concentration of 2.23 mg/ml with DAR 3 (HRMS), 100% monomer purity and 60% overall yield.

SEC-HRMS: wt/charge=149407 (unconjugated mAb); mass/charge=150619 (D1); mass/charge=151831 (D2); mass/charge=153044 (D3); mass/charge=154256 (D4); mass/charge=155468 (D5); mass/charge=156685 (D6); mass/charge=157899 (D7); mass/charge=159014 (D8).

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Synthesis of Examples 33-35: Glutaryl-Val-sulfo-PEG4Lys-C52-benzylamine, Glutaryl-Val-sulfo-PEG4Lys-C52-benzylamine NHS ester and corresponding ADC

- 131 045708

Compound 70: di-tert-butyl-4,7,10,13-tetraoxahexadecane-1,16-dioate.

To a solution of triethylene glycol (5 mL, 37.5 mmol) in THF (15 mL) was added sodium (18 mg, 783.0 μmol). The reaction medium was stirred for 2 hours at room temperature, then added dropwise over 5 minutes. tert-butyl acrylate (14 ml, 95.58 mmol).

The reaction medium was stirred for 20 hours at RT, then quenched with brine (300 ml) and extracted twice with AtOAc (300 ml). The combined organic phases were dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 200 g silica gel (heptane/EtOAc gradient elution) to give 8.05 g of compound 70 as a colorless oil (53%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 1.40 (s, 18 H); 2.40 (t, J=6.5 Hz, 4H); 3.48 (m, 12H); 3.58 (t, J=6.5 Hz, 4H). LCMS (A): ES mass/charge=407 [M+H]+; mass/charge=429 [M+Na] ⁺ ; Rt=1.32 min.

Compound 71: 4,7,10,13-tetraoxahexadecane-1,16-dioic acid.

Formic acid (40 mL, 1.04 mol) was added to compound 70 (8.05 g, 19.8 mmol). The reaction medium was stirred for 1 day at RT, concentrated in vacuo and co-evaporated with AtOAc (20 ml) to obtain 5.9 g of compound 71 as a colorless oil (qty).

1H NMR (300 MHz, δ in ppm, DMSO-d6): 2.44 (t, J=6.5 Hz, 4H); 3.48(s,8H); 3.50(s, 4H); 3.59 (d, J=6.5 Hz, 4 H); 12.25 (wide m, 2H).

Compound 72: 3-oxo-1-phenyl-2,6,9,12,15-pentaoxaoctadecane-18-oic acid.

To a solution of compound 71 (5.9 g, 20.05 mmol) in DMF (50 ml) was added DIEA (3.6 ml, 20.67 mmol) and added dropwise over 10 min. a solution of benzyl bromide (2.43 ml, 20.05 mmol) in DMF (50 ml). The reaction medium was stirred for 20 hours at room temperature, quenched with 1N. HCl (500 ml) and extra

- 132 045708 were digested twice with EtOAc (200 ml). The combined organic phases were dried over MgSO ₄ , filtered, concentrated in vacuo and purified by two successive flash chromatography runs on 400 g and 100 g silica gel (DCM/MeOH gradient elution) to give 2.7 g of compound 72 as pale green oils (35%).

^1H NMR (300 MHz, δ in ppm, DMSO-d6): 2.42 (m, 2H); 2.60 (t, J=6.5 Hz, 2H); 3.47(s,6H); 3.49(s, 6H); 3.59 (m, 2H); 3.66 (d, J=6.5 Hz, 2H); 5.10(s, 2H); 7.29 to 7.41 (m, 5 H).

Compound 73: Sodium 2-aminoethanesulfonate.

To a solution of taurine (2 g, 15.98 mmol) in H ₂ O (30 ml) was added sodium bicarbonate (1.34 g, 15.98 mmol). The reaction medium was stirred for 2 hours at RT, then concentrated in vacuo. The crude product was dissolved in H ₂ O (100 ml) and lyophilized to obtain 2.5 g of compound 73 as a white powder (qty.).

IR spectrum in the case of a KBr tablet; main absorption bands in reciprocal centimeters: 1458; 1344; 1212; 1184; 1046; 1037; 742; 737; 598; 576; 533 and 523.

Compound 74: Sodium 3,18-dioxo-1-phenyl-2,6,9,12,15-pentaoxa-19-azagenicosan-21-sulfonate

To a solution of compound 72 (1 g, 2.69 mmol) in DMF (40 ml) was added NHS (367 mg, 3.13 mmol) and EDC (565 μl, 3.12 mmol). The reaction medium was stirred for 20 hours at RT, then a solution of compound 73 (1.9 g, 12.91 mmol) in H ₂ O (20 ml) and sodium bicarbonate (219 mg, 2.61 mmol) was added. The reaction medium was stirred for 2 hours at room temperature, then acidified to pH 3 with Amberlite IR120 (H) (CAS number [78922-04-0], filtered, concentrated in vacuo and purified by flash chromatography on 150 g silica gel (DCM/MeOH/H ₂ O) to obtain 824 mg of compound 74 as a colorless solid 61%).

LCMS (A): ES mass/charge=490 [M-N] ^- ; mass/charge=492 [M+N]+; t _R =1.1 min.

Compound 75: Sodium 1-carboxy-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecane-18-sulfonate.

A solution of compound 74 (820 mg, 1.60 mmol) in MeOH/H ₂ O 8:2 (30 ml) was filtered through 0.22 μm and subjected to hydrogenolysis on a 10% Pd/C cartridge using an H-cube system at 20° WITH. The reaction medium was concentrated in vacuo to obtain 530 mg of compound 75 as yellow gum (78%).

¹ H NMR (300 MHz, δ in ppm, DMSO-d6): 2.27 (t, J=6.6 Hz, 2H); 2.40 (t, J=6.6 Hz, 2H); 2.52 (partially hidden m, 2H); 3.28 (partially hidden m, 2H); 3.49 (m, 12H); 3.59 (m, 4H); 7.72 (wide t, J=6.4 Hz, 1H); 12.08 (wide m, 1H).

Compound 76: (5S,8S)-8-carboxy-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,14,29-tetraoxo2,17,20,23,26-pentaoxa- Sodium 4,7,13,30-tetraazadotriacontane-32-sulfonate.

To a solution of compound 75 (300 mg, 708.5 μmol) in DMF (15 ml) was added NHS (98 mg, 851.5 μmol) and EDC (150 μl, 855.1 μmol). The reaction medium was stirred for 20 hours at RT, then a solution of compound 64 (416 mg, 825.4 µmol) in DMF (15 ml) and a solution of sodium bicarbonate (180 mg, 2.13 mmol) in H2O (10 ml). The reaction medium was stirred for 1 h at RT, acidified to pH 3 with Amberlit IR-120 (H) (CAS number [78922-04-0]), filtered, washed with DMF (5 ml), concentrated in vacuo and purified by flash chromatography on 100 g silica gel (gradient elution DCM/MeOH/acetic acid) to obtain 447 mg of colorless varnish. This varnish was dissolved in THF/H2O 2:1 (120 ml), acidified to pH 3.3 with 0.1N. HCl (approximately 4 ml) and concentrated in vacuo to obtain 354 mg of compound 76 as a white translucent solid (57%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d6): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d, J=7.0 Hz, 3H); 1.20 to 1.41 (m, 4H); 1.50 to 1.74 (m, 2H); 1.98(m, 1H); 2.26 (t, J=6.8 Hz, 2H); 2.28 (t, J=6.8 Hz, 2H); 2.52 (hidden m, 2H); 3.00 (m, 2H); 3.28 (partially hidden m, 2H); 3.45 (m, 12H); 3.58 (t, J=6.8 Hz, 4H); 3.91 (dd, J=7.0 and 9.2 Hz, 1H); 4.14 (m, 1H); 4.19 to 4.31 (m, 3H); 7.32 (m, 2H); 7.37 (d, J=9.2 Hz, 1H); 7.41 (dt, J=1.0 and 7.8 Hz, 2H); 7.75 (m, 3H); 7.80 (t, J=6.0 Hz, 1H); 7.89 (d, J=7.8 Hz, 2H); 8.07 (d, J=8.0 Hz, 1H); 12.49 (m, 1H). LCMS (A): ES mass/charge=849 [M-N] ^- ; mass/charge=851 [M+N]+; Rt=1.44 min.

Compound 77: (25S,28S)-28-amino-25-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3 -chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en16-yl)ethyl)oxirane-2- yl)benzyl)carbamoyl)-29-methyl-4,19,27-trioxo-7,10,13,16-tetraoxa-3,20,26-triazatriacontane-1-sulfonate sodium.

To a solution of (E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethylphenyl)oxiranyl]-ethyl}-10-(3chloro-4- methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 106 mg , 151.8 µmol) in DMF (5 ml) a solution of compound 76 (225 mg, 257.7 µmol) in DMF (5 ml), HOBt (30 mg, 213.1 µmol) and EDC (40 µl, 226, 0 µmol). The reaction medium was stirred for 2 hours at room temperature, then piperidine (134 μl, 1.36 mmol) was added and stirring was carried out for 2 hours. The reaction medium was concentrated in vacuo and purified by two successive runs of flash chromatography at 50 g and 4 g of silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 73 mg of compound 77 as a white solid (36%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.78 (d, J=6.8 Hz, 6H); 0.91 (d, J=7.0 Hz, 3H); 0.93 (d, J=7.0 Hz,

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3H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.18 to 1.45 (m, 5 H); 1.51 to 1.72 (m, 4H); 1.80 (m, 1H); 2.05 (m, 1H); 2.23 to 2.35 (m, 5 H); 2.56 (m, 2H); 2.68(m, 2H); 2.95 to 3.04 (m, 5 H); 3.32(m, 3H); 3.48 (m, 12H); 3.57 (t, J=6.7 Hz, 2H); 3.59 (t, J=6.7 Hz, 2H); 3.66 (d, J=6.0 Hz, 1H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.20 to 4.36 (m, 4H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 5.12 (m, 1H); 5.80 (dd, J=2.0 and 15.2 Hz, 1H); 6.47 (ddd, J=3.8, 11.3 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.24 (m, 5 H); 7.29 (d, J=2.4 Hz, 1H); 7.76 (t, J=6.0 Hz, 1H); 7.86 (t, J=8.0 Hz, 1H); 7.93 (wide m, 3H); 8.38 (d, J=8.0 Hz, 1H); 8.49 (d, J=8.0 Hz, 1H); 8.59 (t, J=6.0 Hz, 1H). LCMS (A): ES mass/charge=655 [M+2H] ²⁺ ; mass/charge=1306 [M-N] ^- ; mass/charge=1308 [M+N] ⁺ ; Rt=1.07 min.

Example 33: (25S,28S)-33-carboxy-25-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3 -chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en16-yl)ethyl)oxirane-2- yl)benzyl)carbamoyl)-28-isopropyl-4,19,27,30-tetraoxo-7,10,13,16-tetraoxa3,20,26,29-tetraazatritriacontane-1-sulfonate sodium.

Glutaric anhydride (11 mg, 96.4 µmol) was added to a solution of compound 77 (73 mg, 54.9 µmol) in DMF. The reaction medium was stirred for 20 hours at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/MeOH/H ₂ O) to obtain 40 mg of the compound of Example 33 as a white varnish (50 %).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.81 (s, 9H); 1.04 (d, J=7.0 Hz, 3H); 1.38(s,9H); 1.50 (dd, J=3.0 and 14.5 Hz, 1H); 1.58 (dd, J=9.1 and 14.5 Hz, 1H); 2.38 to 2.48 (partially hidden m, 3H); 4.04 (m, 1H); 4.19 (m, 2H); 4.30 (m, 1H); 4.49(s, 2H); 4.91(m, 1H); 5.80 (d, J=16.0 Hz, 1H); 6.17 (dd, J=8.4 and 16.0 Hz, 1H); 6.43 (d, J=16.0 Hz, 1H); 6.70 (m, 1H); 7.23 to 7.44 (m, 8 H); 7.68 (m, 2H); 7.79 (d, J=8.3 Hz, 1H); 7.88 (d, J=7.8 Hz, 2H). LCMS (A): ES mass/charge=712 [M+2H] ²⁺ ; mass/charge=1420 [M-N]-; mass/charge=1422 [M+N] ⁺ ; Rt=1.57 min.

Example 34: (25S,28S)-25-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4 -methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran2-yl)benzyl) carbamoyl)-34-((2,5-dioxopyrrolidin-1-yl)oxy)-28-isopropyl-4,19,27,30,34-pentaoxo7,10,13,16-tetraoxa-3,20,26, 29-tetraazatetratriacontane-1-sodium sulfonate.

To a solution of the compound of Example 33 (3.57 mg, 2.47 µmol) in DMA (93 µl) was added NHS (27.2 µl, 2.72 µmol) and EDC (111 µl, 2.22 µmol). The reaction medium was stirred for 48 hours at RT: LCMS analysis indicated the presence of the expected compound from Example 34 at a concentration of 95% with 5% residual compound from Example 33. This solution was used, for example, for the conjugation and preparation of the compound from example 35.

Example 35: hu2H11_R35-74-Ex34.

The general method described previously was used to prepare the compound of Example 35. 11.16 mg of hu2H11_R35-74 was reacted with 53 μl of a 10 mM solution of the compound of Example 34 in DMA (7 eq.) for 2 hours 30 minutes, then added 15 μl of a 10 mM solution of the compound from example 34 in DMA (2 equiv.) for 2 hours 30 minutes. After purification on PD-10 and Nap-10 columns in buffer B with pH 6.5 + 5% NMP, 9.5 mg of the compound from example 34 was obtained as a colorless transparent solution at a concentration of 1.9 mg/ml with a DAR of 3.8 (HRMS), monomeric purity of 97.5% and overall yield of 82%.

SEC-HRMS: wt/charge=149330 (unconjugated mAb); mass/charge=150744 (D1); mass/charge=152147 (D2); mass/charge=153554 (D3); mass/charge=154959 (D4); mass/charge=156366 (D5); mass/charge=157771 (D6); mass/charge=159172 (D7); mass/charge=160578 (D8).

Synthesis of Examples 36 and 37: Glutaryl-Val-PEG4Lys-PABA-C52-benzylamine and NHS-glutaryl-Val-PEG4Lys-PABA-C52-benzylamine ester

⁰ 78 ⁰

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Compound 78: (9H-fluoren-9-yl)methyl-((208,23S)-20-((4-(hydroxymethyl)phenyl)carbamoyl)-24methyl-14,22-dioxo-2,5,8,11 -tetraoxa-15,21-diazapentacosan-23-yl)carbamate.

To a solution of compound 61 (180 mg, 257.2 μmol) in MeCN (35 ml) under Ar atmosphere was added DIEA (130 μl, 771.7 μmol), 4-aminobenzyl alcohol (48 mg, 385.8 μmol), HOAt ( 41 mg, 295.8 µmol), HATU (147 mg, 385.8 µmol). The reaction medium was stirred for 17 hours at RT, then MeOH (30 ml) and silica were added, the mixture was concentrated in vacuo and purified by flash chromatography on 30 g of silica gel (DCM/MeOH gradient elution) to give 94 mg of compound 78 as a beige solid (46%).

¹ H NMR (300 MHz, δ in ppm, DMSO-d6): 0.85 (d, J=7.0 Hz, 3H); 0.88 (d, J=7.0 Hz, 3H); 1.18 to 1.45 (m, 4H); 1.52 to 1.70 (m, 2H); 1.99 (m, 1H); 2.27 (m, 2H); 3.00 (m, 2H); 3.22(s, 3H); 3.35 to 3.51 (m, 12H); 3.55 (t, J=6.8 Hz, 2H); 3.90 (dd, J=7.2 and 9.1 Hz, 1H); 4.18 to 4.56 (m, 4H); 4.42 (d, J=5.9 Hz, 2H); 5.07 (t, J=5.9 Hz, 1H); 7.22 (d, J=8.9 Hz, 2H); 7.31 (dt, J=1.8 and 7.8 Hz, 2H); 7.40 (m, 3H); 7.53 (d, J=8.9 Hz, 2H); 7.73 (m, 3H); 7.88 (d, J=7.8 Hz, 2H); 8.02 (d, J=8.2 Hz, 1H); 9.92(s, 1H).

Compound 79: (9H-fluoren-9-yl)methyl-((20S,23S)-24-methyl-2O-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)- 14,22-dioxo-2,5,8,11-tetraoxa-15,21-diazapentacosan-23yl)carbamate.

To a solution of compound 78 (94 mg, 118.9 µmol) in DMF, bis-(4-nitrophenyl)carbonate (112 mg, 357.1 µmol) and DIEA (40 µl, 237.2 µmol) were added. The reaction medium was stirred for 20 h at RT, concentrated in vacuo, diluted with DCM (10 ml) and washed with H2O (10 ml). The organic phase was dried over MgSO ₄ , filtered, concentrated in vacuo and purified by flash chromatography on 12 g silica gel (DCM/MeOH gradient elution) to give 55 mg of compound 79 as a brown solid (48%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.86 (d, J=7.0 Hz, 3H); 0.88 (d, J=7.0 Hz, 3H); 1.18 to 1.45 (m, 4H); 1.55 to 1.77 (m, 2H); 1.99 (m, 1H); 2.27 (t, J=6.8 Hz, 2H); 3.00 (m, 2H); 3.21(s, 3H); 3.40 to 3.52 (m, 12H); 3.57 (t, J=6.8 Hz, 2H); 3.91(m, 1H); 4.20 to 4.41 (m, 4H); 5.24(s, 2H); 7.31 (t, J=7.8 Hz, 2H); 7.35 to 7.48 (m, 5 H); 7.58 (d, J=9.2 Hz, 2H); 7.63 (d, J=8.8 Hz, 2H); 7.74 (m, 2H); 7.80 (t, J=6.0 Hz, 1H); 7.90 (d, J=7.8 Hz, 2H); 8.10 (d, J=8.0 Hz, 1H); 8.32 (d, J=9.2 Hz, 2H); 10.12(s, 1H). LCMS (A): ES mass/charge=956 [M+H]+; mass/charge=978 [M+Na] ⁺ ; mass/charge=1000 [M-H+HCO2H]-; Rt=1.45 min.

Compound 80: 4-((S)-20-((S)-2-amino-3-methylbutanamido)-14-oxo-2,5,8,11-tetraoxa-15azagenicosanamido)benzyl-4-((2R, 3R)-3-((S)-1-((3S, 10R, 16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl6,6-dimethyl-2,5,9,12 -tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzylcarbamate.

To a solution of (E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethylphenyl)oxiranyl]-ethyl}-1O-(3

- 135 045708 chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12tetraone (the synthesis of which is described in WO2011001052, compound 77, 19 mg, 27.2 µmol) in DCM (2.5 ml) a solution of compound 79 (58 mg, 60.7 µmol) in DCM (2.5 ml) and DIEA (15 µl, 86.6 µmol) was added ). The reaction medium was stirred for 20 h at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/MeOH gradient elution) to give 38 mg of the Fmoc-protected intermediate as a clear varnish (92% ).

¹ H NMR (400 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=6.7 Hz, 6H); 0.86 (d, J=7.0 Hz, 3H); 0.88 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.03 (d, J=7.0 Hz, 3H); 1.12(s, 3H); 1.20 to 1.45 (m, 5 H); 1.50 to 1.75 (m, 4H); 1.80 (m, 1H); 2.00 (m, 1H); 2.27 (m, 3H); 2.69 (m, 2H); 2.92 to 3.04 (m, 5 H); 3.21(s, 3H); 3.32 (hidden m, 1H); 3.35 to 3.51 (m, 12H); 3.56 (t, J=6.6 Hz, 2H); 3.80(s, 3H); 3.87 (d, J=2.2 Hz, 1H); 3.91 (dd, J=7.1 and 9.1 Hz, 1H); 4.15 to 4.42 (m, 7H); 4.90 (dd, J=3.7 and 9.8 Hz, 1H); 4.97(s, 2H); 5.10 (m, 1H); 5.79 (dd, J=2.0 and 15.2 Hz, 1H); 6.48 (ddd, J=3.8, 11.1 and 15.2 Hz, 1H); 7.04 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.34 (m, 10H); 7.41 (m, 3H); 7.58 (d, J=8.6 Hz, 2H); 7.70 to 7.80 (m, 4H); 7.89 (d, J=7.8 Hz, 2H); 8.06 (d, J=7.6 Hz, 1H); 8.35 (d, J=8.3 Hz, 1H); 10.03(s, 1H). LCMS (A): ES mass/charge=698; mass/charge=1514 [M+H]+; mass/charge=1558 [M-H+HCO ₂ N]-; Rt=1.56 min.

To a solution of this intermediate (35 mg, 23.1 µmol) in DCM (5 ml) was added piperidine (20 µl, 201.5 µmol). The reaction medium was stirred for 1 hour 30 minutes. at room temperature, then DMF (1 ml) was added and stirring was carried out for 1 hour 30 minutes. Piperidine (2 μl) was then added; the reaction medium was stirred for 1 h at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/MeOH gradient elution) to give 25 mg of compound 80 as a clear varnish (83%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.78 (d, J=6.7 Hz, 6H); 0.82 (d, J=7.0 Hz, 3H); 0.89 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.11(s, 3H); 1.22 to 1.48 (m, 5 H); 1.51 to 1.75 (m, 6H); 1.80 (m, 1H); 1.98(m, 1H); 2.28(m, 3H); 2.69 (m, 2H); 2.92 to 3.05 (m, 7H); 3.15 (m, 1H); 3.21(s, 3H); 3.32 (hidden m, 1H); 3.35 to 3.51 (m, 12H); 3.57 (t, J=6.7 Hz, 2H); 3.80(s, 3H); 3.87 (d, J=2.2 Hz, 1H); 4.19 (d, J=6.4 Hz, 2H); 4.23 (ddd, J=3.9, 8.3 and 11.8 Hz, 1H); 4.43 (m, 2H); 4.90 (dd, J=3.7 and 9.8 Hz, 1H); 4.98(s, 2H); 5.10 (m, 1H); 5.80 (dd, J=2.0 and 15.2 Hz, 1H); 6.48 (ddd, J=3.9, 11.3 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8H); 7.59 (d, J=8.6 Hz, 2H); 7.79 (t, J=6.0 Hz, 1H); 7.82 (t, J=6.4 Hz, 1H); 8.21(m, 1H); 8.39 (d, J=8.3 Hz, 1H); 10.13(s, 1H). LCMS (A): ES mass/charge=646.5; mass/charge=1292 [M+H]+; Rt=1.05 min.

Example 36: (20S,23S)-20-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10- (3-chloro-4-methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl) oxiran2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8,11-tetraoxa-15,21,24-triazanonacosane-29-oic acid.

To a solution of compound 80 (25 mg, 19.3 μmol) in DCM (5 ml) was added glutaric anhydride (4 mg, 19.3 μmol). The reaction medium was stirred for 2 hours 30 minutes. at room temperature, then glutaric anhydride (4 mg, 19.3 µmol) and DMF (0.5 ml) were added and stirring was carried out for 26 hours. The reaction medium was concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/MeOH gradient elution) to obtain 10 mg of the compound from Example 36 as a white varnish (37%).

Example 37: (20S,23S)-2,5-dioxopyrrolidin-1-yl-20-((4-((((4-((2R,3R)-3-((S)-1-((3S ,10R,16S,E)-10(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec -13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-carbamoyl)oxy)methyl)phenyl)-carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8, 11-tetraoxa-15,21,24-triazanonacosane-29-oate

To a solution of the compound of Example 36 (10 mg, 7.11 µmol) in THF (2 ml) was added DSC (2 mg, 7.65 µmol) and DIEA (1.2 µl, 7.26 µmol). The reaction medium was stirred for 20 hours at RT, concentrated in vacuo and purified by two sequential flash chromatography runs on 0.6 g and 2 g silica gel (DCM/iPrOH gradient elution) to give 3.3 mg of compound from example 37 in the form of white varnish (31%).

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Synthesis of Examples 38 and 39: Glutaryl-Val-PEG24Lys-PABA-C52-benzylamine and NHS-glutaryl-Val-PEG24Lys-PABA-C52-benzylamine ester

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Compound 81: (9H-fluoren-9-yl)methyl-((80S,83S)-80-((4-(hydroxymethyl)phenyl)carbamoyl)-84methyl-74,82-dioxo-2,5,8,11 ,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa75,81-diazapentaoctacontan-83-yl )carbamate.

To a solution of compound 65 (360 mg, 229.8 µmol) in MeCN (70 ml) was added DIEA (77 µl, 456.6 µmol), 4-aminobenzyl alcohol (43 mg, 342.2 µmol), HOAt (36 mg, 264.5 µmol) and HATU (109 mg, 278.1 µmol). The reaction medium was stirred for 2 h at RT, concentrated in vacuo and purified by flash chromatography on 25 g silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 307 mg of compound 81 as a white solid ( 80%).

¹ H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.85 (d, J=7.0 Hz, 3H); 0.88 (d, J=7.0 Hz, 3H); 1.21 to 1.42 (m, 4H); 1.54 to 1.78 (m, 2H); 1.99 (m, 1H); 2.27 (t, J=6.8 Hz, 2H); 3.00 (m, 2H); 3.22(s, 3H); 3.40 to 3.52 (m, 92H); 3.54 (t, J=6.8 Hz, 2H); 3.91 (dd, J=6.9 and 9.0 Hz, 1H); 4.18 to 4.32 (m, 3H); 4.39 (m, 1H); 4.42 (d, J=5.9 Hz, 2H); 5.08 (t, J=5.9 Hz, 1H); 7.22 (d, J=8.9 Hz, 2H); 7.32 (dt, J=1.2 and 7.8 Hz, 2H); 7.41 (m, 3H); 7.53 (d, J=8.9 Hz, 2H); 7.75 (m, 3H); 7.89 (d, J=7.8 Hz, 2H); 8.02 (d, J=8.2 Hz, 1H); 9.92(s, 1H). LCMS (d): ES mass/charge=827; mass/charge=1671 [M+H]+; Rt=2.94 min.

- 138 045708

Compound 82: (9H-fluoren-9-yl)methyl-((80S,83S)-84-methyl-80-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)- 74,82-dioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53, 56,59,62,65, 68,71-tetracosaoxa-75,81-diazapentaoctacontan-83-yl)carbamate.

To a solution of compound 81 (300 mg, 179.4 µmol) in DCM (10 ml) was added bis-(4nitrophenyl)carbonate (450 mg, 1.48 mmol) and DIEA (60 µl, 361.2 µmol). The reaction medium was stirred at room temperature. overnight, then bis-(4-nitrophenyl) carbonate (80 mg, 263.1 µmol) was added and stirring was carried out for 4 hours. At this time, bis-(4-nitrophenyl) carbonate (80 mg, 263.1 µmol) and stirring was carried out overnight. The reaction medium was concentrated in vacuo and purified by flash chromatography on 80 g silica gel (gradient elution DCM/MeOH/H2O) to obtain 325 mg of compound 82 as a white varnish (98%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d, J=7.0 Hz, 3H); 1.20 to 1.45 (m, 4H); 1.58 to 1.76 (m, 2H); 1.99 (m, 1H); 2.27 (t, J=6.8 Hz, 2H); 3.00 (m, 2H); 3.22(s, 3H); 3.40 to 3.70 (m, 94H); 3.91 (dd, J=7.2 and 9.1 Hz, 1H); 4.19 to 4.32 (m, 3H); 4.39 (m, 1H); 5.24(s, 2H); 7.31 (dt, J=1.2 and 7.8 Hz, 2H); 7.40 (m, 4H); 7.57 (d, J=9.2 Hz, 2H); 7.62 (d, J=8.6 Hz, 2H); 7.73 (m, 3H); 7.89 (d, J=7.8 Hz, 2H); 8.07 (d, J=7.8 Hz, 1H); 8.31 (d, J=9.2 Hz, 2H); 10.10(s, 1H). LCMS (d): ES mass/charge=827; mass/charge=1836 [M+H]+; Rt=3.38 min.

Compound 83: 4-((S)-80-((S)-2-amino-3-methylbutanamido)-74-oxo-2,5,8,11,14,17,20,23,26,29, 32,35, 38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azagenoctacontanamido)-benzyl-4-((2R,3R)-3-(( S)-1((3S,10R, 16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4 -dioxa8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzylcarbamate.

To a solution of (E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethylphenyl)oxiranyl]-ethyl}-10-(3chloro-4- methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (the synthesis of which is described in WO2011001052, compound 77, 79 mg , 113.1 µmol) in DCM (5 ml), a solution of compound 82 (325 mg, 177.5 µmol) in DCM (5 ml) and DIEA (63 µl, 365.6 µmol) was added. The reaction medium was stirred for 20 hours at RT, then compound 82 (34 mg, 18.6 µmol) was added and stirring was carried out for 4 hours. The reaction medium was concentrated in vacuo and purified using two sequential flash chromatography operations on 25 g silica gel and 15 g diol-modified silica gel (DCM/MeOH gradient elution) to give 152 mg of crude product, which was purified by reverse phase chromatography on a C ₁₈ 10 µm, 250x40 mm column (MeCN/H2O gradient elution) to give 78 mg Fmoc-protected intermediate containing 16% compound 83 as a white powder (29%).

1H NMR (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.8 Hz, 6H); 0.85 (d, J=7.0 Hz, 3H); 0.88 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.20 to 1.43 (m, 5 H); 1.50 to 1.74 (m, 4H); 1.79 (m, 1H); 1.99 (m, 1H); 2.25 (m, 3H); 2.68(m, 2H); 2.93 to 3.04 (m, 5 H); 3.22(s, 3H); 3.32 (hidden m, 2H); 3.40 to 3.76 (m, 94H); 3.80(s, 3H); 3.87 (d, J=2.0 Hz, 1H); 3.90 (dd, J=7.5 and 9.1 Hz, 1H); 4.19 (d, J=6.5 Hz, 2H); 4.20 to 4.33 (m, 4H); 4.39 (m, 1H); 4.90 (dd, J=3.9 and 9.9 Hz, 1H); 4.93(s, 2H); 5.11(m, 1H); 5.79 (dd, J=1.8 and 15.2 Hz, 1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.36 (m, 10H); 7.41 (m, 3H); 7.48 (d, J=8.6 Hz, 2H); 7.70 to 7.80 (m, 5 H); 7.89 (d, J=7.8 Hz, 2H); 8.08 (d, J=8.0 Hz, 1H); 8.35 (d, J=8.0 Hz, 1H); 10.04 (s, 1H).

To a solution of this intermediate (75 mg, 31.3 µmol) in DMF (5 ml) was added piperidine (25 µl, 253.2 µmol). The reaction medium was stirred for 1 h 30 min at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (gradient elution DCM/MeOH/H2O) to obtain 58 mg of compound 83 as a white varnish (85 %).

1H NMR (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.9 Hz, 9 H); 0.88 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.05 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.15 to 1.50 (m, 7H); 1.52 to 1.73 (m, 4H); 1.80 (m, 1H); 1.93 (m, 1H); 2.27 (m, 3H); 2.69 (m, 2H); 2.95 to 3.04 (m, 6H); 3.23(s, 3H); 3.32 (m, 1H); 3.40 to 3.55 (m, 92H); 3.57 (t, J=6.8 Hz, 2H); 3.80(s, 3H); 3.87 (d, J=2.2 Hz, 1H); 4.20 (d, J=6.5 Hz, 2H); 4.24 (ddd, J=3.9, 8.2 and 12.0 Hz, 1H); 4.42 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.98(s, 2H); 5.10 (m, 1H); 5.79 (d, J=15.8 Hz, 1H); 6.47 (ddd, J=3.8, 6.0 and 11.5 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.31 (m, 8 H); 7.58 (d, J=8.6 Hz, 2H); 7.78 (m, 2H); 8.10 (d, J=8.6 Hz, 1H); 8.36 (d, J=8.1 Hz, 1H); 10.10(s, 1H). LCMS (d): ES mass/charge=725; mass/charge=1087 [M+2N] ²⁺ ; Rt=1.07 min.

Example 38: (80S,83S)-80-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10- (3-chloro-4-methoxybenzyl)-3isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl) oxiran2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20, 23,26,29,32 ,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanoneoctacontane-89-oic acid.

To a solution of compound 83 (58 mg, 26.7 µmol) in DCM (2 ml) was added glutaric anhydride (5.5 mg, 48.2 µmol). The reaction medium was stirred for 3 hours at RT, concentrated in vacuo and purified by two sequential flash chromatography runs on 4 g and 12 g silica gel (gradient elution DCM/MeOH/H2O) to obtain 37 mg of the compound of example 38 in the form of white varnish (61%).

- 139 045708

1H NMR (500 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=6.8 Hz, 6H); 0.93 (d, J=7.0 Hz, 3H); 0.97 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.20 to 1.45 (m, 5 H); 1.51 to 1.75 (m, 6H); 1.80 (m, 1H); 2.00 (m, 1H); 2.13 to 2.33 (m, 7H); 2.69 (m, 2H); 2.95 to 3.05 (m, 5 H); 3.23(s, 3H); 3.32 (m, 1H); 3.40 to 3.58 (m, 94H); 3.80(s, 3H); 3.87 (d, J=2.2 Hz, 1H); 4.15 to 4.38 (m, 4H); 4.32 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.98(s, 2H); 5.10 (m, 1H); 5.79 (d, J=15.2 Hz, 1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.60 (d, J=8.6 Hz, 2H); 7.78 (t, J=6.5 Hz, 1H); 7.90 (wide d, J=8.6 Hz, 2H); 8.20 (m, 1H); 8.38 (d, J=8.6 Hz, 2H); 10.08 (m, 1H); 12.10 (m, 1H). LCMS (d): ES mass/charge=698; mass/charge=2287 [M+H]+;Rt=3.13 min.

Example 39: (8OS,83S)-2,5-dioxopyrrolidin-1-yl-8O-((4-((((4-((2R,3R)-3-((S)-1-((3S ,10R,16S,E)-1O(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexade -13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-83-isopropyl-74, 82,85-trioxo-2,5,8,11, 14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84triazanoneoctacontane-89- oat.

To a solution of the compound of Example 38 (29 mg, 12.7 µmol) in THF (3 ml) was added DSC (3.6 mg, 13.8 µmol) and DIEA (2.1 µl, 12.9 µmol). The reaction medium was stirred for 20 h at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/iPrOH gradient elution) to obtain 6.9 mg of the compound of Example 39 as a clear varnish (23 %).

¹ H NMR (500 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=6.8 Hz, 6H); 0.93 (d, J=7.0 Hz, 3H); 0.97 (d, J=7.0 Hz, 3H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.11(s, 3H); 1.13 to 1.42 (m, 5H); 1.52 to 1.88 (m, 7H); 1.98(m, 1H); 2.28 (m, 5 H); 2.53 to 2.72 (m, 4H); 2.80(s, 4H); 2.96 to 3.05 (m, 5 H); 3.23(s, 3H); 3.32 (m, 1H); 3.60 to 3.65 (m, 94H); 3.80(s, 3H); 3.87 (d, J=2.2 Hz, 1H); 4.15 to 4.28 (m, 4H); 4.33 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.98(s, 2H); 5.10 (m, 1H); 5.79 (d, J=15.8 Hz, 1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8 H); 7.60 (d, J=8.6 Hz, 2H); 7.78 (m, 2H); 7.90 (d, J=8.5 Hz, 1H); 8.07 (m, 2H); 8.37 (d, J=8.5 Hz, 2H); 9.95(s, 1H). LCMS (D): ES mass/charge=698; mass/charge=2384 [M+N] ⁺ ; Rt=3.19 min.

Synthesis of Examples 40 and 41: DBCO-glutaryl-Val-PEG24Lvs-PABA-C52-benzylamine and the corresponding ADC ________________________

Compound 84: 5-[[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]amino]-5oxopentanoic acid.

To a solution of DBCO-amine (50 mg, 180.9 µmol) in DCM (2 ml) was added glutaric anhydride (21.1 mg, 180.9 µmol). The reaction medium was stirred for 2 h at RT, concentrated in vacuo and purified by flash chromatography on 4 g silica gel (DCM/MeOH gradient elution) to give 32.6 mg of compound 84 (45%).

1H NMR (400 MHz, δ in ppm, DMSO- _d6 ): 1.59 (m, 2H); 1.81(m, 1H); 1.94 (t, J=7.4 Hz, 2H); 2.11 (t, J=7.4 Hz, 2H); 2.40 (m, 1H); 2.91(m, 1H); 3.09 (m, 1H); 3.62 (d, J=14.2 Hz, 1H); 5.13 (d, J=14.2 Hz, 1H); 7.38 to 7.53 (m, 6H); 7.56 to 7.67 (m, 3H); 12.05 (wide m, 1H).

Example 40: DBCO-glugaryl-Val-PEO24Lys-PABA-C52-benzylamine.

To a solution of compound 83 (22 mg, 10.1 µmol) in DCM (2 ml) was added compound 84 (5.14 mg, 13.2 µmol), EDC (1.8 µl, 10.1 µmol) and HOBt (1 .4 mg, 10.1 µmol). Stir the reaction medium

- 140 045708 li at k.t. overnight, concentrated in vacuo and purified by reverse phase chromatography on a _C18 -modified 5 µm, 150 mm x 30 mm column (gradient elution MeCN/0.2 N ammonium acetate pH 5.6) to give 9.2 mg of the compound from Example 40 as a white powder (36%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.78 (d, J=6.8 Hz, 6H); 0.80 to 0.90 (m, 6H); 1.00(s, 3H); 1.05 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.20 to 1.41 (m, 5 H); 1.52 to 1.72 (m, 6H); 1.75 to 2.00 (m, 5 H); 2.09 (m, 2H); 2.27 (m, 3H); 2.40 (m, 1H); 2.68(m, 2H); 2.88 to 3.12 (m, 7H); 3.23(s, 3H); 3.28 to 3.58 (partially hidden m, 95 H); 3.60 (d, J=11.0 Hz, 1H); 3.80(s, 3H); 3.88 (d, J=2.0 Hz, 1H); 4.11 to 4.28 (m, 4H); 4.33 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97(s, 2H); 5.04 (d, J=11.0 Hz, 1H); 5.11(m, 1H); 5.80 (d, J=15.2 Hz, 1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.31 (m, 6H); 7.32 to 7.50 (m, 6H); 7.53 to 7.70 (m, 4H); 7.80 (m, 3H); 8.08 (d, J=8.9 Hz, 1H); 8.36 (d, J=8.4 Hz, 1H); 9.95(s, 1H). LCMS (D): ES mass/charge=849 [M+3H] ³⁺ ; mass/charge=1273 [M+2N] ²⁺ ; Rt=5.29 min.

Example 41: hu2H11_R35-74-Ex40.

194 mg of hu2H11_R35-74 in buffer A was reacted with 627 μl of a solution of 2,5-dioxopyrrolidin-1yl-4-azidobutanoate (CAS number [943858-70-6]) at 22.0 mM in DMA (10 equiv.) so so that the final antibody concentration is 10.5 mg/ml and the percentage of DMA in buffer A is 5%. After stirring for 2 hours at room temperature. the mixture was purified by gel filtration using a Sephadex™ G25 matrix (Hiprep 26/10 desalting column, GEHealthcare) pre-equilibrated in buffer A. Fractions containing monomeric modified antibody were pooled and filtered through a Steriflip® filter block (Durapore PVDF membrane ® 0.22 µm, Millipore) yielding 173 mg of modified antibody at a concentration of 5.75 mg/ml with a linker to antibody ratio of 5.2 (HRMS), monomeric purity of 97% and overall yield of 89%.

The general method described previously was used to prepare the compound of Example 41. 45 mg of modified hu2H11_R35-74 was reacted with 604 μl of a 6.89 mM solution of the compound of Example 40 in DMA (14 eq.) for 4 hours 30 minutes, then 604 μl of a 10 mM solution of the compound of Example 40 in DMA (14 eq.) was added and stirring was carried out overnight at room temperature. After purification on Superdex 200 pg in buffer B with pH 6.5 + 20% NMP, concentration on Amicon Ultra-15, gel filtration on PD-10 in buffer B with pH 6.5 + 5% NMP and filtration on PVDF- A 0.22 μm filter provided 28 mg of the compound from Example 41 as a colorless, clear solution at a concentration of 1.6 mg/mL with a DAR of 3.44 (HRMS), 99.6% monomeric purity, and 62% yield.

SEC-HRMS: mass/charge=152041 (D1); mass/charge=154678 (D2); mass/charge= 157332 (D3); mass/charge=159992 (D4); mass/charge=162648 (D5); mass/charge=165303 (D6); mass/charge=168004 (D7).

Synthesis of Example 42: Sulfo-Val-PEG4Lys-C52-benzylamine

Compound 85: Sodium 2-(4-((3-(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4oxobutanamido)ethane-1-sulfonate.

To a solution of compound 13 (0.958 g, 2.68 mmol) in DMF (5 ml) was added NHS (390.3 mg, 3.32 mmol) and EDC (604.2 μl, 3.4 mmol). The reaction medium was stirred for 2 hours at room temperature, then stored for 2 days at -20°C. After returning to room temperature the reaction medium was stirred for 2 h at room temperature, then EDC (60 μl, 340 μmol) and a solution of NHS (20 mg, 170.3 μmol) in DMF (3 ml) were added. The reaction medium was stirred at room temperature. overnight, then a solution of compound 73 (1.96 g, 13.3 mmol) was added

- 141 045708 in H2O (10 ml) and sodium bicarbonate (225.2 mg, 2.68 mmol). The reaction medium was stirred for 1 h at room temperature, diluted with H2O (6 ml), acidified to pH 3 with Amberlite IR-120 (H) (CAS number [78922-04-4]), filtered, concentrated in vacuo and purified by flash chromatography on silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 270 mg of compound 85 as a colorless foam (21%).

1H NMR (400 MHz, δ in ppm, DMSO-d ₆ ): 50:50 mixture of conformers; 1.39 (s, 4.5 H); 1.40 (s, 4.5 H); 2.27 (t, J=7.0 Hz, 4H); 2.38 (t, J=7.0 Hz, 2H); 3.53 (m, 3H); 2.68 (t, J=7.0 Hz, 1H); 3.25 to 3.61 (m, 6H); 4.55 to 4.58 (m, 2H); 5.18 to 5.36 (m, 2H); 5.90 (m, 1H); 7.70 (t, J=6.8 Hz, 1H); 10.53 (wide m, 1H). LCMS (A): ES mass/charge=409; mass/charge=463 [M-N] ^- ; mass/charge=465 [M+N]+; Rt=1.13 min.

Compound 86: Sodium 2-(4-((3-(allyloxy)-3-oxopropyl)(2-carboxyethyl)amino)-4-oxobutanamido)ethane-1-sulfonate.

To a solution of compound 85 (60 mg, 123.3 µmol) in DCM (4 ml) was added TFA (100 µl, 1.25 mmol). The reaction medium was stirred for 2 hours at RT, then TFA (100 µL, 1.25 mmol) was added and stirring was carried out for 2 days at RT. TFA (50 µl, 0.625 mmol) was then added, the reaction stirred at RT, concentrated in vacuo and co-evaporated with toluene (3x) to give 60 mg of compound 86 as a clear varnish (qt).

Compound 87: 2-(4-((3-(allyloxy)-3-oxopropyl)(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3oxopropyl)amino)-4-oxobutanamido)ethane-1 -sodium sulfonate

To a solution of compound 86 (52.94 mg, 123 µmol) in DCM (5 ml) was added EDAC (26.0 µl, 147.6 µmol), NHS (14.75 mg, 123 µmol) and DMF (0.5 ml ). The reaction mixture was stirred for 4 hours at RT, then EDC (10 µL, 45.7 µmol) was added and stirring was carried out at RT. during the night. The reaction medium was stored at -20°C and was used, for example, for the synthesis of compound 88.

Compound 88: (20S,23S)-28-(2-allyloxy-2-carboxyethyl)-20-((4-((2R,3R)-3-((S)-1-((3S,10R,

16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13 -en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-23-isopropyl-14,22,25,29,32-pentaoxo-2,5,8,11-tetraoxa-15,21, Sodium 24,28,33-pentaazapentatriacontane-35-sulfonate.

To a solution of compound 62 (50 mg, 43.7 μmol) in DCM (4 ml) under Ar atmosphere was added dropwise the reaction medium containing compound 87 (23.06 mg, 43.7 μmol). The reaction medium was stirred at room temperature. for 15 min, concentrated in vacuo and purified by flash chromatography on 15 g silica gel (gradient elution DMC/MeOH/H ₂ O) to obtain 26 mg of compound 88 as a white varnish (38%).

Example 42: (20S,23S)-28-(2-carboxyethyl)-20-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)- 10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en16-yl)ethyl) oxiran-2-yl)benzyl)carbamoyl)-23-isopropyl-14,22,25,29,32-pentaoxo-2,5,8,11-tetraoxa15,21,24,28,33-pentaazapentatriacontane-35-sulfonate sodium

Dimedone (2.57 mg, 18.0 µmol) and tetrakis(triphenylphosphine)palladium(0) (5.3 mg, 4.5 µmol) were added to a solution of compound 88 (14 mg, 9 µmol) in THF. The reaction medium was stirred for 1 hour at room temperature, then dimedone (2.57 mg, 18.0 µmol) and tetrakis(triphenylphosphine)palladium(0) (5.3 mg, 4.5 µmol) were added and stirring was carried out at k.t. during the night. At this time, dimedone (2.57 mg, 18.0 µmol) and tetrakis(triphenylphosphine)palladium(0) (5.3 mg, 4.5 µmol) were added. The reaction medium was stirred for 4 hours at RT, filtered, concentrated in vacuo and purified by flash chromatography on 4 g of silica gel (gradient elution DCM/MeOH/H ₂ O) to obtain 7 mg of the compound of example 42 as white varnish (51%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 50:50 mixture of conformers; 0.78 (d, J=6.8 Hz, 6H); 0.81 (d, J=6.8 Hz, 6H); 0.86 (m, 6H); 1.00(s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.11(s, 3H); 1.15 to 1.41 (m, 5H); 1.53 to 1.70 (m, 4H); 1.80 (m, 1H); 2.00 (m, 1H); 2.11 to 2.58 (partially hidden m, 13 H); 2.70 (m, 2h); 2.92 to 3.05 (m, 5 H); 3.23(s, 3h); 3.25 to 3.58 (partially hidden m, 23 H); 3.81(s, 3H); 3.88 (d, J=2.0 Hz, 1H); 4.10 to 4.32 (m, 5 H); 4.91 (dd, J=3.8 and 9.9 Hz, 1H); 5.10 (m, 1H); 5.80 (d, J=15.2 Hz, 1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.06 (d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 6H); 7.67 to 7.93 (m, 3H); 8.18 to 8.60 (m, 4H). LCMS (E): ES mass/charge=747.5 [M+2H] ²⁺ ; mass/charge=1491 [M-N] ^- ; mass/charge=1493 [M+H]+; Rt=4.74 min.

- 142 045708

Synthesis of Example 43: Sulfo-Val-glucose-Gln-C52-benzylamine

example 43

Compound 89: (6S,9S)-14-(3-(allyloxy)-3-oxopropyl)-6-((4-((2R,3R)-3-((S)-1-((3S,10R , 16S,E)-10-(3chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13 - en16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-9-isopropyl-3,8,11,15,18-pentaoxo-1 -((2R,3 S,4S,5R,6R)-3 ,4,5-tpuhydroxy-6-methoxytetrahydro-2H-pyran-2-yl)-2,7,10,14,19-pentaazagenicosan-21-sodium sulfonate.

To a solution of compound 50 (55 mg, 49.9 µmol) in DMF (3 ml) was added a solution of compound 87 in DMF (1.66 ml at 30 mmol, 49.9 µmol). The reaction medium was stirred for 18 h at RT, filtered through 0.45 µm, washed twice with DMF (0.8 ml) and purified by reverse phase chromatography on a C ₁₈ 5 µm, 30x150 mm (gradient) column eluting with MeCN/0.2N ammonium acetate pH 5.6) to give 19.8 mg of compound 89 as a white solid (26%).

1H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): mixture of conformers 60:40; 0.78 (d, J=6.8 Hz, 6H); 0.81(d,

J=6.8 Hz, 6H); 0.81 to 0.87 (m, 6H); 0.99(s, 3H); 1.03 (d, J=7.1 Hz, 3H); 1.12(s, 3H); 1.30 (m, 1H); 1.52 to

1.62(m, 2H); 1.80 (m, 3H); 1.85 to 2.00 (m, 3H); 2.10 to 2.30 (m, 5 H); 2.35 to 2.48 (m, 2H); 2.53 (m, 6H);

2.68(m, 2H); 2.88 to 3.15 (m, 9H); 3.28(m, 3H); 3.38(m, 3H); 3.40 to 3.58 (m, 5 H); 3.80(s, 3H); 3.87(d)

J=2.3 Hz, 1H); 4.01 (d, J=8.0 Hz, 1H); 4.10 to 4.24 (m, 6H); 4.55 (m, 1.2H); 4.57 (0.8 H); 4.91 (dd, J=3.8 and 9.9 Hz, 1H); 4.95 (d, J=5.0 Hz, 1H); 5.01 (d, J=5.0 Hz, 1H); 5.06 (d, J=5.0 Hz, 1H); 5.10 (m, 1H); 5.18 to 5.35 (m, 2H); 5.80 (dd, J=2.0 and 15.2 Hz, 1H); 5.92(m, 1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.06 (d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.23 (m, 5 H); 7.29 (d, J=2.4 Hz, 1H); 7.69 (t, J=6.8 Hz, 1H); 7.88 (t, J=6.8 Hz, 1H); 7.98 (d, J=9.0 Hz, 0.4H); 8.04 (d, J=7.0 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6 H); 8.14 (d, J=7.0 Hz, 0.6 H); 8.32 (t, J=7.0 Hz, 1H); 8.38 (d, J=8.0 Hz, 1H). LCMS (A): ES mass/charge=746.5 [M+2H] ²⁺ ; mass/charge=1490 [MN] ^- ; mass/charge=1492 [M+N] ⁺ ; Rt=1.4 min.

Example 43: (6S,9S)-14-(2-carboxyethyl)-6-((4-((2R,3R)-3-((S)-1-((3S,1OR,16S,E)- 10-(3-chloro-4methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en16-yl)ethyl) oxiran-2-yl)benzyl)carbamoyl)-9-isopropyl-3,8,11,15,18-pentaoxo-1 -((2R,3S,4S,5R,6R)3,4,5-trihydroxy-6 -methoxytetrahydro-2H-pyran-2-yl)-2,7,10,14,19-pentaazagenicosan-21-sodium sulfonate.

To a solution of compound 89 (19 mg, 12.6 µmol) in DMF (1 ml) was added barbituric acid (5.94 mg, 37.7 µmol) and a solution of tetrakis-(triphenylphosphine) palladium(0) (0.74 mg, 0.63 µmol) in DMF (0.5 ml). The reaction medium was stirred for 3 hours at room temperature, then barbituric acid was added

- 143 045708 tu (5.94 mg, 37.7 µmol) and a solution of tetrakis(triphenylphosphine)palladium(0) (0.74 mg, 0.63 µmol) in DMF (0.5 ml). The reaction medium was stirred at room temperature. overnight, diluted with DMF, filtered through 0.45 µm and purified by two successive reverse phase chromatography runs on a C ₁₈ 5 µm, 30x150 mm column (gradient elution MeCN/0.2N ammonium acetate pH 5, 6) to obtain 4.7 mg of the compound from Example 43 as a white solid (25%).

¹ H NMR (500 MHz, δ in ppm, DMSO-d ₆ ): 0.80 (m, 6H); 0.85 (m, 6H); 0.99(s, 3H); 1.03 (d, J=7.3 Hz, 3H); 1.11(s, 3H); 1.33 (m, 1H); 1.58 (m, 2H); 1.78 (m, 2H); 1.90 (m, 2H); 2.03 to 2.55 (partially hidden m, 11H); 2.70 (m, 2H); 2.90 to 3.58 (partially hidden m, 19H); 3.18(s, 3H); 3.81(s, 3H); 3.88(s, 1H); 4.00 to 4.40 (m, 8 H); 4.90 (m, 1H); 4.99 (m, IH); 5.80 (d, J=15.2 Hz, 1H); 6.46 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.22 (m, 4H); 7.29 (d, J=2.4 Hz, 1H); 7.31 (m, 1H); 8.30 to 8.70 (m, 5H). LCMS (A): ES mass/charge=1448 [M-N] ^- ; mass/charge=1450 [M+N]+; Rt=1.58 min.

For illustrative purposes, two conjugates derived from WO2011/001052, ADC1 and ADC2, depicted below, and one conjugate described in PCT/EP2016/076603, ADC3, depicted below, were also tested.

Physicochemical results.

Conjugates of formula (V) were subjected to stability studies assessing aggregation (as judged by monomeric purity) and loss of DAR upon removal of the organic co-solvent used in the formulation and under accelerated stress conditions (heating for 10 days at 40° C.).

Stability in the absence of NMP.

ADC, which was stored in 10 mM phosphate, 140 mM NaCl pH 6.5, 5% NMP, filtered on a SEC column to remove NMP, formulated in 10 mM phosphate, 70 mM NaCl, 5% sucrose pH 6.5 and analyzed using HRMS and SEC HPLC to determine DAR and monomer purity.

- 144 045708

Table I 5% NMP without NMP ADAR (%) A% monomer (%) protein extraction DAR %monomer DAR %monomer ADC1 4.1 99 3.25 88.7 -21% -10% quantity ADC3 3.5 99.7 3.5 99.5 0% -0.2% And. O. Note 3 3.4 99.8 3.2 99.7 -6% -0.1% 88% Note 6 4.05 99 3.4 99 -16% 0% 75% Note 16* 2.5 99.8 2.3 99.8 -8% 0% 73% Note 19 2.8 97.9 2.4 97.8 -14% -0.1% 72% Note 23* 2.9 100 2.4 100 -17% 0% 76% Note 26 3.9 100 3.9 100 0% 0% And. O. Note 29 4 98.4 3.9 100 -2.5% 0% 76% Note 32 2.8 95.1 2.9 94.1 0 -eleven% 82%

* After 10 months at 4°C, significant aggregation was observed for approx. 16 and approx. 23 (-22% and -19%, respectively, in terms of monomer purity), so both batches were purified by gel filtration on Superdex 200 pg before stability studies were performed.

Stability after 10 days at 40°C in phosphate buffer.

ADC in 10 mM phosphate, 70 mM NaCl, 5% sucrose pH 6.5 was heated at 40°C for 10 days and analyzed by HRMS and SEC HPLC to determine DAR and monomeric purity.

Table II in to After 10 days at 40°C ADAR (%) A% monomer (%) protein extraction DAR %monomer DAR %monomer ADC1 3.25 88.7 3.0 85.7% -8% -3% 56% ADC3 3.5 99.5 3 85 -14% -15% 46% Note 3 3.2 99.7 2.9 92 -9% -8% 96% Note 6 3.4 99 3.3 97 -3% -2% 91% Note 16 2.3 99.8 1.8 93.8 -22% -6% 89% Note 19 2.4 97.8 1.8 92.9 -25% -5% quantity Note 23 2.4 100 2.4 91.6 0% -8% 91% Note 26 3.9 100 3.6 100 -8% 0% And. O. Note 29 3.9 100 3.9 98.7 0% -1% 97% Note 32 2.9 94.1 2.7 89.8 -7% -5% 95% Note 41 3.3 97.9 3.05 75.3 -7.5% -23% quantity

Evaluation of drug release after treatment of conjugates of formula (V) with cathepsin B.

Drug release by cathepsin B was assessed in vitro by incubating ADC (1.5 μg/ml) in the presence of human cathepsin B (Calbiochem no. 219362, 40 μg/ml) in acetate buffer pH 5 at 37°C for 24 h and drug detection using UPLC-MSSIR-DAD-ELSD. The slope angle was determined based on kinetic parameters over 1 hour, half-life was determined based on kinetic parameters over 24 hours.

- 145 045708

Table III inclination angle ti/ ₂ (h)

ADC1 34.3 2 ADC2 14.1 15 Note 16 12.8 >24 Note 19 22.3 3.5 Note 23 19.3 6 Note 32 33.5 2

Conjugates of formula (V) based on cryptophycin were cleaved by human cathepsin B with kinetics similar to those of control ADCs.

Pharmacological results.

The conjugates of formula (V) were subjected to pharmacological tests to determine their antitumor effect.

Evaluation of inhibition of proliferation of the MDA-MB-231 cell line by conjugates of formula (V).

MDA-MB-231 cells in their exponential growth phase were trypsinized and resuspended in their culture medium (DMEM/F12 Gibco no. 21331, 10% FCS Gibco no. 10500-056, 2 nM glutamine Gibco no. 25030). The cell suspension was seeded into 96-well Cytostar culture plates (GE Healthcare Europe, no. RPNQ0163) throughout the culture medium containing serum at a density of 5000 cells/well. After incubation for 4 hours, serial dilutions of ADC were added to the wells in concentrations decreasing from 10 ^-7 to 10 ^-7 M (in triplicate for each concentration). Cells were cultured at 37°C in an atmosphere containing 5% CO ₂ in the presence of ADC for 3 days. On day 4, 10 μl of ¹⁴ C-thymidine solution (0.1 μCi/well, Perkin Elmer #NEC56825000) was added to each well. ¹⁴ C-thymidine incorporation was measured 96 h after the start of the experiment using a Microbeta radioactivity counter (Perkin Elmer). Data were expressed as percentage survival by determining the ratio of the reduced amount obtained for ADC-treated cells to the amount obtained for control well cells (treated with culture medium only). In some experiments, unconjugated antibody was added to the wells at a concentration of 1 μM at the beginning of the experiment and proliferation inhibition was measured as described previously.

Table IV 1C ₅₀ (pM), MDA-MB-231 Coefficient selectivity ADC only in the presence of unconjugated antibody ADC1 42 4571 109 ADC2 46 9377 204 Note 3 19 5602 295 Note 6 22 7417 337 Note 16 39 13908 357 Note 19 29 18243 629 Note 23 37 9022 243 Note 26 27 33689 1248 Note 29 67 > 100000 > 1493 Note 32 25 7953 318 Note 35 25 11272 451 Note 41 34 5043 148

Conjugates of formula (V) based on cryptophycin, as well as ADC1 and ADC2, were found to inhibit the proliferation of the MDA-MB-231 cell line with an IC ₅₀ ranging from 19 pM to 67 pM and a selectivity ratio of ADC only and ADC+unconjugated antibody from 109 to > 1493.

Determination of MTD of conjugates of formula (V) after a single intravenous administration in SCID mice.

MTD was defined as the maximum dose that does not cause a loss of 15% body weight within 3

- 146045708 consecutive days for an individual mouse or loss of 20% body weight in 1 day or death. It was assessed following a single intravenous (i.v.) bolus injection into 3 female SCID mice and over a period of 28 days post-treatment.

The cryptophycin conjugates of formula (V) tested exhibited MTD in SCID mice ranging from 30 mg/kg to >40 mg/kg.

Evaluation of in vivo antitumor activity of conjugates of formula (V) against MDAMB-231 in SCID mice after a single intravenous administration.

In vivo antitumor activity was assessed at 3 dose levels against measurable MDA-MB-231 mammary xenografts implanted subcutaneously in female SCID mice. Control groups were left untreated. Conjugates were administered via a single intravenous bolus injection, with the day of treatment indicated on each graph by an arrow (▼).

To evaluate the antitumor activity of the conjugates, animals were weighed twice a week and tumors were measured with a caliper twice a week. The body weight of the animals included the mass of tumors. Tumor volume was calculated using the formula: mass (mm3) = [length (mm) x width (mm) ² ]/2. Primary efficacy endpoints were ΔT/ΔC, mean percent regression, partial and complete regression (PR and CR). Changes in tumor volume for each treatment group (T) and control group (C) were calculated for each tumor by subtracting the tumor volume on the first treatment day (staging day) from the tumor volume on the specified observation day. The mean ΔΤ value was calculated for the treatment group, and the mean ΔC value was calculated for the control group. Then the ratio ΔΤ/ΔΟ was calculated and expressed as a percentage: ΔT/ΔС=(delta T/delta C)x100.

The percentage of tumor regression was defined as the % reduction in tumor volume in the treatment group on the designated observation day (t) compared with its volume on the first day of the first treatment (t0). The % regression was calculated at a specific time point and for each animal. The mean % regression for the group was then calculated. % regression (at time t) = ((volume _t0 - volume _t )/volume _t0 )x100. Regressions were defined as partial (PR) if the tumor volume was reduced to 50% of the tumor volume at the beginning of treatment, and as complete (CR) when the tumor volume could not be measured (0 mm ³ ). Tumor-free survivors (TFS) were defined as animals with undetectable tumors at the end of the study (>100 days after last treatment).

Evaluation of antitumor activity in vivo approx. 6 relative to MDA-MB-231 in SCID mice after a single intravenous administration.

- 147 045708 niyu k

Evaluation of antitumor activity in vivo approx. 16, approx. 19, approx. 23 and approx. 32 relative to MDA-MB-231 in SCID mice after a single intravenous dose of 2.5 mg/kg.

Table VII Dose (mg/kg) Average AT/AC value in % (D59) Average % regression Regressions PR CR TFS Control - - - - - - ADC2 2.5 <0 14% - - - Note 16 2.5 <0 92% 6 3 - Note 19 2.5 <0 100% 6 5 2 Note 23 2.5 <0 100% 6 6 1 Note 32 2.5 <0 100% 6 6 2 and antitumor activity in vivo approx. 16, approx. 19, approx. 23 and approx. 32 -MB-231 in SCID mice after a single intravenous dose of 1.25 Table VIII Dose (mg/kg) Average AT/AC value in % (D50) Average % regression Regressions PR CR TFS Control - - - - - - ADC2 1.25 14 - - - - Note 16 1.25 7 - - - - Note 19 1.25 1 - 1 1 - Note 23 1.25 0 - 1 - - Note 32 1.25 9 - 2 1 -

in relation toEvaluation of antitumor activity in vivo approx. 26 in relation to MDA-MB-231 for SCID-

mice after a single intravenous injection. Dose (mg/kg) Average AT/AC value in % (D36) Average % regression Regressions Table IX PR CR TFS Control - - - - - - ADC2 2.5 8 - 1 - 1 Note 26 5 <0 100% 6 6 2 2.5 <0 100% 6 4 1 1.25 10 - 1 - -

Evaluation of antitumor activity in vivo approx. 29 relative to MDA-MB-231 in SCID mice after a single intravenous administration.

Table X Dose (mg/kg) Average AT/AC value in % (D27) Average % regression Regressions PR CR TFS Control - - - - - - 4 <0 100% 6 6 6 Note 29 2 <0 100% 6 6 4 1 0 — — — —

- 148 045708

Evaluation of antitumor activity in vivo approx. 35 relative to MDA-MB-231 in SCID mice after a single intravenous administration.

Table XI Dose (mg/kg) Average AT/AC value in % (D27) Average % regression Regressions PR CR TFS Control - - - - - - 4 <0 100% 6 6 4 Note 35 2 <0 100% 6 4 1 1 3 — — — —

Evaluation of antitumor activity in vivo approx. 41 relative to MDA-MB-231 v SCID mice after a single intravenous injection.

All tested cryptophycin ADCs of the present invention, as well as ADC2, exhibited antitumor activity at doses ranging from 1 mg/kg to 5 mg/kg.

Therefore, it is obvious that the compounds (V) of the present invention have anticancer activity.

Accordingly, in other aspects, the present invention also relates to the use of cryptophycin-based conjugates of formula (V) as anticancer agents.

Accordingly, in other aspects, the present invention also relates to the use of cryptophycin-based conjugates of formula (V) for the preparation of a medicament for the treatment of cancer, for example breast cancer.

According to other aspects, the present invention also provides conjugates of formula (V) in accordance with the present invention for use in the treatment of cancer.

According to other aspects, the present invention also provides drugs that contain at least one conjugate of formula (V).

These drugs are used therapeutically, especially in the treatment of cancer, such as breast cancer.

According to other aspects, the present invention relates to pharmaceutical compositions containing as an active substance the conjugate of formula (V) in accordance with the present invention. These pharmaceutical compositions contain an effective dose of at least one conjugate of formula (V) according to the present invention and also at least one pharmaceutically acceptable excipient.

Thus, in accordance with another aspect, the present invention relates to a pharmaceutical composition containing at least one conjugate of formula (V) in accordance with the present invention, as well as at least one pharmaceutically acceptable excipient.

Said excipients are selected according to the pharmaceutical form and the required route of administration from the usual excipients known to the person skilled in the art.

According to other aspects, the present invention also provides a method of treating the above pathologies, such as cancer, for example, breast cancer, which comprises administering to a patient an effective dose of a conjugate of formula (V) in accordance with the present invention.

Claims (22)

CLAIM 1. Compound of formula (I) RCG1-LP _(IX where RCG1 is a reactive chemical group that is reactive with a chemical group present on the antibody; P is -OH or activated O, which is selected from the group consisting of wherein each Gl is independently selected from the group consisting of halogen and nitro; and M is Na+, K+ or Cs+; L is a linker of formula (II) ABOUT _; A : L2 (AA)W L1 _nn where L1 is represented by formula (III) y represents 0; z represents 0; J ₁ represents CA ₁ or N; J2 represents CA ₁ or N; J3 is CA ₁ or N; J ₄ represents CA ₁ or N; A ₁ represents hydrogen or (C ₁ -C6)alkyl; A2 represents hydrogen or (C ₁ -C6)alkyl; A3 represents hydrogen or (C ₁ -C6)alkyl; A4 represents hydrogen or (C1-C6)alkyl and ALK is a (C ₁ -C ₁₂ )alkylene group, (AA) _w is a sequence of w amino acids linked to each other through peptide bonds, where the amino acids, (AA) _w , are independently selected from the group consisting of an unsubstituted amino acid, ( AAns), and substituted amino acid, (AA) _s ; w represents 2; each (AA _ns ) is a natural or unnatural amino acid selected from the group consisting of alanine, γ-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine, asparagine, aspartic acid, citrulline, cysteine, α,α-dimethyl-γ-aminobutyric acid, 3,3-dimethyl-γ-aminobutyric acid, glutamine, glutamic acid, glycine, homocysteine, selenocysteine, homoselenocysteine, histidine, isoleucine, leucine, lysine, ε-acetyllysine, methionine , ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; each (AAs) is independently represented by formula (VI) ^and x ( _VI ) where T represents trivalent (C ₁ -C ₈ )alkyl; U represents -C(O)-, -C(O)NH-, -C(O)N(alkyl)-, -C(O)O-, -NH-, -N(alkyl)-, -NHC (O)-, -N(alkyl)С(О)-, -NHC(NH)NH-, -NHC(O)NH-, -NHS(O) ₂ -, -N(alkyl)S(О) ₂ -, -O-, -OS(O)-, -OS(O)O-, -OR(O)-, -P(O)(OH)-, -P(O)(OH)O-, - OP(O)(OH)O-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ NH-, -S(O) ₂ N(alkyl)- or -Se-; And A9 represents a straight or branched, saturated or unsaturated, optionally substituted C ₁ -C ₁₆₀ hydrocarbon chain, with at least one methylene unit regardless - 150 045708 simo replaced by -NH-C(=O)-, -C(=O)NH-, -CH(OH)-, -CH(SO ₃ H)-, -CH(O-alkyl)- or - C(=O)-, or heterocycloalkyl group containing from one to three heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom, and from 2 to 8 carbon atoms, where the heterocycloalkyl group is replaced by at least one substituent, identical or different , selected from -OH and -O-alkyl, it should be understood that each A9 containing the SO3H functional group may be in salt forms; And L2 represents a (C1-C6)alkyl group, a C(=O)-( _C1 - _C6 )alkyl group, a (C1- _C6 )alkylNA ₇ -( _C1 - _C6 )alkyl group; A ₇ is a straight or branched, saturated or unsaturated, optionally substituted C1-C ₁₆₀ hydrocarbon chain, with at least one methylene unit independently replaced by -NHC(=O)-, -C(=O)NH-, -CH( SO ₃ H)-, -O- or -C(=O)-, it should be understood that each A7 containing the SO3H functional group may be in salt forms; And RCG1 is selected from the following list: (i) a reactive group R _a Z _a -C(=O), for which Z _a represents a single bond, -O- or -NH and Ra represents a hydrogen atom, a (C1-C ₆ )alkyl group, (C ₃ -C ₇ )cycloalkyl group, (C ₂ -C ₆ )alkenyl group, aryl group containing from 6 to 10 carbon atoms, heteroaryl group containing from one to five heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom, and from 2 to 10 carbon atoms or a (C ₃ -C ₇ )heterocycloalkyl group containing from one to three heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom, where the aryl group, heteroaryl group and/or (C ₃ -C ₇ The heterocycloalkyl group is optionally substituted with 1 to 5 atoms/groups selected from a halogen atom, a (C1- _C6 )alkyl group, a (C1- _C6 )alkoxy group, a hydroxyl group, an oxo group, a nitro group and a cyano group; f (ii) one of the following reactive groups: maleimido group, haloacetamido-^21 C1, Br or Ig group ⁰ , wherein _R21 represents a hydrogen atom or a (C1- _C6 )alkyl group; Cl-; N3-; HO-; HS-; activated disulfide; H2N-; IICW- group or activated C=C group; O-alkylhydroxylamine or Pictet-Spengler reaction substrate; provided that: (1) if P is -OH, then x is 0, y is 0 and z is 0; and (2) if P is an activated group O, then x is 1, y is 0 and z is 0; (3) at least one amino acid (AA)W is a substituted amino acid (AA) _{s of} formula (VI); and (4) the substituted amino acid (AA) _s of formula (VI) is not selected from the group consisting of alanine, γ-aminobutyric acid, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine, asparagine , aspartic acid, citrulline, cysteine, α,α-dimethyl-γ-aminobutyric acid, 3,3-dimethyl-γ-aminobutyric acid, glutamine, glutamic acid, glycine, homocysteine, selenocysteine, homoselenocysteine, histidine, isoleucine, leucine, lysine , ε-acetyllysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. 2. A compound of formula (I) according to claim 1, wherein the substituted amino acids AAs are characterized by formula (VI) as defined in claim 1, wherein T represents 'g or the group U represents -NH-C(=O)- or -C(=O)NH-; A ₉ represents -[(CH ₂ ) ₂ -O] _b -CH ₃ where b is an integer ranging from 1 to 50. - 151 045708 3. A compound of formula (I) according to claim 1, wherein the sequence (AAs)w containing at least one substituted amino acid AAs is selected from the list - 152 045708 4. The compound of formula (I) according to claim 1, wherein (AA)w contains at least one substituted amino acid AAs, and L2 represents a (C ₁ -C6)alkyl group; C(=O)-(C ₁ -C ₆ )alkyl group or (C ₁ -C ₆ )alkyl-NA ₇ -(C ₁ -C ₆ )alkyl group, in which A ₇ represents a -C(=O) group )-(CH2)2-C(=O)-NH-(CH2)2-SO3H; it should be understood that each A ₇ containing a SO ₃ H functional group may be in salt forms. 5. A compound of formula (I) according to claim 1, where A ₇ represents a group -C(=O)-[(CH ₂ ) ₂ -O] ₄ -CH ₃ ; group -C(=O)-[(CH2)2-O]7-CH3; group -C(=O)-[(CH2)2-O]24-CH3; group -C(=O)-(CH2)2-C(=O)-NH-(CH2)2SO3H; group -C(=O)-(CH2)2-C(=O)-NH-[(CH2)2-O-]4-(CH2)2-C(=O)-NH-(CH2)2- SO3H; it should be understood that each A ₇ containing a SO ₃ H functional group may be in salt forms. - 153 045708 6. The compound of formula (I) according to claim 1, wherein L2 represents 7. The compound of formula (I) according to claim 1, where L2 contains A ₇ representing the group C(=O)-[(CH2)2-O]4-CH3; group C(=O)-[(CH2)2-O]7-CH3; group C(=O)-[(CH2)2-O]24-CH3; group C(=O)-(CH ₂ ) ₂ -C(=O)-NH-(CH ₂ ) ₂ -SO ₃ H; group C(=O)-(CH ₂ ) ₂ -C(=O)-NH-[(CH ₂ ) ₂ -O] ₄ -(CH ₂ ) ₂ -C(=O)-NH(CH ₂ ) ₂ -SO ₃ H; it should be understood that each A ₇ containing a SO ₃ H functional group may be in salt forms. 8. A compound of formula (I) according to any one of claims 1 to 7, wherein said linker L is selected from the following list: - 154 045708 - 155 045708 - 156 045708 HE 9. A compound of formula (I) according to any one of claims 1 to 8, where RCG1 is selected from the following list: (i) a group R _a Z _a -C(=O), in which Z _a represents -O-, and Ra represents hydrogen, (C ₁ -C6)alkyl, (C2-C6)alkenyl, (C3-C ₇ )cycloalkyl, (C3- _C7 )heterocycloalkyl containing from one to three heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom, aryl containing from 6 to 10 carbon atoms, or heteroaryl containing from one to five heteroatoms, selected from a nitrogen atom, a sulfur atom and an oxygen atom, and from 2 to 10 carbon atoms, wherein (C3- _C7 )heterocycloalkyl, aryl or heteroaryl are unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group , consisting of halogen, CN, NO2, alkyl, OH, O(alkyl) and =O; - 157 045708 (ii) one of the following groups: COOH 10. Payload with cryptophycin of formula (IV) where R1 represents a (C1-C6)alkyl group; R2 and _R3 independently represent a hydrogen atom or a (C1-C6)alkyl group; or alternatively, R2 and _R3 form, together with the carbon atom to which they are attached, a ( _C3 -C6)cycloalkyl or ( _C3 -C6)heterocycloalkyl group containing from one to three heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom; R4 and R5 independently represent a hydrogen atom, a (C1-C6)alkyl group, a -( _C1 - _C6 )alkyl-NH( _R12 ) group, a -(C1- _C6 )alkyl-OH group, a -(C ₁ -C ₆ )alkyl-SH or -(C1C ₆ )alkyl-CO ₂ H group; or alternatively, R ₄ and R ₅ form, together with the carbon atom to which they are attached, a -(C3-C6)cycloalkyl or -(C3-C6)heterocycloalkyl group containing from one to three heteroatoms selected from a nitrogen atom, a sulfur and oxygen atom; X represents O or N(R6); R6 represents a hydrogen atom or a -(C1- _C6 )alkyl group; R ₇ and R ₈ independently represent a hydrogen atom or a -(C1-C ₆ )alkyl group, a -(C1-C ₆ )alkyl-CO ₂ H group or a -(C ₁ -C ₆ )alkyl-N group (C ₁ -C ₆ )alkyl ₂ ; or alternatively, R ₇ and R ₈ form, together with the carbon atom to which they are attached, a -(C ₃ -C ₆ )cycloalkyl group or a -(C ₃ -C ₆ )heterocycloalkyl group containing from one to three heteroatoms selected of a nitrogen atom, a sulfur atom and an oxygen atom; each R9 independently represents a hydrogen atom, -OH, (C ₁ -C ₄ )alkoxy, a halogen atom, -NH ₂ , -NH(C ₁ -C ₆ )alkyl, -N(C ₁ -C ₆ )alkyl ₂ ,-NH(C ₁ -C ₆ )cycloalkyl or (C ₃ -C ₆ )heterocycloalkyl group containing from one to three heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom; each R ₁₀ represents independently a hydrogen atom and a (C1- _C4 )alkyl group; Y represents -NR ₁₁ -(C ₁ -C ₆ )alkylene-, where -(C1 -C ₆ )alkylene- is linked to a phenyl ring, and -NR11- is linked directly to L1 of formula (II); wherein Y is located in the ortho-(o), meta-(m) or para-(p) position of the phenyl ring; R11 and _R12 independently represent hydrogen or -(C1- _C6 )alkyl; L is defined in formula (I) as defined in claim 1, and is a linker of formula (II) as defined in any of claims 1 to 9; RCG1 is a reactive chemical group as defined in claim 1 present at the end of linker L, wherein RCG1 is reactive with a chemical group present on the antibody. 11. Payloads with cryptophycin of formula (IV) according to claim 10, characterized by the following structure: where R1, _{R2 , R3} , _R4 , R5, R6, R7, _{R8 ,} R9, _R10 , R11, _R12 , RCG1, X, Y and L are defined in formula (IV), as indicated in clause 10. - 158 045708 12. Payload with cryptophycin of formula (IV) according to claim 10, which are selected from the following list: HE is he - 159 045708 - 160 045708 - 161 045708 - 162 045708 where R1, R2, _R3 , R4, R5, R6, R7, _R8 , _R9 , _R10 , R11, _R12 , X, Y and L are defined in formula (IV), as specified in paragraph 10 or 11 ; 13. Conjugate of formula (V) - 163 045708 G is the reaction product between RCG1 and RCG2; where RCG2 is an orthogonal reactive chemical group present on the antibody, Ab, and Ab is an antibody. 14. Conjugate of formula (V) according to claim 13, characterized by the following structure: where R _b R ₂ , R ₃ , R ₄ , R5, R6, R ₇ , R8, R9, R ₁₀ , Rn, R ₁₂ , X, Y, L, G, RCG1, RCG2 and Ab are as in the formula (V) specified in paragraph 13. 15. Conjugate of formula (V) according to claim 13 or 14, where RCG2 is selected from: (I) ε-amino groups located on the side chains of lysine residues that are present on the surface of the antibody; (ii) α-amino groups of the N-terminal amino acids of the heavy and light chains of the antibody; (iii) antibody hinge region saccharide groups; (iv) cysteine thiols formed by reduction of intrachain disulfide bonds of the antibody, or thiols of engineered antibody cysteines; (v) amide groups located on the side chains of glutamine residues that are present on the surface of the antibody; and (vi) aldehyde groups introduced into the antibody using a formylglycine-forming enzyme. 16. The conjugate of formula (V) according to claim 13 or 14, wherein if RCG1 is an N-hydroxysuccinimidyl ester, RCG2 is an NH ₂ group; if RCG1 represents a maleimido functional group, a haloacetamido functional group, a chlorine atom or an activated disulfide, RCG2 represents a -SH group; if RCG1 is a N ₃ group, RCG2 is a C=CH group or an activated C=C group; if RCG1 represents an OH or NH ₂ group, RCG2 represents a carboxylic acid functional group or an amide functional group; if RCG1 is an SH group, RCG2 is a maleimido functional group, a haloacetamido functional group or an activated disulfide functional group; if RCG1 is a C=CH group or an activated C=C group, RCG2 is a N ₃ group; while RCG1 is an O-alkylhydroxylamine functional group or a Pictet-Spengler reaction substrate, RCG2 is an aldehyde or ketone functional group. 17. A conjugate of formula (V) according to any one of claims 13 to 16, wherein G is selected from the group consisting of - 164 045708 18. The conjugate of formula (V) according to claim 13, which is selected from the following list: - 165045708 where Ab represents an antibody. 19. A method for producing a conjugate of formula (V) as defined in any one of claims 13 to 18, comprising at least the steps of: (i) contacting and causing the reaction of a buffered or unbuffered aqueous solution of an antibody, unmodified or modified with a modifying agent, and a payload solution with cryptophycin of formula (IV) according to any one of claims 10 to 12, wherein the chemical group RCG1 in the cryptophycin payload of formula (IV) is reactive with the RCG2 chemical groups present on the antibody, whether or not the RCG2 chemical groups have been introduced by means of a modifying agent, so as to attach the cryptophycin payload of formula (IV) to the antibody by formation of covalent bonds; and then (ii) separating or not separating the conjugate formed in step (i) from the cryptophycin payload of formula (IV), and/or from the unreacted antibody, and/or from any aggregates that may have formed. 20. A medicinal product, where it contains at least one conjugate of formula (V) according to any one of claims 13-18. 21. Pharmaceutical composition, where it contains at least one conjugate of formula (V) according to any one of claims 13 to 18, as well as at least one pharmaceutically acceptable excipient. 22. Use of conjugates of formula (V) according to any one of claims 13 to 18 as anticancer agents.