Sulfonylmaleimide-based linkers and corresponding conjugates

Abstract

The present invention relates to a linker or a salt thereof of the following formula (I): (I). The present invention relates to a linker-drug conjugate or a salt thereof of the following formula (II): (II). The present invention also relates to a binding unit-drug conjugate such as an antibody-drug conjugate or a salt thereof of the following formula (III) or (IV): (III), (IV), and a pharmaceutical composition comprising the binding unit-drug conjugate, and its use for treating cancer.

Description

Technical Field

[0001] The present invention relates to sulfonylmaleimide-based linkers for preparing conjugates by covalently linking a drug molecule to a binding unit, advantageously an antibody, such as antibody-drug conjugates (ADCs). Background Art

[0002] ADCs are controlled mixtures of different drug-loaded species (drug molecules per antibody (DAR) are 0 to 8) and have a typical average DAR of 3.5 or 4. Uncoupled species are generally inactive and compete with drug-loaded species for antigen binding. In addition, species with a DAR greater than 4 have been shown to result in lower tolerance, higher plasma clearance, and reduced efficacy. Most ADCs currently on the market and in clinical trials have common structural features, such as sulfosuccinimide linkages, which are formed by the reaction of a sulfhydryl group with an alkylmaleimide. This type of chemical is widely used because the reaction of maleimide and sulfhydryl groups is very rapid and quantitative under physiological conditions (both original species are not in large excess). However, the formation of sulfosuccinimide under physiological conditions is slowly reversible. ADCs containing alkylmaleimides may cause measurable drug loss during long-term circulation. The pharmacological consequences of this maleimide elimination (via the reverse Michael reaction) in ADCs include reduced antitumor activity due to reduced exposure of the drug in antibody-conjugated form, and greater toxicity due to untargeted release of the drug and linker. This has been described for both cysteine-linked ADCs and lysine-linked ADCs via the thioether linker SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate).

[0003] The present invention therefore relates to compounds for conjugating drugs to binding units, the resulting conjugates being more stable and more effective. Summary of the Invention

[0004] Therefore, the present invention relates to a linker of the following formula (I):

[0005]

[0006] The following formula (Ia) is preferred:

[0007]

[0008] or a salt thereof,

[0009] in:

[0010] -X1 and X2 independently represent H, a halogen atom, a (C1-C6) alkoxy group, an optionally substituted aryloxy group, or -O-(CH2CH2O) rH(-O-PEG), provided that X1 and X2 do not represent H at the same time;

[0011] -L1 expression L1'-(CO-Z') z’ A group wherein L1' is -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, heteroarylene, cycloalkanediyl, -(CH2) n -Arylene-, -(CH2) n -heteroarylene-, -(CH2) n -cycloalkanediyl-, -arylene-(CH2) p -, -heteroarylene-(CH2) p -, -cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-(CH2) p -、-(CH2) n -heteroarylene-(CH2) p -、-(CH2) n -cycloalkanediyl-(CH2) p -、-(CH2CH2O) m -CH2-CH2-arylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-heteroarylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-CH2-CH2-(OCH2CH2) m -、-(CH2) n -heteroarylene-CH2-CH2-(OCH2CH2) m -, or -(CH2) n -cycloalkanediyl-CH2-CH2-(OCH2CH2) m -;

[0012] - each W independently represents an amino acid unit;

[0013] -Y is -PAB-CO-(Z) z -, where PAB is (The oxygen of the PAB unit is connected to CO-(Z) z );

[0014] -Z is -NR4-(CH2) u-NR5- or -NR4-(CH2) u -NR5-CO-, or -NR4-(CH2) u -NR5-CO-(CH2) v -or-NR4-(CH2) u -NR5-CO-(CH2) v -CO-(NR4 group is connected to the CO group of PAB-CO);

[0015] -Z' is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO-(CH2) v -(NR4 group is connected to the CO group of CO-Z');

[0016] - R4 and R5 are independently H or (C1-C6) alkyl;

[0017] -c is 0 or 1, preferably 1;

[0018] -m is an integer from 1 to 15;

[0019] -n is an integer from 1 to 6;

[0020] -p is an integer from 1 to 6;

[0021] -q is 0, 1 or 2, preferably 2;

[0022] - r is an integer from 1 to 24, in particular an integer from 1 to 12;

[0023] -u is an integer from 1 to 6;

[0024] -v is an integer from 1 to 6;

[0025] -w is an integer from 0 to 5, preferably 0 or 2;

[0026] -y is 0 or 1 (preferably, y is 0 when w is 0, and y is 0 or 1 when w is an integer from 1 to 5);

[0027] -z is 0 or 1;

[0028] -z' is 0 or 1, especially 0; and

[0029] -When y=z=1 and Z is -NR4-(CH2) u -NR5-, or when c=w=y=0, z'=1 and Z' is -NR4-(CH2) u When -NR5-, X3 represents H, and in other cases, X3 represents OH, NH2 or a leaving group.

[0030] The leaving group is more particularly a halogen atom, a radical of the formula -OSO2-R LG sulfonic acid group, N-succinimidyloxy group, 4-nitrophenoxy group, pentafluorophenoxy group or N-benzotriazoleoxy group, wherein R LG represents a (C1-C6)alkyl group, an aryl group, an aryl-(C1-C6)alkyl group or a (C1-C6)alkyl-aryl group, said group being optionally substituted by one or more halogen atoms (for example, a fluorine atom).

[0031] Preferably, the compound of formula (I) is not a compound of formula (I) wherein:

[0032] -X1 is Cl, X2 is H, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0033] -X1 is Cl, X2 is Cl, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0034] -X1 is Cl, X2 is H, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0035] -X1 is Cl, X2 is H, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0036] -X1 is Cl, X2 is H, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0037] -X1 is Cl, X2 is H, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Br;

[0038] -X1 is Cl, X2 is H, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is 1;

[0039] -X1 is H, X2 is Cl, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0040] -X1 is H, X2 is Br, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0041] -X1 is H, X2 is Br, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0042] -X1 is Cl, X2 is Cl, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0043] -X1 is Cl, X2 is Cl, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0044] -X1 is Cl, X2 is Cl, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0045] -X1 is Cl, X2 is Br, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0046] -X1 is Cl, X2 is Br, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0047] -X1 is Cl, X2 is Br, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl; or

[0048] -X1 is Br, X2 is Br, q is 0, L1 is c is 0, w is 0, y is 0 and X3 is Cl;

[0049] The dotted line represents L1 and The wavy line represents the connection point between L1 and X3.

[0050] Said compounds are disclosed in WO 2007 / 001932 or US 4,127,687, but not as linkers more particularly intended for the preparation of conjugates, such as ADCs.

[0051] The present invention also relates to a linker-drug conjugate of the following formula (II):

[0052]

[0053] The following formula (IIa) is preferred:

[0054]

[0055] or a salt thereof,

[0056] in:

[0057] -X1 and X2 independently represent H, a halogen atom, a (C1-C6) alkoxy group, an optionally substituted aryloxy group, or -O-(CH2CH2O) rH, provided that X1 and X2 do not simultaneously represent H;

[0058] -L1 expression L1'-(CO-Z') z’ A group wherein L1' is -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, heteroarylene, cycloalkanediyl, -(CH2) n -Arylene-, -(CH2) n -heteroarylene-, -(CH2) n -cycloalkanediyl-, -arylene-(CH2) p -, -heteroarylene-(CH2) p -, -cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-(CH2) p -、-(CH2) n -heteroarylene-(CH2) p -、-(CH2) n -cycloalkanediyl-(CH2) p -、-(CH2CH2O) m -CH2-CH2-arylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-heteroarylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-CH2-CH2-(OCH2CH2) m -、-(CH2) n -heteroarylene-CH2-CH2-(OCH2CH2) m -, or -(CH2) n -cycloalkanediyl-CH2-CH2-(OCH2CH2) m -;

[0059] - each W independently represents an amino acid unit;

[0060] -Y is -PAB-CO-(Z) z -, where PAB is (The oxygen of the PAB unit is connected to CO-(Z) z );

[0061] -Z is -NR4-(CH2) u -NR5- or -NR4-(CH2)u -NR5-CO-, or -NR4-(CH2) u -NR5-CO-(CH2) v -or-NR4-(CH2) u -NR5-CO-(CH2) v -CO-(NR4 group is connected to the CO group of PAB-CO);

[0062] -Z' is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO-(CH2) v -(NR4 group is connected to the CO group of CO-Z');

[0063] - R4 and R5 are independently H or (C1-C6) alkyl;

[0064] -Q represents the drug moiety;

[0065] -c is 0 or 1, preferably 1;

[0066] -m is an integer from 1 to 15;

[0067] -n is an integer from 1 to 6;

[0068] -p is an integer from 1 to 6;

[0069] -q is 0, 1 or 2, preferably 2;

[0070] - r is an integer from 1 to 24, in particular an integer from 1 to 12;

[0071] -u is an integer from 1 to 6;

[0072] -v is an integer from 1 to 6;

[0073] -w is an integer from 0 to 5, preferably 0 or 2;

[0074] -y is 0 or 1 (preferably, y is 0 when w is 0, and y is 0 or 1 when w is an integer from 1 to 5);

[0075] -z is 0 or 1; and

[0076] -z' is 0 or 1, especially 0.

[0077] The present invention also relates to a binding unit-drug conjugate of the following formula (III) or (IV):

[0078]

[0079]

[0080] or a salt thereof, preferably a pharmaceutically acceptable salt thereof,

[0081] in:

[0082] - the binding unit is a peptide, a protein (e.g., an engineered protein), an antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof;

[0083] -L1 expression L1'-(CO-Z') z’ A group wherein L1' is -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, heteroarylene, cycloalkanediyl, -(CH2) n -Arylene-, -(CH2) n -heteroarylene-, -(CH2) n -cycloalkanediyl-, -arylene-(CH2) p -, -heteroarylene-(CH2) p -, -cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-(CH2) p -、-(CH2) n -heteroarylene-(CH2) p -、-(CH2) n -cycloalkanediyl-(CH2) p -、-(CH2CH2O) m -CH2-CH2-arylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-heteroarylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-CH2-CH2-(OCH2CH2) m -、-(CH2) n -heteroarylene-CH2-CH2-(OCH2CH2) m -, or -(CH2) n -cycloalkanediyl-CH2-CH2-(OCH2CH2) m -;

[0084] - each W independently represents an amino acid unit;

[0085] -Y is -PAB-CO-(Z) z -, where PAB is (The oxygen of the PAB unit is connected to CO-(Z) z );

[0086] -Z is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO-, or -NR4-(CH2) u -NR5-CO-(CH2) v -or-NR4-(CH2) u -NR5-CO-(CH2) v -CO-(NR4 group is connected to the CO group of PAB-CO);

[0087] -Z is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO-(CH2) v -(NR4 group is connected to the CO group of CO-Z');

[0088] - R4 and R5 are independently H or (C1-C6) alkyl;

[0089] -Q represents the drug moiety;

[0090] -c is 0 or 1, preferably 1;

[0091] -m is an integer from 1 to 15;

[0092] -n is an integer from 1 to 6;

[0093] -p is an integer from 1 to 6;

[0094] -s is an integer from 1 to 8;

[0095] -u is an integer from 1 to 6;

[0096] -v is an integer from 1 to 6;

[0097] -w is an integer from 0 to 5, preferably 0 or 2;

[0098] -y is 0 or 1 (preferably, y is 0 when w is 0, and y is 0 or 1 when w is an integer from 1 to 5);

[0099] -z is 0 or 1; and

[0100] -z' is 0 or 1, especially 0.

[0101] According to a preferred embodiment, the binding unit is an IGF-1R antibody, a HER2 antibody (eg, trastuzumab) or an antigen-binding fragment thereof.

[0102] The present invention also relates to the use of a linker of formula (I) or a drug-linker conjugate of formula (II) (preferably wherein q=2) for covalently linking a drug to a binding unit, such as an antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof. Thus, the covalent link is formed via the linker moiety.

[0103] Indeed, compounds of formula (I) or (II), preferably wherein q = 2, can be used to covalently link a drug to a binding unit, such as an antibody (eg, a monoclonal antibody) or an antigen-binding fragment thereof.

[0104] Compounds of formula (I) or (II) with q = 0 or 1 can also be used as synthetic intermediates for preparing compounds of formula (I) or (II) with q = 2. Therefore, the present invention also relates to compounds of formula (I) or (II) as defined above with q = 0 or 1 as synthetic intermediates.

[0105] The present invention also relates to a method for preparing a linker of formula (I) or a conjugate of formula (II), (III) or (IV).

[0106] The present invention also relates to a pharmaceutical composition comprising a binding unit-drug conjugate of formula (III) or (IV) and at least one pharmaceutically acceptable excipient.

[0107] The present invention also relates to a binding unit-drug conjugate of formula (III) or (IV), or a pharmaceutical composition comprising a binding unit-drug conjugate of formula (III) or (IV) and at least one pharmaceutically acceptable excipient, for use in treating cancer.

[0108] The present invention also relates to the use of a binding unit-drug conjugate of formula (III) or (IV) in the preparation of a medicament intended for the treatment of cancer.

[0109] The present invention also relates to a method for treating cancer, comprising administering to a person in need thereof an effective amount of a binding unit-drug conjugate of formula (III) or (IV) or a pharmaceutical composition comprising a binding unit-drug conjugate of formula (III) or (IV) and at least one pharmaceutically acceptable excipient.

[0110] definition

[0111] For the purposes of the present invention, the term "pharmaceutically acceptable" is intended to mean that it is used in the preparation of pharmaceutical compositions and is generally safe and non-toxic for pharmaceutical use.

[0112] The term "pharmaceutically acceptable salts", within the scope of the present invention, is intended to mean salts of pharmaceutically acceptable compounds as defined above, which possess the pharmacological activity of the corresponding compounds.

[0113] Pharmaceutically acceptable salts include:

[0114] (1) Acid addition salts formed with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid; such as acetic acid, benzenesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid and trifluoroacetic acid; and

[0115] (2) Salts formed when an acidic proton present in a compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or salts formed by coordination with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.

[0116] The term "halogen" as used herein refers to a fluorine, bromine, chlorine or iodine atom.

[0117] The term "(C1-C6) alkyl" used in the present invention refers to a monovalent straight or branched saturated hydrocarbon chain containing 1 to 6 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0118] The term "(C1-C6)alkoxy" as used in the present invention refers to a (C1-C6)alkyl group as defined above that is bonded to the molecule via an oxygen atom, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexoxy, and the like.

[0119] The term "(C2-C6) alkenyl" as used in the present invention refers to a straight or branched monovalent unsaturated hydrocarbon chain containing 2 to 6 carbon atoms and containing at least one double bond, including but not limited to ethenyl, propenyl, butenyl, pentenyl, hexenyl, etc.

[0120] The term "cycloalkanediyl" used in the present invention refers to a divalent saturated hydrocarbon ring advantageously having 4 to 10 carbon atoms, especially 5 or 6 carbon atoms, including but not limited to cyclopentanediyl, cyclohexanediyl, etc. Preferably, it is cyclohexanediyl.

[0121] The term "aryl" as used in the present invention refers to a monovalent aromatic hydrocarbon group preferably containing 6 to 10 carbon atoms and containing one or more fused rings, such as phenyl or naphthyl. Advantageously, it is phenyl.

[0122] The term "aryl-(C1-C6)alkyl" as used in the present invention refers to a (C1-C6)alkyl group as defined above substituted by an aryl group as defined above. In particular, it may be a benzyl group.

[0123] The term "(C1-C6)alkyl-aryl" as used in the present invention refers to an aryl group as defined above substituted by a (C1-C6)alkyl group as defined above. In particular, it may be tolyl (CH3Ph).

[0124] The term "aryloxy" as used herein refers to an aryl group as defined above bound to the molecule via an oxygen atom, including but not limited to phenoxy.

[0125] The term "arylene" as used in the present invention refers to a divalent aromatic hydrocarbon group preferably containing 6 to 10 carbon atoms and containing one or more fused rings, such as phenylene or naphthylene. Advantageously, it is phenylene.

[0126] The term "heteroarylene" as used in the present invention refers to a divalent aromatic group comprising one or more (especially one or two) fused hydrocarbon rings, wherein one or more (especially one to four, advantageously one to three) carbon atoms are each replaced by a heteroatom selected from a sulfur atom, an oxygen atom and a nitrogen atom, preferably selected from an oxygen atom and a nitrogen atom, more preferably a nitrogen atom. Advantageously, it is a divalent 1,2,3-triazole, for example a divalent 1H-1,2,3-triazole.

[0127] The term "leaving group" as used in the present invention refers to a chemical group that can be easily replaced by a nucleophilic reagent (e.g., an amine or alcohol with a functional group NH or OH, respectively) in a nucleophilic substitution reaction. The leaving group can be, in particular, a halogen atom, a sulfonic acid group, an N-succinimidyloxy group, a 4-nitro-phenoxy group, a pentafluorophenoxy group, or an N-benzotriazoloxy group. The sulfonic acid group is in particular the group -OSO2-R LG , where R LG represents (C1-C6)alkyl, aryl, aryl-(C1-C6)alkyl or (C1-C6)alkyl-aryl, said group being optionally substituted by one or more halogen atoms (e.g. fluorine atoms). The sulfonic acid group may in particular be methanesulfonic acid (OMs, CH3-S(O2)O-), trifluoromethanesulfonic acid (OTf, CF3-S(O)2O-) or toluenesulfonic acid (OTs, p-Me-C6H4-S(O)2O-). The leaving group may in particular be Cl, Br, I, OTf, OMs, OTf, N-succinimidyloxy, 4-nitro-phenoxy or N-benzotriazolyloxy.

[0128] The term "trialkylsilyl" as used herein refers to the group -SiAlk1Alk2Alk3, wherein Alk1, Alk2 and Alk3 are identical or different and represent a (C1-C6)-alkyl group as defined above. For example, it may be trimethylsilyl or triethylsilyl.

[0129] The term "protected form" of a molecule means that at least one OH or NH function present on said molecule is protected by an O-protecting group or an N-protecting group, respectively.

[0130] The term "protecting group" used in the present invention refers to a chemical group that selectively blocks a reactive site in a multifunctional compound, thereby allowing a chemical reaction to be selectively performed on another unprotected reactive site.

[0131] The term "O-protecting group" as used herein refers to a substituent that protects a hydroxyl group (OH) from undesirable reactions during synthesis, such as those disclosed in "Greene's Protective Groups In Organic Synthesis" (4th edition, 2007, John Wiley & Sons, Hoboken, New Jersey). The hydroxyl group protected by the O-protecting group can be, for example, an ether, ester, carboxylate, acetal, or the like. In particular, the O-protecting group may be a (C1-C6)alkyl group optionally substituted by one or more (especially 1 to 3) halogen atoms (e.g. chlorine atoms), such as a methyl, ethyl, tert-butyl or 2,2,2-trichloroethyl group; an aryl-(C1-C6)alkyl group (e.g. benzyl), the aryl moiety being optionally substituted by one or more methoxy groups, such as benzyl (Bn) or p-methoxybenzyl (PMB); a trityl derivative of the formula -CAr1Ar2Ar3, such as trityl (also known as trityl-Tr), (4-methoxyphenyl)diphenylmethyl (also known as methoxytrityl-NMT) or bis-(4-methoxyphenyl)benzyl (also known as dimethoxytrityl-DMT); a group of the formula -CH2OR GP2 Or -CH2SR GP2 Substituted methyl (especially -CH2OR GP2 ), such as methoxymethyl (MOM), benzyloxymethyl, 2-methoxyethoxymethyl (MEM), 2-(trimethylsilyl)ethoxymethyl or methylthiomethyl; formula -CH2CH2OR GP2 or -CH2CH2SR GP2 Substituted ethyl (especially -CH2CH2OR GP2 ), such as ethoxyethyl (EE); formula -SiR GP3 R GP4 R GP5silyl, such as trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldimethylsilyl (TBS or TBDMS) and tert-butyldiphenylsilyl (TBDPS); formula -CO-R GP6 A carbonyl group such as acetyl (Ac), pivaloyl (Piv or Pv) or benzoyl (Bz), or a group of formula -CO2-R GP7 carbonyl groups, such as allyloxycarbonyl (Alloc) or 9-fluorenylmethyloxycarbonyl (Fmoc); or tetrahydropyranyl ( )(THP) or tetrahydrofuranyl ( );

[0132] wherein Ar1, Ar2 and Ar3 independently represent an aryl group (eg, a phenyl group), which is optionally substituted with one or more methoxy groups; R GP2 represents a (C1-C6)alkyl group (e.g., methyl or ethyl) optionally substituted by an aryl group (e.g., phenyl), a (C1-C6)alkoxy group (e.g., methoxy) or a trialkylsilyl group (e.g., SiMe3); R GP3 、R GP4 and R GP5 R independently represents (C1-C6) alkyl or aryl (eg phenyl); GP6 and R GP7 Each independently represents a (C1-C6)alkyl group, a (C2-C6)alkenyl group, an aryl group, an aryl-(C1-C6)alkyl group or a 9-fluorenylmethyl group.

[0133] The term "N-protecting group" used in the present invention refers to a group that protects an amine functional group (especially a primary amine functional group) from undesirable reactions during the synthesis process. Commonly used N-protecting groups are disclosed in "Greene's Protective Groups In Organic Synthesis" (4th edition, 2007, John Wiley & Sons, Hoboken, New Jersey). The amine functional group protected by the N-protecting group can be a carbamate, an amide, a sulfonamide, an N-alkyl derivative, an aminoacetal derivative, an N-benzyl derivative, an imine derivative, an enamine derivative or an N-heteroatom derivative. In particular, the N-protecting group may be formyl; aryl (e.g. phenyl), which is optionally substituted with one or more methoxy groups, such as p-methoxyphenyl (PMP); aryl-(C1-C6)alkyl (e.g. benzyl), the aryl portion of which is optionally substituted with one or more methoxy groups, such as benzyl (Bn), p-methoxybenzyl (PMB) or 3,4-dimethoxybenzyl (DMPM); -CO-R GP1, such as acetyl (Ac), pivaloyl (Piv or Pv), benzoyl (Bz) or p-methoxybenzylcarbonyl (Moz); -CO2-R GP1 , such as tert-butyloxycarbonyl (Boc), trichloroethoxycarbonyl (TROC), allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz or Z) or 9-fluorenylmethoxycarbonyl (Fmoc); -SO2-R GP1 , such as phenylsulfonyl, toluenesulfonyl (Ts or Tos) or 2-nitrobenzenesulfonyl (also known as nosyl-Nos or Ns); etc.

[0134] where R GP1 represents a (C1-C6)alkyl group optionally substituted by one or more halogen atoms (e.g. F or Cl); a (C2-C6)alkenyl group, such as allyl; an aryl group (e.g. phenyl), which is optionally substituted by one or more groups selected from OMe (methoxy) and NO2 (nitro); an aryl-(C1-C6)alkyl group (e.g. benzyl), the aryl part of which is optionally substituted by one or more methoxy groups; or a 9-fluorenylmethyl group.

[0135] The terms "antibody," "antibodies," "ab," "Ab," "MAb," or "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies, isolated, engineered, or recombinant antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, or multispecific antibodies (e.g., bispecific antibodies), and antibody fragments thereof, so long as they exhibit the desired biological activity.

[0136] The term "recombinant antibody" refers to an antibody produced by expression of recombinant DNA in living cells. The recombinant antibodies according to the present invention are obtained by using genetic recombination laboratory methods well known to those skilled in the art to produce DNA sequences that do not exist in organisms.

[0137] The term "antigen-binding fragment" of an antibody according to the present invention is intended to mean any peptide, polypeptide or protein that retains the ability to bind to the antibody's target (also commonly referred to as antigen).

[0138] "Binding", "binds", etc. refer to the formation of a relatively stable complex between an antibody or any antigen-binding fragment thereof and an antigen under physiological conditions. Specific binding can be achieved by at least about 1×10 -6 The antibody can be characterized by the equilibrium dissociation constant M. Methods for determining whether two molecules bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, radiolabeled assays, and the like. For the avoidance of doubt, this does not mean that the antibody cannot bind or interfere with another antigen at a low level. However, as an embodiment, the antibody only binds to the antigen.

[0139] The expression "IGF-1R antibody" used in the present specification should be interpreted similarly to "anti-IGF-1R antibody" and refers to an antibody capable of binding to IGF-1R.

[0140] The expression "HER2 antibody" used in this specification should be interpreted similarly to "anti-HER2 antibody" and refers to an antibody capable of binding to HER2.

[0141] The term half maximal effect concentration (EC 50 ) corresponds to the concentration of a drug, antibody, or toxicant that elicits a response halfway between baseline and maximum after a specified exposure time. It is often used as a measure of drug efficacy. Thus, the EC of a graded dose-response curve is 50 The EC value of the quantitative dose-response curve is the concentration of compound at which 50% of the compound's maximal effect is observed. 50 The concentration of compound at which 50% of the population exhibits a response after a specified exposure duration. Concentration measurements typically follow an S-shaped curve, rising rapidly with relatively small changes in concentration. It can be determined mathematically by deriving a best-fit curve.

[0142] As a preferred embodiment, the EC determined in the present invention 50 Characterize the efficacy of antibodies binding to IGF-1R ECD exposed on human tumor cells. EC was determined using FACS analysis. 50 Parameters. EC 50 The parameter reflects the antibody concentration at which 50% of maximal binding to human IGF-1R expressed on human tumor cells is achieved. Each EC was calculated using a four-parameter regression curve fitting program (Prism software). 50 The value was used as the midpoint of the dose-response curve. This parameter was chosen to represent physiological / pathological conditions.

[0143] The term "epitope" is the region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope can include determinants that are chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, an epitope can have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0144] As used herein, the term "monoclonal antibody" or "Mab" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies in the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. The monoclonal antibody can be produced by a single clone of a B cell or a hybridoma. Monoclonal antibodies can also be recombinant, i.e., produced by protein engineering or chemical synthesis. Monoclonal antibodies can also be isolated from phage antibody libraries. In addition, in contrast to the preparation of polyclonal antibodies, which typically include various antibodies directed against various determinants or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. Monoclonal antibodies herein include mouse, chimeric, and humanized antibodies.

[0145] The term "chimeric antibody" refers to an antibody comprising the natural variable regions (light and heavy chains) of an antibody derived from a given species and an antibody that is combined with the constant regions of the light and heavy chains of an antibody of a species that is heterologous to the given species. Chimeric antibodies can be prepared using recombinant genetics techniques. For example, chimeric antibodies can be produced by cloning recombinant DNA comprising a promoter and a sequence encoding the variable regions of a non-human (especially mouse) source monoclonal antibody, and a sequence encoding the constant regions of a heterologous species (preferably human) antibody. The chimeric antibody according to the present invention encoded by one of the recombinant genes can be, for example, a mouse-human chimera, the specificity of which is determined by the variable regions derived from mouse DNA, while its isotype is determined by the constant regions derived from human DNA.

[0146] The term "humanized antibody" refers to an antibody comprising a CDR region derived from a non-human antibody, wherein the other parts of the antibody molecule are derived from one (or more) human antibody. In addition, some skeleton fragment residues (called FR) can be modified to maintain binding affinity. Humanized antibodies or their fragments can be prepared by techniques known to those skilled in the art. The humanized antibodies are preferably used in methods related to in vitro diagnosis or in vivo prevention and / or treatment. Other humanization techniques are also known to those skilled in the art, such as PDL's "CDR implantation" technology described in patents and patent applications EP 0 451 216, EP 0 682 040, EP 0 939 127, EP 0 566 647, US 5,530,101, US 6,180,370, US 5,585,089, US 5,693,761. Reference may also be made to US Patents 5,639,641, 6,054,297, 5,886,152, and 5,877,293.

[0147] Unless otherwise indicated in this specification, complementarity determining regions or CDRs refer to the hypervariable regions of immunoglobulin heavy and light chains as defined according to the IMGT numbering system.

[0148] However, CDRs may also be defined according to the Kabat numbering system (Kabat et al., Sequences of proteins of immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Herein, the terms "CDR" and "CDRs" are used to denote one or more or even all regions, as appropriate, comprising the majority of the amino acid residues responsible for the binding affinity of an antibody to an antigen or an epitope it recognizes. To simplify the reading of this application, the CDRs according to Kabat are not defined. However, it will be apparent to those skilled in the art that the definition of CDRs according to Kabat can be used to define the CDRs according to IMGT.

[0149] In the sense of the present invention, "identity" or "percent identity" between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotide or amino acid residues between the two sequences being compared, obtained after optimal alignment, which percentage is purely statistical, with the differences between the two sequences being randomly distributed along their lengths. Traditionally, the comparison of two nucleic acid or amino acid sequences is performed by comparing the optimally aligned sequences, which can be performed segmentally or by using an "alignment window." In addition to manual alignment, optimal alignment of the sequences to be compared can be performed by the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48:443], the search similarity method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444], or by computer software that uses these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or the comparison software BLAST NR or BLAST P).

[0150] The percent identity is calculated by determining the number of positions where the amino acid nucleotides or residues are identical between the two sequences (preferably between the two complete sequences), dividing the number of identical positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0151] For example, the BLAST program "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol., 1999, Lett. 174: 247-250) (available on the website http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html) can be used with the default parameters (in particular the parameters "open gap penalty": 5, "extension gap penalty": 2; the matrix selected is, for example, the "BLOSUM 62" matrix proposed by the program); the program directly calculates the percentage identity between the two sequences to be compared.

[0152] The expression "back-mutation" or "back mutation" refers to a mutation or substitution of a human residue present in the germline with the corresponding residue originally present in the murine sequence.

[0153] The terms "nucleic acid," "nucleic acid sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence" are used interchangeably herein to refer to a precise sequence of nucleotides, modified or unmodified, that defines a fragment or region of a nucleic acid, whether or not it contains non-natural nucleotides, and may be double-stranded DNA, single-stranded DNA, or a transcription product of said DNA.

[0154] The term "peptide" refers to a chain of amino acid monomers linked to each other by peptide (amide) bonds. Covalent peptide bonds (amides) are formed by the reaction of the carboxyl (COOH) group of one amino acid with the amino (NH2) group of another amino acid. The term peptide includes oligopeptides and polypeptides.

[0155] The term "protein" is an assembly of one or more peptides as defined above that have undergone post-translational modifications and protein folding so as to arrange them in a biologically functional manner.

[0156] The term "amino acid" as used in the present invention refers to natural α-amino acids in D or L form (e.g., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val)), as well as unnatural amino acids. Acids (e.g., β-alanine, allylglycine, tert-leucine, 3-aminoadipic acid, 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 2-aminobutyric acid, 4-amino-1-carboxymethylpiperidine, 1-amino-1-cyclobutanecarboxylic acid, 4-aminocyclohexaneacetic acid, 1-amino-1-cyclohexanecarboxylic acid, (1R,2R)-2-aminocyclohexanecarboxylic acid, (1R,2S)-2-aminocyclohexanecarboxylic acid, (1S,2R)-2-aminocyclohexanecarboxylic acid, (1S,2S)-2-aminocyclohexanecarboxylic acid, 3-aminocyclohexanecarboxylic acid, 4-aminocyclohexanecarboxylic acid, (1R,2R)-2-aminocyclopentanecarboxylic acid, (1R,2S )-2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 4-(2-aminoethoxy)-benzoic acid, 3-aminomethylbenzoic acid, 4-aminomethylbenzoic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 1-aminoindan-1-carboxylic acid, 4-aminomethyl-phenylacetic acid, 4-aminophenylacetic acid, 3-amino-2-naphthoic acid, 4-aminophenylbutyric acid, 4-amino-5-(3-indolyl)-pentanoic acid, (4R,5S)-4-amino-5-methylheptanoic acid, (R)-4-amino-5-methylhexanoic acid, (R)-4-amino-6-methylthiohexanoic acid, (S)-4-amino pentanoic acid, (R)-4-amino-5-phenylpentanoic acid, 4-aminophenylpropionic acid, (R)-4-aminopimelic acid, (4R,5R)-4-amino-5-hydroxyhexanoic acid, (R)-4-amino-5-hydroxypentanoic acid, (R)-4-amino-5-(p-hydroxyphenyl)-pentanoic acid, 8-aminooctanoic acid, (2S,4R)-4-amino-pyrrolidine-2-carboxylic acid, (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, azetidine-2-carboxylic acid, (2S,4R)-4-benzyl-pyrrolidine-2-carboxylic acid, (S)-4,8-diaminooctanoic acid, tert-butylglycine, γ-carboxyglutamic acid, β-cyclohexylalanine, citrulline, 2,3-Diaminopropionic acid, hippuric acid, homocyclohexylalanine, moleucine, homophenylalanine, 4-hydroxyproline, indoline-2-carboxylic acid, isodocosanoic acid, α-methylalanine, niacin, norleucine, norvaline, octahydroindole-2-carboxylic acid, ornithine, penicillamine, phenylglycine, 4-phenyl-pyrrolidine-2-carboxylic acid, pipecolic acid, propargylglycine, 3-pyridylalanine, 4-pyridylalanine, 1-pyrrolidine-3-carboxylic acid, sarcosine, statins, tetrahydroisoquinoline-1-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or tranexamic acid).

[0157] Detailed description

[0158] Connector part

[0159] The linker moiety according to the invention enables the covalent attachment of an antibody to at least one drug moiety.

[0160] The linker moiety may be "non-cleavable" or "cleavable."

[0161] In a preferred embodiment, it consists in a "cleavable" linker moiety that facilitates the release of the drug in the cell.

[0162] For example, in some embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., in a lysosome, endosome, or pit). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). Typically, the peptidyl linker comprises at least two consecutive amino acids or at least three consecutive amino acids. The cleavage agent can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs in target cells. For example, a peptidyl linker that can be cleaved by the sulfhydryl-dependent protease cathepsin-B, which is highly expressed in cancer tissue, can be used (e.g., a linker comprising Phe-Leu or Gly-Phe-Leu-Gly). In a specific embodiment, the peptidyl linker that can be cleaved by an intracellular protease comprises or is Val-Cit, Phe-Lys, or Val-Ala. One advantage of using intracellular proteolysis to release the drug is that the drug is usually attenuated during conjugation, and the serum stability of the conjugate is usually very high.

[0163] Group -L1-(CO) c - represents the stretching unit of the linker part which must be present. Group -L1-(CO) c - is the formula -L1'-(CO-Z') z’ -(CO) c-, wherein z' and c are 0 or 1, for example, when z' is 0, -L1'-(CO) c Preferably, when at least one of w and y is not 0, then z' is 0, and in other cases, z' is 0 or 1.

[0164] L1' represents -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, heteroarylene, cycloalkanediyl, -(CH2) n -Arylene-, -(CH2) n -heteroarylene-, -(CH2) n -cycloalkanediyl-, -arylene-(CH2) p -, -heteroarylene-(CH2) p -, -cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-(CH2) p -、-(CH2) n -heteroarylene-(CH2) p -、-(CH2) n -cycloalkanediyl-(CH2) p -、-(CH2CH2O) m -CH2-CH2-arylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-heteroarylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-CH2-CH2-(OCH2CH2) m -、-(CH2) n -heteroarylene-CH2-CH2-(OCH2CH2) m -, or -(CH2) n -cycloalkanediyl-CH2-CH2-(OCH2CH2) m More particularly, the arylene group is a phenylene group; the cycloalkanediyl group is a cyclohexanediyl group, such as p-cyclohexanediyl; and the heteroarylene group is a divalent 1,2,3-triazole group, such as a divalent 1H-1,2,3-triazole group.

[0165] According to a particular embodiment, L1' represents -(CH2) n -、-(CH2CH2O) m-CH2-CH2-, arylene, -cycloalkanediyl-, -(CH2) n -Arylene-, -Arylene-(CH2) n -、-(CH2) n -cycloalkanediyl-, -cycloalkanediyl-(CH2) n -、 More particularly, the arylene group is phenylene; and the cycloalkanediyl group is cyclohexanediyl, such as p-cyclohexanediyl.

[0166] According to another particular embodiment, L1' represents -(CH2) n -or-(CH2CH2O) m -CH2-CH2-, especially -(CH2) n -, for example -(CH2)5-.

[0167] When z'=1, the L1' stretcher moiety may be completely comprised of a stretcher moiety CO-Z', wherein Z' is -CO-NR4-(CH2) u -NR5- or -CO-NR4-(CH2) u -NR5-CO-(CH2) v R4 and R5 are independently H or (C1-C6)alkyl, such as H or Me. u and v are independently an integer from 1 to 6, such as an integer from 1 to 4, especially 1 or 2, such as 2.

[0168] (W) w represents the amino acid unit of the linker.

[0169] The amino acid unit of the linker can be cleaved enzymatically, including but not limited to, by tumor-associated proteases, to release the drug.

[0170] The selectivity of the amino acid units can be designed and optimized for enzymatic cleavage by specific tumor-associated proteases. Suitable units are units whose cleavage is catalyzed by proteases (cathepsins B, C and D and plasmin).

[0171] (W) w It may be absent (w=0) or may be a dipeptide, tripeptide, tetrapeptide or pentapeptide unit (w=1, 2, 3, 4 or 5), wherein the amino acids forming the peptides may differ from one another.

[0172] Therefore, (W) w It can be expressed as follows: (W1) w1 (W2) w2 (W3) w3 (W4) w4 (W5) w5, wherein each W1 to W5 independently represents an amino acid unit, and each w1 to w5 is 0 or 1.

[0173] In some embodiments, the Amino Acid unit (W) w Amino acid residues (eg, naturally occurring amino acid residues) as well as minor amino acids and non-naturally occurring amino acid analogs (eg, citrulline) can be included.

[0174] Amino acid unit (W) w The amino acid residues include, but are not limited to, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine protected or unprotected with acetyl or formyl groups, arginine, arginine protected or unprotected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl groups, and citrulline. Exemplary amino acid linker components preferably include dipeptides or tripeptides.

[0175] Exemplary dipeptides include: Val-Cit, Ala-Val, Ala-Ala, Val-Ala, Lys-Lys, Cit-Cit, Val-Lys, Ala-Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -nitro-Arg, preferably Val-Cit or Val-Ala.

[0176] Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys, Gly-Phe-Lys.

[0177] Exemplary tetrapeptides include: Gly-Phe-Leu-Gly (SEQ ID NO. 53), Ala-Leu-Ala-Leu (SEQ ID NO. 54).

[0178] Exemplary pentapeptides include: Pro-Val-Gly-Val-Val (SEQ ID NO. 55).

[0179] According to a particular embodiment, (W) w It can be a dipeptide (ie, w=2), such as Val-Cit or Val-Ala, preferably Val-Cit, or the linker lacks an amino acid unit (w=0). When the linker lacks an amino acid unit, it preferably also lacks a spacer unit Y (y=0).

[0180] According to a preferred embodiment, w=0 (ie (W) w is a single bond) or w=2(ie (W) w is a dipeptide), so (W) w Can be selected from:

[0181]

[0182]

[0183] And especially for Val-Cit,

[0184] in

[0185] Asterisks indicate spacer units (Y) y connection point; and

[0186] The wavy line indicates -L1-(CO) c - (CO if c=1, L1 if c=0) connection point.

[0187] Y represents a spacer unit of the linker.

[0188] There are two general types of Spacer units: self-immolative and non-self-immolative.

[0189] For non-self-cleavable spacer units, after enzymatic cleavage of the amino acid unit from the antibody-drug conjugate, some or all of the spacer units remain bound to the drug. Examples of non-self-cleavable spacer units include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units. In order to release the drug, an independent hydrolysis reaction should occur within the target cell to cleave the bond of the glycine-drug unit.

[0190] The self-cleavable spacer unit can release the drug without a separate hydrolysis step. In these embodiments, (Y) is the residue of a p-aminobenzyl alcohol unit (PAB), which is bound to (W) via the nitrogen atom of the PAB group. w The linker comprising the PAB moiety can also be considered as a traceless linker.

[0191] In the present invention, the spacer unit (Y) is -PAB-CO-(Z) z -, where PAB is (the oxygen of the PAB unit is linked to the carbonyl group), also known as -p-aminobenzyl-O-CO-, and y=1 or the linker lacks a spacer unit (y=0).

[0192] When z=1, the spacer -p-aminobenzyl-O-CO- can be completely provided with a spacer Z, wherein the spacer Z is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO- or -NR4-(CH2) u -NR5-CO-(CH2) v -or-NR4-(CH2) u -NR5-CO-(CH2) v -CO-. R4 and R5 are independently H or (C1-C6)alkyl, such as H or Me. u and v are independently an integer from 1 to 6, such as an integer from 1 to 4, especially 1 or 2, such as 2.

[0193] Advantageously, y is 0 when w is 0 and y is 0 or 1 when w is an integer from 1 to 5, which means that the spacer unit Y may be present only when the amino acid unit W is present.

[0194] Preferably, when w is 0, y is 0, and when w is an integer from 1 to 5, y is 1, which means that when an amino acid unit (W) is present w When the spacer unit Y exists, when the amino unit (W) w When not present, the interval unit Y does not exist.

[0195] According to a particular embodiment, the group -L1-(CO) c -(W) w -(Y) y - represents -(CH2) n -、-(CH2) n -CO-, -(CH2CH2O) m -CH2-CH2-, -(CH2CH2O) m -CH2-CH2-CO-, -CH2-p-cyclohexyl-CO-, -aryl-(CH2) n -、-(CH2) n -Val-Cit-p-aminobenzyl-O-CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-, -(CH2CH2O) m -CH2-CH2-Val-Cit-p-aminobenzyl-O-CO-, -(CH2CH2O) m -CH2-CH2-CO-Val-Cit-p-aminobenzyl-O-CO-, -CH2-p-cyclohexyl-CO-Val-Cit-p-aminobenzyl-O-CO-, -aryl-(CH2) n-Val-Cit-p-aminobenzyl-O-CO-, -aryl-CO-Val-Cit-p-aminobenzyl-O-CO-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-, -(CH2CH2O) m -CH2-CH2-Val-Ala-p-aminobenzyl-O-CO-, -(CH2CH2O) m -CH2-CH2-CO-Val-Ala-p-aminobenzyl-O-CO-, -CH2-p-cyclohexyl-CO-Val-Ala-p-aminobenzyl-O-CO-, -aryl-(CH2) n -Val-Ala-p-aminobenzyl-O-CO-, -aryl-CO-Val-Ala-p-aminobenzyl-O-CO-, -(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2CH2O) m -CH2-CH2-Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2CH2O) m -CH2-CH2-CO-Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-、-CH2-p-cyclohexyl-CO-Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -aryl-(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-、-Aryl-CO-Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2CH2O) m -CH2-CH2-Val-Ala-p-aminobenzyl-O-CO-NH-(CH2)u -NH-, -(CH2CH2O) m -CH2-CH2-CO-Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -CH2-p-cyclohexyl-CO-Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -aryl-(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-、-Aryl-CO-Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2CH2O) m -CH2-CH2-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2CH2O) m -CH2-CH2-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -CH2-p-cyclohexyl-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -aryl-(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -Aryl-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2CH2O) m -CH2-CH2-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2)u -NCH3-CO-, -(CH2CH2O) m -CH2-CH2-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -CH2-p-cyclohexyl-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -aryl-(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -Aryl-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-,

[0196]

[0197]

[0198] Group -L1-(CO) c -(W) w -(Y) y - can also represent -(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2CH2O) m -CH2-CH2-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2CH2O) m -CH2-CH2-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -CH2-p-cyclohexyl-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-CH2-p-cyclohexyl-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -aryl-(CH2) n -CO-NCH3-(CH2)u -NCH3-CO-(CH2) v -, -aryl-(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -CO-NCH3-(CH2) u -NCH3-, -(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-, -(CH2CH2O) m -CH2-CH2-CO-NCH3-(CH2) u -NCH3-, -(CH2CH2O) m -CH2-CH2-CO-NCH3-(CH2) u -NCH3-, -(CH2CH2O) m -CH2-CH2-CO-NCH3-(CH2) u -NCH3-CO-, -CH2-p-cyclohexyl-CO-NCH3-(CH2) u -NCH3-, -CH2-p-cyclohexyl-CO-NCH3-(CH2) u -NCH3-CO-, -aryl-(CH2) n -CO-NCH3-(CH2) u -NCH3-, -aryl-(CH2) n -CO-NCH3-(CH2) u -NCH3-, -aryl-(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-, -(CH2) n -CO-NH-(CH2) u -NH-, -(CH2) n -CO-NH-(CH2) u -NH-CO-, -(CH2CH2O) m -CH2-CH2-CO-NH-(CH2) u -NH-, -(CH2CH2O) m -CH2-CH2-CO-NH-(CH2) u -NH-CO-, -CH2-p-cyclohexyl-CO-NH-(CH2) u -NH-, -CH2-p-cyclohexyl-CO-NH-(CH2) u-NH-CO-, -aryl-(CH2) n -CO-NH-(CH2) u -NH-, -aryl-(CH2) n -CO-NH-(CH2) u -NH-CO-, -(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2CH2O) m -CH2-CH2-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2CH2O) m -CH2-CH2-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2CH2O) m -CH2-CH2-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2CH2O) m -CH2-CH2-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -CH2-p-cyclohexyl-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v-、-CH2-p-cyclohexyl-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -aryl-(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -, -aryl-(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -Aryl-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-Aryl-CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2CH2O) m -CH2-CH2-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2CH2O) m -CH2-CH2-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u-NCH3-CO-(CH2) v -CO-, -(CH2CH2O) m -CH2-CH2-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2CH2O) m -CH2-CH2-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -CH2-p-cyclohexyl-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-CH2-p-cyclohexyl-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -aryl-(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -, -aryl-(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -Aryl-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -, or -aryl-CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-.

[0199] According to another particular embodiment, the group -L1-(CO) c -(W) w -(Y) y - represents -(CH2) n -、-(CH2) n -CO-, -(CH2) n -Val-Cit-p-aminobenzyl-O-CO-, -(CH2) n-CO-Val-Cit-p-aminobenzyl-O-CO-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-, -(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, or -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, wherein n and u are as defined previously, and in particular n=5 and u=2.

[0200] Group -L1-(CO) c -(W) w -(Y) y - can also represent -(CH2) n -CO-NH-(CH2) u -NH-, -(CH2) n -CO-NH-(CH2) u -NH-CO-, -(CH2) n -CO-NCH3-(CH2) u -NCH3-, -(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-, -(CH2)n -CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -, or -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, wherein n, u and v are as defined previously, and in particular n=5 and u=v=2.

[0201] According to a particular embodiment, the group -L1-(CO) c -(W)w -(Y) y - represents -(CH2) n -CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NH-(CH2) u -NH-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, or -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-, wherein n and u are as defined previously, and in particular n=5 and u=2.

[0202] Group -L1-(CO) c -(W) w -(Y) y - can also represent -(CH2) n -CO-NH-(CH2) u -NH-, -(CH2) n -CO-NH-(CH2) u -NH-CO-, -(CH2) n -CO-NCH3-(CH2) u -NCH3-, -(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-, -(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -、-(CH2) n -CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v-、-(CH2) n -CO-Val-Cit-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -, or -(CH2) n -CO-Val-Ala-p-aminobenzyl-O-CO-NCH3-(CH2) u -NCH3-CO-(CH2) v -CO-, wherein n, u and v are as defined previously, and in particular n=5 and u=v=2.

[0203] Group Preferred It is a functional group that reacts with a binding unit (e.g., an antibody) to connect a drug moiety thereto, and the reaction is due to the sulfhydryl group present on the binding unit. The sulfhydryl group can be produced by reducing the intramolecular disulfide bond (if present) of the binding unit, particularly in an antibody. Alternatively, the sulfhydryl group can be generated by reacting the amino group of the lysine moiety of the binding unit with 2-iminothiolane or other sulfhydryl generating reagents. In a specific embodiment, the binding unit (e.g., an antibody) is engineered to carry one or more lysines. More preferably, the binding unit (e.g., an antibody) can be engineered to carry one or more cysteines (see ThioMabs).

[0204] X1 and X2 independently represent H, a halogen atom (such as Cl or Br), a (C1-C6) alkoxy group, an optionally substituted aryloxy group, or -O-(CH2CH2O) r H, provided that X1 and X2 do not represent H at the same time. More particularly, the aryloxy group is optionally substituted by one or more groups (e.g. one) selected from halogen, CN, NO2 and an aryloxy group (e.g. phenoxy) optionally substituted by one or more halogen atoms (e.g. fluorine atoms). In particular, the aryloxy group is optionally substituted by one or more (e.g. one) groups selected from CN, NO2 and pentafluorophenoxy, especially optionally substituted by CN. In particular, the aryloxy group can be a phenoxy group.

[0205] According to a specific embodiment, X1 and X2 independently represent H, a halogen atom (such as Cl or Br), an optionally substituted (C1-C6) alkoxy or aryloxy group, with the proviso that X1 and X2 do not represent H at the same time. More particularly, the aryloxy group is optionally substituted by one or more groups (such as one) selected from halogen, CN, NO2 and an aryloxy group (such as a phenoxy group) optionally substituted by one or more halogen atoms (such as a fluorine atom). In particular, the aryloxy group is optionally substituted by one or more (such as one) groups selected from CN, NO2 and pentafluorophenoxy, in particular optionally substituted by CN. In particular, the aryloxy group can be a phenoxy group.

[0206] According to another particular embodiment, X1 and X2 independently of one another represent H, Cl, Br, methoxy or phenoxy substituted by CN, in particular H, Cl or Br, with the proviso that X1 and X2 do not simultaneously represent H.

[0207] Advantageously, X1 and X2 are the same and are not H, or one of X1 and X2 is H and the other is not H. When X1 and / or X2 are not H, they are halogen atoms (e.g. Cl or Br), (C1-C6) alkoxy, optionally substituted aryloxy, or -O-(CH2CH2O) r H; in particular a halogen atom (eg Cl or Br), a (C1-C6)alkoxy group or an optionally substituted aryloxy group; preferably Cl, Br, a methoxy group or a phenoxy group substituted by CN; in particular Cl or Br.

[0208] q represents 0, 1 or 2. Preferably, q represents 2.

[0209] X3 represents a functional group (when y=z=1 and Z is -NR4-(CH2) u -NR5-, which optionally has a terminal nitrogen of Z, or when c=w=y=0 and z'=1 and Z' is -NR4-(CH2) u -NR5-, which optionally has a terminal nitrogen at Z'), is intended to react with the drug (QH or Q-OH) to ultimately covalently link the drug to the binding unit (eg, antibody).

[0210] It is also conceivable to first introduce the spacer unit Y and the amino acid unit (W) on the drug portion before attaching the stretcher unit bearing the sulfonylmaleimide functional group. w In this case, compounds of formula (I) in which w=y=0 will be used (i.e. comprising only the Stretcher unit and the sulfonylmaleimide functional group), and in this case X3 represents a molecule to be combined with the amino acid unit (W). w The reactive functional group or the spacer unit Y is already attached to the drug unit.

[0211] When y=z=1 and Z is -NR4-(CH2) u -NR5-, or when c=w=y=0, z'=1 and Z' is -NR4-(CH2) u -NR5- (and forming an NH functional group with the terminal nitrogen of the Z or Z' group), X3 represents H, while in other cases, X3 represents OH, NH2 or a leaving group, for example OH or a leaving group. The leaving group can be a halogen atom (for example Cl, Br, I), a sulfonic acid group (for example OTf, OMs, OTs), N-succinimidyloxy, 4-nitro-phenoxy, pentafluorophenoxy or N-benzotriazoloxy. In particular, when y=z=1 and Z is -NR4-(CH2) u -NR5-, or when c=w=y=0, z'=1 and Z' is -NR4-(CH2) u In the case of -NR5-, X3 represents H, while in other cases X3 may more particularly be OH, Cl or N-succinimidyloxy.

[0212] Drug section

[0213] The drug moiety (Q) is the residue of drug QH or drug Q-OH.

[0214] The medicine according to the present invention can be any medicine useful in human or veterinary therapy, especially medicine for treating cancer. It can be especially cytotoxic agent. Advantageously, the medicine comprises a functional group capable of connecting the medicine to a connexon portion. It can also be envisioned that such a functional group is added to the medicine for connection. The functional group can be, for example, OH, SH, NH or COOH, and will react with the X3 end of the connexon to connect the medicine to the connexon portion. The coupling reaction can be, for example, nucleophilic substitution (for example, reaction of OH, SH, NH or COOH with X3=leaving group), peptide coupling (for example, reaction of COOH with X3=NH2 or ZX3 or Z'X3 ending with NH), esterification (reaction between COOH and OH), Mitsunobu reaction, etc.

[0215] The drug moiety Q can be, for example:

[0216] - a residue of an auristatin derivative, such as a monomethyl auristatin F (MMAF) residue (attached via its terminal NH or COOH group), a monomethyl auristatin E (MMAE) residue (attached via its terminal NH or OH group), a monomethyl dolastatin-10 residue (attached via its terminal NH group) or a residue of a derivative thereof, such as a drug moiety of formula (C) as defined below;

[0217]

[0218] - an anthracycline residue, for example a residue of daunorubicin, doxorubicin, epirubicin or idarubicin (attached via the NH2 group or the OH group of -COCH2OH), or a derivative thereof, for example a residue of 2-pyrrolinodoxorubicin or pro-2-pyrrolinodoxorubicin (attached via the OH group of -COCH2OH), or a residue of PNU-159682 (attached via the OH group of -COCH2OH) or a derivative thereof; in particular a residue of doxorubicin (attached via the OH group of -COCH2OH) OH group of -COCH2OH), a 2-pyrrolidoxorubicin residue, a pre-2-pyrrolidoxorubicin residue (linked via the OH group of -COCH2OH) or a PNU-159682 residue (linked via the OH group of -COCH2OH) or a residue of a derivative of PNU-159682, in particular as shown below; preferably a PNU-159682 residue (linked via the OH group of -COCH2OH) or a residue of a derivative of PNU-159682 (linked via the COOH) as shown below;

[0219]

[0220]

[0221]

[0222] - a residue of camptothecin or a derivative thereof, for example a residue of SN-38 (attached via its OH group);

[0223]

[0224] - a residue of a tubulysin, for example a residue of tubulysin A, tubulysin B, tubulysin C or tubulysin D (attached via a COOH group or an OH group, where present);

[0225]

[0226]

[0227] - a calicheamicin residue, such as esperamicin or calicheamicin gamma 1, or a derivative thereof such as N-acetyldimethylhydrazine calicheamicin (attached via its hydrazine moiety);

[0228]

[0229]

[0230] - a residue of a maytansinoid, for example a residue of maytansine (also known as maitansine), or a derivative thereof such as the residue of DM1 or DM4 (attached via an SH group); in particular a residue of DM1 or DM4 (attached via an SH group);

[0231]

[0232] - a duocarmycin residue, for example a residue of duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA or CC-1065 (attached via a CONH2 group); in particular a residue of CC-1065 (attached via a CONH2 group);

[0233]

[0234] - an amanitine residue (attached via an OH, NH, COOH or CONH2 group, in particular an OH group), for example a residue of α-amanitine, β-amanitine, γ-amanitine or ε-amanitine; in particular a residue of α-amanitine (in particular attached via a CH2OH group);

[0235]

[0236]

[0237] - a pyrrolobenzodiazepine (PBD) residue, for example an anthramycin residue (attached via its OH or NH2 group) or a SGD-1882 residue (attached via its NH2 group); in particular a SGD-1882 residue (attached via its NH2 group);

[0238]

[0239] - the residue of an immune checkpoint agonist, for example, advantageously a residue of a STING (stimulator of interferon genes) agonist of formula (D) as defined below (attached via OH, SH or NH), or a residue of an IDO (indoleamine 2,3-dioxygenase) inhibitor, for example, a residue of epacadostat (INCB024360) or BMS-986205.

[0240] Advantageously, the drug moiety Q is:

[0241] - a residue of an auristatin derivative, for example a residue of MMAF (attached via its terminal NH or COOH group), a residue of MMAE (attached via its terminal NH or OH group) or a residue of monomethyldolastatin-10 (attached via its terminal NH group) or a residue of a drug moiety of formula (C) as defined below;

[0242] - a residue of a STING agonist, in particular a residue of formula (D) as defined below; or

[0243] - an anthracycline residue, such as defined above, preferably a residue of PNU-159682 or a derivative thereof, as shown below:

[0244]

[0245] The drug moiety Q is in particular the residue of an anthracycline, for example as defined above, and preferably the residue of PNU-159682 or a derivative thereof, as shown below:

[0246]

[0247] According to a first embodiment, the residue of the auristatin derivative has the following formula (C):

[0248]

[0249] in:

[0250] -R1 is H or OH,

[0251] -R2 is (C1-C6)alkyl (e.g., methyl), COOH, COO-((C1-C6)alkyl) (e.g., COOMe) or thiazolyl (e.g., thiazol-2-yl),

[0252] - R3 is H or (C1-C6)alkyl (e.g. methyl), in particular (C1-C6)alkyl,

[0253] -X4 is O or NR9,

[0254] -R9 is H or (C1-C6) alkyl (e.g. methyl), and

[0255] -t is an integer from 1 to 8, in particular an integer from 1 to 6, advantageously an integer from 1 to 4, preferably 1 or 2.

[0256] According to a specific embodiment:

[0257] -R1 is OH, R2 is (C1–C6) alkyl, such as methyl; or

[0258] -R1 is H, R2 is thiazolyl such as thiazol-2-yl, COO-(C1–C6)alkyl such as COOMe or COOH.

[0259] Advantageously, R1 is H, R2 is thiazolyl such as thiazol-2-yl, COO-(C1-C6)alkyl such as COOMe, or COOH. Preferably, R1 is H, R2 is COOH or COOMe, in particular COOH.

[0260] t is an integer from 1 to 8, in particular an integer from 1 to 6, advantageously an integer from 1 to 4, preferably 1 or 2.

[0261] Advantageously, R3 is (C1-C6)alkyl, preferably methyl.

[0262] According to a particular embodiment, R1 is H, R2 is COOH or COOMe (preferably COOH), R3 is methyl, and t is 1 or 2.

[0263] Advantageously, X4 is NR9, wherein R9 is H or (C1-C6)alkyl, preferably H or methyl.

[0264] In a preferred embodiment:

[0265] -R1 is H, R2 is COOH, R3 is methyl, X4 is NR9, R9 is methyl and t is 1 or 2, or

[0266] -R1 is H, R2 is COOH, R3 is methyl, X4 is NR9, R9 is H and t is 1 or 2.

[0267] According to a preferred embodiment, the X4 group is relative to (CH2) t The group is located in the para position on the benzene ring.

[0268] Advantageously, the residue of the auristatin of formula (C) is selected from the following moieties:

[0269]

[0270]

[0271] The preparation of the auristatin derivatives is disclosed in, for example, WO 2014 / 174064 or WO 2015 / 162293.

[0272] According to a second embodiment, the STING agonist has the following formula (X):

[0273]

[0274] in:

[0275] -X11 and X 21 are independently O or S, preferably O,

[0276] -X 12 and X 22 are independently OH or SH, preferably SH,

[0277] -A 11 and A 21 are independently groups of the formula:

[0278]

[0279] Preferred in:

[0280] Z1 is OR 11 or NR 11 R 12 , where R 11 and R 12 H, R 13 or COR 13 , where R 13 is (C1–C6)alkyl, aryl or aryl(C1–C6)alkyl,

[0281] Z2 is H or NR 21 R 22 , where R 21 and R 22 H, R 23 or COR 23 , where R 23 is (C1–C6)alkyl, aryl or aryl(C1–C6)alkyl,

[0282] Z3 is N or CR 33 , preferably N, wherein R 33 is H or a halogen atom (such as F or Cl), and

[0283] Z4 is H or (C1–C6) alkyl,

[0284] -A 12 and A 22 are independently H, OH or F, and

[0285] -A2 is H, or A2 and A 22 connected to each other, where A2 is CH2 and A 22 It is O.

[0286] When Z1 is OH or Z4 is H, the following tautomeric forms can be obtained:

[0287]

[0288] According to a particular embodiment, the STING agonist has one of the following formulae:

[0289]

[0290] where X 11 、X 21 、X 12 、X 22 、A 11 、A 21 、A 12 、A 22 and A2 are as defined above or below.

[0291] According to another particular embodiment, the STING agonist has one of the following formulae:

[0292]

[0293] where X 11 、X 21 、X 12 、X 22 、A 11 、A 21 、A 12 、A 22 and A2 are as defined above or below.

[0294] Advantageously, X 11 and X 21 are all O. Advantageously, X 12 and X 22 At least one of them is SH, and preferably X 12 and X 22 Preferably, X 11 and X 21 All are O, and X 12 and X 22 All are SH.

[0295] In particular, R 11 and R 12 are H, and advantageously, R 11 、R 12 、R 21 and R 22 Both are H.

[0296] Z3 is especially N. Advantageously, Z1 is OH or NH2; Z2 is H or NH2; Z3 is N; and Z4 is H.

[0297] Preferably, A 11 and A 21 independently selected from (cytosine), (adenine), (Adenine-6-benzamide), (2,6-diaminopurine), (hypoxanthine), (guanine) and (guanine-2-isobutyramide); more preferably selected from cytosine, adenine, adenine-6-benzamide, 2,6-diaminopurine, hypoxanthine, guanine and guanine-2-isobutyramide; most preferably selected from adenine, hypoxanthine and guanine.

[0298] It is in particular ADU-S100 having the following formula:

[0299]

[0300] Alternatively, it may in particular be one of the compounds specifically disclosed in Lioux et al., J. Med. Chem., 2016, 59(22), pp 10253–10267.

[0301] The preparation of the STING agonist is disclosed in, for example, WO2014 / 179335, WO2016 / 096174, WO2016 / 145102, WO2017 / 106740 or WO2018 / 100558.

[0302] The STING agonist is linked to the linker moiety via the SH, OH or NH groups present on the molecule, i.e., via the group X 12 (OH or SH), X 22 (OH or SH), when R 11 and R 12 At least one of them is H(OH, NHR 11 or NHR 12 ) through Z1, or when R 21 and R 22 At least one of them is H(NHR 21 or NHR 22 ) are connected through Z2. Preferably, X 12 and X 22 At least one of the SH groups is SH, and the STING agonist is linked via the SH group.

[0303] Thus, the residue of the STING agonist advantageously has the following formula (D), (D-1), (D-2), (D-3), (D-1a), (D-2a), or (D-3a):

[0304]

[0305]

[0306] in:

[0307] -X 11 and X 21 As defined above,

[0308] -X 12 and X 22 As defined above or O or S,

[0309] -A 11 and A 21 As defined above, groups are independently of the formula:

[0310]

[0311] Preferred in:

[0312] Z1 is as defined above or is O or NR 11 ,

[0313] Z2 is as defined above or is NR 21 ,

[0314] Z3 is as defined above, and

[0315] Z4 is as defined above,

[0316] -A 12 and A 22 as defined above, and

[0317] -A2 is as defined above,

[0318] in:

[0319] When X 12 When it is O or S, then X 22 Not O and not S, Z1 is not O and not NR 11 , Z2 is not NR 21 , and the residues of the STING agonist are expressed by X 12 connected to the rest of the molecule;

[0320] When X 22 When it is O or S, then X 12 Not O and not S, Z1 is not O and not NR 11 , Z2 is not NR 21 , and the residues of the STING agonist are expressed by X 22 connected to the rest of the molecule;

[0321] When Z1 is O or NR11 When X 12 Not O and not S, X 22 Not O and not S, Z2 is not NR 21 , and the residues of the STING agonist are connected to the rest of the molecule via Z1;

[0322] When Z2 is NR 21 When X 12 Not O and not S, X 22 Not O and not S, Z1 is not O and not NR 11 , and the residues of the STING agonist are connected to the rest of the molecule via Z2.

[0323] Combined unit part

[0324] The binding unit is a peptide, a protein (eg, an engineered protein), an antibody (eg, a monoclonal antibody), or an antigen-binding fragment thereof.

[0325] Preferably, the binding unit according to the present invention is an antibody or an antigen-binding fragment thereof. Therefore, the binding unit-drug conjugate according to the present invention is an antibody-drug conjugate (ADC). In one embodiment, the antibody of the present invention is composed of a recombinant antibody. In another embodiment, the antibody of the ADC of the present invention is composed of a chemically synthesized antibody.

[0326] More specifically, the molecule is composed of a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0327] In one embodiment, the "antigen-binding fragment" is selected from Fv, scFv (sc means single chain), Fab, F(ab')2, Fab', scFv-Fc fragment or diabody, or any fragment whose half-life can be extended by chemical modification, such as the addition of polyalkylene glycol (e.g., polyethylene glycol) ("PEGylation") (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" means polyethylene glycol), or by incorporation into liposomes, and the fragment has at least one characteristic CDR of an antibody according to the present invention. Preferably, the "antigen-binding fragment" will comprise or consist of a partial sequence of the heavy or light variable chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100, more preferably at least 1 / 10 of the affinity of the antibody from which it is derived for the target. More preferably, the "antigen-binding fragment" will comprise or consist of at least three CDRs CDR-H1, CDR-H2 and CDR-H3 of the heavy variable chain and three CDRs CDR-L1, CDR-L2 and CDR-L3 of the light variable chain of the antibody from which it is derived.

[0328] According to a preferred embodiment, the binding unit is an IGF-1R antibody, a HER2 antibody or an antigen-binding fragment thereof.

[0329] More specifically, the HER2 antibody is trastuzumab.

[0330] In one embodiment of the present application, the antibody is an IGF-1R antibody, and the epitope of the antibody is preferably located in the extracellular domain of human IGF-1R (also called IGF-1R ECD).

[0331] In a particular embodiment, the antibody or any antigen-binding fragment thereof is capable of binding to ECF-1R, wherein the ECF-1R 50 Between 10×10 -10 to 1×10 -10 More preferably, it is between 8×10 -10 to 2×10 -10 between.

[0332] Competition for binding to IGF-1R can be determined by any method or technique known to those skilled in the art, such as, but not limited to, radiometric methods, Biacore, ELISA, flow cytometry, etc. "Competing for binding to IGF-1R" refers to competition of at least 20%, preferably at least 50%, more preferably at least 70%.

[0333] Binding to the same epitope can be determined by any method or technique known to those skilled in the art, such as, but not limited to, radiometric methods, Biacore, ELISA, flow cytometry, etc. "Binding to the same epitope as IGF-1R" refers to a competition of at least 20%, preferably at least 50%, more preferably at least 70%.

[0334] As described above, contrary to common knowledge, the present invention focuses on specific IGF-1R antibodies that exhibit high internalization capacity after binding to IGF-1R. As used herein, an "internalized" or "internalized" antibody (both expressions are similar) is an antibody that is taken up by (meaning "enters") the cell after binding to IGF-1R on mammalian cells. Such antibodies are useful as part of an ADC, as they guide or direct the attached cytotoxic agent into the targeted cancer cell. Once internalized, the cytotoxic agent triggers cancer cell death.

[0335] Advantageously, the IGF-1R antibodies according to the present invention all present identical sequences for CDR-H2, CDR-H3 and CDR-L2, and differ for the other three CDRs. This observation appears to be consistent with the common general knowledge concerning antibody binding specificity, with CDR-H3 being described as being the most important and involved in epitope recognition.

[0336] The key to the success of ADC therapy is believed to be target antigen specificity and internalization of the antigen-antibody complex into cancer cells. Obviously, non-internalized antigens are less effective in delivering cytotoxic agents than internalized antigens. The internalization process is variable across antigens and depends on multiple parameters that can be affected by the antibody.

[0337] In ADCs, the drug moiety possesses cytotoxic activity, while the antibody used is responsible for specificity against cancer cells and serves as a vehicle for entry into cells to properly initiate cytotoxicity. Therefore, to improve ADCs, the antibody should exhibit high internalization into the targeted cancer cells. The efficiency of antibody-mediated internalization varies significantly depending on the targeted epitope. Selecting an effective internalizing IGF-1R antibody requires extensive experimental data, not only investigating IGF-1R downregulation but also the effects of the IGF-1R antibody after internalization into cells.

[0338] In one embodiment, the internalization of the antibody according to the ADC of the present invention can be assessed by immunofluorescence or FACS (flow cytometry) (as exemplified herein below) or any method or process known to those skilled in the art. In a preferred embodiment, the antibody according to the ADC of the present invention is capable of inducing at least 30%, preferably 50%, and more preferably 80% internalization upon binding to IGF-1R.

[0339] The IGF-1R / antibody complex is internalized after the antibody binds to the ECD of the IGF-1R and induces a decrease in the amount of IGF-1R on the cell surface. The decrease can be quantified by any method known to those skilled in the art, such as, but not limited to, Western blotting, FACS, and immunofluorescence.

[0340] In one embodiment, the reduction (thereby reflecting internalization) can be preferably measured by FACS and expressed as the difference or delta between the mean fluorescence intensity (MFI) measured after 4 hours of antibody incubation at 4°C and the MFI after 4 hours of antibody incubation at 37°C.

[0341] As a non-limiting example, the Δ is determined based on the MFI obtained for untreated cells and cells treated with the antibody using i) breast cancer MCF7 cells after 4 hours of incubation with the antibodies described herein, and ii) a secondary antibody labeled with Alexa 488. This parameter is defined as calculated by the following formula: Δ(MFI 4℃ –MFI 37℃ ).

[0342] This difference between MFIs reflects downregulation of IGF-1R, as MFI is directly proportional to IGF-1R expressed on the cell surface.

[0343] In an advantageous aspect, the antibody triggers a Δ(MFI) of at least 280, preferably at least 400, on MCF-7 cells. 4℃ –MFI 37℃ ) of the antibody composition.

[0344] In more detail, the above-mentioned Δ can be measured according to the following procedure, which must be considered as an illustrative and non-limiting example:

[0345] a) treating and incubating tumor cells of interest with the antibodies of the present invention in cold (4° C.) or warm (37° C.) complete culture medium;

[0346] b) treating the treated cells of step a) with a secondary antibody, and in parallel treating untreated cells;

[0347] c) measuring the MFI (indicating the amount of IGF-1R present on the surface) of treated and untreated cells using a labeled secondary antibody capable of binding to the antibody of the invention; and

[0348] d) Δ was calculated by subtracting the MFI obtained from the treated cells from the MFI obtained from the untreated cells.

[0349] From this ΔMFI, the percentage of internalization can be determined as:

[0350] 100×(MFI4℃ -MFI 37℃ ) / MFI 4℃ .

[0351] Preferably, the antibody of the ADC according to the present invention exhibits an internalization percentage on MCF7 ranging from 50% to 99%, 70% to 90%, preferably from 75% to 87%.

[0352] A particular advantage of the antibodies described herein relies on their internalization rate.

[0353] It is generally known that for ADCs, it is desirable that the antibody used exhibits a rapid internalization rate, preferably within 24 hours, more preferably within 12 hours, and even more preferably within 6 hours after administration of the antibody.

[0354] In the present invention, the internalization rate, also referred to as the reduction of cell surface bound antibodies or the decay of cell surface antibodies, is expressed as t1 / 2 (half-life) and corresponds to the time required to obtain a 50% reduction in ΔMFI (this aspect will be clearly understood with reference to the following examples).

[0355] A particular advantage is that the antibodies of the ADC of the present invention have a t1 / 2 between 5 and 25 minutes, preferably between 10 and 20 minutes.

[0356] According to a particular embodiment of the present invention, the antibody comprises three heavy chain CDRs of sequences SEQ ID Nos. 1, 2 and 3 and three light chain CDRs of sequences SEQ ID Nos. 4, 5 and 6.

[0357] According to a particular embodiment of the present invention, the antibody comprises three heavy chain CDRs and three light chain CDRs, the heavy chain CDRs comprising or consisting of the following sequences: SEQ ID Nos. 1, 2 and 3, or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID Nos. 1, 2 or 3; the light chain CDRs comprising or consisting of the following sequences: SEQ ID Nos. 4, 5 and 6, or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID Nos. 4, 5 or 6.

[0358] According to a particular embodiment of the present invention, the binding unit is an antibody or an antigen-binding fragment thereof capable of binding to human IGF-1R, and the human IGF-1R is selected from:

[0359] i) an antibody comprising three heavy chain CDRs having CDR-H2 of SEQ ID No. 2 and CDR-H3 of SEQ ID No. 3, and three light chain CDRs having CDR-L2 of SEQ ID No. 5;

[0360] ii) an antibody that competes with the antibody of i) for binding to IGF-1R; and

[0361] iii) an antibody that binds to the same IGF-1R epitope as the antibody of i).

[0362] According to a particular embodiment of the present invention, the binding unit is an antibody or an antigen-binding fragment thereof capable of binding to human IGF-1R, and the human IGF-1R is selected from:

[0363] i) an antibody comprising three heavy chain CDRs of sequences SEQ ID No. 1, 2 and 3 and three light chain CDRs of sequences SEQ ID No. 4, 5 and 6;

[0364] ii) an antibody that competes with the antibody of i) for binding to IGF-1R; and

[0365] iii) an antibody that binds to the same IGF-1R epitope as the antibody of i).

[0366] In another embodiment, the antibody or any antigen-binding fragment thereof comprises three heavy chain CDRs comprising the sequences of SEQ ID Nos. 1, 2, and 3 and three light chain CDRs comprising the sequences of SEQ ID Nos. 4, 5, and 6.

[0367] The IMGT unique number has been defined to compare variable domains regardless of antigen receptor, chain type or species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT unique numbering, conserved amino acids always occupy the same position, such as cysteine ​​23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine ​​104 (2nd-CYS), and phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides standardized delimitations for framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104, and FR4-IMGT: 118 to 128) and complementarity determining regions (CDR1-IMGT: positions 27 to 38, CDR2-IMGT: 56 to 65, and CDR3-IMGT: 105 to 117). Since gaps represent unoccupied positions, the CDR-IMGT length (shown between brackets and separated by dots, e.g., [8.8.13]) is key information. IMGT unique numbers are used in the 2D graphical representation, called IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].

[0368] For amino acid sequences that exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity to a reference amino acid sequence, preferred examples include those comprising the reference sequence, a modification (especially deletion, addition or substitution, truncation or extension of at least one amino acid). In the case of substitution of one or more consecutive or non-consecutive amino acids, substitutions are preferred in which the substituted amino acids are replaced by "equivalent" amino acids. Here, the expression "equivalent amino acid" refers to any amino acid that may replace one of the structural amino acids, but which does not alter the biological activity of the corresponding antibody and the specific examples defined below.

[0369] Equivalent amino acids can be determined based on their structural homology to the amino acid they replace, or based on comparative testing of biological activities between various antibodies that may be generated.

[0370] As non-limiting examples, Table 1 below summarizes possible substitutions that may be made without resulting in a significant modification of the biological activity of the corresponding modified antibody; reverse substitutions may naturally be made under the same conditions.

[0371] Table 1

[0372] Original residue replace Ala(A) Val,Gly,Pro Arg(R) Lys,His Asn(N) Gln Asp(D) Glu Cys(C) Ser Gln(Q) Asn Glu(E) Asp Gly(G) Ala His(H) Arg Ile(I) Leu Leu(L) Ile,Val,Met Lys(K) Arg Met(M) Leu Phe(F) Tyr Pro(P) Ala Ser(S) Thr,Cys Thr(T) Ser Trp(W) Tyr Tyr(Y) Phe,Trp Val(V) Leu,Ala

[0373] A particular aspect of the present invention is that the antibodies do not bind to the insulin receptor (IR). This aspect is significant because the antibodies described herein will not have any negative impact on the IR (meaning insulin metabolism).

[0374] In another embodiment, another advantageous aspect of the antibody is that it can bind not only to human IGF-1R, but also to monkey IGF-1R, and more particularly to cynomolgus monkey IGF-1R. This aspect is also significant because it will facilitate the toxicity assessment required for clinical trials.

[0375] In another embodiment, the antibody consists of a monoclonal antibody.Monoclonal antibodies herein include murine, chimeric, and humanized antibodies, as described below.

[0376] The antibodies are preferably derived from murine hybridomas deposited at the French Collection of Cultures of Microorganisms (CNCM, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France), obtained by fusion of splenocytes / lymphocytes of Balb / C immunized mice and cells of the myeloma Sp 2 / O-Ag 14 cell line.

[0377] In one embodiment, the IGF-1R antibody consists of a murine antibody, which is then referred to as m[antibody name].

[0378] In one embodiment, the IGF-1R antibody consists of a chimeric antibody, which is then referred to as c[antibody name].

[0379] In one embodiment, the IGF-1R antibody consists of a humanized antibody, which is then referred to as hz[antibody name].

[0380] For the avoidance of doubt, in the following description, the expressions "IGF-1R antibody" and "[antibody name]" are analogous and encompass (unless otherwise indicated) murine, chimeric, and humanized forms of the IGF-1R antibody or "[antibody name]" described above. Where necessary, the prefixes m- (for murine), c- (for chimeric), or hz- (for humanized) are used.

[0381] For greater clarity, Table 2 below illustrates the CDR sequences of preferred antibodies according to the IMGT definition.

[0382] Table 2

[0383]

[0384]

[0385] It will be apparent to those skilled in the art that any combination of the six CDRs described above should be considered part of the present invention.

[0386] As can be seen from this Table 2, the CDR-H2, CDR-H3 and CDR-L2 of all antibodies described herein have identical sequences, a property which is particularly significant as described above.

[0387] According to a specific aspect, the antibody is a murine antibody, characterized in that the antibody further comprises light chain and heavy chain constant regions derived from an antibody of a species xenogeneic to the mouse, in particular human.

[0388] According to another specific aspect, the antibody is a chimeric (c) antibody, characterized in that the antibody further comprises light chain and heavy chain constant regions derived from an antibody of a species xenogeneic to the mouse, particularly human.

[0389] Chimeric antibodies are antibodies that contain the native variable regions (light and heavy chains) derived from an antibody of a given species and the constant regions of the light and heavy chains of an antibody of a species that is different from the given species.

[0390] Chimeric antibodies can be prepared using recombinant genetics techniques. For example, chimeric antibodies can be produced by cloning recombinant DNA comprising a promoter and a sequence encoding the variable region of a non-human (especially mouse) monoclonal antibody, and a sequence encoding the constant region of an antibody from a heterologous species (preferably human). The chimeric antibody according to the ADC of the present invention encoded by one of the recombinant genes can be, for example, a mouse-human chimera, the specificity of the antibody being determined by the variable region derived from mouse DNA and the isotype being determined by the constant region derived from human DNA.

[0391] According to one embodiment of the present invention, the antibody is selected from:

[0392] a) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 7, 2 and 3 and three light chain CDRs of SEQ ID Nos. 9, 5 and 11;

[0393] b) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 7, 2 and 3 and three light chain CDRs of SEQ ID Nos. 10, 5 and 11;

[0394] c) an antibody comprising three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3 and three light chain CDRs of sequence SEQ ID No. 9, 5 and 12; and

[0395] d) an antibody comprising the three heavy chain CDRs of SEQ ID Nos. 8, 2 and 3 and the three light chain CDRs of SEQ ID Nos. 9, 5 and 11.

[0396] In a preferred but non-limiting embodiment, the antibody is selected from:

[0397] a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 13 or any sequence showing at least 80% identity to SEQ ID No. 13 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;

[0398] b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 14 or any sequence showing at least 80% identity to SEQ ID No. 14 and three light chain CDRs of sequences SEQ ID Nos. 10, 5 and 11;

[0399] c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 15 or any sequence showing at least 80% identity to SEQ ID No. 15 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12;

[0400] d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 16 or any sequence showing at least 80% identity to SEQ ID No. 16 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and

[0401] e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 17 or any sequence showing at least 80% identity to SEQ ID No. 17 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12.

[0402] “Any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID No. 13 to 17” refers to a sequence that exhibits the three heavy chain CDRs SEQ ID Nos. 1, 2 and 3 and, in addition, exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to the complete sequences SEQ ID No. 13 to 17, outside the sequences corresponding to said CDRs (i.e., SEQ ID No. 1, 2 and 3).

[0403] According to one embodiment of the present invention, the antibody is selected from:

[0404] a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 13 and three light chain CDRs of sequence SEQ ID Nos. 9, 5 and 11;

[0405] b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 14 and three light chain CDRs of sequence SEQ ID Nos. 10, 5 and 11;

[0406] c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 15 and three light chain CDRs of sequence SEQ ID Nos. 9, 5 and 12;

[0407] d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 16 and three light chain CDRs of sequence SEQ ID Nos. 9, 5 and 11; and

[0408] e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 17 and three light chain CDRs of sequence SEQ ID Nos. 9, 5 and 12.

[0409] In another preferred but non-limiting embodiment, the antibody is selected from:

[0410] a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 18 or any sequence showing at least 80% identity to SEQ ID No. 18 and three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;

[0411] b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 19 or any sequence showing at least 80% identity to SEQ ID No. 19 and three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;

[0412] c) an antibody comprising a light chain variable domain of sequence SEQ ID No. 20 or any sequence showing at least 80% identity to SEQ ID No. 20 and three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;

[0413] d) an antibody comprising a light chain variable domain of sequence SEQ ID No. 21 or any sequence showing at least 80% identity to SEQ ID No. 21 and the three heavy chain CDRs of sequences SEQ ID Nos. 8, 2 and 3; and

[0414] e) an antibody comprising a light chain variable domain of sequence SEQ ID No. 22 or any sequence showing at least 80% identity to SEQ ID No. 22 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.

[0415] “Any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID Nos. 18 to 22” refers to sequences that exhibit the three light chain CDRs SEQ ID Nos. 4, 5 and 6, respectively, and, in addition, exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity with the complete sequences SEQ ID Nos. 18 to 22, excluding the sequences corresponding to said CDRs (i.e., SEQ ID Nos. 4, 5 and 6).

[0416] According to one embodiment of the present invention, the antibody is selected from:

[0417] a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 18 and three heavy chain CDRs of sequence SEQ ID Nos. 7, 2 and 3;

[0418] b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 19 and three heavy chain CDRs of sequence SEQ ID Nos. 7, 2 and 3;

[0419] c) an antibody comprising a light chain variable domain of sequence SEQ ID No. 20 and three heavy chain CDRs of sequence SEQ ID Nos. 7, 2 and 3;

[0420] d) an antibody comprising a light chain variable domain of sequence SEQ ID No. 21 and three heavy chain CDRs of sequence SEQ ID Nos. 8, 2 and 3; and

[0421] e) an antibody comprising a light chain variable domain of sequence SEQ ID No. 22 and three heavy chain CDRs of sequence SEQ ID Nos. 7, 2 and 3.

[0422] According to one embodiment of the present invention, the antibody is selected from:

[0423] a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 13 or any sequence showing at least 80% identity to SEQ ID No. 13 and a light chain variable domain of sequence SEQ ID No. 18 or any sequence showing at least 80% identity to SEQ ID No. 18;

[0424] b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 14 or any sequence showing at least 80% identity to SEQ ID No. 14 and a light chain variable domain of sequence SEQ ID No. 19 or any sequence showing at least 80% identity to SEQ ID No. 19;

[0425] c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 15 or any sequence showing at least 80% identity to SEQ ID No. 15 and a light chain variable domain of sequence SEQ ID No. 20 or any sequence showing at least 80% identity to SEQ ID No. 20;

[0426] d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 16 or any sequence showing at least 80% identity to SEQ ID No. 16 and a light chain variable domain of sequence SEQ ID No. 21 or any sequence showing at least 80% identity to SEQ ID No. 21; and

[0427] e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 17 or any sequence showing at least 80% identity thereto and a light chain variable domain of sequence SEQ ID No. 22 or any sequence showing at least 80% identity thereto.

[0428] The chimeric antibodies described herein can also be characterized by their constant domains, and more particularly, the chimeric antibodies can be selected or designed, for example, but not limited to, IgG1, IgG2, IgG3, IgM, IgA, IgD or IgE. More preferably, in the context of the present invention, the chimeric antibodies are IgG1 or IgG4.

[0429] According to one embodiment of the present invention, the antibody is a chimeric antibody in IgG1 format comprising the variable domains VH and VL as described above. More preferably, the chimeric antibody comprises the constant domain of VH of sequence SEQ ID No. 43 and the kappa domain of VL of sequence SEQ ID No. 45.

[0430] According to one embodiment of the present invention, the antibody is a chimeric antibody in IgG4 format comprising the variable domains VH and VL as described above. More preferably, the chimeric antibody comprises the constant domain of VH of sequence SEQ ID No. 44 and the kappa domain of VL of sequence SEQ ID No. 45.

[0431] In another preferred but non-limiting embodiment, the antibody is selected from:

[0432] a) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No. 23 or any sequence showing at least 80% identity to SEQ ID No. 23 and a light chain of sequence SEQ ID No. 28 or any sequence showing at least 80% identity to SEQ ID No. 28;

[0433] b) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No. 24 or any sequence showing at least 80% identity with SEQ ID No. 24 and a light chain of sequence SEQ ID No. 29 or any sequence showing at least 80% identity with SEQ ID No. 29;

[0434] c) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No. 25 or any sequence showing at least 80% identity to SEQ ID No. 25 and a light chain of sequence SEQ ID No. 30 or any sequence showing at least 80% identity to SEQ ID No. 30;

[0435] d) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No. 26 or any sequence showing at least 80% identity to SEQ ID No. 26 and a light chain of sequence SEQ ID No. 31 or any sequence showing at least 80% identity to SEQ ID No. 31; and

[0436] e) an antibody comprising or consisting of the heavy chain of sequence SEQ ID No. 27 or any sequence showing at least 80% identity to SEQ ID No. 27 and the light chain of sequence SEQ ID No. 32 or any sequence showing at least 80% identity to SEQ ID No. 32.

[0437] For greater clarity, the following Table 3 illustrates the sequences of the VH and VL of preferred chimeric antibodies, respectively.

[0438] Table 3

[0439]

[0440]

[0441] According to another specific aspect of the present invention, the antibody is a humanized antibody, characterized in that the constant regions of the light chain and heavy chain are derived from the λ or κ region and the γ-1, γ-2 or γ-4 region, respectively, of human antibodies.

[0442] Humanized antibodies or fragments thereof can be prepared by techniques known to those skilled in the art. The humanized antibodies are preferably used for methods relating to in vitro diagnosis or in vivo prevention and / or treatment. Other humanization techniques are also known to those skilled in the art, such as the "CDR implantation" technology described in PDL patents EP 0 451 216, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761. U.S. Patents 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also be cited.

[0443] In a preferred embodiment, the antibody comprises a heavy chain variable domain (VH) having:

[0444] i) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID Nos. 7, 2 and 3, respectively, and

[0445] ii) FR1, FR2 and FR3 derived from human germline IGHV1-46*01 (SEQ ID No. 46), and

[0446] iii) FR4 derived from human germline IGHJ4*01 (SEQ ID No. 48).

[0447] In a preferred embodiment, the antibody comprises a light chain variable domain (VL) having:

[0448] i) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID Nos. 9, 5 and 11, respectively, and

[0449] ii) FR1, FR2 and FR3 derived from human germline IGKV1-39*01 (SEQ ID No. 47), and

[0450] iii) FR4 derived from human germline IGKJ4*01 (SEQ ID No. 49).

[0451] In a preferred but non-limiting embodiment, the antibody comprises:

[0452] a) a heavy chain having CDR-H1, CDR-H2 and CDR-H3 of SEQ ID Nos. 7, 2 and 3, respectively, and FR1, FR2 and FR3 derived from human germline IGHV1-46*01 (SEQ ID No. 46), and FR4 derived from human germline IGHJ4*01 (SEQ ID No. 48); and

[0453] b) a light chain having CDR-L1, CDR-L2 and CDR-L3 of SEQ ID Nos. 9, 5 and 11, respectively, and FR1, FR2 and FR3 derived from human germline IGKV1-39*01 (SEQ ID No. 47), and FR4 derived from human germline IGKJ4*01 (SEQ ID No. 49).

[0454] In one embodiment, the antibody comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33 and a light chain variable domain (VL) of sequence SEQ ID No. 35. The humanized antibody is hereinafter referred to as hz208F2 ("Variant 1" or "Var. 1").

[0455] In another embodiment, the antibody comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33, wherein the sequence SEQ ID No. 33 comprises at least one backmutation selected from residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95.

[0456] In another embodiment, the antibody comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33, wherein the sequence SEQ ID No. 33 comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 back mutations selected from residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95.

[0457] For greater clarity, preferred back mutations are listed in Table 4 below.

[0458] Table 4

[0459] Residue number 20 34 35 38 48 50 59 61 mouse M I Y K L W K N people V M H R M I S A

[0460] Residue number 62 70 72 74 76 77 79 82 95 mouse E L A K S N A F F people Q M R T T S V E Y

[0461] In one embodiment, the antibody comprises a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein the sequence SEQ ID No. 35 comprises at least one backmutation selected from residues 22, 53, 55, 65, 71, 72, 77 and 87.

[0462] In one embodiment, the antibody comprises a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein the sequence SEQ ID No. 35 comprises 2, 3, 4, 5, 6, 7 or 8 back mutations selected from residues 22, 53, 55, 65, 71, 72, 77 or 87.

[0463] In another embodiment, the antibody comprises:

[0464] a) a heavy chain variable domain (VH) of sequence SEQ ID No. 33, wherein the sequence SEQ ID No. 33 comprises at least one backmutation selected from residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82, and 95; and

[0465] b) a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said sequence SEQ ID No. 35 comprises at least one backmutation selected from residues 22, 53, 55, 65, 71, 72, 77 and 87.

[0466] For greater clarity, preferred back mutations are listed in Table 5 below.

[0467] Table 5

[0468] Residue number 22 53 55 65 71 72 77 87 mouse S R H R Y S N F people T S Q S F T S Y

[0469] In this embodiment, the antibody comprises all of the above-mentioned back mutations and corresponds to an antibody comprising a heavy chain variable domain (VH) of sequence SEQ ID No. 34 and a light chain variable domain (VL) of sequence SEQ ID No. 36. The humanized antibody is hereinafter referred to as hz208F2 ("Variant 3" or "Var. 3").

[0470] In another embodiment, the present invention encompasses all humanized forms comprised between variant 1 and variant 3. In other words, the antibody corresponds to an antibody comprising a heavy chain variable domain (VH) of the "consensus" sequence SEQ ID No. 41 and a light chain variable domain (VL) of the "consensus" sequence SEQ ID No. 42. The humanized antibody as a whole is hereinafter referred to as hz208F2 ("Variant 2" or "Var. 2").

[0471] In a preferred but non-limiting embodiment, the antibody is selected from:

[0472] a) an antibody comprising a heavy chain variable domain having the sequence SEQ ID No. 33 or any sequence having at least 80%, preferably at least 85%, 90%, 95% and 98% identity thereto and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and

[0473] b) an antibody comprising a heavy chain variable domain having the sequence SEQ ID No. 34 or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity thereto and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11.

[0474] “Any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID No. 33 or 34” refers to a sequence that exhibits the three heavy chain CDRs SEQ ID Nos. 1, 2 and 3 and, in addition, exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to the complete sequence SEQ ID No. 33 or 34, excluding the sequences corresponding to said CDRs (i.e., SEQ ID Nos. 1, 2 and 3).

[0475] In one embodiment of the invention, the antibody is selected from:

[0476] a) an antibody comprising a heavy chain variable domain having the sequence of SEQ ID No. 33 or any sequence having at least 80% identity to SEQ ID No. 33 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and

[0477] b) an antibody comprising a heavy chain variable domain having the sequence of SEQ ID No. 34 or any sequence having at least 80% identity to SEQ ID No. 34 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11.

[0478] If no sequence or sequence showing at least 80% identity with a particular sequence is indicated in the relevant paragraphs of this specification, it must be understood that the sequence shows at least 80%, preferably 85%, 90%, 95% and 98% identity with the reference sequence. Regardless of whether these sequences contain CDR sequences, it is intended to mean that the sequence shows at least these CDRs are identical to the CDRs of the reference sequence, and the 80%, preferably 85%, 90%, 95% and 98% identity with the entire sequence must be calculated from the remaining sequence outside the sequences corresponding to these CDRs.

[0479] In a preferred but non-limiting embodiment, the antibody is selected from:

[0480] a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 35, or any sequence having at least 80%, preferably at least 85%, 90%, 95% and 98% identity thereto, and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3; and

[0481] b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 36, or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity thereto, and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.

[0482] “Any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID No. 35 or 36” refers to a sequence that exhibits the three light chain CDRs SEQ ID Nos. 4, 5 and 6 and, in addition, exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to the complete sequence SEQ ID No. 35 or 36, excluding the sequences corresponding to said CDRs (i.e., SEQ ID Nos. 4, 5 and 6).

[0483] In one embodiment of the invention, the antibody is selected from:

[0484] a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 35, or any sequence having at least 80% identity to SEQ ID No. 35, and three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3; and

[0485] b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 36, or any sequence having at least 80% identity to SEQ ID No. 36, and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.

[0486] The humanized antibodies described herein can also be characterized by constant domains, and more particularly, the humanized antibodies can be selected or designed, for example, but not limited to, IgG1, IgG2, IgG3, IgM, IgA, IgD or IgE. More preferably, in the context of the present invention, the humanized antibodies are IgG1 or IgG4.

[0487] According to one embodiment of the present invention, the antibody is a humanized antibody in IgG1 format comprising the variable domains VH and VL as described above. More preferably, the humanized antibody comprises the constant domain of VH of sequence SEQ ID No. 43 and the kappa domain of VL of sequence SEQ ID No. 45.

[0488] According to one embodiment of the present invention, the antibody is a humanized antibody in IgG4 format comprising the variable domains VH and VL as described above. More preferably, the humanized antibody comprises the constant domain of VH of sequence SEQ ID No. 44 and the kappa domain of VL of sequence SEQ ID No. 45.

[0489] In another embodiment of the present invention, the antibody is selected from:

[0490] a) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No. 37 or any sequence having at least 80% identity thereto and a light chain of sequence SEQ ID No. 39 or any sequence having at least 80% identity thereto; and

[0491] b) an antibody comprising or consisting of the heavy chain of sequence SEQ ID No. 38 or any sequence with at least 80% identity thereto and the light chain of sequence SEQ ID No. 40 or any sequence with at least 80% identity thereto.

[0492] For greater clarity, Table 6a below illustrates non-limiting examples of sequences of VH and VL of variant 1 (Var. 1) and variant 3 (Var. 3) of the humanized antibody hz208F2. It also includes the consensus sequence of variant 2 (Var. 2).

[0493] Table 6a

[0494]

[0495] In another preferred but non-limiting embodiment, the antibody is selected from:

[0496] a) an antibody comprising a heavy chain variable domain and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11, wherein the sequence of the heavy chain variable domain is selected from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80, or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80;

[0497] b) an antibody comprising a light chain variable domain and three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3, wherein the sequence of the light chain variable domain is selected from SEQ ID Nos. 57 or 60, or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID Nos. 57 or 60;

[0498] c) an antibody comprising a heavy chain variable domain and a light chain variable domain, the sequence of the heavy chain variable domain being selected from the group consisting of SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80, or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity thereto; and the sequence of the light chain variable domain being selected from the group consisting of SEQ ID Nos. 57 or 60, or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity thereto.

[0499] In another embodiment of the present invention, the antibody is selected from:

[0500] a) an antibody comprising a heavy chain sequence of SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80, or any sequence at least 80% identical to SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80; and a light chain sequence of SEQ ID No. 57 or any sequence at least 80% identical to SEQ ID No. 57; and

[0501] b) an antibody comprising a heavy chain sequence of SEQ ID Nos. 56, 64, 68 and 78, or any sequence at least 80% identical to SEQ ID Nos. 56, 64, 68 or 78; and a light chain sequence of SEQ ID No. 60 or any sequence at least 80% identical to SEQ ID No. 60.

[0502] In another embodiment of the present invention, the antibody is selected from:

[0503] a) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 58 or any sequence having at least 80% identity to SEQ ID No. 58, and the light chain sequence of SEQ ID No. 59 or any sequence having at least 80% identity to SEQ ID No. 59;

[0504] b) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 58 or any sequence having at least 80% identity to SEQ ID No. 58, and the light chain sequence of SEQ ID No. 61 or any sequence having at least 80% identity to SEQ ID No. 61;

[0505] c) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 63 or any sequence having at least 80% identity to SEQ ID No. 63, and the light chain sequence of SEQ ID No. 59 or any sequence having at least 80% identity to SEQ ID No. 59;

[0506] d) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 65 or any sequence with at least 80% identity to SEQ ID No. 65, and the light chain sequence of SEQ ID No. 59 or any sequence with at least 80% identity to SEQ ID No. 59;

[0507] e) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 65 or any sequence having at least 80% identity to SEQ ID No. 65, and the light chain sequence of SEQ ID No. 61 or any sequence having at least 80% identity to SEQ ID No. 61;

[0508] f) an antibody comprising, or consisting of, a heavy chain sequence of SEQ ID No. 67 or any sequence having at least 80% identity to SEQ ID No. 67, and a light chain sequence of SEQ ID No. 59 or any sequence having at least 80% identity to SEQ ID No. 59;

[0509] g) an antibody comprising, or consisting of, a heavy chain sequence of SEQ ID No. 69 or any sequence with at least 80% identity to SEQ ID No. 69, and a light chain sequence of SEQ ID No. 59 or any sequence with at least 80% identity to SEQ ID No. 59;

[0510] h) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 69 or any sequence with at least 80% identity to SEQ ID No. 69, and the light chain sequence of SEQ ID No. 61 or any sequence with at least 80% identity to SEQ ID No. 61;

[0511] i) an antibody comprising, or consisting of, a heavy chain sequence of SEQ ID No. 71 or any sequence having at least 80% identity to SEQ ID No. 71, and a light chain sequence of SEQ ID No. 59 or any sequence having at least 80% identity to SEQ ID No. 59;

[0512] j) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 73 or any sequence having at least 80% identity to SEQ ID No. 73, and the light chain sequence of SEQ ID No. 59 or any sequence having at least 80% identity to SEQ ID No. 59;

[0513] k) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 75 or any sequence with at least 80% identity to SEQ ID No. 75, and the light chain sequence of SEQ ID No. 59 or any sequence with at least 80% identity to SEQ ID No. 59;

[0514] l) an antibody comprising, or consisting of, a heavy chain sequence of SEQ ID No. 77 or any sequence having at least 80% identity to SEQ ID No. 77, and a light chain sequence of SEQ ID No. 59 or any sequence having at least 80% identity to SEQ ID No. 59;

[0515] m) an antibody comprising, or consisting of, a heavy chain sequence of SEQ ID No. 79 or any sequence having at least 80% identity to SEQ ID No. 79, and a light chain sequence of SEQ ID No. 59 or any sequence having at least 80% identity to SEQ ID No. 59;

[0516] n) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 79 or any sequence having at least 80% identity to SEQ ID No. 79, and the light chain sequence of SEQ ID No. 61 or any sequence having at least 80% identity to SEQ ID No. 61; and

[0517] o) an antibody comprising, or consisting of, the heavy chain sequence of SEQ ID No. 81 or any sequence with at least 80% identity to SEQ ID No. 81, and the light chain sequence of SEQ ID No. 59 or any sequence with at least 80% identity to SEQ ID No. 59.

[0518] In other words, the antibody may be an antibody comprising:

[0519] a) a heavy chain sequence selected from the group consisting of SEQ ID Nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81, or any sequence at least 80% identical to SEQ ID Nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81; and

[0520] b) a light chain sequence selected from the group consisting of SEQ ID Nos. 59 and 61 or any sequence having at least 80% identity to SEQ ID Nos. 59 and 61.

[0521] In one embodiment of the invention, the antibody is selected from:

[0522] a) a heavy chain sequence selected from the group consisting of SEQ ID Nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81, or any sequence at least 80% identical to SEQ ID Nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81; and

[0523] b) a light chain sequence selected from the group consisting of SEQ ID Nos. 59 and 61 or any sequence having at least 80% identity to SEQ ID Nos. 59 and 61.

[0524] For greater clarity, Table 6b below illustrates non-limiting examples of sequences (variable domain and full length) of the VH and VL of different variants of the humanized antibody hz208F2.

[0525] Table 6b

[0526]

[0527]

[0528] According to another aspect of the present invention, the antibody is an antibody selected from the following: i) an antibody produced by hybridomas I-4757, I-4773, I-4775, I-4736 or I-4774 deposited at the CNCM on May 30, 2013, June 26, 2013, June 26, 2013, April 24, 2013 and June 26, 2013, respectively, by the Pasteur Institute in France; or ii) an antibody that competes with the antibody of i) for binding to IGF-1R; or iii) an antibody that binds to the same IGF-1R epitope as the antibody of i).

[0529] According to a particular aspect, the binding unit is an antibody or antigen-binding fragment thereof as described above, which is used as an addressing vehicle for delivering a cytotoxic agent to a host target site, wherein the host target site consists of an epitope located in IGF-1R, preferably the extracellular domain of IGF-1R, more preferably human IGF-1R (SEQ ID No. 50), even more preferably the extracellular domain of human IGF-1R (SEQ ID No. 51), even more preferably the N-terminus of the extracellular domain of human IGF-1R (SEQ ID No. 52), or any natural variant sequence thereof.

[0530] In a preferred embodiment, the host target site is a target site of a mammalian cell, more preferably a target site of a human cell, and more preferably a target site of a cell that expresses IGF-1R naturally or through genetic recombination.

[0531] In another embodiment, the host target site is a target site in a cell of a patient, preferably a human suffering from cancer, preferably a cancer expressing IGF-1R or a cancer associated with IGF-1R.

[0532] Cancers expressing IGF-1R or cancers associated with IGF-1R specifically include cancers in which tumor cells express or overexpress all or part of IGF-1R on their surface.

[0533] IGF-1R antibodies that can be used as binding units in the present invention are in particular described in WO2015 / 162291 / WO2015 / 162292 or WO2015 / 162293.

[0534] Linker molecules

[0535] The linker of formula (I) according to the present invention (preferably wherein q=2) can be used to covalently link a drug to a binding unit, such as an antibody (eg a monoclonal antibody) or an antigen-binding fragment thereof.

[0536] To this end, the sulfonylmaleimide portion of the linker can react with the sulfhydryl moiety present on the binding unit, while the X3 end of the linker can react with the functional group (QH or Q-OH) present on the drug.

[0537] Linker molecules can be prepared according to various synthetic methods exemplified in the experimental section.

[0538] When X1 and X2 are independently selected from H and Cl, and at least one is Cl, the linker according to the present invention can be prepared by reacting a disulfide compound of the formula L-NHCO-CH2CH2-SS-CH2CH2-CONH-L with a chlorinating agent (e.g., SO2Cl2), wherein L represents L1-(CO) c -(W) w -(Y) y -X3, optionally in protected form.

[0539] When X1 and X2 are independently selected from H and Br, and at least one is Br, the linker according to the present invention can be represented by the formula The compound is prepared by reaction with a brominating agent (such as Br2), wherein L represents L1-(CO) c -(W) w -(Y) y -X3, optionally in protected form.

[0540] When at least one of X1 and X2 is (C1-C6)alkoxy, optionally substituted aryloxy, or -O-(CH2CH2O) r When H, the linker according to the present invention can be formed by reacting with the corresponding linker of formula (I) (wherein at least one of X1 and X2 is Cl or Br, optionally in protected form) a -OH alcohol (where R arepresents (C1-C6)alkyl, optionally substituted aryl, or -(CH2CH2O) r H) Prepared by nucleophilic substitution reaction.

[0541] It is also contemplated to form sulfonylmaleimide moieties wherein a truncated linker moiety (e.g. wherein L = L1-(CO) c -X6, wherein an X6 functional group such as NH2, OH or a leaving group, optionally in protected form) is grafted thereon, and the synthesis of the linker is completed after formation of the sulfonylmaleimide moiety, in particular as illustrated below in the synthesis of drug-linker conjugates.

[0542] In addition, an oxidation step may be performed to convert S(O) q The group is converted to the desired oxidation state (i.e. preferably q=2). The oxidation step is well known to those skilled in the art. The oxidizing agent used may be, for example, mCPBA, RuO4 or RuCl3 / NaIO4.

[0543] Further protection / deprotection steps may be performed in the above methods, and said steps and their reaction conditions are well known to those skilled in the art.

[0544] The linker obtained can be separated from the reaction medium by methods well known to those skilled in the art, for example by extraction, evaporation of the solvent or by precipitation or crystallization followed by filtration.

[0545] If necessary, the linker can also be purified by methods well known to those skilled in the art, such as recrystallization, distillation, silica gel column chromatography, or high performance liquid chromatography (HPLC).

[0546] Drug-linker conjugates

[0547] The drug-linker conjugate of formula (II) according to the present invention (preferably wherein q=2) can be used to covalently link the drug to a binding unit, such as an antibody (eg, a monoclonal antibody) or an antigen-binding fragment thereof.

[0548] To this end, the sulfonylmaleimide portion of the drug-linker conjugate is capable of reacting with the sulfhydryl moiety present on the binding unit.

[0549] Drug-linker conjugates can be prepared according to various synthetic methods. In practice, the linker of formula (I) is capable of reacting with the drug (QH or Q-OH) to form a conjugate. However, other possibilities can be envisioned in which the linker is gradually formed on the drug molecule, i.e., first the first portion of the linker is grafted onto the drug, and the resulting compound is reacted with a truncated linker molecule to form the drug-linker conjugate.

[0550] Thus, the following non-limiting synthetic routes can be used to prepare the drug-linker conjugates of formula (II) according to the present invention, although other synthetic routes are contemplated.

[0551] In all of these synthetic routes, further protection / deprotection / substitution steps may be performed, which steps and their reaction conditions are well known to those skilled in the art.

[0552] The obtained drug-linker conjugate can be separated from the reaction medium by methods well known to those skilled in the art, for example by extraction, evaporation of the solvent or by precipitation or crystallization followed by filtration.

[0553] If necessary, the drug-linker conjugate can be purified by methods well known to those skilled in the art, such as recrystallization, distillation, silica gel column chromatography, or high performance liquid chromatography (HPLC).

[0554] Synthesis route I represented by Scheme I:

[0555]

[0556] Plan I

[0557] As described in detail below, the terminal sulfonylmaleimide moiety can be formed from a group already present on the drug to which a precursor linker moiety is grafted.

[0558] Step 1: 3-(2-chlorocarbonyl-ethyldisulfide)-propionyl chloride and H2N-L1-(CO) c -(W) w -(Y) y -Q (i.e., a drug molecule to which a portion of the linker has been grafted). The reaction can be carried out in the presence of a base (e.g., trimethylamine). The reaction can be carried out in a solvent (e.g., DCM), particularly at a temperature between 0°C and room temperature.

[0559] 3-(2-Chlorocarbonyl-ethyldisulfide)-propionyl chloride can be prepared from 3,3'-disulfanyldipropionic acid by known methods for forming acid chlorides, such as by reaction with (COCl). The reaction can be carried out in a solvent such as DCM, especially at room temperature. A catalytic amount of DMF may be added.

[0560] It is also conceivable, for example, to react 3-(2-chlorocarbonyl-ethyldisulfide)-propionyl chloride with an optionally protected form of a compound of the formula H2N-L1-(CO) c -X3 and in a subsequent step complete the synthesis of the linker moiety grafted with Q, in particular according to one of the other synthetic routes described below.

[0561] Step 2: The molecule obtained in step 1 can be subjected to cyclization and chlorination in the presence of SO2Cl2, especially when SO2Cl2 is in great excess (e.g., 5 to 10 eq., e.g., about 9 eq.). The reaction can be carried out in a solvent (e.g., DCM), especially at room temperature.

[0562] The chlorine atom can be converted into another X1 or X2 group (other than H) by well known methods, for example by nucleophilic substitution.

[0563] Step 3: If necessary, the molecule obtained in step 2 is oxidized to obtain the drug-linker of formula (IIa). The oxidation step can be carried out under conditions well known to those skilled in the art, in particular in the presence of mCPBA (e.g., 10 eq.). The reaction can be carried out in a solvent (e.g., DCM), in particular at room temperature.

[0564] Synthesis route II represented by Scheme II:

[0565]

[0566] Option II

[0567] Direct coupling can be performed between the drug (QH or Q-OH) and the linker of formula (I), the conditions of which depend on X3 and the nature of the functional groups present on the drug.

[0568] The coupling can be a substitution, such as a nucleophilic substitution or a Mitsunobu reaction, and the reaction conditions of the chemical reaction are well known to those skilled in the art.

[0569] When X3 = OH and at least y = 1, w ≠ 0 or c = 1 (i.e., the terminal functional group of the linker of formula (I) is COOH) and QH comprises an NH functional group, the coupling between the linker of formula (I) and the drug (QH) can be a peptide coupling well known to those skilled in the art. The terminal COOH functional group can also be converted into an acyl chloride COCl, which can then react with a nucleophilic functional group (e.g., NH or OH) present on the drug (QH).

[0570] When X3=NH2; or X3=H, y=z=1 and Z=-NR4-(CH2) u -NR5-; or X3=H, c=w=y=0, z'=1 and Z' is -NR4-(CH2) u When -NR5- (i.e., the terminal functional group of the linker of formula (I) is NH) and Q-OH comprises a COOH functional group, the coupling between the linker of formula (I) and the drug (Q-OH) can be a peptide coupling well known to those skilled in the art. The terminal COOH functional group of the drug can also be converted into an acyl chloride, COCl, which can then react with the NH functional group.

[0571] If desired, an additional oxidation step can be performed to convert S(O) q The group is converted to the desired oxidation state (i.e. preferably q=2). The oxidation step is well known to those skilled in the art. The oxidizing agent used may be, for example, mCPBA, RuO4 or RuCl3 / NaIO4.

[0572] Synthesis route III represented by schemes IIIa and IIIb:

[0573]

[0574] Scheme IIIa (where c=1)

[0575]

[0576] Scheme IIIb (where w≠0)

[0577] As shown in Schemes IIIa and IIIb, peptide coupling can also be performed between a truncated linker with a COOH functional group and a drug moiety grafted to another portion of the linker. The terminal COOH functional group of the truncated linker can also be converted into an acyl chloride, COCl, which can then react with the NH functional group of another reactant. The reaction conditions for this reaction are well known to those skilled in the art.

[0578] If desired, an additional oxidation step can be performed to convert S(O) q The group is converted to the desired oxidation state (ie preferably q=2), for example in the presence of mCPBA, RuO4 or RuCl3 / NaIO4.

[0579] Synthesis route IV represented by Scheme IV:

[0580]

[0581] Option IV

[0582] The coupling between the sulfonylmaleimide moiety and the drug to which the rest of the linker has been grafted can also be carried out as shown in Scheme IV, wherein X5 represents OH or a leaving group as defined above.

[0583] The coupling may be a substitution, such as a nucleophilic substitution.

[0584] If desired, an additional oxidation step can be performed to convert S(O) q The group is converted to the desired oxidation state (ie preferably q=2), for example in the presence of mCPBA, RuO4 or RuCl3 / NaIO4.

[0585] Synthesis Route V represented by Schemes Va and Vb:

[0586]

[0587] Plan Va

[0588]

[0589] Plan Vb

[0590] When the linker comprises a heteroarylene moiety having a divalent 1H-1,2,3-triazole, the heteroarylene can be formed by a click chemistry reaction between an azide and an alkyne under conditions well known to those skilled in the art, as shown in Schemes Va and Vb above, wherein L2 represents -(CH2) n or -(CH2CH2O) m -CH2-CH2-, L3 represents -(CH2) p -or-(CH2CH2O) m -CH2-CH2-, L2 and L3 are not -(CH2CH2O) m -CH2-CH2- group.

[0591] If desired, an additional oxidation step can be performed to convert S(O) q The group is converted to the desired oxidation state (ie preferably q=2), for example in the presence of mCPBA, RuO4 or RuCl3 / NaIO4.

[0592] Binding unit-drug conjugates

[0593] Binding unit-drug conjugates, such as antibody-drug conjugates, can be prepared by the following methods:

[0594] 1) forming a sulfhydryl function on the binding unit, in particular by reducing disulfide bonds; and

[0595] 2) reacting the binding unit with the sulfhydryl functional group with a drug-linker conjugate, thereby covalently linking the drug moiety to the binding unit by reacting the sulfonylmaleimide functional group with the sulfhydryl functional group.

[0596] This method is shown below in Scheme VI.

[0597]

[0598] Plan VI

[0599] Pharmaceutical composition

[0600] The pharmaceutical composition according to the present invention comprises a binding unit-drug conjugate of formula (III) or (IV) and at least one pharmaceutically acceptable excipient.

[0601] The pharmaceutical composition of the present invention can be used for enteral (e.g. oral) or parenteral (e.g. intravenous) administration, preferably oral or intravenous administration. The active ingredient can be given to animals, preferably mammals, including humans, in a unit form for administration mixed with conventional pharmaceutical excipients.

[0602] For oral administration, the pharmaceutical composition may be in solid or liquid (solution or suspension) form.

[0603] Solid composition can be in the form of tablet, gelatin capsule, powder, granule etc.In tablet, can before tabletting, active component and pharmaceutical carrier such as gelatin, starch, lactose, magnesium stearate, talcum powder, gum arabic etc. are mixed.Tablet can further be coated, especially with sucrose or other suitable material coating, or can be processed in the mode of extending or delaying activity.In powder or granule, active component can be mixed or granulated with dispersant, wetting agent or suspending agent and flavoring agent or sweetener.In gelatin capsule, active component can be introduced in soft or hard gelatin capsule in the form of powder or granule (for example mentioned before) or in the form of liquid composition (for example mentioned below).

[0604] The liquid composition can comprise the active ingredient and a sweetener, flavoring agent or suitable coloring agent in a solvent such as water. The liquid composition can also be obtained by suspending or dissolving a powder or granules as described above in a liquid such as water, fruit juice, milk. It can be, for example, a syrup or elixir.

[0605] For parenteral administration, the composition may be in the form of an aqueous suspension or solution, which may contain a suspending agent and / or a wetting agent. The composition is advantageously sterile. It may be in the form of an isotonic solution (particularly compared to blood).

[0606] The parenteral compositions will advantageously comprise a physiologically acceptable vehicle, generally based on an isotonic saline solution, ie 0.9% NaCl in water (normal saline). Water-immiscible cosolvents such as ethanol, glycerol, propylene glycol or n-lactamide may also be used.

[0607] The parenteral composition of the present invention may further comprise one or more additives, such as suspending agents, wetting agents, preservatives, antioxidants, chelating agents, buffers, tonicity regulators, etc. Such additives are conventional to those skilled in the art.

[0608] The suspending agent may be alginate, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, gum (such as gum arabic, tragacanth or xanthan gum), gelatin, carrageenan, polyvinyl pyrrolidone, or the like.

[0609] The wetting agent may be glycerol, propylene glycol or a nonionic surfactant such as lecithin, polysorbate or poloxamer.

[0610] The preservative may be benzyl alcohol, phenol, cresol, chlorobutanol, parabens (eg, methyl paraben, propyl paraben or propyl paraben), benzalkonium chloride, benzethonium chloride, or the like.

[0611] The antioxidant may be ascorbic acid, citric acid, acetylcysteine, sulfite (bisulfite, metabisulfite), monothioglycerol, sodium formaldehyde sulfoxylate, thiourea, tocopherol, and the like.

[0612] The chelating agent may be an ethylenediaminetetraacetic acid (EDTA) salt.

[0613] The buffer can be acetate, citrate, tartrate, phosphate, triethanolamine (TRIS), etc.

[0614] The tonicity regulator can be glucose, glycerol, sodium chloride, glycerin, mannitol, etc.

[0615] The binding unit-drug conjugates of the present invention can be used in pharmaceutical compositions at a dosage of 0.01 mg to 1000 mg per day, with only one dose per day or multiple doses per day, for example, twice a day in equal doses. The daily dosage is advantageously between 5 mg and 500 mg, and more advantageously between 10 mg and 200 mg. However, it may be necessary to use dosages outside these ranges, as will be appreciated by those skilled in the art.

[0616] Treating cancer

[0617] The Binding unit-drug conjugates of Formula (III) or (IV) or pharmaceutical compositions comprising the Binding unit of Formula (III) or (IV) are useful for treating cancer, particularly when the drug moiety (Q) they comprise is the residue of a drug (QH) useful for treating cancer (e.g., a cytotoxic agent).

[0618] Binding unit-drug conjugates, such as antibody-drug conjugates (ADCs), combine the binding specificity of a binding unit (eg, an antibody) with the potency of a drug (eg, a cytotoxic agent).

[0619] The use of binding unit-drug conjugates, such as ADCs, allows for the local delivery of drugs that, if administered in unconjugated form, would result in unacceptable levels of toxicity to normal cells. In other words, maximum efficacy with minimal toxicity is sought.

[0620] The cancer can be, for example, but not limited to, prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, glioblastoma, colon cancer, gastric cancer, kidney cancer, pancreatic cancer, head and neck cancer, or any other cancer associated with expression of the antigen targeted by the antibody on tumor cells.

[0621] The invention is illustrated by the following non-limiting examples and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0622] Figure 1A 、 Figure 2 、 Figure 3A 、 Figure 4A 、 Figure 5A 、 Figure 6A 、 Figure 7A 、 Figure 21 and Figure 22A Shows the mass spectrum of the drug-linker conjugate according to the present invention.

[0623] Figure 1B 、 Figure 3B 、 Figure 4B 、 Figure 5B 、 Figure 6B 、 Figure 7B 、 Figure 8 and Figure 9 represents the drug-linker conjugate according to the present invention 1 H-NMR spectrum.

[0624] Figure 10 and Figure 11 Shows the mass spectrum of the drug-somatostatin conjugate according to the present invention.

[0625] Figure 12 Figure 2 shows SDS-PAGE analysis of Ab1 antibody (1) and purified ADCs according to the present invention (ADC1-A (2), ADC1-B (3), ADC1-C (4), ADC1-D (5), ADC1-E (6), ADC1-F (7), and ADC1-G (8)) under reducing and non-reducing conditions. The bands observed on the gel correspond to fully bridged antibody (i.e., LHHL); partially bridged (i.e., HHL, HH, HL); and unbridged (i.e., H and L).

[0626] Figure 13 SEC analysis of the Abl antibody and ADCs according to the present invention (ADC1-A, ADC1-B, ADC1-C, ADC1-D, ADC1-E, ADC1-F, and ADC1-G) is shown.

[0627] Figure 14A 、 Figure 14B 、 Figure 14C and Figure 14DADC m / z spectra before deconvolution are shown for ADCs according to the present invention: (A) ADC1-A, (B) ADC1-B, (C) ADC1-C, and (D) ADC-1D, respectively.

[0628] Figure 15A 、 Figure 15B and Figure 15C Represents the DAR distribution after Maxent deconvolution of (A) ADC1-A, (B) ADC1-B, and (C) ADC1-D, respectively.

[0629] Figure 16A and Figure 16B Shown are native mass spectrometry analyses of ADCs: (A) reference ADC Ref-A, (B) ADC1-C according to the present invention.

[0630] Figure 17A 、 Figure 17B and Figure 17C Represents the results of in vitro stability studies showing the percentage of total antibody (100%) and ADC at each time point (D0, D3, D7, and D14) for (1) human, (2) cynomolgus monkey, (3) mouse, and (4) rat sera, respectively, for (A) reference ADC Ref-B, (B) ADC1-C, and (C) ADC1-E.

[0631] Figure 18A and Figure 18B In vitro cytotoxicity evaluation of different ADCs in NCI-H2122 (A) and MCF-7 (B) cells, respectively.

[0632] Figure 19 and Figure 20 Represents the in vivo activity of ADC1-C and the reference ADC Ref-A in an ovarian cancer model.

[0633] Figure 22B shows the TOF-MS spectrum of the drug-linker conjugate according to the present invention.

[0634] Figure 23A Shown are SEC analyses of Ab1 antibody and ADC according to the present invention synthesized using PNU-159682 derivatives.

[0635] Figure 23B Shown are SEC analyses of Ab2 antibody and ADC according to the present invention synthesized using PNU-159682 derivatives.

[0636] Figure 24: Figure 24A 、 Figure 24B 、 Figure 24C and Figure 24DADC m / z spectra before ADC deconvolution according to the present invention are shown: (A) hz208F2-F562524, (B) c9G4-F562524, (C) hz208F2-F562616, and (D) c9G4-F562646, respectively.

[0637] Figure 25: Figure 25A 、 Figure 25B 、 Figure 25C and Figure 25D 1 and 2 show the DAR distributions of the ADCs according to the present invention after Maxent deconvolution: (A) hz208F2-F562524, (B) c9G4-F562524, (C) hz208F2-F562616, and (D) c9G4-F562646, respectively.

[0638] Figure 26: Figure 26A and Figure 26B Figure 5 shows the in vitro cytotoxicity evaluation of ADC hz208F2-F562524 and the corresponding control ADC c9G4-F562524 in NCI-H2122 (A) and MCF-7 (B) cells, respectively.

[0639] Figure 27: Figure 27A and Figure 27B Figure 5 shows the in vitro cytotoxicity evaluation of ADC hz208F2-F562646 and the corresponding control ADC c9G4-F562646 in NCI-H2122 (A) and MCF-7 (B) cells, respectively.

[0640] Figure 28 Shown are the in vivo activities of ADC hz208F2-F562524 and the corresponding control ADC c9G4-F562524 in an ovarian cancer model. DETAILED DESCRIPTION

[0641] Example

[0642] abbreviation

[0643] ACN: acetonitrile

[0644] ADC: Antibody-Drug Conjugate

[0645] aq: water-based

[0646] BBO: Broadband Observation

[0647] BCA: Bicinchoninic acid

[0648] CDR: complementarity determining region

[0649] DAR: drug-antibody ratio

[0650] DCM: dichloromethane

[0651] DIPEA: N,N-diisopropylethylamine

[0652] DMF: dimethylformamide

[0653] DMSO: dimethyl sulfoxide

[0654] EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0655] EDTA: Ethylenediaminetetraacetic acid

[0656] eq: equivalent

[0657] ES: electrospray

[0658] ESI: electrospray ionization

[0659] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0660] HOBt: 1-hydroxybenzotriazole

[0661] HIC: Hydrophobic Interaction Chromatography

[0662] HPLC: High Performance Liquid Chromatography

[0663] HRMS: High-resolution mass spectrometry

[0664] LBA: Ligand Binding Assay

[0665] LC: Liquid chromatography

[0666] LCMS: Liquid chromatography-mass spectrometry

[0667] mCPBA: meta-chloroperbenzoic acid

[0668] Ms:Methylsulfonyl

[0669] MS: mass spectrometry

[0670] NMR: Nuclear Magnetic Resonance

[0671] PBS: Phosphate buffered saline

[0672] Q-TOF: Quadrupole-Time of Flight

[0673] Rf: retarding factor

[0674] rt: room temperature

[0675] sat.: saturated

[0676] SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

[0677] SEC: Size Exclusion Chromatography

[0678] TBME: tert-butyl methyl ether

[0679] TCEP: tris(2-carboxyethyl)phosphine

[0680] TEA: triethylamine

[0681] TFA: trifluoroacetic acid

[0682] THF: Tetrahydrofuran

[0683] TLC: Thin layer chromatography

[0684] TOF: Time of Flight

[0685] Ts: Tosyl

[0686] UV: Ultraviolet

[0687] Experimental steps:

[0688] All reactions requiring anhydrous conditions were performed in an oven-dried apparatus under nitrogen. Anhydrous solvents were contained in sealed bottles under an inert atmosphere. All reagents were used as received. 430 and Grace On X2, when using silica gel (50μm) Column chromatography was performed on a pre-coated silica column (Merck silica gel 60F 254 TLC analysis was performed on aluminum sheets with a 254 nm UV lamp for visualization. The samples were analyzed at room temperature using a Bruker 500 MHz Ascend HPLC equipped with a BBO Prodigy probe (5 mm). TM Proton recording in CDCl3 and DMSO ( 1 H) and carbon ( 13 C) NMR spectrum. Using Topspin TM 3.2 The software interprets the spectrum. Chemical shift (δ H and δ C ) is reported in parts per million (ppm) relative to CDCl3 ( 1 H NMR 7.26, 13 C NMR 77.0, the center signal of the triplet) or DMSO ( 1 HNMR 2.50, 13C NMR 37.9, the center signal of the septet) was used as reference. COSY and HSQC experiments assisted in the assignments. Coupling constants (J: adjacent protons, J cis :Sequentially adjacent protons, J o : adjacent proton, J m : meta protons) are given in Hertz to the nearest ±0.1 Hz. Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), triplet of triplets (t.of t.), multiplet (m), and broad (b), where applicable. Electrospray ionization (ES+) was used in Mass spectra (m / z) were recorded on a ZQ Mass Detector spectrometer with source temperature of 120°C, desolvation temperature of 350°C, capillary voltage of 3.20 kV, cone voltage of 25 V, extraction voltage of 5 V, Rf lens voltage of 0.5 V, and MS scan range of 100–2000. HPLC analysis was performed using X-Bridge Shield RP18 3.5μm (3.0mm×30mm) column and X-Bridge Shield RP18 3.5μm (3.0mm×20mm) pre-column, in HPLC Analyses were performed on an Alliance 2695 equipped with MassLynx 4.1 software and a Waters 2996 PDA UV / Vis detector at the appropriate wavelength for the sample being analyzed. t ) are given in minutes, accurate to 0.01 minutes. Two elution methods were used:

[0689] Table 7. Elution method for LC 1

[0690] Time (minutes) Solvent A (water + 0.1% formic acid)% Solvent B (ACN + 0.1% formic acid)% 0.00 97.0 3.0 2.25 0.0 100.0 2.50 0.0 100.0 2.60 97.0 3.0 3.00 97.0 3.0

[0691] Table 8. Elution method 2 for LC

[0692] Time (minutes) Solvent A (water + 0.1% formic acid)% Solvent B (ACN + 0.1% formic acid)% 0.00 50.0 50.0 2.10 25.0 75.0 2.25 0.0 100.0 2.50 0.0 100.0 2.75 97.0 3.0 3.00 97.0 3.0

[0693] The retention time given by method 1 is expressed as R t,1 Indicates that the retention time given by method 2 is expressed as R t,2 express.

[0694] 1. Synthesis of Linkers

[0695] Synthesis route example of oxoisothiazolone:

[0696]

[0697] I.1.3,3'-Disulfanediyldipropionyl chloride

[0698]

[0699] I.2. At 0°C, under an inert atmosphere, 3,3'-disulfanediyldipropionic acid (4 g, 0.019 mol, 1 eq.) was suspended in anhydrous DCM (100 mL), and anhydrous DMF (300 μL) was added followed by oxalyl chloride (7.24 g, 0.057 mol, 3 eq.). The solution became clear. The mixture was left at room temperature for 3 hours until it became clear and no more gas formation was observed. The crude product was evaporated and kept under reduced pressure for another 30 minutes to remove the residues of oxalyl chloride. A yellow oil (4.70 g, 100%) was obtained. The crude product was used without further purification; R t,1 (MeOH in): 2.13; MS ES+m / z: 206.86.

[0700] I.3. Dibenzyl 6,6'-((3,3'-disulfanediylbis(propionyl))bis(azadiyl))dihexanoate

[0701]

[0702] 6-(Benzyloxy)-6-oxohexyl-1-ammonium 4-toluenesulfonate (12.20 g, 0.031 mol, 2.2 eq.) was suspended in anhydrous DCM (75 mL) at 0°C under an inert atmosphere with vigorous stirring in an ice bath. TEA (15.72 mL, 0.113 mol, 8 eq.) was added to the solution. Freshly prepared 3,3'-disulfanediyldipropionyl chloride (3.88 g, 0.014 mol, 1 eq.) in DCM (25 mL) was slowly added dropwise to the solution maintained at 0°C. Stirring was continued for 24 hours while the solution reached room temperature. Water (50 mL) was added, and the mixture was transferred to a separatory funnel. The organic layer was separated and washed with brine (1 x 100 mL), followed by 1 M HCl (1 x 100 mL), a saturated solution of NaHCO3 salt (2 x 100 mL), and brine (1 x 100 mL). The combined aqueous layers were extracted with DCM (2 x 100 mL). The organic layer was dried over MgSO4, filtered, and evaporated to dryness to afford a yellow solid, which was triturated in MeOH to afford dibenzyl 6,6'-((3,3'-disulfanediylbis(propionyl))bis(azadiyl))dihexanoate (6.0 g, 66%) as a light yellow powder. 1H NMR (500MHz, CDCl3), δ7.35 (m, 10H), 6.0 (s, 2H), 5.11 (s, 4H), 3.25 (q, J = 5.9Hz, 4 H),3.00(t,J=7.0Hz,4H),2,55(t,J=7.10Hz,4H),2,.36(t,J=7.30Hz,4H),1.66(t of t., J=8.10Hz, 4H), 1.52 (t of t., J=8.10Hz, 4H), 1.35 (t of t, J=8.52Hz, 4H); 13 C NMR (500 MHz, CDCl3), δ 173.5 (2-NH-C=O), 170.9 (-OC=O), 136.0 (2C of aromatic ring) quat ),128.6(2H-C 芳香 ),128.3(HC 芳香 ),128.2(2H-C 芳香 ),66.2(2-CH2-O-),39.4(2-CH2-N),35.8(2-CH2-COO-),34.3(2-CH2-S-),34.1(2-CH2-CNO-),29.1(2C),26,3(2C),24,4(2C); R t,1 (in MeOH): 2.50; MS ES+M / Z: 617.00.

[0703] I.4. Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate

[0704]

[0705] Dibenzyl 6,6'-((3,3'-disulfanediylbis(propionyl))bis(azadiyl))dihexanoate (2.50 g, 4.05 mmol, 1 eq.) was dissolved in anhydrous DCM (20.3 mL). SO2Cl2 (97% pure, 2.96 mL, 0.036 mol, 9 eq.) was added dropwise to the solution, and the mixture was stirred at room temperature under an inert atmosphere for 5 hours. The solution became clear and turned light yellow. Subsequently, the solution was washed with water (2 x 100 mL) and brine (1 x 100 mL). The combined aqueous layers were extracted with DCM (2 x 100 mL). The organic layer was dried over MgSO4 and filtered, then concentrated under reduced pressure and purified by column chromatography. Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate (0.824 g, 29.9%) and benzyl 6-(3-oxoisothiazol-2(3H)-yl)hexanoate were obtained as light yellow oil. 1H NMR(CDCl3), δ7.35(m,5H),6.25(s,1H),5.11(s,2H),3.72(t,J=7.34Hz,2H),2.37(t,J=7.39Hz,2H),1.69(m,4H),1.38(m,2H); 13 C NMR (500 MHz, CDCl3), δ 173.2 (-OC=O), 166.9 (-NC=O), 145.6 (Cl-HC=CH-), 136.0 (aromatic ring C quat .),128.6-128.3(5H-C 芳香 ),114.8(Cl-HC=CH-),66.2(-CH2-O-),43.5(-CH2-N-),34.0(-CH2-C=O),29.4,25.9,24.4; R t,1 (ACN in): 2.35; MS ES+m / z: 339.84.

[0706] I.5. Benzyl 6-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0707]

[0708] Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate (802 g, 3.58 mmol) was diluted in anhydrous DCM (25 mL). 3-Chlorobenzeneperoxy acid (1.2 eq.) was added. The solution was stirred at room temperature under an inert atmosphere for 48 hours. The solution was then diluted with DCM and treated with a 10% aqueous Na2S2O3 solution. The organic phase was then extracted with a saturated NaHCO3 solution (2 x 100 mL) and then with brine (1 x 100 mL). The combined aqueous layers were extracted with DCM (2 x 100 mL). The organic layer was dried over MgSO4, filtered, then concentrated under reduced pressure and purified by column chromatography. Benzyl 6-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate (753 mg) was obtained as a colorless oil.

[0709] I.6. Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0710]

[0711] Standard procedure for the oxidation of monochloride compounds:

[0712] Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate (1.11 g, 0.0036 mol, 1 eq.) was dissolved in anhydrous DCM (7 mL). 3-Chlorobenzeneperoxyacid (2.69 g, 0.0109 mol, 3 eq.) was added. The solution was stirred at room temperature under an inert atmosphere for 48 hours. The solution was then diluted with DCM and treated with a 10% aqueous Na2S2O3 solution. The organic phase was then extracted with a saturated NaHCO3 salt solution (2 x 100 mL) and then with brine (1 x 100 mL). The combined aqueous layers were extracted with DCM (2 x 100 mL). The organic layer was dried over MgSO4, filtered, then concentrated under reduced pressure and purified by column chromatography. Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (0.760 g, 64.3%) was obtained as a light yellow oil. 1 H NMR(CDCl3), δ7.36(m,5H),6.68(s,1H),5.11(s,2H),3.67(t,J=7.68Hz,2H),2.37(t,J=7.27Hz,2H),1.78(t of t,J=7.73Hz,2H),1.70(t of t,J=7.38Hz,2H),1.40(m,2H); 13 CNMR (500MHz, CDCl3), δ173.2(-OC=O), 157.0(-NC=O), 144.6(Cl-HC=CH-), 136.0(aromatic ring C quat .),128.6-128.2(HC 芳香 ),123.6(Cl-HC=CH-),66.2(-CH2-O),40.3(-CH2-N-),34.0(-CH2-C=O),27.9,26.0,24.2; R t,1 (ACN): 2.49; MS ES+M / Z: 371.83.

[0713] Example 1. 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid

[0714]

[0715] Starting from benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (0.445 g, 77%), a standard method for benzyl ester deprotection was followed to obtain 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid as a pale white powder. 1H NMR (CDCl3), δ 10.89 (br. s, 1H), 6.70 (s, 1H), 3.70 (t, J = 7.43 Hz, 2H), 2.38 (t, J = 7.38 Hz, 2H), 1.81 (t of t., J = 7.60 Hz, 2H), 1.69 (t of t, J = 7.71 Hz, 2H), 1.43 (split t of t, J = 7.75 Hz, J = 3.31 Hz, 2H); 13 R t,1 (ACN in): 1.98; MS ES+m / z: 349.89.

[0716] I.7. Benzyl 6-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)hexanoate

[0717]

[0718] Dibenzyl 6,6'-((3,3'-disulfanediylbis(propionyl))bis(azadiyl))dihexanoate (3.21 g, 0.0052 mol, 1 eq.) was dissolved in anhydrous DCM (26 mL). SO2Cl2 (97% pure, 3.80 mL, 0.047 mol, 9 eq.) was added dropwise to the solution, and the mixture was stirred at room temperature under an inert atmosphere for 24 hours. The solution became clear and turned light yellow. Subsequently, the mixture was washed with water (2 × 100 mL) and brine (1 × 100 mL). The combined aqueous layers were extracted with DCM (2 × 100 mL). The organic layer was dried over MgSO4 and filtered, then concentrated under reduced pressure and purified by column chromatography. Benzyl 6-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)hexanoate (1.391 g, 35.7%) was obtained as a light yellow oil. 1 H NMR(CDCl3), δ7.35(m,5H),5.11(s,2H),3.79(t,J=7.18Hz,2H),2.37(t,J=7.33Hz,2H),1.70(m,4H),1.38(m,2H); 13 CNMR (500MHz, CDCl3), δ173.2(-OC=O), 161.9(-NC=O), 138(-CH2-O-), .3(Cl-CS-), 135.6(aromatic ring C quat.),128.6-128.3(5H-C 芳香 ),115.1(Cl-CC=O),66.2,44.9(-CH2-N-),33.9(-CH2-C=O),29.1,25.9,24.3; R t,1 (ACN in): 2.50; MS ES+m / z: 373.75.

[0719] I.8. ​​Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0720]

[0721] Standard procedure for the oxidation of dichloride compounds:

[0722] To a stirred solution of benzyl 6-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)hexanoate (1.94 g, 0.0052 mol, 1 eq.) in water:DCM:ACN (2:1:1, 1 mL) was added ruthenium trichloride monohydrate (16 mg, 0.07 mmol, 0.013 eq.) in one portion. Sodium periodate (3.33 g, 0.0156 mol, 3 eq.) was then added over 5 minutes, and the resulting mixture was stirred at room temperature under an inert atmosphere for 90 minutes. The solid was filtered, and the filtrate was diluted with water (50 mL), extracted with EtOAc (2 x 100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The obtained grey solid was then purified using column chromatography to obtain benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (0.930 g, 44.2%) as a light yellow oil. 1 H NMR(CDCl3), δ7.36(m,5H),5.12(s,2H),3.72(t,J=7.53Hz,2H),2.73(t,J=7.50Hz,2H),1.80(t of t,J=7,53Hz,2H),1.70(t of t,J=7.70Hz),1.41(m,2H); 13 C NMR (500 MHz, CDCl3), δ 173.1 (-OC=O-), 154.1 (NC=O), 138.0 (Cl-C-SO2-), 136.0 (aromatic ring C quat. ),130.7 1(Cl-CC=O),128.6-128.3(5H-C 芳香 ),66.2(-CH2-O-),41.1(-CH2-N-),33.9(-CH2-C=O),27.9,26.0,24.2; R t,1(ACN in): 2.59; MS ES+m / z: 405.76.

[0723] Example 2. 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid

[0724]

[0725] Standard procedure for benzyl ester deprotection:

[0726] Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (0.930 g, 2.23 mmol, 1 eq.) was diluted in anhydrous DCM (11.5 mL). Methanesulfonic acid (1.5 mL, 0.023 mol, 10 eq.) was added. The solution was stirred at room temperature under an inert atmosphere for 24 hours. The solution was then diluted with DCM and treated with water (50 mL). The organic layer was extracted with water (2 x 100 mL) and then with brine (1 x 100 mL). The combined aqueous layers were extracted with DCM (2 x 100 mL). The organic layer was dried over MgSO4 and concentrated under reduced pressure, then purified by column chromatography. 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (0.563 g, 78%) was obtained as a pale white powder. 1 H NMR (500MHz, CDCl3), δ = 3.76 (t, J = 7.34Hz, 2H), 2.38 (t, J = 7.32Hz, 2H), 1.83 (t of t, J = 7.65Hz, 2H), 1.70 (t of t, J = 7.77Hz, 2H), 1.45 (t of t, J=7.54Hz, J=3.31Hz, 2H); 13 R t,1 (ACN in): 2.09; MS ES+m / z: 315.76.

[0727] I.9. Benzyl 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0728]

[0729] To a solution of benzyl 6-(1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (obtained from benzyl 6-(3-oxoisothiazol-2(3H)-yl)hexanoate following the standard procedure for the oxidation of the monochloride compound) (3 g, 8.89 mmol, 1.00 equiv) in CCl4 (40 mL) was added Br2 (1.2 mL, 19.58 mmol, 2.2 equiv) dropwise over 30 min at ambient temperature with stirring and stirred at 75 °C overnight. The reaction mixture was concentrated under vacuum and diluted with CHCl3 (40 mL), followed by the addition of pyridine (0.9 g). The resulting solution was stirred at ambient temperature for 30 min and then quenched by the addition of 50 mL of saturated NaHCO3 solution. The resulting mixture was washed with saturated sodium carbonate (2×50 mL) and 50 mL of brine. The resulting mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:5) to obtain 0.4 g (10.8%) of benzyl 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate as a light yellow oil. LC-MS (ES, m / z): 416 [M+H] + ,433[M+NH4] + ; 1 H-NMR (400 Mhz, chloroform-d) δ 7.42-7.28 (m, 2H), 5.12 (s, 1H), 3.69 (t, J = 7.4 Hz, 1H), 2.38 (t, J = 7.4 Hz, 1H), 1.86-1.64 (m, 2H), 1.47-1.34 (m, 1H).

[0730] Example 3. 6-(4-Bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid

[0731]

[0732] To a solution of benzyl 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (1 g, 2.40 mmol, 1.00 equiv) in dioxane (10 mL) was added 4N HCl (10 mL) dropwise with stirring at 0°C. The resulting solution was stirred at room temperature for 2 days. The resulting mixture was concentrated under vacuum and extracted with dichloromethane (3×50 mL). The combined organic layers were washed with brine (2×100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column purification with DCM / MeOH (10:1) to give 100 mg (13%) of 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid as a white solid. LC-MS (ES, m / z): 308 [M+NH4] + ,326 / 328[M+H]+ ; 1 H-NMR (300 MHz, chloroform-d) δ7.58 (s, 1H), 3.75 (t, J = 7.4 Hz, 2H), 2.41 (t, J = 7.4 Hz, 2H), 1.78 (dq, J = 34.6, 7.4 Hz, 4H), 1.47 (t, J = 7.7 Hz, 2H).

[0733] Example of synthetic route for synthesis of linker of X1=OR:

[0734]

[0735] I.10. Benzyl 6-(5-(4-cyanophenoxy)-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0736]

[0737] At 0 ℃, a solution of 4-hydroxybenzonitrile (76 mg, 0.639 mmol) in THF (2 mL) was added to a mixture of NaH (0.639 mmol) in THF (2 mL). After stirring for 30 minutes, 6-(5-chloro-1-oxidation-3-oxoisothiazol-2 (3H)-yl) benzyl hexanoate (250 mg, 0.703 mmol) was added to THF (2 mL). The reaction mixture was then stirred at room temperature for 18 hours. The reaction mixture was diluted with AcOEt and NH4Cl (10% aqueous solution) was added. The organic phase was then washed with salt water and dried with MgSO4, filtered and concentrated. The crude product was purified by silica gel column using a DCM / MeOH mixture (80 / 20) to obtain 6-(5-(4-cyanophenoxy)-1-oxidation-3-oxoisothiazol-2 (3H)-yl) benzyl hexanoate (210 mg, 75% yield) as a colorless oil. LC-MS(ES,m / z): 439.0[M+H]+; 1 H-NMR (300 MHz, chloroform-d) δ 7.79 (m, 2H), 7.35 (m, 7H), 5.59 (s, 1H), 5.12 (s, 2H), 3.69 (m, 2H), 2.37 (m, 2H), 1.71 (m, 4H), 1.39 (m, 2H).

[0738] I.11. Benzyl 6-(5-(4-cyanophenoxy)-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0739]

[0740] Following the standard procedure for the oxidation of monochloride compounds, starting from benzyl 6-(5-(4-cyanophenoxy)-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate, the product was obtained as a colorless oil (136 mg, 48% yield). LC-MS (ES, m / z): 455.0 [M+H] +; 1 H-NMR (300 Mhz, chloroform-d) δ 7.82 (m, 2H), 7.40 (m, 2H), 7.35 (m, 5H), 5.63 (s, 1H), 5.12 (s, 2H), 3.65 (m, 2H), 2.38 (m, 2H), 1.79 (m, 2H), 1.70 (m, 2H), 1.41 (m, 2H).

[0741] Example 4. 6-(5-(4-cyanophenoxy)-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid

[0742]

[0743] Following the standard procedure for benzyl ester deprotection, starting from benzyl 6-(5-(4-cyanophenoxy)-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate, the product was obtained as a white solid (38 mg, 35% yield). LC-MS (ES, m / z): 365.0 [M+H]+; 1 H-MR (300 MHz, CHLOROFORM-d) 7.82 (m, 2H), 7.41 (m, 2H), 5.65 (s, 1H), 3.67 (m, 2H), 2.38 (m, 2H), 1.80 (m, 2H), 1.69 (m, 2H), 1.44 (m, 2H).

[0744] I.12. Benzyl 6-(5-methoxy-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0745]

[0746] A mixture of benzyl 6-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate (270 mg, 0.759 mmol) in methanol (5 mL) and triethylamine (113 μL, 0.835 mmol) was stirred at room temperature for 18 hours. The volatiles were then removed under vacuum, and the residue was purified on a silica gel column using cyclohexane / AcOEt (1 / 1) to give benzyl 6-(5-methoxy-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate (107 mg, 40%) as a yellow oil. LC-MS (ES, m / z): 352.0 [M+H]+; 1H-NMR (300 MHz, chloroform-d) δ7.36 (m, 5H), 5.57 (s, 1H), 5.11 (s, 2H), 4.04 (s, 3H), 3.60 (m, 2H), 2.37 (m, 2H), 1.75 (m, 2H), 1.69 (m, 2H), 1.39 (m, 2H).

[0747] I.13. Benzyl 6-(5-methoxy-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0748]

[0749] Following the standard procedure for the oxidation of monochloride compounds, starting from benzyl 6-(5-methoxy-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate, the product was obtained as a white solid (62 mg, 36% yield). LC-MS (ES, m / z): 368.0 [M+H] +; 1 H-NMR (300 MHz, chloroform-d) δ7.36 (m, 5H), 5.57 (s, 1H), 5.11 (s, 2H), 4.04 (s, 3H), 3.60 (m, 2H), 2.37 (m, 2H), 1.75 (m, 2H), 1.69 (m, 2H), 1.39 (m, 2H).

[0750] Example 5. 6-(5-methoxy-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid

[0751]

[0752] Following the standard procedure for benzyl ester deprotection, starting from benzyl 6-(5-methoxy-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate, the product was obtained as a white solid (37 mg, 67% yield). HRMS (ES, m / z): [M+H] found 278.0691, calculated 278.0698; 1 H-NMR (300 MHz, chloroform-d) δ 5.60 (s, 1H), 4.05 (s, 3H), 3.62 (m, 2H), 2.36 (m, 2H), 1.77 (m, 2H), 1.68 (m, 2H), 1.42 (m, 2H).

[0753] I.14. Benzyl 4-(aminomethyl)cyclohexane-1-carboxylate 4-toluenesulfonate

[0754]

[0755] A mixture of 4-(aminomethyl)cyclohexanecarboxylic acid (10 g, 63.61 mmol, 1 eq), benzyl alcohol (55.03 g, 508.87 mmol, 52.91 mL, 8 eq) and TsOH.H2O (12.70 g, 66.79 mmol, 1.05 eq) in toluene (50 mL) was stirred at 140°C for 16 hours, and the water of condensation and toluenesulfonic acid monohydrate was collected using a Dean and Stark apparatus. After refluxing for several hours, the reaction became clear. The clear reaction mixture was poured into TBME (500 mL), and the resulting white solid was filtered off, washed with TBME (200 mL), and dried under vacuum. Benzyl 4-(aminomethyl)cyclohexanecarboxylate 4-toluenesulfonate (26.6 g, 63.40 mmol, 99.68% yield) was obtained as a white solid. 1 H NMR (400MHz, methanol-d4) δppm 7.71(d,J=8.16Hz,2H)7.28-7.40(m,5H)7.23(d,J=7.94Hz,2H)5.11(s,2H)2.77(d,J=7.06Hz,2H)2.29-2.39(m,4H)1.99-2.08(m,2H)1.85(br d,J=11.25Hz,2H)1.53-1.65(m,1H)1.43(qd,J=12.97,3.20Hz,2H)1.06(qd,J=12.75,3.20Hz,2H).

[0756] I.15. 4,4'-(((3,3'-Disulfanediylbis(propionyl))bis(azadiyl))bis(methylene))bis(cyclohexane-1-carboxylic acid dibenzyl ester)

[0757]

[0758] At 0 ° C, EDCI (13.32 g, 69.48 mmol, 2.2 eq) was added to a solution of 3-(2-carboxyethyldisulfide)propionic acid (6.64 g, 31.58 mmol, 1 eq), HOBt (9.39 g, 69.48 mmol, 2.2 eq) and TEA (12.78 g, 126.33 mmol, 17.58 mL, 4 eq) in DCM (300 mL). 4-(aminomethyl)benzyl cyclohexanecarboxylate 4-toluenesulfonic acid (26.5 g, 63.17 mmol, 2 eq) was then added at this temperature. The mixture was stirred at 0-20 ° C for 4 hours. TLC (petroleum ether: ethyl acetate = 2: 1, Rf = 0.5) showed that the reaction was complete. The mixture was poured into a saturated NaHCO solution (100 mL) and H2O (100 mL) and the organic layer was separated. The aqueous layer was extracted with DCM (200 mL). The combined organic layers were washed with H2O (100 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was dissolved in DCM (50 mL) and petroleum ether was added very slowly until a white precipitate formed. The mixture was filtered, washed with petroleum ether, and dried in vacuo. Benzyl 4-[[3-[[3-[(4-benzyloxycarbonylcyclohexyl)methylamino]-3-oxopropyl]disulfanyl]propionylamino]methyl]cyclohexanecarboxylate (19 g, 28.40 mmol, 89.94% yield) was obtained as a white solid. 1 H NMR (400MHz, chloroform-d) δppm 7.27-7.41 (m, 10H) 6.05 (br s,2H)5.11(d,J=1.54Hz,4H)3.10-3.17(m,4H)2.96-3.02(m,4H)2.54-2.63(m,4H)2.30(td,J=12.24,1.76Hz,2H)2.04(br d,J=12.57Hz,4H)1.85(br d,J=12.79Hz,4H)1.38-1.54(m,6H)0.99(q,J=12.72Hz,4H).

[0759] I.16. Benzyl 4-((5-chloro-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0760]

[0761] I.17. Benzyl 4-((4,5-dichloro-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0762]

[0763] To a solution of benzyl 4-[[3-[[3-[(4-benzyloxycarbonylcyclohexyl)methylamino]-3-oxopropyl]disulfanyl]propionylamino]methyl]cyclohexanecarboxylate (10 g, 14.95 mmol, 1 eq) in DCM (100 mL) was added dropwise sulfuryl chloride (10.09 g, 74.75 mmol, 7.47 mL, 5 eq) at 0°C. The mixture was stirred at 0-20°C for 12 hours. A clear solution was obtained after the addition of sulfuryl chloride. TLC (petroleum ether:ethyl acetate = 2:1, Rf (main) = 0.5) indicated that the reaction was complete. The mixture was poured into H2O (30 mL) and extracted with DCM (50 mL×2). The combined organic layers were washed with H2O (50 mL), brine (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. Purification on a silica gel column gave benzyl 4-[(3-oxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (2.1 g, 3.42 mmol, 11.44% yield, 54% purity) as a brown solid, benzyl 4-[(5-chloro-3-oxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (7.1 g, 18.82 mmol, 62.96% yield, 97% purity) as an off-white solid, and benzyl 4-[(4,5-dichloro-3-oxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (0.4 g, 869.31 μmol, 2.91% yield, 87% purity) as a brown oil.

[0764] I.18. Benzyl 4-((5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0765]

[0766] At 0 ° C, under N2, to a mixture of 4-[(5-chloro-3-oxoisothiazol-2-yl)methyl]cyclohexanecarboxylic acid benzyl ester (2 g, 5.01 mmol, 1 eq) in H2O (20 mL), ACN (10 mL) and DCM (10 mL) was added RuCl3.H2O (22.58 mg, 100.14 μmol, 0.02 eq) and NaIO4 (6.43 g, 30.04 mmol, 1.66 mL, 6 eq) in one portion. The mixture was then heated to 20 ° C and stirred for 16 hours. TLC showed that the reaction was complete (petroleum ether: ethyl acetate = 2: 1, Rf-p1 = 0.6). The mixture was filtered, the filtrate was concentrated by nitrogen flow, and the solid that appeared again was filtered, and the filtrate was concentrated by nitrogen. The residue was purified by preparative TLC (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether: ethyl acetate = 2:1) to obtain benzyl 4-[(5-chloro-1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (1.4 g, 3.17 mmol, yield 63.25%, purity 90%) as a white solid.

[0767] Example 6. 4-((5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylic acid

[0768]

[0769] To a mixture of benzyl 4-[(5-chloro-1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (0.1 g, 226.20 μmol, 1 eq) in DCM (5 mL) was added MsOH (217.39 mg, 2.26 mmol, 161.03 μL, 10 eq) at 30 ° C under N2. The mixture was stirred at 30 ° C for 16 hours. TLC showed that the reaction was complete. LCMS (ET17992-54-P1A) showed that the expected MS was detected. The mixture was poured into ice water (5 mL) and stirred for 5 minutes. The aqueous phase was extracted with DCM (3 mL×2). The combined organic phase was washed with brine (3 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative TLC (column height: 250 mm, diameter: 100 μm, 100-200 mesh silica gel, petroleum ether: ethyl acetate = 2:1) to give 4-[(5-chloro-1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylic acid (0.02 g, 64.99 μmol, yield 28.73%) as a colorless oil; LC-MS (ES, m / z): 306.0 [MH] - ; 1H-NMR (300MHz, DMSO-D6) δ12.01(bs,1H),7.62(s,1H),3.45(m,2H),2.11(m,1H),1.90(m,2H),1.76(m,2H),1.73(m,1H),1.23(m,2H),0.96(m,2H).

[0770] I.19. Benzyl 4-((4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0771]

[0772] To a mixture of benzyl 4-[(4,5-dichloro-3-oxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (1.2 g, 2.70 mmol, 1 eq) in H2O (20 mL), DCM (10 mL) and ACN (10 mL) was added RuCl3.H2O (12.16 mg, 53.96 μmol, 0.02 eq) and NaIO4 (3.46 g, 16.19 mmol, 896.96 μL, 6 eq) at 0°C under N2. The mixture was stirred at 20°C for 2 hours. TLC showed that the reaction was complete (petroleum ether:ethyl acetate=2:1, Rf-p1=0.7). The mixture was poured into ice water (30 mL) and stirred for 5 minutes. The aqueous phase was extracted with ethyl acetate (20 mL×2). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether: ethyl acetate = 5:1 to 3:1) to obtain benzyl 4-[(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (0.8 g, 1.67 mmol, yield 61.73%, purity 90%) as a white solid.

[0773] Example 7. 4-((4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylic acid

[0774]

[0775] To a mixture of benzyl 4-[(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (0.8 g, 1.67 mmol, 1 eq) in DCM (20 mL) was added MsOH (1.60 g, 16.65 mmol, 1.19 mL, 10 eq) in one portion at 30 ° C. under N2. The mixture was stirred at 30 ° C. for 16 hours. LCMS showed that the reaction was complete. The mixture was poured into ice water (5 mL) and concentrated under reduced pressure, after which a solid appeared. The solution was filtered and triturated with EtOAc (2 mL x 3), and the filter cake was dried under vacuum to obtain 4-[(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylic acid (0.170 g, 486.86 μmol, 29.23% yield, 98% purity) as a white solid; LC-MS (ES, m / z): 364.0 [M+Na] + ; 1 H-NMR (300MHz, DMSO-D6) δ11.99(bs,1H),3.49(m,2H),2.11(m,1H),1.88(m,2H),1.79(m,2H),1.66(m,1H),1.23(m,2H),0.96(m,2H).

[0776] I.20. Dibenzyl 3,3'-(((3,3'-disulfanediylbis(propionyl))bis(azadiyl))bis(4,1-phenylene))dipropionate

[0777]

[0778] At 0 ° C, EDCI (1.31 g, 6.82 mmol, 2.2 eq) was added to a solution of 3-(2-carboxyethyldisulfide)propionic acid (651.56 mg, 3.10 mmol, 1 eq), HOBt (921.15 mg, 6.82 mmol, 2.2 eq) and TEA (1.25 g, 12.39 mmol, 1.73 mL, 4 eq) in DCM (50 mL). 4-(aminomethyl)benzyl cyclohexanecarboxylate 4-toluenesulfonic acid (2.6 g, 6.20 mmol, 2 eq) was then added at this temperature. The mixture was stirred at 0-20 ° C for 12 hours. TLC (petroleum ether: ethyl acetate = 2: 1, Rf = 0.5) showed that the reaction was complete. The mixture was poured into a saturated NaHCO solution (20 mL) and H2O (20 mL) and the organic layer was separated. The aqueous layer was extracted with DCM (50 mL). The combined organic layers were washed with H2O (50 mL), brine (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. Benzyl 4-[[3-[[3-[(4-benzyloxycarbonylcyclohexyl)methylamino]-3-oxopropyl]disulfanyl]propionylamino]methyl]cyclohexanecarboxylate (1.1 g, 1.64 mmol, 53.07% yield) was obtained as a white solid. 1 H NMR (400MHz, chloroform-d) δppm 7.28-7.41 (m, 10H) 5.96 (br s,2H)5.10(s,4H)3.13(t,J=6.39Hz,4H)2.98(t,J=6.84Hz,4H)2.57(t,J=6.95Hz,4H)2.23-2.34(m,2H)2.03(br d,J=12.79Hz,4H)1.84(br d,J=12.13Hz,4H)1.37-1.63(m,6H)0.91-1.05(m,4H).

[0779] I.21. Benzyl 3-(4-(5-chloro-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0780]

[0781] To a solution of benzyl 3-[4-[3-[[3-[4-(3-benzyloxy-3-oxopropyl)phenylamino]-3-oxopropyl]disulfanyl]propionylamino]phenyl]propanoate (10 g, 14.60 mmol, 1 eq) in DCM (200 mL) was added dropwise thionyl chloride (5.91 g, 43.80 mmol, 4.38 mL, 3 eq) at 25° C. under N 2 . The solution was stirred at 25° C. for 8 hours. The color of the solution changed from colorless to black after the addition of thionyl chloride, and then to yellow after 1 hour. TLC (petroleum ether:ethyl acetate=2:1, Rf=0.60) showed that the starting material had been consumed and two new major spots had been generated. The residue was poured into ice water (200 mL) and then concentrated in vacuo to remove DCM. After concentration, the aqueous phase was extracted with ethyl acetate (200 mL × 3), and the combined organic phases were washed with water (200 mL × 1), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 5: 1 to 1: 1) to give benzyl 3-[4-(3-oxoisothiazol-2-yl)phenyl]propanoate (2.5 g, 7.37 mmol, 50.47% yield) 1 H NMR (400 MHz, methanol-d4) δ = 8.56 (d, J = 6.2 Hz, 1H), 7.44-7.39 (m, 2H), 7.37-7.26 (m, 7H), 6.30 (d, J = 6.4 Hz, 1H), 5.09 (s, 2H), 2.98 (t, J = 7.4 Hz, 2H), 2.78-2.66 (m, 2H); and benzyl 3-[4-(5-chloro-3-oxoisothiazol-2-yl)phenyl]propanoate (5 g, 13.37 mmol, 91.60% yield) was obtained as a yellow solid; 1 H NMR (ET17992-22-P2A) confirmed ET17992-22-P2. 1 H NMR (400 MHz, methanol-d4) δ = 7.42-7.36 (m, 2H), 7.35-7.25 (m, 7H), 6.49-6.45 (m, 1H), 5.08 (s, 2H), 2.99-2.91 (m, 2H), 2.69 (t, J = 7.5 Hz, 2H).

[0782] I.22. Benzyl 3-(4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0783]

[0784] At 25 ℃, to 3-[4-(5-chloro-3-oxoisothiazol-2-yl)phenyl]benzyl propionate (1.4g, 3.74mmol, 1eq) in H o (12mL), DCM (6mL) and ACN (6mL) in a single addition NaIO (4.81g, 22.47mmol, 1.25mL, 6eq), then use N purge the mixture three times. Then, under N, add RuCl .H o (42.21mg, 187.24μmol, 0.05eq). The mixture is stirred at 25 ℃ for 12 hours. The mixture becomes turbid and the color becomes gray. The residue is poured into ethyl acetate (100mL) and then filtered. The filtrate is concentrated in vacuo. The residue was purified by column chromatography (SiO 2 , petroleum ether: ethyl acetate = 10: 1 to 4: 1) to give benzyl 3-[4-(5-chloro-1,1,3-trioxoisothiazol-2-yl)phenyl]propanoate (460 mg, 1.08 mmol, 28.75% yield, 95% purity) as a yellow solid; 1 H NMR (ET17992-63-P1A) confirmed ET17992-63-P1. 1 H NMR (400 MHz, chloroform-d) δ = 7.41-7.30 (m, 7H), 6.84 (s, 1H), 5.13 (s, 2H), 3.04 (t, J = 7.6 Hz, 2H), 2.72 (t, J = 7.7 Hz, 2H).

[0785] Example 8. 3-(4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoic acid

[0786]

[0787] To a solution of benzyl 3-[4-(5-chloro-1,1,3-trioxoisothiazol-2-yl)phenyl]propanoate (460 mg, 1.13 mmol, 1 eq) in DCM (15 mL) was added dropwise methanesulfonic acid (1.09 g, 11.33 mmol, 806.87 μL, 10 eq) at 10 ° C. The solution was then heated to 35 ° C and stirred for 12 hours. The residue was washed with water (15 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was poured into water (20 mL) and then filtered. The filter cake was dissolved in DCM (5 mL), and then petroleum ether (30 mL) was poured into the residue. The solution was stirred at 10° C. for 2 minutes and then filtered. The filter cake was dried in vacuo to give 3-[4-(5-chloro-1,1,3-trioxoisothiazol-2-yl)phenyl]propanoic acid (162 mg, 501.81 μmol, 44.27% yield, 97.8% purity) as a white solid; LC-MS (ES, m / z): 313.9 [MH] - ; 1 H-NMR (300MHz, DMSO-D6) δ12.18(bs,1H),7.81(s,1H),7.45(m,2H),7.37(m,2H),2.89(m,2H),2.58(m,2H).

[0788] I.23. Benzyl 3-(4-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0789]

[0790] At 0 ° C, under N2, to a mixture of 4-[(3-oxoisothiazol-2-yl)methyl]benzyl cyclohexanecarboxylate (1.2 g, 3.32 mmol, 1 eq) in water (20 mL), ACN (10 mL) and DCM (10 mL) was added RuCl3.H2O (14.95 mg, 66.33 μmol, 0.02 eq) and NaIO4 (4.26 g, 19.90 mmol, 1.10 mL, 6 eq) in one portion. The mixture was then heated to 20 ° C and stirred for 16 hours. The mixture was filtered and the filtrate was concentrated by a nitrogen stream. A solid appeared again and was filtered, and the filtrate was concentrated by nitrogen. The residue was purified by preparative TLC (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether: ethyl acetate = 2:1) to obtain benzyl 4-[(1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (0.45 g, 1.11 mmol, yield 33.60%, purity 90%) as a colorless oil; 1H NMR (400 MHz, CHLOROFORM-d) δ = 7.53 (dd, J = 3.2, 8.7 Hz, 2H), 7.17 (ddd, J = 3.2, 7.6, 9.0 Hz, 2H), 6.97-6.89 (m, 2H), 5.56 (br s, 1H), 4.12-4.06 (m, 4H), 3.92 (s, 5H), 3.55 (q, J = 5.1 Hz, 5H).

[0791] I.24. Benzyl 3-(4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0792]

[0793] To a mixture of benzyl 3-[4-(4,5-dichloro-3-oxoisothiazol-2-yl)phenyl]propanoate (650 mg, 1.59 mmol, 1 eq) and NaIO4 (1.36 g, 6.37 mmol, 352.86 μL, 4 eq) in H2O (10 mL), DCM (5 mL), and ACN (5 mL) was added RuCl3.H2O (7.18 mg, 31.84 μmol, 0.02 eq) in one portion at 0°C under N2. The mixture was stirred at 20°C for 2 hours. The residue was extracted with ethyl acetate (30 mL x 2). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=30:1 to 5:1) to give benzyl 3-[4-(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)phenyl]propanoate (180 mg, 25.68% yield) as a yellow solid; 1 H NMR (400 MHz, chloroform Md) δ = 7.43-7.29 (m, 9H), 5.13 (s, 2H), 3.05 (t, J = 7.6 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H).

[0794] Example 9. 3-(4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoic acid

[0795]

[0796] To a solution of benzyl 3-[4-(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)phenyl]propanoate (180 mg, 408.81 μmol, 1 eq) in DCM (5 mL) was added methanesulfonic acid (392.89 mg, 4.09 mmol, 291.03 μL, 10 eq) dropwise at 10 ° C. The solution was stirred at 35 ° C for 12 hours. The residue was poured into water (5 mL) and then filtered. The filter cake was washed with water (10 mL × 3) and then dried in vacuo. The residue was dissolved in dichloromethane:methanol (5.5 mL, v / v=10:1) and then purified by preparative TLC (ethyl acetate, Rf=0.26) to give 3-[4-(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)phenyl]propanoic acid (57.64 mg, 156.83 μmol, 38.36% yield, 95.278% purity) as a white solid; LC-MS (ES, m / z): 347.9 [MH]-; 1 H NMR (400MHz, DMSO-d6) δ = 12.22 (br s, 1H), 7.51-7.40 (m, 4H), 2.90 (br t, J = 7.6Hz, 2H), 2.60 (br t, J = 7.6Hz, 2H).

[0797] I.25. Dibenzyl 4,4'-((3,3'-disulfanediylbis(propionyl))bis(azadiyl))dibenzoate

[0798]

[0799] To a solution of 3-(2-carboxyethyldisulfanyl)propionic acid (6.01 g, 28.60 mmol, 1 eq) and pyridine (14.93 g, 188.77 mmol, 15.24 mL, 6.6 eq) in DMF (120 mL) was added EDCI (12.06 g, 62.92 mmol, 2.2 eq) and benzyl 4-aminobenzoate (13 g, 57.20 mmol, 2 eq) at 10 ° C. The mixture was then stirred at 50 ° C for 12 hours. The residue was poured into ice water (200 mL) and stirred for 20 minutes. The aqueous phase was extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated NaCl (200 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was then recrystallized from petroleum ether: DCM = 50:1 to obtain a solid. The solid was washed three times with petroleum (150 mL×3) and then dried in vacuo to give benzyl 4-[3-[[3-(4-benzyloxycarbonylphenylamino)-3-oxopropyl]disulfanyl]propionylamino]benzoate (14 g, crude) as a white solid; 1H NMR (400MHz, DMSO-d6) δ = 10.37 (s, 2H), 8.02-7.83 (m, 4H), 7.72 (d, J = 8.8Hz, 4H),7.48-7.32(m,10H),5.31(s,4H),3.05-2.98(m,4H),2.81-2.75(m,4H).

[0800] I.26. Benzyl 4-(5-chloro-3-oxoisothiazol-2(3H)-yl)benzoate

[0801]

[0802] I.27. Benzyl 4-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)benzoate

[0803]

[0804] To a solution of benzyl 4-[3-[[3-(4-benzyloxycarbonylphenylamino)-3-oxopropyl]disulfanyl]propionylamino]benzoate (9.4 g, 14.95 mmol, 1 eq) in DCM (120 mL) was added sulfonyl chloride (10.09 g, 74.75 mmol, 7.47 mL, 5 eq) dropwise at 0°C. The mixture was stirred at 0-20°C for 12 hours. After stirring for a few minutes, the mixture became clear. TLC (petroleum ether:ethyl acetate = 2:1) indicated that the reaction was complete. The mixture was poured into H2O (200 mL) and extracted with DCM (200 mL x 2). The combined organic layers were washed with H2O (200 mL x 2), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 1:1) to give benzyl 4-(3-oxoisothiazol-2-yl)benzoate (1.2 g, 3.43 mmol, 11.47% yield, 89% purity) as an off-white solid; 1 H NMR (400 MHz, chloroform-d) δ ppm 8.09-8.27 (m, 3H), 7.63-7.82 (m, 2H), 7.32-7.52 (m, 5H), 6.34 (br d, J=6.36 Hz, 1H), 5.39 (s, 2H); benzyl 4-(5-chloro-3-oxoisothiazol-2-yl)benzoate (3.5 g, 10.01 mmol, 33.49% yield, 98.92% purity) was obtained as an off-white solid; 1H NMR (400 MHz, chloroform-d) δ ppm 8.15 (d, J = 8.60 Hz, 2H) 7.69 (d, J = 8.82 Hz, 2H) 7.33-7.49 (m, 4H) 6.38 (s, 1H) 5.38 (s, 2H), and benzyl 4-(4,5-dichloro-3-oxoisothiazol-2-yl)benzoate (2.8 g, 7.19 mmol, 24.06% yield, 97.68% purity) was obtained as an off-white solid; 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.18 (d, J = 8.60 Hz, 2H) 7.71 (d, J = 8.60 Hz, 2H) 7.33-7.50 (m, 4H) 5.39 (s, 2H).

[0805] I.28. Benzyl 4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoate

[0806]

[0807] To a mixture of 4-(5-chloro-3-oxoisothiazol-2-yl)benzoic acid benzyl ester (1 g, 2.89 mmol, 1 eq) in H O (10 mL), ACN (5 mL) and DCM (5 mL) was added RuCl .H O (13.04 mg, 57.84 μmol, 0.02 eq) and NaIO (2.47 g, 11.57 mmol, 640.97 μL, 4 eq) at 0° C. under N . The mixture was then heated to 20° C. and stirred for 2 hours. The residue was filtered and the filtrate was poured into water (40 mL). The aqueous phase was extracted with ethyl acetate (50 mL×1). The organic phase was washed with saturated NaCl (30 mL×3), dried over anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 15:1 to 5:1) to give benzyl 4-(5-chloro-1,1,3-trioxoisothiazol-2-yl)benzoate (500 mg, 1.32 mmol, 45.77% yield) as a yellow oil; 1 H NMR (400 MHz, CHLOROFORM-d) δ = 8.27-8.21 (m, 2H), 7.61-7.55 (m, 2H), 7.49-7.34 (m, 5H), 6.87 (s, 1H), 5.40 (s, 2H).

[0808] Example 10. 4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoic acid

[0809]

[0810] To a solution of benzyl 4-(5-chloro-1,1,3-trioxoisothiazol-2-yl)benzoate (450 mg, 1.19 mmol, 1 eq) in DCM (20 mL) was added dropwise methanesulfonic acid (1.14 g, 11.91 mmol, 847.94 μL, 10 eq) at 10 ° C. The solution was stirred at 35 ° C for 10 hours. The residue was concentrated in vacuo to remove DCM. The residue was then dissolved in ethyl acetate (10 mL), and the organic phase was washed with water (20 mL × 5), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in methanol (3 mL), and petroleum ether (30 mL) was poured into the residue. The solution was stirred at 10° C. for 2 minutes, then filtered, and the filter cake was dried in vacuo to obtain 4-(5-chloro-1,1,3-trioxoisothiazol-2-yl)benzoic acid (112.56 mg, 379.48 μmol, yield 31.86%, purity 96.986%) as a white solid; LC-MS (ES, m / z): 285.9 [MH]-; 1 H NMR (400MHz, DMSO-d6) δ = 13.33 (br s, 1H), 8.17-8.11 (m, 2H), 7.87 (d, J = 1.5Hz, 1H), 7.67-7.61 (m, 2H).

[0811] I.29. Benzyl 4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoate

[0812]

[0813] To a mixture of 4-(4,5-dichloro-3-oxoisothiazol-2-yl)benzoic acid benzyl ester (1 g, 2.63 mmol, 1 eq) in H O (10 mL), ACN (5 mL) and DCM (5 mL) was added RuCl .H O (11.86 mg, 52.60 μmol, 0.02 eq) and NaIO (2.25 g, 10.52 mmol, 582.91 μL, 4 eq) at 0° C. under N . The mixture was then heated to 20° C. and stirred for 2 hours. The residue was filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (SiO 2 , petroleum ether: ethyl acetate = 20: 1 to 5: 1) to give benzyl 4-(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)benzoate (130 mg, 315.35 μmol, 11.99% yield) as a white solid; 1 H NMR (400 MHz, chloroform-d) δ = 8.25 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.49-7.34 (m, 5H), 5.41 (s, 2H).

[0814] Example 11. 4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoic acid

[0815]

[0816] To a solution of 4-(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)benzyl benzoate (130 mg, 315.35 μmol, 1 eq) in DCM (5 mL) was added methanesulfonic acid (303.07 mg, 3.15 mmol, 224.49 μL, 10 eq) dropwise at 10 ° C. The solution was stirred at 10 ° C for 5 minutes, and then the solution was heated to 35 ° C and stirred for 10 hours. The color of the solution changed from colorless to yellow. The reaction solution was poured into water (20 mL) and then filtered. The filter cake was washed three times with water (10 mL × 3) and DCM (10 mL × 3), respectively. The filter cake was then dried in vacuo. The residue was dispersed with DCM (10 mL), and then petroleum ether (30 mL) was poured into the residue. The mixture was stirred at 10° C. for 2 minutes and then filtered. The filter cake was dried in vacuo to obtain 4-(4,5-dichloro-1,1,3-trioxoisothiazol-2-yl)benzoic acid (93.4 mg, 282.97 μmol, yield 89.73%, purity 97.590%) as a white solid; LC-MS (ES, m / z): 319.9 [MH]-; 1 H NMR (400MHz, DMSO-d6) δ = 8.16 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H).

[0817] I.30. Benzyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate 4-toluenesulfonate

[0818]

[0819] A mixture of 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoic acid (4 g, 18.08 mmol, 1 eq), phenylmethanol (15.64 g, 144.63 mmol, 15.04 mL, 8 eq), and TsOH.H2O (3.61 g, 18.98 mmol, 1.05 eq) in toluene (30 mL) was stirred at 140°C for 8 hours, with the condensed water collected using a Dean-Stark apparatus. After refluxing for several hours, the reaction became clear. TLC (dichloromethane:methanol = 10:1, Rf = 0.3) indicated that the reaction was complete. The clear reaction mixture was poured into TBME:petroleum ether (1:1, 50 mL), and the clear solution was removed. The residue was washed twice with TBME:petroleum ether (1:1, 50 mL) and dried in vacuo. The crude product, benzyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoate 4-toluenesulfonic acid (8.9 g, crude product), was obtained as a yellow oil; 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.76 (br d, J = 8.07 Hz, 2H) 7.33-7.38 (m, 5H) 7.15 (d, J = 7.95 Hz, 2H) 5.11 (s, 2H) 3.72 (q, J = 6.11 Hz, 4H) 3.53-3.64 (m, 8H) 3.11-3.24 (m, 2H) 2.53-2.69 (m, 2H) 2.30-2.41 (m, 1H) 2.34 (s, 3H).

[0820] I.31. Benzyl 3-[2-[2-[2-[3-[[3-[2-[2-[2-(3-benzyloxy-3-oxo-propyloxy)ethoxy]ethoxy]ethylamino]-3-oxopropyl]disulfanyl]propionylamino]ethoxy]ethoxy]ethoxy]propanoate

[0821] To a mixture of 3-(2-carboxyethyldisulfanyl)propanoic acid (1.90 g, 9.04 mmol, 1 eq), HOBt (2.69 g, 19.88 mmol, 2.2 eq), and TEA (4.57 g, 45.18 mmol, 6.29 mL, 5 eq) in DCM (100 mL) was added EDCI (3.81 g, 19.88 mmol, 2.2 eq). Benzyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoate 4-toluenesulfonic acid (8.74 g, 18.07 mmol, 2 eq) was then added to the solution. The mixture was stirred at 20°C for 12 hours. TLC (petroleum ether:ethyl acetate = 2:1, Rf = 0.25) indicated that the reaction was complete. MeOH was added, and the solution was concentrated under reduced pressure and dried in vacuo. The residue was poured into H2O (20 mL) and extracted with EtOAc (50 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by preparative TLC (petroleum ether: ethyl acetate = 1: 1, Rf = 0.5) to give 3-[2-[2-[2-[3-[[3-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy]ethoxy]ethylamino]-3-oxo-propyl]disulfanyl]propionylamino]ethoxy]ethoxy]ethoxy]benzyl propionate (6.52 g, 7.94 mmol, 87.81% yield, 97% purity) as a yellow oil; 1 H NMR (400MHz, chloroform-d) δppm 7.29-7.42 (m, 10H) 6.42 (br s,2H)5.15(s,4H)3.79(t,J=6.42Hz,4H)3.53-3.69(m,18H)2.92-3.00(m,4H)2.62-2.71(m,4H)2.53-2.62(m,4H).

[0822] I.32. Benzyl 3-(2-(2-(2-(5-chloro-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoate

[0823]

[0824] To a solution of benzyl 3-[2-[2-[2-[3-[[3-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy]ethoxy]ethylamino]-3-oxopropyl]disulfanyl]propionylamino]ethoxy]ethoxy]ethoxy]propanoate (6.4 g, 8.03 mmol, 1 eq) in DCM (50 mL) was added dropwise thionyl chloride (4.34 g, 32.12 mmol, 3.21 mL, 4 eq) in DCM (10 mL) at 0°C. The mixture was stirred at 0-10°C for 12 hours. TLC (petroleum ether:ethyl acetate = 0:1, Rf = 0.15, 0.35) indicated that the reaction was complete. The mixture was poured into ice / water (100 mL) and extracted with DCM (200 mL x 2). The combined organic layers were washed with H2O (100 mL x 2) and brine (100 mL), and dried over Na2SO4. Filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1 to 0:1) to give benzyl 3-[2-[2-[2-(3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (820 mg, 1.66 mmol, 10.33% yield, 80% purity) as a brown oil; 1 H NMR (400 MHz, chloroform-d) δ ppm 8.06 (d, J = 6.17 Hz, 1H) 7.30-7.41 (m, 5H) 6.24 (d, J = 6.39 Hz, 1H) 5.15 (s, 2H) 3.95-4.03 (m, 2H) 3.79 (t, J = 6.39 Hz, 2H) 3.68-3.75 (m, 2H) 3.59-3.68 (m, 8H) 2.63-2.70 (m, 2H) and benzyl 3-[2-[2-[2-(5-chloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (2.5 g, 4.30 mmol, 26.79% yield, 74% purity) as a colorless oil; 1 H NMR (400 MHz, chloroform-d) δ ppm 7.28-7.43 (m, 5H) 6.25 (s, 1H) 5.15 (s, 2H) 3.92-3.98 (m, 2H) 3.77-3.81 (m, 2H) 3.59-3.71 (m, 10H) 2.66 (t, J = 6.39 Hz, 2H).

[0825] I.33. Benzyl 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoate

[0826]

[0827] To a mixture of benzyl 3-[2-[2-[2-(5-chloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (1 g, 2.33 mmol, 1 eq) and NaIO (1.99 g, 9.30 mmol, 515.57 μL, 4 eq) in H O (20 mL), DCM (10 mL), and ACN (10 mL) was added RuCl .H O (26.22 mg, 116.30 μmol, 0.05 eq) in one portion at 20° C. under N . The mixture was then stirred at 20° C. for 1 hour. The residue was extracted with ethyl acetate (20 mL×2). The combined organic phases were dried over anhydrous Na SO , filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 , petroleum ether: ethyl acetate = 5: 1 to 1: 1) to give benzyl 3-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (560 mg, 1.21 mmol, 52.12% yield, 100% purity) as a purple oil; 1 H NMR (400 MHz, chloroform-d) δ = 7.42-7.29 (m, 5H), 6.70 (s, 1H), 5.15 (s, 2H), 3.92-3.86 (m, 2H), 3.77 (td, J = 6.0, 11.9 Hz, 4H), 3.68-3.58 (m, 8H), 2.67 (t, J = 6.5 Hz, 2H).

[0828] Example 12. 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoic acid

[0829]

[0830] To a solution of benzyl 3-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (560 mg, 1.21 mmol, 1 eq) in DCM (30 mL) was added dropwise methanesulfonic acid (1.17 g, 12.12 mmol, 863.06 μL, 10 eq) at 10 ° C. The mixture was heated to 35 ° C and stirred for 10 hours. The color of the solution turned yellow. The residue was washed with water (30 mL × 3), and the organic phase was then dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative TLC (ethyl acetate:ethyl acetate:methanol:acetic acid=40:8:1, Rf=0.77) to give 3-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]propanoic acid (95.61 mg, 240.16 μmol, 19.81% yield, 93.389% purity) as a colorless oil; LC-MS (ES, m / z): 372.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ = 12.13 (br s, 1H), 7.64 (s, 1H), 3.86-3.74 (m, 2H), 3.61 (td, J = 6.0, 16.9Hz, 4H), 3.54-3.46 (m, 8H), 2.43 (t, J = 6.4Hz, 2H).

[0831] I.34. Benzyl 3-(2-(2-(2-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoate

[0832]

[0833] To a solution of benzyl 3-[2-[2-[2-(5-chloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (1.6 g, 3.72 mmol, 1 eq) in DCM (30 mL) was added dropwise sulfuryl chloride (1.00 g, 7.44 mmol, 744.17 μL, 2 eq) at 0°C. The mixture was stirred at 0-10°C for 12 hours. A clear light yellow solution was obtained after the addition of sulfuryl chloride. TLC (ethyl acetate:petroleum ether=2:1, Rf=0.5) indicated that the reaction was complete. The mixture was concentrated in vacuo to give a crude product. The residue was poured into H2O (50 mL) and extracted with DCM (50 mL×2). The combined organic layers were washed with H2O (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=1:2 to 2:1) to give benzyl 3-[2-[2-[2-(4,5-dichloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (1.06 g, 1.76 mmol, 47.17% yield, 76.9% purity) as a colorless oil; 1 H NMR (400MHz, chloroform-d) δppm 7.28-7.40(m,4H)5.13(s,2H)3.98-4.04(m,2H)3.77(t,J=6.39Hz,2H)3.67-3.72(m,2H)3.57-3.67(m,8H)2.65(t,J=6.39Hz,2H).

[0834] I.35. Benzyl 3-(2-(2-(2-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoate

[0835]

[0836] To a mixture of benzyl 3-[2-[2-[2-(4,5-dichloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (1 g, 2.15 mmol, 1 eq) and NaIO4 (1.84 g, 8.61 mmol, 477.32 μL, 4 eq) in H2O (20 mL), CH3CN (10 mL) and DCM (10 mL) was added RuCl3.H2O (7.28 mg, 32.30 μmol, 0.015 eq) at 0°C under N2. The mixture was stirred at 0-10°C for 2 hours. TLC indicated that the reaction was complete. The mixture was diluted with EtOAc (50 mL) and filtered to remove insoluble solids. The organic layer was separated and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate=1:1, Rf=0.6) to give benzyl 3-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (830 mg, 1.61 mmol, yield 74.96%, purity 96.533%) as a colorless oil; 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.28-7.41 (m, 5H) 5.15 (s, 2H) 3.91-3.97 (m, 2H) 3.75-3.83 (m, 4H) 3.59-3.68 (m, 8H) 2.66 (t, J = 6.50 Hz, 2H).

[0837] Example 13. 3-(2-(2-(2-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoic acid

[0838]

[0839] To a solution of benzyl 3-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (820.00 mg, 1.65 mmol, 1 eq) in DCM (10 mL) was added dropwise methanesulfonic acid (1.59 g, 16.52 mmol, 1.18 mL, 10 eq) at 10 ° C. The solution was then heated at 35 ° C and stirred for 10 hours. The residue was diluted with DCM (20 mL), and the solution was then washed with water (15 mL × 3). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (ethyl acetate:acetic acid=250:1, Rf=0.55) to give 3-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoic acid (249.4 mg, 602.76 μmol, 36.49% yield, 98.180% purity) as a yellow oil; LC-MS (ES, m / z): 406.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ = 12.16 (br s, 1H), 3.85 (t, J = 5.5Hz, 2H), 3.65 (br t, J = 5.4Hz, 2H), 3.61-3.45 (m, 10H), 2.43 (t, J = 6.3Hz, 2H).

[0840] I.36. Benzyl 1-amino-3,6,9,12,15,18-hexaoxaheneicosane-21-oate 4-toluenesulfonate

[0841]

[0842] A mixture of benzyl alcohol (2.45 g, 22.64 mmol, 2.35 mL, 8 eq), 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (1 g, 2.83 mmol, 1 eq), and TsOH.HO (565.16 mg, 2.97 mmol, 1.05 eq) in toluene (30 mL) was stirred at 140° C. for 14 hours with a Dean-Stark trap. After several hours, the mixture turned from cloudy to clear. The residue was concentrated in vacuo to remove toluene, and then TBME (50 mL) was poured into the residue and stirred for 1 minute. Then, the supernatant was removed and dried to give benzyl 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate 4-toluenesulfonic acid (1.45 g, crude product) as a yellow oil; 1H NMR (400 MHz, CHLOROFORM-d) δ = 7.80 (d, J = 8.1 Hz, 2H), 7.67-7.46 (m, 2H), 7.39-7.31 (m, 5H), 7.15 (d, J = 7.8 Hz, 2H), 5.13 (s, 2H), 3.96-3.83 (m, 2H), 3.75-3.50 (m, 22H), 3.24-3.14 (m, 2H), 2.63 (t, J = 6.2 Hz, 2H), 2.34 (s, 3H).

[0843] I.37. 23,30-Dioxo-4,7,10,13,16,19,34,37,40,43,46,49-dodeca-26,27-dithia-22,31-diazapentadocandioic acid dibenzyl ester

[0844]

[0845] To a mixture of 3-(2-carboxyethyldisulfanyl)propanoic acid (47.41 mg, 225.47 μmol, 1 eq), TEA (91.26 mg, 901.86 μmol, 125.53 μL, 4 eq), HOBt (91.40 mg, 676.40 μmol, 3 eq), and EDCI (129.67 mg, 676.40 μmol, 3 eq) in DCM (5 mL) at 25°C was added benzyl 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate 4-toluenesulfonic acid (277.65 mg, 450.93 μmol, 2 eq). After the addition, the mixture was stirred at 25°C for 8 hours. TLC (ethyl acetate:methanol = 3:1, Rf = 0.33) indicated complete consumption of the starting material and the formation of a new major spot. The residue was poured into saturated NaCl (10 mL) and stirred for 2 minutes. The aqueous phase was then extracted with DCM (5 mL × 3). The combined organic phases were washed with saturated NaCl (10 mL × 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (chromatographic column: Waters Xbridge 150×25 5u; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B%: 32%-62%, 12 minutes) to give benzyl 3-[2-[2-[2-[2-[2-[2-[3-[[3-[2-[2-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-3-oxopropyl]disulfanyl]propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (70 mg, 65.96 μmol, 29.25% yield) as a colorless oil;1 H NMR (400MHz, chloroform-d) δ = 7.41-7.29 (m, 10H), 6.50 (br s,2H),5.14(s,4H),3.78(t,J=6.5Hz,4H),3.67-3.61(m,40H),3.59-3.55(m,4H),3.4 5(q,J=5.1Hz,4H), 2.97(t,J=7.2Hz,4H), 2.66(t,J=6.5Hz,4H), 2.60(t,J=7.1Hz,4H).

[0846] I.38. Benzyl 1-(5-chloro-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-olate

[0847]

[0848] To a solution of benzyl 3-[2-[2-[2-[2-[2-[2-[3-[[3-[2-[2-[2-[2-[2-[2-(3-benzyloxy-3-oxo-propyloxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-3-oxopropyl]disulfanyl]propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (5.5 g, 5.18 mmol, 1 eq) in DCM (60 mL) was added sulfonyl chloride (3.50 g, 25.91 mmol, 2.59 mL, 5 eq) dropwise at 0° C. The mixture was stirred at 0-20° C. for 12 hours. TLC (ethyl acetate:methanol=10:1, Rf=0.3, 0.5) indicated that the reaction was complete. The mixture was poured into ice / water (10 mL) and extracted with DCM (20 mL×2). The combined organic layers were washed with H2O (20 mL x 2) and brine (20 mL), and dried over Na2SO4. Filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1 to 0:1) to give benzyl 3-[2-[2-[2-[2-[2-(3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1.4 g, 2.29 mmol, 22.05% yield, 86.148% purity) as a brown oil; 1H NMR (400 MHz, chloroform-d) δ ppm 8.02 (d, J = 6.17 Hz, 1H) 7.22-7.32 (m, 5H) 6.17 (br d, J = 6.17 Hz, 1H) 5.07 (s, 2H) 3.92 (br t, J = 4.30 Hz, 2H) 3.51-3.73 (m, 24H) 2.58 (t, J = 6.39 Hz, 2H) and benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (3.1 g, 4.44 mmol, 42.85% yield, 80.532% purity) as a colorless oil; 1 HNMR (400 MHz, CHLOROFORM-d) δ ppm 7.30-7.40 (m, 5H) 6.26 (s, 1H) 5.15 (s, 2H) 3.93-3.99 (m, 2H) 3.78 (t, J = 6.50 Hz, 2H) 3.60-3.72 (m, 23H) 2.66 (t, J = 6.50 Hz, 2H).

[0849] I.39 1-(5-Chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-oic acid benzyl ester

[0850]

[0851] To a mixture of benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1 g, 1.78 mmol, 1 eq) and NaIO4 (1.52 g, 7.12 mmol, 394.34 μL, 4 eq) in H2O (20 mL), DCM (10 mL), and acetonitrile (10 mL) was added RuCl3.H2O (20.05 mg, 88.96 μmol, 0.05 eq) in one portion at 20°C under N2. The mixture was then stirred at 20°C for 1 hour. The residue was extracted with ethyl acetate (20 mL×2). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 , petroleum ether: ethyl acetate = 1: 1 to ethyl acetate: methanol = 10: 1) to give benzyl 3-[2-[2-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (520 mg, 849.06 μmol, 47.72% yield, 97% purity) as a yellow oil; 1H NMR (400 MHz, CHLOROFORM-d) δ = 7.44-7.29 (m, 5H), 6.72 (s, 1H), 5.14 (s, 2H), 3.92-3.86 (m, 2H), 3.80-3.73 (m, 4H), 3.69-3.59 (m, 20H), 2.66 (t, J = 6.4 Hz, 2H).

[0852] Example 14. 1-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-oic acid

[0853]

[0854] To a solution of benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (520 mg, 875.32 μmol, 1 eq) in DCM (30 mL) was added dropwise methanesulfonic acid (841.23 mg, 8.75 mmol, 623.13 μL, 10 eq) at 10°C. The solution was heated to 35°C and stirred for 10 hours. The residue was washed with water (30 mL x 3), and the organic phase was then dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (ethyl acetate:methanol:acetic acid = 40:8:1, Rf = 0.58). The residue was purified again by preparative HPLC (chromatographic column: Nano-micro Kromasil C18 100×30 mm 5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 1%-30%, 10 min) to give 3-[2-[2-[2-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (55.93 mg, 106.24 μmol, yield 12.14%, purity 95.726%) as a colorless oil; LC-MS (ES, m / z): 504.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ = 12.30-11.96 (m, 1H), 7.64 (s, 1H), 3.83-3.76 (m, 2H) ,3.65-3.58(m,4H),3.54-3.52(m,2H),3.52-3.48(m,18H),2.44-2.42(m,2H).

[0855] I.40. Benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0856]

[0857] To a solution of benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1.3 g, 2.31 mmol, 1 eq) in DCM (30 mL) was added thionyl chloride (624.34 mg, 4.63 mmol, 462.47 μL, 2 eq) dropwise at 20° C. The solution was stirred at 20° C. for 2 hours. The solution turned yellow. The residue was poured into ice water (30 mL) and stirred for 30 minutes. The DCM phase was washed with water (50 mL×6), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by preparative TLC (ethyl acetate:methanol=10:1, Rf=0.50) to give benzyl 3-[2-[2-[2-[2-[2-(4,5-dichloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (800 mg, 1.21 mmol, 52.19% yield, 90% purity) as a yellow oil; 1 H NMR (400 MHz, CHLOROFORM-d) δ = 7.40-7.29 (m, 5H), 5.15 (s, 2H), 4.04 (t, J = 4.7 Hz, 2H), 3.78 (t, J = 6.4 Hz, 2H), 3.72 (t, J = 4.7 Hz, 2H), 3.69-3.63 (m, 16H), 3.62 (s, 4H), 2.66 (t, J = 6.5 Hz, 2H).

[0858] I.41. Benzyl 1-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-olate

[0859]

[0860] To a mixture of benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (600 mg, 1.01 mmol, 1 eq) and NaIO4 (860.56 mg, 4.02 mmol, 222.94 μL, 4 eq) in H2O (20 mL), DCM (10 mL), and ACN (10 mL) was added RuCl3.H2O (11.34 mg, 50.29 μmol, 0.05 eq) in one portion at 0°C under N2. The mixture was stirred at 0°C for 2 minutes, then heated to 25°C and stirred for 1 hour. The residue was poured into ethyl acetate (30 mL) and filtered. The filtrate was extracted with ethyl acetate (30 mL×3). The combined organic phases were concentrated in vacuo. The residue was purified by preparative TLC (ethyl acetate, Rf=0.50) to give benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (220 mg, 350.03 μmol, 34.80% yield, 100% purity) as a colorless oil; 1 H NMR (400 MHz, CHLOROFORM-d) δ = 7.40-7.27 (m, 4H), 5.15 (s, 2H), 3.99-3.90 (m, 2H), 3.78 (t, J = 6.2 Hz, 4H), 3.70-3.58 (m, 20H), 2.66 (t, J = 6.4 Hz, 2H).

[0861] Example 15. 1-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-oic acid

[0862]

[0863] To a solution of benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (220 mg, 350.03 μmol, 1 eq) in methanol (5 mL) was added dropwise methanesulfonic acid (504.60 mg, 5.25 mmol, 373.78 μL, 15 eq) at 10°C. The solution was then heated to 40°C and stirred for 20 hours. The residue was diluted with DCM (20 mL), and the solution was then washed with water (15 mL x 3). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC (chromatographic column: Nano-microKromasil C18 100×30 mm 5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 25%-55%, 10 minutes) to give 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (53.79 mg, 98.63 μmol, yield 28.18%, purity 98.724%) as a yellow oil; LC-MS (ES, m / z): 538.2 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ = 12.13 (br s,1H),3.89-3.81(m,2H),3.65(t,J=5.5Hz,2H),3.59(t,J=6.4Hz,2H),3.56-3.48(m,20H),2.43(t,J=6.4Hz,2H).

[0864] Example 16.

[0865]

[0866] The product 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (12.58 mg, 0.045 mmol), DCM (2 mL) and DMF (10 μL) were added to a flask under argon. The mixture was cooled to 0 ° C and then oxalyl dichloride (11.65 μL, 0.136 mmol) was added dropwise. The mixture was warmed to room temperature and stirred until complete conversion was observed by LCMS (LCMS tracking was performed by adding to a dry MeOH aliquot to form a methyl ester). The crude product was evaporated under vacuum. The residue was dissolved in DCM and dried under vacuum again to give a yellow solid. The crude product can be used in the next step without further purification.

[0867] 2. Synthesis of Drug-Linker Conjugates

[0868] Example A. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0869]

[0870] Standard steps for drug-linker synthesis:

[0871] At room temperature, 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (205 mg, 0.73 mmol) (Example 1), dichloromethane (10 mL) and DMF (100 μL) were added to a flask under nitrogen. The mixture was cooled to 0° C. using an ice bath, and then oxalyl chloride (190.4 μL, 2.18 mmol) was added. The mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was evaporated in vacuo. The residue was dissolved in CH2Cl2 and dried under vacuum again to give 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride as a yellow solid. Into a vial at room temperature under N2 were introduced (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid, (S)-2-((2R,3R)-3- ((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid compound and 2,2,2-trifluoroacetic acid (1:1) (111 mg, 0.102 mmol) and dichloromethane (3.7 mL). The mixture was cooled to 0°C and DIPEA (70.9 μL, 0.406 mmol) was added. The reaction mixture was stirred at 0°C for 10 minutes, and then a solution of 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride (36.6 mg, 0.122 mmol) in DCM (248 mg of acid chloride in 2 mL of DCM) was added dropwise. The mixture was stirred at 0°C for 1 hour and 15 minutes. The reaction was terminated by adding trifluoroacetic acid (32.9 μL, 0.426 mmol), acetonitrile (2.1 mL), and water (0.3 mL) to the mixture at 0°C.The crude product was concentrated in vacuo, and the residue was purified by preparative HPLC (X-Bridge C18 column (100×30) using a gradient of ACN and water containing 0.1% TFA as the mobile phase) to give ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-(6-(5-chloro-1,1-dioxo-3-oxoisothiazol-2(3H)-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine. The mass spectrum of the drug-linker conjugate was shown in Figure 5. 1 H-NMR spectra are Figure 1A and Figure 1B shown.

[0872] Example B. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-(6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0873]

[0874] It was synthesized according to the standard procedure for drug-linker synthesis using 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 2) and ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine compound and 2,2,2-trifluoroacetic acid (1:1) as starting materials.

[0875] The mass spectrum of the drug-linker conjugate is as follows Figure 2 shown.

[0876] Example C. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0877]

[0878] It was obtained according to the standard procedure for drug-linker synthesis using 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 1) and ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutyramido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine as starting materials.

[0879] The mass spectra of the drug-linker conjugate and 1 H-NMR spectra are Figure 3A and Figure 3B shown.

[0880] Example D. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0881]

[0882] It was obtained according to the standard procedure for drug-linker synthesis using 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 2) and ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine as starting materials.

[0883] The mass spectra of the drug-linker conjugate and 1 H-NMR spectrum Figure 4A and Figure 4B shown.

[0884] Example E. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propionamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine 2,2,2-trifluoroacetate

[0885]

[0886] It was synthesized according to the standard procedure for drug-linker synthesis using 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoic acid (Example 12) and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid as starting materials.

[0887] The mass spectra of the drug-linker conjugate and 1 H-NMR spectrum Figure 5A and Figure 5B shown.

[0888] Example F. ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-(3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propionamido)-3-methylbutanamido)-N-methylpropionamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-D-phenylalanine

[0889]

[0890] It was synthesized according to the standard procedure for drug-linker synthesis using 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoic acid (Example 12) and ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-amino-3-methylbutanamido)-N-methylpropionamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-D-phenylalanine as starting materials.

[0891] The mass spectra of the drug-linker conjugate and 1 H-NMR spectrum Figure 6A and Figure 6B shown.

[0892] Example G. ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-N-methylpropanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-D-phenylalanine

[0893]

[0894] It was synthesized according to the standard procedure for drug-linker synthesis using 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 1) and ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-amino-3-methylbutanamido)-N-methylpropionamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-D-phenylalanine as starting materials.

[0895] The mass spectra of the drug-linker conjugate and 1 H-NMR spectrum Figure 7A and Figure 7B shown.

[0896] Example H. 4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxamido)ethyl)carbamate

[0897]

[0898] Example H was synthesized according to the following synthetic route:

[0899]

[0900] I.42. (2S,4S)-2,5,12-Trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid

[0901] To a mixture of methanol (15 mL) and water (10 mL) in a flask was added PNU-159682 (52 mg, 0.081 mmol). A solution of NaIO4 (34.7 mg, 0.162 mmol) in water (5 mL) was added. The reaction mixture was stirred at room temperature until complete conversion was observed by LCMS. The solvent was removed in vacuo to afford 1.42 as a red solid, which was used directly in the next step.

[0902] I.43. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((((2-aminoethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0903] In a flask under argon, (9H-fluorene-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (1 g, 1.662 mmol) and bis(4-nitrophenyl)carbonate (1.011 g, 3.32 mmol) were added in DMF (0.443 mol / L). The mixture was then cooled to 0°C and DIPEA (639 μL, 3.66 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 18 hours. The crude mixture was concentrated in vacuo. The crude product was dissolved in a 1:1 mixture of EtO / EtOAc and filtered. The precipitate was washed with EtO, 5% citric acid, HO, and then washed with EtO again to give a yellow solid. The solid was purified by automated silica gel column chromatography (100 DCM:0 MeOH to 80 DCM:20 MeOH) to give 345 mg of (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate as a white solid in 27.1% yield.

[0904] To a solution of the previous product (150 mg, 0.196 mmol) in DMF (6 mL) at 0° C., HOBt (34.4 mg, 0.254 mmol) and pyridine (63.3 μL, 0.782 mmol) were added. After 5 minutes, tert-butyl (2-aminoethyl)carbamate 1-2 (40.7 mg, 0.254 mmol) in DMF (1.5 mL) was added to the mixture, followed by DIPEA (102 μL, 0.587 mmol). The mixture was allowed to warm to room temperature and stirred for 2 hours. The crude product was concentrated in vacuo to give a white solid, which was purified by automated silica gel column chromatography (100 DCM:0 MeOH to 80 DCM:20 MeOH) to afford 129 mg of 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl tert-butylethane-1,2-diyldicarbamate (white solid) in 84% yield.

[0905] In a flask, in DCM (6 mL), the previous product (154 mg, 0.195 mmol) was placed. The mixture was cooled at 0°C, and TFA (753 μL, 9.77 mmol) was added and stirred at 0°C until complete conversion was observed by LCMS. The crude mixture was concentrated in vacuo to give 1.43 as a white solid (quantitative yield).

[0906] I.44. 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamido)ethyl)carbamate

[0907] In a flask, 1.42 (50.9 mg, 0.081 mmol), 1.43 (78.0 mg, 0.097 mmol) and DMF (8 mL) were added, followed by HATU (30.8 mg, 0.081 mmol) and DIPEA (56.7 μ L, 0.324 mmol). The reaction mixture was stirred at room temperature for 18 hours. Piperidine (80 μ L, 0.811 mmol) was then added to the mixture. The reaction mixture was stirred for 1 hour (until complete conversion was observed by LCMS). The mixture was concentrated under vacuum. Immediately, the crude product obtained was purified by automatic silica gel column chromatography (100 DCM: 0 MeOH / NH3 aqueous solution to 85 DCM: 25 MeOH / NH3 aqueous solution) to give 20 mg of 1.44 (red oil), in a yield of 23%.

[0908] Example H

[0909] In a flask under N2, in DCM (1mL) and DMF (10 μL), 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 1) (7.86 mg, 0.028 mmol) was added. The mixture was cooled to 0 ° C. and then oxalyl chloride (7.28 μL, 0.085 mmol) was added dropwise. The mixture was warmed to room temperature and stirred until complete conversion was observed by LCMS (LCMS tracking was performed by adding to dry MeOH aliquots to form methyl esters). The crude mixture was evaporated in vacuo. The residue was dissolved in DCM and dried in vacuo again to give 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride (quantitative yield) as a yellow solid. The crude material can be used in the next step without further purification.

[0910] At room temperature, I.44 (20 mg, 0.019 mmol) is introduced into DCM (2 mL) in a flask under N2. The mixture is cooled to 0 ° C, and DIPEA (12.96 μ L, 0.074 mmol) is added. The mixture is stirred at 0 ° C for 10 minutes, then the product (8.40 mg, 0.028 mmol) of the previous step diluted in DCM (1 mL) is added. The mixture is then stirred at 0 ° C for 2 hours (until complete conversion is observed by LCMS). The crude mixture is concentrated in vacuo and purified by automatic silica gel column chromatography (100 DCM: 0 MeOH to 85 DCM: 15 MeOH) to obtain 6.85 mg of Example H (also referred to as compound F562524) as a red solid, in a yield of 27%.

[0911] The drug-linker conjugate 1 H-NMR spectrum Figure 8 shown.

[0912] Example 1. 4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)ethyl)ethane-1,2-diylbis(methylcarbamate)

[0913]

[0914] Example 1 was synthesized according to the following synthetic route:

[0915]

[0916] I.45. 2-Oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl (perfluorophenyl) carbonate

[0917] PNU-159682 (12 mg, 0.0180 mmol) and DMF (1.5 mL) were added to a flask under argon. The mixture was cooled to 0°C and bis(perfluorophenyl)carbonate (36.9 mg, 0.094 mmol) was added. DIPEA (9.80 μL, 0.056 mmol) in DMF (0.5 mL) was then slowly added over 5 minutes. The mixture was stirred at 0°C for 3 hours (conversion observed by LCMS). The crude mixture was concentrated in vacuo and purified by automated silica gel column chromatography (100 DCM:0 [80 DCM:20 MeOH] to 50 DCM:50 [80 DCM:20 MeOH]) to afford 5.52 mg of I.45 as a red oil in a 36% yield.

[0918] I.46. (9H-Fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate 2,2,2-trifluoroacetate

[0919] In a flask under argon, in DMF (0.443 mol / L), (9H-fluorene-9-yl) methyl ((S) -1- (((S) -1- ((4- (hydroxymethyl) phenyl) amino) -1- oxo -5- ureidopentan-2-yl) amino) -3-methyl -1- oxobutan-2-yl) carbamate (1g, 1.662mmol) and bis (4-nitrophenyl) carbonate (1.011g, 3.32mmol) were added. Then, the mixture was cooled to 0 ° C and DIPEA (639 μ L, 3.66mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 18 hours. The crude mixture was concentrated in vacuo, dissolved in Et O / EtOAc (1 / 1) and filtered. The precipitate was washed with Et O, 5% citric acid, H O, and then washed with Et O again to give a yellow solid. By automatic silica gel column chromatography (100DCM:0MeOH to 80DCM:20MeOH) purification of the solid, 345mg white solid was obtained, yield 27.1%. At 0 ° C, HOBt (27.0mg, 0.200mmol) and pyridine (49.8 μ L, 0.616mmol) were added to a solution of DMF (6mL) of the compound (118mg, 0.154mmol). After 5 minutes, a solution of tert-butyl methyl (2- (methylamino) ethyl) carbamate (37.7mg, 0.200mmol) in DMF (1.5mL) was added to the mixture, followed by DIPEA (81.0 μ L, 0.462mmol). The mixture was then heated to room temperature and stirred for 2 hours (until complete conversion was observed by LCMS). The crude mixture was concentrated in vacuo to give a yellow oil, which was purified by automated silica gel column chromatography (100 DCM:0 MeOH to 80 DCM:20 MeOH) to afford 103 mg of a white solid in 82% yield.

[0920] In a flask was placed a solution of the product (198 mg, 0.243 mmol) in DCM (12 mL). The mixture was cooled to 0 ° C and TFA (935 μ L, 12.13 mmol) was added and stirred at 0 ° C for 4 hours (until complete conversion was observed by LCMS). The crude mixture was concentrated in vacuo to give 220 mg of I.46 as a clear yellow solid (quantitative yield).

[0921] I.47 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl)ethane-1,2-diylbis(methyl carbamate)

[0922] At room temperature, to DMF (1mL) solution of product 1.45 (19mg, 0.022mmol), add DMF solution (1mL) of product 1.46 (22.2mg, 0.027mmol) and DIPEA (15.59 μ L, 0.089mmol).The reaction mixture is stirred at room temperature for 3 hours (until complete conversion is observed by LCMS).Then, piperidines (22.09 μ L, 0.223mmol) is added to the mixture.The reaction mixture is stirred for 1 hour (complete conversion is observed by LCMS).The crude mixture is concentrated in vacuo, and purified by automatic silica gel column chromatography (100DCM:0MeOH / NH (9 / 1) to 75DCM:25MeOH / NH (9 / 1)) to obtain 10mg of red oily 1.47, 39% yield.

[0923] Example 1.

[0924] In a flask under N2, in DCM (1mL) and DMF (10 μL), 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 1) (7.86 mg, 0.028 mmol) was added. The mixture was cooled to 0 ° C. and then oxalyl chloride (7.28 μL, 0.085 mmol) was added dropwise. The mixture was warmed to room temperature and stirred until complete conversion was observed by LCMS (LCMS tracking was performed by adding to dry MeOH aliquots to form methyl esters). The crude mixture was evaporated in vacuo. The residue was dissolved in DCM and dried in vacuo again to give 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride (quantitative yield) as a yellow solid. The crude material can be used in the next step without further purification.

[0925] At room temperature, N2, product 1.47 (10 mg, 0.0086 mmol) in DCM (2 mL) was introduced into a flask. The mixture was cooled to 0 ° C and DIPEA (6.0 μ L, 0.034 mmol) was added. The mixture was stirred at 0 ° C for 10 minutes, and the previous product (3.90 mg, 0.013 mmol) diluted in DCM (1 mL) was then added. The mixture was then stirred at 0 ° C for 2 hours (until complete conversion was observed by LCMS). The crude product was concentrated in vacuo and purified by automatic silica gel column chromatography (100 DCM: 0 MeOH to 85 DCM: 15 MeOH) to give Example I as a red solid (2.45 mg, 19% yield).

[0926] The drug-linker conjugate 1 H-NMR spectrum Figure 9 shown.

[0927] Example J.

[0928]

[0929] Example J was synthesized according to the following synthetic route:

[0930]

[0931] Compound I.50 was prepared according to the following synthetic route:

[0932]

[0933] Compound I.48:

[0934] In a flask under argon, 2-([1,1'-biphenyl]-4-yl)propan-2-ol (1 g, 4.71 mmol) and pyridine (0.465 mL, 5.75 mmol) were added to DCM (5 mL). The mixture was then cooled to 0 ° C. and phenyl chloroformate (0.662 mL, 5.28 mmol) in dry DCM (2.4 mL) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 18 hours (checked by LCMS). The crude product was concentrated in vacuo. The solid mixture was dissolved in DCM and washed 3 times with brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give 880 mg of the desired compound I.48 as a white solid in a 56% yield. LCMS (ESI): 333.40 (MH+).

[0935] Compound I.49:

[0936] At 0 ° C, in a flask under argon containing N, N'-dimethyl-1,2-ethylenediamine (2791 μL, 26.2 mmol), N-ethyl-N-isopropylpropan-2-amine (305 μL, 1.748 mmol) and DMF (4 mL), a solution of 2-([1,1'-biphenyl]-4-yl)propan-2-ylphenyl carbonate (581 mg, 1.748 mmol) in DMF (1.5 mL) was added. The reaction mixture was warmed to room temperature and stirred for 24 hours (checked by LCMS). The crude product was concentrated in vacuo, and the residue was purified by automatic column chromatography (Interchim, solid deposition): DCM / MeOH: 9 / 1. The desired fractions were concentrated in vacuo to give 433 mg of the desired compound I.49 as a yellow oil with a yield of 76%. LCMS (ESI): 327.43 (MH+).

[0937] Compound 1.50:

[0938] At 0 ° C, in a flask containing bis(trichloromethyl)carbonate (157 mg, 0.531 mmol) and toluene (4.3 mL) under argon, a solution of 2-([1,1'-biphenyl]-4-yl)propan-2-ylmethyl(2-(methylamino)ethyl)carbamate (433 mg, 1.326 mmol) and triethylamine (368 μL, 2.65 mmol) in toluene (2.9 mL) was added. The reaction mixture was warmed to room temperature and stirred for 1 hour (checked by LCMS). The solution was filtered, the solvent was concentrated in vacuo, and the residue was purified by automatic column chromatography (Interchim, solid deposition): cyclohexane / ethyl acetate: 7 / 3. The desired fractions were concentrated in vacuo to give 166 mg of the desired compound I.50 as a white solid in 33% yield. LCMS (ESI): 405.60 (MH+).

[0939] Compound I.52 was prepared according to the following synthetic route:

[0940]

[0941] Compound I.51:

[0942] In the presence of (8S,10S)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydronaphthacene-5,12-dione (50 mg, 0 To a flask under argon containing 2-([1,1'-biphenyl]-4-yl)propan-2-yl(2-((chlorocarbonyl)(methyl)amino)ethyl)(methyl)carbamate (91 mg, 0.234 mmol)) was added a solution of 2-([1,1'-biphenyl]-4-yl)propan-2-yl(2-((chlorocarbonyl)(methyl)amino)ethyl)(methyl)carbamate (91 mg, 0.234 mmol)) in DCM (0.5 mL). The mixture was stirred at 25° C. in the dark for 5 days. The solution was filtered, the solvent was concentrated in vacuo, and the residue was used in the next step without further purification.

[0943] Compound I.52:

[0944] To a solution of product 1.51 in DCM (1 mL) in an ice bath was added a solution of dichloroacetic acid (96 μL, 1.169 mmol) in 0.5 mL of DCM. The solution was stirred at room temperature for 2 hours. The solvent was concentrated in vacuo, and the residue was purified by automated column chromatography (Interchim, solid deposition): DCM / MeOH: 9 / 1. The desired fractions were concentrated in vacuo to give 13 mg of the desired compound 1.52 as a red solid in a 22% yield. LCMS (ESI): 756.76 (MH+).

[0945] Compound I.53:

[0946] In a flask under argon was added (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (250 mg, 0.485 mmol), bis(perfluorophenyl)carbonate (382 mg, 0.970 mmol) and DMF (4 mL). The mixture was then cooled to 0° C. and N-ethyl-N-isopropylpropan-2-amine (127 μL, 0.727 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 2 hours (checked by LCMS). The crude product was concentrated in vacuo. The crude product was purified by automated column chromatography (Interchim, solid deposition): DCM / MeOH: 9 / 1. The desired fractions were concentrated in vacuo to give 281 mg of the desired compound I.53 as a yellow oil in 80% yield. LCMS (ESI): 726.65 (MH+).

[0947] Compound I.54:

[0948] In a flask under argon was added 2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethylmethyl(2-(methylamino)ethyl)carbamate (78 mg, 0.103 mmol), 1-hydroxybenzotriazole (27.9 mg, 0.206 mmol), N,N'-diisopropylethylamine (35.1 μL, 0.206 mmol) and DMF (2 mL). The mixture was then cooled to 0° C. and (9H-fluoren-9-yl)methyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-((((perfluorophenoxy)carbonyl)oxy)methyl)phenyl)amino)propan-2-yl)amino)butan-2-yl)carbamate (112 mg, 0.155 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 2 hours (checked by LCMS). The crude product was concentrated in vacuo. The crude product was purified by automated column chromatography (Interchim, solid deposition): DCM / MeOH: 9 / 1. The desired fractions were concentrated in vacuo to give 77 mg of the desired compound I.54 as a red oil in 58% yield. LCMS (ESI): 1298.0 (MH+).

[0949] Compound I.55:

[0950] In a flask under argon was added 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)benzyl(2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl)ethane-1,2-diylbis(methylcarbamate) (77.7 mg, 0.060 mmol) and DMF (2 mL). Then, the mixture was cooled to 0°C and morpholine (259 μL, 2.99 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 2 hours (checked by LCMS). The crude product was concentrated in vacuo. The crude product was purified by automatic column chromatography (Interchim, solid deposition): DCM / MeOH: 9 / 1. The desired fractions were concentrated in vacuo to give 32 mg of the desired compound I.55 as a red oil in a 50% yield. LCMS (ESI): 1075.80 (MH+).

[0951] Example J.

[0952]

[0147] 4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)benzyl(2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl)ethane-1,2-diylbis(carbamate) (32 mg, 0.030 mmol, 1 eq) was introduced in dichloromethane (2 mL) at 25 °C. The mixture was cooled to 0°C and N-ethyl-N-isopropylpropan-2-amine (20.74 μL, 0.119 mmol) was added. The mixture was stirred at 0°C for 10 minutes, and then Example 16 diluted in dichloromethane (2 mL) was added. The mixture was then stirred at 0°C for 2 hours (until complete conversion was observed by LCMS). The crude product was concentrated in vacuo and purified by automated column chromatography (Interchim, 12 g, solid deposition): DCM / MeOH: 9 / 1. The desired fractions were concentrated in vacuo to give 17.4 mg of the desired Example J (also known as Compound F562646) as a red oil in 40% yield. LCMS (ESI): 1338.41 (MH+).

[0953] The mass spectrum of the drug-linker conjugate is as follows Figure 21 shown.

[0954] Example K.

[0955]

[0956] Example K was synthesized according to the following synthetic route:

[0957]

[0958] Compound I.56:

[0959] In a flask under argon, starting material SM1 (100 mg, 0.135 mmol) and DMF (1 mL) were added. The mixture was cooled to 0 ° C. and then a solution of 3-bromopropionic acid (22.79 mg, 0.149 mmol) and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (40.5 μL, 0.271 mmol) in DMF (0.5 mL) was added dropwise. The mixture was then warmed to room temperature and stirred until complete conversion was observed by LCMS. The crude product was concentrated in vacuo and purified by automatic column chromatography (Interchim, 12 g, solid deposition): DCM / MeOH: 80 / 20. The desired fraction was concentrated in vacuo to give 108 mg of the desired compound I.56 as a white solid in 98% yield. LCMS (ESI): 811.35 (MH+).

[0960] Compound I.57:

[0961] In a flask under argon, compound I.56 (87.0 mg, 0.107 mmol) and DCM (2 mL) were added. The mixture was then cooled to 0 ° C and N-hydroxysuccinimide (13.59 mg, 0.118 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (30.9 mg, 0.161 mmol) were added. The reaction mixture was warmed to room temperature and stirred for 2 hours (LCMS check). The crude product was concentrated in vacuo. The crude material was used in the next step without further purification.

[0962] Compound I.58:

[0963] In a flask under argon, compound I.57 (97 mg, 0.107 mmol) and DCM (1 mL) were added, followed by dropwise addition of a solution of compound I.49 in 1 mL of DCM and N,N'-diisopropylethylamine (37.3 μL, 0.214 mmol). The mixture was stirred until complete conversion was observed by LCMS. The crude product was concentrated in vacuo and used in the next step without further purification.

[0964] Compound 1.59:

[0965] In a flask under argon, compound I.58 (58 mg, 0.052 mmol) and DCM (1 mL) were added. The mixture was cooled to 0 ° C., and then dichloroacetic acid (86 μL, 1.037 mmol) was added dropwise. The mixture was then warmed to room temperature and stirred until complete conversion was observed by LCMS. The crude product was concentrated in vacuo and purified by automatic column chromatography (Interchim, 12 g, solid deposition): DCM / MeOH: 80 / 20. The desired fractions were concentrated in vacuo to obtain 39 mg of the desired compound I.59 as a white solid in 86% yield. LCMS (ESI): 882.6 (MH+).

[0966] Example K.

[0967] At 25 ° C, in a flask under argon, compound I.59 was introduced in dichloromethane (1 mL). The mixture was cooled to 0 ° C, and N-ethyl-N-isopropylpropan-2-amine (15.83 μL, 0.091 mmol) was added. The mixture was stirred at 0 ° C for 10 minutes, and then Example 16 diluted in dichloromethane (2 mL) was added. The mixture was then stirred at 0 ° C for 2 hours (until complete conversion was observed by LCMS). The crude product was concentrated in vacuo and purified by preparative HPLC (HCOOH conditions) to give 6 mg of the desired Example K as a white solid with a yield of 22%. LCMS (ESI): 1165.37 (M+Na) + .

[0968] The mass spectrum and TOF-MS spectrum of the drug-linker conjugate are shown in Figure 22A and Figure 22B .

[0969] 3. Coupling with somatostatin

[0970] VI1. Reaction of Somatostatin with Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0971]

[0972] 1 mg of lyophilized somatostatin (m 准确=1636.72) was dissolved in 4 mL of buffer (57.5% NaH2PO4 20 mM, pH 6.5, 40% ACN, 2.5% DMF) to a concentration of 153 μM (0.25 mg / mL). 33.5 mg of TCEP was dissolved in 4 mL of buffer (57.5% NaH2PO4 20 mM, pH 6.5, 40% ACN, 2.5% DMF). 3 μL of TCEP solution (1.1 eq) was added to 300 μL of somatostatin solution (1 eq). The solution was stirred at 37°C for 1 hour. Commercial somatostatin: R t,1 (ACN):1.57;MSES+:M +3 / 3=546.4,M +2 / 2=819.2. Disulfide bond-reduced somatostatin: R t,1 (ACN medium):1.50;MS ES+:M +3 / 3=547.2,M +2 / 2 = 820.3. 5 mg of benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (I.6) was dissolved in 800 μL of ACN. 3 μL of benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (1.1 eq) was added to the somatostatin solution. The solution was stirred at 37°C. t,1 (ACN medium):1.66;MS ES+:M +3 / 3=637.6,M +2 / 2=956.0.

[0973] The same reaction was performed in pH 8 buffer (57.5% NaH2PO4 20 mM, pH 8, 40% ACN, 2.5% DMF).

[0974] The mass spectrum of the obtained conjugate is as follows Figure 10 shown.

[0975] VI2. Reaction of Somatostatin with Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0976]

[0977] 1 mg of lyophilized somatostatin (m 准确=1636.72) was dissolved in 4 mL of buffer (57.5% NaH2PO4 20 mM, pH 6.5, 40% ACN, 2.5% DMF) to a concentration of 153 μM (0.25 mg / mL). 33.5 mg of TCEP was dissolved in 4 mL of buffer (57.5% NaH2PO4 20 mM, pH 6.5, 40% ACN, 2.5% DMF). 3 μL of TCEP solution (1.1 eq) was added to 300 μL of somatostatin solution (1 eq). The solution was stirred at 37°C for 1 hour. Commercial somatostatin: R t,1 (ACN):1.57;MSES+:M +3 / 3=546.4,M +2 / 2=819.2. Disulfide bond-reduced somatostatin: R t,1 (ACN medium):1.50;MS ES+:M +3 / 3=547.2,M +2 / 2=820.3. 5.4 mg of benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (I.7) was dissolved in 800 μL of ACN. 3 μL of benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (1.1 eq.) solution was added to the somatostatin solution. The solution was stirred at 37°C. t,1 (ACN medium):1.65;MS ES+:M +3 / 3=637.3,M +2 / 2=955.5.

[0978] The same reaction was performed in pH 8 buffer (57.5% NaH2PO4 20 mM, pH 8, 40% ACN, 2.5% DMF).

[0979] The mass spectrum of the obtained conjugate is as follows Figure 11 shown.

[0980] 4. Conjugation with monoclonal antibodies

[0981] ADC Synthesis, Purification, and Characterization

[0982] The steps described below are applicable to chimeric, humanized and human IgG1 formats. It must be understood that for any other format, such as IgG2, IgG4 etc., a person skilled in the art will be able to adapt the steps using general knowledge.

[0983] Ab1 is an anti-IGF1R IgG1 monoclonal antibody. This antibody corresponds to antibody 208F2 of WO2015162291 (see Table 3, page 36), wherein the three light chain CDRs have sequences of SEQ ID Nos. 9, 5, and 11; the three heavy chain CDRs have sequences of SEQ ID Nos. 7, 2, and 3; the light chain variable domain has sequence of SEQ ID No. 18; and the heavy chain variable domain has sequence of SEQ ID No. 13.

[0984] The Ab2 antibody is an unrelated chimeric (IgG1) antibody directed against a bacterial protein, the outer membrane protein A of Escherichia coli, and is designated c9G4 (Haeuw JF and Beck A. Proteomics for development of immunotherapies, In Proteomics: Biomedical and Pharmaceutical Applications, Kluwer Academic Publishers, Hondermarck H. ed., 2014, pp. 243-278; WO2015162291).

[0985] The antibody (1-5 mg / mL) was partially reduced with TCEP hydrochloride at 37°C for 2-4 hours in 10 mM borate buffer (pH 8.4, containing 150 mM NaCl and 2 mM EDTA). Typically, 6-20 molar equivalents of TCEP were used to achieve a target DAR of approximately 4. Partial reduction of the antibody was confirmed by SDS-PAGE analysis under non-reducing conditions. The antibody concentration was then adjusted to 1 mg / mL with 10 mM borate buffer (pH 8.4, containing 150 mM NaCl, 2 mM EDTA, 6% sucrose), and a 5-20 molar excess of drug-linker conjugate (10 mM DMSO solution) was added to the antibody.

[0986] Seven examples of drug-linker conjugates according to the present invention were conjugated to Ab1:

[0987] - Example A and Example B give ADC1-A and ADC1-B (non-cleavable linker), respectively;

[0988] - Example C, Example D, Example E, Example F and Example G give ADC1-C, ADC1-D, ADC1-E, ADC1-F and ADC1-G (cleavable linker), respectively.

[0989] The final DMSO concentration was adjusted to 10% to maintain drug solubility in aqueous media during the conjugation process. The reaction was carried out at room temperature or 37°C for 1-4 hours. Excess drug was quenched by adding 2.5 mol N-acetylcysteine ​​per mol of drug and incubating at room temperature for 1 hour.

[0990] After overnight dialysis at 4°C with 25 mM His buffer (pH 6.5, containing 150 mM NaCl and 6% sucrose), the rebridged antibody-drug conjugate was purified using commercially available chromatography columns and ultrafiltration units using methods known to those skilled in the art. The purified ADC was sterile filtered through a 0.2 μm filter and stored at 4°C.

[0991] The drug was further analyzed by SDS-PAGE under reducing and non-reducing conditions to confirm drug conjugation, and SEC analysis was performed on a TSKG3000 SWXL analytical column to determine the monomer content and aggregated form. Figure 13 ) It was deduced that the content of aggregated form was less than 5%, as shown in Table 9.

[0992] Table 9. Content of aggregated forms

[0993] Ab / ADC monomer% Ab1 99.6 ADC1-A 99.0 ADC1-B 99.3 ADC1-C 98.1 ADC1-D 99.5 ADC1-E 99.4 ADC1-F 99.6 ADC1-G 95.2

[0994] SDS-PAGE analysis confirmed the formation of fully bridged antibody H2L2 ( Figure 12 However, other species corresponding to partially bridged antibodies were also detected (H2L, H2, and HL). It is important to note that these species were only visible when the samples were heat treated under reducing conditions prior to the run to ensure that the heavy and light chains (H and L) were completely dissociated (not connected by an intact interchain bridge).

[0995] Protein concentration was determined by using a BCA assay using IgG as a standard. The DAR of each purified ADC was estimated by HIC using a TSK-butyl-NPR column. It was between 3.5 and 4.3 (Table 10). The HIC curve showed that for most of the synthesized ADCs, no DAR0 was observed, and a main peak of DAR4 was observed. In fact, only ADC1-C and E showed trace amounts of DAR0. In addition, for ADC1-A, B, C, and G, only DAR3, DAR4, and DAR5 were observed. Except for ADC1-D, the main peak was DAR4. Compared to second-generation ADCs, these ADCs have higher homogeneity, as shown in Table 10.

[0996] Table 10. DAR distribution estimated by HIC using the TSK-Butyl-NPR column

[0997]

[0998] use (brentuximab vedotin) was used as a reference because it uses a second-generation maleimide linker coupled to the cysteines of the antibody. It is the best representative example of a second-generation ADC; the technology described in this claim can be considered third-generation.

[0999] 4.2. Analysis of ADCs by native mass spectrometry

[1000] All chemicals were purchased from Sigma-Aldrich: ammonium acetate (A1542), cesium iodide (21004), 2-propanol (I9516). IgGZERO (A0-IZ1-010) enzyme was obtained from Genovis. Ultrapure water system (Sartorius, Germany) to prepare aqueous solutions.

[1001] Prior to native MS experiments, ADC1-A to ADC1-G were deglycosylated by incubating with one unit of IgGZERO per microgram of ADC at 37°C for 30 minutes. The IgGZERO was then concentrated using a microconcentrator (Vivaspin, 10-kD cutoff, Sartorius, The ADC was buffer exchanged with 150 mM ammonium acetate solution (pH 6.9) through six concentration / dilution cycles. Protein concentration was determined by UV absorbance using a NanoDrop spectrophotometer (Thermo Fisher Scientific, France). Non-denaturing (native) mass spectrometry analysis of the ADC was performed on a Q-TOF (Synapt G2 HDMS, Waters, Manchester, UK) mass spectrometer operating in positive ion mode, all coupled to a chip-based automated nanoelectrospray device (Triversa Nanomate, Advion, Ithaca, USA). Analysis was performed over the m / z range of 1000-10000. Samples were diluted in 150 mM NH4OAc (pH 6.9) and injected at 10 μM. External calibration was performed using singly charged ions generated by a solution of 2 g / L cesium iodide in 2-propanol / water (50 / 50 v / v).

[1002] The nanoelectrospray voltage was set to 1.75 kV, the nitrogen nanoflow was set to 0.75 psi, the cone voltage was set to 180 V, and the back pressure was set to 6 mbar.

[1003] Figure 14 shows an example of a non-deconvoluted mass spectrum.

[1004] The MaxEnt software of MassLynn 4.1 (Waters, Manchester, UK) was used.TM After deconvolution, the algorithm determined the DAR distribution (Figure 15). The software parameters were optimized for each spectrum.

[1005] The average DAR value ( FIG. 15 ) was calculated by using the following formula (where j is the maximum drug loading).

[1006]

[1007] The results were derived from the relative peak intensities of each charge state in the original spectra and are shown in Table 11 below.

[1008] Table 11. MaxEnt using Mass Lynx 4.1 TM DAR distribution calculated by the algorithm

[1009]

[1010] Figure 16 compares the DAR distributions of two different ADCs, which were determined from the raw spectra after mass deconvolution, namely:

[1011] -ADC1-C according to the present invention, which is prepared from Ab1 antibody and drug-linker conjugate C ( Figure 16B ),and

[1012] - Reference ADC Ref-A, a comparative ADC synthesized from the same antibody (Ab1) and a drug-linker conjugate corresponding to drug-linker conjugate C, wherein the sulfonylmaleimide moiety ( ) is replaced by a maleimide moiety ( ) instead of ( Figure 16A ).

[1013] For ADCs synthesized by linking drugs to antibodies using classical maleimide chemistry, a heterogeneous distribution ranging from DAR 0 to DAR 8 was observed ( Figure 16A ), whereas the ADCs generated using the sulfonylmaleimide chemistry according to the present invention were highly uniform with 75% DAR 4, no DAR 0 / 2 and 6 / 8 species ( Figure 16B ). These results are summarized in Table 12 below.

[1014] Table 12. MaxEnt using MassLynx 4.1 TM DAR distribution calculated by the algorithm

[1015]

[1016] 4.3. In vitro stability of ADCs in sera from four mammals using a ligand binding assay

[1017] In order to confirm the increase of stability, in vitro stability study was carried out. It includes incubating ADC at 37 DEG C for 14 days. Samples were collected at 0, 3, 7 and 14 days. Each sample (D0, D3, D7 and D14) was then analyzed by LBA to determine the total antibody concentration versus ADC concentration. In practice, the solution of each ADC was prepared in 4 serums (human, cynomolgus monkey, mouse and rat) at a concentration of 100 μg / mL and incubated at 37 DEG C for up to 14 days. Aliquots were then collected at D0, D3, D7 and D14 and stored at -80 DEG C until use. For total Ab and ADC quantification, the plate was shaken and thawed at room temperature, and two LBA assays were performed in parallel. In brief, 2 μg / mL anti-His antibody solutions prepared using 50 μL of PBS 1× were coated onto standard microtiter plates (MSD, Gaithersburg, USA). After incubation at 4°C overnight, the assay plates were treated with blocking buffer (3% MSD Blocker A (MSD, Gaithersburg, USA)) for 1 hour at 37°C. Recombinant His-tagged antigen was then added at a concentration of 2.5 μg / mL in assay buffer for 1 hour at 37°C. After a wash step, the samples were analyzed in duplicate at a 1 / 5000° dilution and incubated at 37°C for 1 hour, while standard ADC was loaded in duplicate onto the assay plates. The detection step was completed using a 1 μg / mL goat anti-human Igκ sulfonyl-tag solution for detection of total Ab, or a sulfonyl-tag labeled mouse monoclonal anti-drug antibody for ADC detection. After incubation at 37°C for 1 hour, detection was achieved using 150 μL of 2× MSD-read T buffer (MSD, Gaithersburg, USA) containing surfactant, followed by reading using an MSD Sector Imager.

[1018] The concentrations of total antibody and ADC were determined at each time point and converted to percentages, with 100% being the quantification of total ADC or antibody at each time point.

[1019] The data of 3 ADCs are as follows Figure 17A 、 Figure 17B and Figure 17C As shown: ADC1-C( Figure 17B ) and ADC1-E( Figure 17C ) and reference ADC Ref-B( Figure 17A ), wherein ADC1-C and ADC1-E, in which the drug has been linked to the Ab1 antibody using sulfonylmaleimide chemistry according to the present invention (linkage is performed via drug-linker conjugates C or E, respectively), are compared to reference ADCRef-B, in which the drug has been linked to the antibody using classic maleimide chemistry.

[1020]

[1021] Drug-linker moiety for preparing reference ADC Ref-B

[1022] As a comparator, a drug-linker using the same payload and non-cleavable linker (the drug-linker of ADCRef-B) was selected. It was coupled to the same antibody using maleimide chemistry. This comparator was chosen to limit the "instability" of the reference ADC in serum, which is the instability of the ADC being uncoupled from the antibody via the reverse Michael reaction. This comparator is therefore more favorable than our construct based on a cleavable linker, which allows for a more substantial improvement in the stability of our drug-linker.

[1023] As expected, a decrease in ADC concentration was observed for the ADC synthesized using classical maleimide chemistry (ADC Ref-B), whereas ADCs generated using sulfonylmaleimide chemistry according to the present invention (ADC1-C and ADC1-E) were surprisingly more stable over 14 days.

[1024] In vitro cytotoxicity of ADCs

[1025] The in vitro cytotoxicity of the ADC according to the present invention was evaluated. To evaluate nonspecific cytotoxicity, the compound was also conjugated to an unrelated chimeric antibody (Ab2, referred to as c9G4) using the same drug-linker conjugate at the same DAR to obtain ADC2-C (Example C), ADC2-E (Example E) and ADC2-F (Example F).

[1026] MCF-7 and NCI-H2122 cells were seeded in complete growth medium in 96-well plates (2500 cells per well). The next day, serial dilutions of the ADC to be tested were added to the corresponding wells and incubated at 37°C for 6 days. Six days after the addition of the ADC, the plates were tested using the Cell Titer Glo assay (PROMEGA) to check cell viability.

[1027] The results obtained are expressed as percentage of survival and are shown in Figure 18A and Figure 18B As expected, ADCs synthesized with irrelevant antibodies had no or minimal cytotoxic activity against MCF-7 and NCI-H2122 cells. In contrast, the ADCs of the present invention: ADC1-C, ADC1-E, and ADC1-F significantly reduced cell viability. The EC values ​​of ADC1-C, ADC1-E, and ADC1-F obtained on NCI-H2122 cells were 50 The values ​​are 7, 61.10 respectively -11 、7,16.10-11 and 3,64.10 -11 M, EC values ​​of ADC1-C, ADC1-E, and ADC1-F obtained on MCF7 cells 50 The values ​​are 1, 0, 4, and 10 respectively. -11 、1,33.10 -11 and 7,39.10 -11 M, indicates potent cytotoxic activity.

[1028] In vivo

[1029] All experimental protocols have been approved by Pierre Fabre's Institutional Animal Care and Use Committee.

[1030] For the ovarian cancer model, 7-week-old female SCID mice (Charles RIVER Laboratories) were subcutaneously implanted with 10.10 6 CaoV3 cells (6 animals per group).

[1031] Treatment was by intravenous administration of ADC1-C according to the present invention, reference ADC Ref-A, or ADC vector, starting when tumors reached approximately 150 mm 3 Animals were treated with one injection (Q1d1) or three injections (once a week) (Q7d3). Tumor volumes (length × width × height × 0.52) were measured at least twice a week using electronic calipers for approximately 25 days after the first injection. The results are shown in Figure 19 (Q1d1 treated animals) and Figure 20 (Q7d3 treated animals).

[1032] from Figure 19 and Figure 20 It can be seen that even after a single injection, the ADC according to the present invention has a tremendous efficacy, resulting in complete tumor regression.

[1033] 5. PNU-159682 derivatives coupled to monoclonal antibodies

[1034] ADC Synthesis, Purification, and Characterization

[1035] Two PNU-159682 derivatives, F562524 (Example J) and F562646 (Example H), were conjugated to antibodies 208F2 (Ab1) and c9G4 (Ab2) under the conditions previously described in Example 4. 208F2 (Ab1) and c9G4 (Ab2) were as described in Example 4. Briefly, the antibodies (1-5 mg / mL) were partially reduced with 6-20 equivalents of TCEP hydrochloride in 10 mM borate buffer (pH 8.4, containing 150 mM NaCl and 2 mM EDTA) at 37°C for 2-4 hours. The antibody concentration was then adjusted to 1 mg / mL with 10 mM borate buffer (pH 8.4, containing 150 mM NaCl, 2 mM EDTA, and 6% sucrose), and a 5-20 molar excess of the drug-linker conjugate (10 mM in DMSO) relative to the antibody was added. The reaction was carried out at room temperature or 37°C in the presence of 10% DMSO for 1-4 hours. Excess drug was quenched by adding 2.5 mol of N-acetylcysteine ​​per mol of drug and incubating at room temperature for 1 hour. After overnight dialysis at 4°C against 25 mM His buffer (pH 6.5, containing 150 mM NaCl and 6% sucrose), the ADC was purified by chromatography or ultrafiltration. The ADC concentration was determined by using a BCA assay with IgG as a standard, and the purified ADC was stored at 4°C after sterile filtration on a 0.2 μm filter.

[1036] The ADC was further analyzed by SDS-PAGE and SEC (TSK G3000 SWXL column) to confirm drug conjugation and re-bridging and to determine the content of monomer and aggregates as previously described in Example 4. The monomer content was approximately 95% (Figure 23 and Table 13).

[1037] Table 13. Monomer content

[1038] ADC monomer% Ab1(208F2) 99.8 Ab1-F562524(208F2-F562524) 94.1 Ab1-F562646(208F2-F562646) 94.8 Ab2(c9G4) 99.7 Ab2-F562524(c9G4-F562524) 94.4 Ab2-F562646(c9G4-F562646) 94.7

[1039] ADC Analysis by Native LC-MS

[1040] ADC was analyzed by native liquid chromatography-mass spectrometry in a UPLC Acquity H Class Bio system coupled to a Synapt G2Si mass spectrometer (Waters). LC separation was performed on two polyhydroxyethyl A columns (Poly-LC, 150×1 mm, 300A, 5 μm) connected in series. The sample was diluted to 0.2 mg / mL with elution buffer (150 mM ammonium acetate). 4 μg of sample was injected and eluted at a flow rate of 40 μL / min. The mass spectrometer was operated in positive mode with a capillary voltage of 2.9 kV. The sample cone was set to 150 V. Analysis was performed in the range of m / z 1000-8000 with a scan time of 1 second. The m / z spectrum before deconvolution is shown in Figure 24. After deconvolution of the MS spectrum, the MaxEnt software of Mass Lynx (Waters) was used. TM The algorithm determines the DAR distribution (Figure 25). The average DAR value is calculated by the following formula (where j is the maximum drug loading):

[1041]

[1042] The results are shown in Table 14 below.

[1043] Table 14. ADC Analysis by Native LC-MS Analysis: DAR Distribution and Average DAR

[1044]

[1045]

[1046] In vitro stability

[1047] ADC hz208F2-F562524 in 200 μg / mL cynomolgus monkey serum was incubated for 14 days at 37° C. Samples were collected on days 0, 3, 7, and 14 and stored at −80° C. until LC-MS analysis to determine the mean DAR.

[1048] Prior to LC-MS analysis, the samples were immunopurified using streptavidin magnetic beads (M-280, Invitrogen) coated with Capture Select anti-human IgG-biotin conjugate (LifeTechnologies, 8 μg antibody / 200 μL beads). The samples were incubated with anti-IgG coated beads at room temperature for 2 hours (100 μL sample / 200 μL beads) and then acid eluted with 40 μL of 0.4% trifluoroacetic acid. 4 μL of 3M Tris / HCL solution (pH 8.8) was added to increase the pH. Prior to LC-MS analysis under native conditions as described above, the immunopurified samples were incubated with 2 μL of IgGZero at 37°C for 30 minutes. After deconvolution of the MS spectra, the samples were analyzed using MaxEnt™ software (Waters) from Mass Lynx. TM The algorithm determines the DAR distribution and calculates the average DAR value using the following formula (where j is the maximum drug loading):

[1049]

[1050] The results are shown below in Table 15. ADC hz208F2-F562524 was highly stable after incubation in cynomolgus monkey serum for up to 14 days in vitro.

[1051] Table 15. In vitro stability study of hz208F2-F562524: DAR distribution and average DAR

[1052]

[1053] In vitro cytotoxicity

[1054] The cytotoxicity of ADC was evaluated in MCF-7 and NCI-H2122 cells. Cells were seeded in complete growth medium on 96-well plates (2500 cells per well). The next day, serial dilutions of the ADC to be tested were added to the corresponding wells and incubated at 37°C for 6 days. Cell viability was determined by measuring ATP using a cell Titer Glo kit (Promega). Luminescence was read using a Mithras plate reader (Berthold). The results obtained are expressed as percentages of survival, as shown in Figures 26 and 27. The survival rate in untreated wells was 100%.

[1055] As expected, the ADCs hz208F2-F562524 and hz208F2-F562646 significantly reduced cell viability. EC values ​​of hz208F2-F562524 and hz208F2-F562646 determined on NCI-H2122 cells 50 The values ​​are 2.48.10 respectively-11 and 1.92.10 -12 M, ECs of hz208F2-F562524 and hz208F2-F562646 obtained on MCF-7 cells 50 The values ​​are 5.86.10 respectively -12 and 9.45.10 -13 M, indicating potent cytotoxic activity. In contrast, the corresponding ADC synthesized with an irrelevant antibody showed less cytotoxic activity against both MCF-7 and NCI-H2122 cells.

[1056] 5.5. In vivo antitumor activity

[1057] Seven-week-old female SCID mice (Charles River Laboratories) were implanted subcutaneously with 10.10 6 Caov3 cells (6 animals per group). Treatment was performed by intravenous administration (Q7d2) of ADC hz208F2-F562524 (0.3 mg / kg), corresponding control ADC c9G4-F562524 (0.3 mg / kg), or vehicle, starting when tumors reached approximately 150 mm 3 About 50 days after the first injection, the tumor volume (length × width × height × 0.52) was measured twice a week using an electronic caliper. The results are shown in Figure 28 Complete tumor regression was observed after two injections of the ADC hz208F2-F562524, whereas no antitumor effect was observed with the control ADC or vehicle.

[1058] 5.6. Conclusion

[1059] The ADCs synthesized from PNU-159682 derivatives using sulfonylmaleimide linker technology were highly homogeneous and stable in serum, and their efficacy was demonstrated in different in vitro and in vivo models.

[1060] 6. Overall Conclusion

[1061] In summary, compared to conventional maleimide-based linkers used in commercially available compounds (e.g., Adcetris), the sulfonylmaleimide-based linker technology described in this invention has better stability in plasma across different species and better efficacy in in vitro models. These properties have translated into significant improvements in in vivo efficacy across different cell lines, especially for a cell line with low antigen expression (CAOV3).

[1062] Better tolerability is also expected, as the ADCs according to the invention are more stable in the circulation, which correlates with improved efficacy and safety margins in human treatment.

[1063]

[1064]

[1065]

[1066]

[1067]

[1068]

[1069]

[1070]

[1071]

[1072]

[1073]

[1074]

[1075]

[1076]

[1077]

[1078]

[1079]

[1080]

[1081]

[1082]

[1083]

[1084]

[1085]

[1086]

[1087]

[1088]

[1089]

[1090]

[1091]

[1092]

[1093]

[1094]

[1095]

[1096]

[1097]

[1098]

[1099]

[1100]

[1101]

[1102]

[1103]

[1104]

[1105]

[1106]

[1107]

[1108]

[1109]

[1110]

[1111]

[1112]

[1113]

[1114]

[1115]

[1116]

[1117]

[1118]

[1119]

[1120]

[1121]

[1122]

[1123]

[1124]

[1125]

[1126]

[1127]

[1128]

[1129]

[1130]

[1131]

[1132]

[1133]

[1134]

[1135]

[1136]

[1137]

[1138]

[1139]

[1140]

[1141]

[1142]

[1143]

[1144]

[1145]

[1146]

[1147]

[1148]

[1149]

[1150]

[1151]

[1152]

[1153]

[1154]

[1155]

[1156]

[1157]

[1158]

[1159]

[1160]

[1161]

[1162]

[1163]

[1164]

[1165]

[1166]

[1167]

[1168]

[1169]

[1170]

Claims

1. A linker of the following formula (I), or a salt thereof, in: - X1 and X2 independently represent H, a halogen atom, a (C1-C6) alkoxy group, or an unsubstituted or substituted phenoxy group, wherein the substituted phenoxy group is substituted by one or more groups selected from halogen, CN, NO2 and a phenoxy group which is unsubstituted or substituted by one or more halogen atoms, provided that X1 and X2 do not represent H at the same time; -L1 expression L1'-(CO-Z') Z’ A group wherein L1' is -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, cycloalkanediyl, -(CH2) n -Arylene-, -(CH2) n -cycloalkanediyl-, -arylene-(CH2) p -, -cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-(CH2) p -、-(CH2) n -cycloalkanediyl-(CH2) p -、-(CH2CH2O) m -CH2-CH2-arylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-CH2-CH2-(OCH2CH2) m -, or -(CH2) n -cycloalkanediyl-CH2-CH2-(OCH2CH2) m -; - each W independently represents an amino acid unit; -Y is PAB-CO-(Z) z -, where PAB is The oxygen of the PAB unit is connected to CO-(Z) z ; -Z is -NR4-(CH2) u -NR5-, -NR4-(CH2) u -NR5-CO-, -NR4-(CH2) u -NR5-CO-(CH2) v -, or -NR4-(CH2) u -NR5-CO-(CH2) v -CO-, wherein the NR4 group is attached to the CO group of PAB-CO; -Z' is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO-(CH2) v -, wherein the NR4 group is connected to the CO group of CO-Z'; - R4 and R5 are independently H or (C1-C6) alkyl; -c is 0 or 1; -m is an integer from 1 to 15; -n is an integer from 1 to 6; -p is an integer from 1 to 6; -q is 2; -u is an integer from 1 to 6; -v is an integer from 1 to 6; -w is an integer from 0 to 2; -y is 0 or 1; -z is 0 or 1; -z' is 0 or 1; and -When y=z=1 and Z is -NR4-(CH2) u -NR5-, or when c=w=y=0, z'=1 and Z' is -NR4-(CH2) u -NR5-, X3 represents H, and in other cases, X3 represents OH, NH2 or a leaving group, wherein the leaving group is a halogen atom, in: -Arylene means phenylene or naphthylene, and -Cycloalkanediyl refers to a divalent saturated hydrocarbon ring having 4 to 10 carbon atoms.

2. The linker according to claim 1, wherein c is 1.

3. The linker according to claim 1, wherein w is 0 or 2.

4. The linker according to claim 1, wherein It has the following formula (Ia): or a salt thereof, in: - X1, X2, X3, L1, W, Y, c and w are as defined in claim 1, and - When w is 0, y is 0, and when w is an integer from 1 to 2, y is 0 or 1. The linker according to claim 4 , wherein at least X 1 or X 2 represents a halogen atom. The linker according to claim 5 , wherein at least X 1 or X 2 represents Br or Cl.

7. The linker according to claim 6, wherein one of X1 and X2 represents Br or Cl, and the other group represents H, Cl or Br.

8. The linker according to claim 1, wherein L1' represents -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, -cycloalkanediyl-, -(CH2) n -Arylene-, -Arylene-(CH2) p -、-(CH2) n -cycloalkanediyl- or -cycloalkanediyl-(CH2) p -.

9. The linker according to claim 8, wherein L1' is -(CH2) n -or-(CH2CH2O) m -CH2-CH2-. 10 . The linker according to claim 9 , wherein L1′ is —(CH 2 ) 5 —.

11. The linker of claim 1, wherein each W is selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, lysine protected by acetyl or formyl, arginine, arginine protected by tosyl or nitro, histidine, ornithine, ornithine protected by acetyl or formyl, and citrulline.

12. The linker according to claim 1, wherein: -w = 0 and (W) w is a key, or -w = 2 and (W) w is Val-Cit or Val-Ala.

13. The linker according to claim 1, wherein: -X1 and X2 are the same and are selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and phenoxy which is unsubstituted or substituted by one or more halogen atoms, or - One of X1 and X2 is H, and the other is selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and phenoxy that is unsubstituted or substituted by one or more halogen atoms.

14. The linker according to claim 13, wherein: -X1 and X2 are the same and are selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and unsubstituted or substituted phenoxy with one or more fluorine atoms, or - One of X1 and X2 is H, and the other is selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and phenoxy that is unsubstituted or substituted by one or more fluorine atoms.

15. The linker according to claim 1, wherein when y=z=1 and Z is -NR4-(CH2) u -NR5-, or when c=w=y=0, z'=1 and Z' is -NR4-(CH2) u When -NR5-, X3 is H, and in other cases X3 is OH or Cl.

16. A linker-drug conjugate of the following formula (II): or a salt thereof, in: - X1 and X2 independently represent H, a halogen atom, a (C1-C6) alkoxy group, or an unsubstituted or substituted phenoxy group, wherein the substituted phenoxy group is substituted by one or more groups selected from halogen, CN, NO2 and a phenoxy group which is unsubstituted or substituted by one or more halogen atoms, provided that X1 and X2 do not represent H at the same time; -L1 expression L1'-(CO-Z') z A group wherein L1' is -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, cycloalkanediyl, -(CH2) n -Arylene-, -(CH2) n -cycloalkanediyl-, -arylene-(CH2) p -, -cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-(CH2) p -、-(CH2) n -cycloalkanediyl-(CH2) p -、-(CH2CH2O) m -CH2-CH2-arylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-CH2-CH2-(OCH2CH2) m -, or -(CH2) n -cycloalkanediyl-CH2-CH2-(OCH2CH2) m -; - each W independently represents an amino acid unit; -Y is PAB-CO-(Z) z -, where PAB is The oxygen of the PAB unit is connected to CO-(Z) z ; -Z is -NR4-(CH2) u -NR5-, -NR4-(CH2) u -NR5-CO-, -NR4-(CH2) u -NR5-CO-(CH2) v -, or -NR4-(CH2) u -NR5-CO-(CH2) v -CO-, wherein the NR4 group is attached to the CO group of PAB-CO; -Z' is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO-(CH2) v -, wherein the NR4 group is connected to the CO group of CO-Z'; - R4 and R5 are independently H or (C1-C6) alkyl; -Q represents the drug moiety; -c is 0 or 1; -m is an integer from 1 to 15; -n is an integer from 1 to 6; -p is an integer from 1 to 6; -q is 2; -u is an integer from 1 to 6; -v is an integer from 1 to 6; -w is an integer from 0 to 2; -y is 0 or 1; -z is 0 or 1; and -z' is 0 or 1, in: -Arylene means phenylene or naphthylene, -Cycloalkanediyl means a divalent saturated hydrocarbon ring having 4 to 10 carbon atoms, and - The drug moiety is selected from: - a residue of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE) or monomethyl dolastatin-10, or a residue of a derivative thereof having the following formula (C): in: -R1 is H or OH, -R2 is (C1-C6) alkyl, COOH, COO-((C1-C6) alkyl) or thiazolyl, -R3 is H or (C1-C6) alkyl, -X4 is O or NR9, - R9 is H or (C1-C6) alkyl, and -t is an integer from 1 to 8; - a residue of daunorubicin, doxorubicin, epirubicin, idarubicin, 2-pyrrolidoxorubicin, pre-2-pyrrolidoxorubicin or PNU-159682, or a residue of the following formula (A) or (B): - Residues of camptothecin or SN-38; - a residue of tubulysin A, tubulysin B, tubulysin C or tubulysin D; - the residue of esperamicin, calicheamicin gamma 1 or N-acetyldimethylhydrazine calicheamicin; - residues of maytansine, DM1 or DM4; - residues of duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA or CC-1065; - residues of α-amanitin, β-amanitin, γ-amanitin or ε-amanitin; - residues of anthramycin or SGD-1882; - The residue of a STING agonist of the following formula (D): in: -X 11 and X 21 are independently O or S, -X 12 and X 22 are independently OH, SH, O or S, -A 11 and A 21 are independently groups of the formula: in: Z1 is OR 11 NR 11 R 12 , O or NR 11 , where R 11 and R 12 Independently of each other, H and R 13 or COR 13 , where R 13 is (C1-C6)alkyl, aryl or aryl(C1-C6)alkyl, Z2 is H, NR 21 R 22 or NR 21 , where R 21 and R 22 Independently of each other, H and R 23 or COR 23 , where R 23 is (C1-C6)alkyl, aryl or aryl(C1-C6)alkyl, Z3 is N or CR 33 , where R 33 is H or a halogen atom, and · Z4 is H or (C1-C6) alkyl, -A 12 and A 22 are independently H, OH or F, and -A2 is H, or A2 and A 22 connected to each other, wherein A2 is CH2 and A 22 For O, in: When X 12 When it is O or S, then X 22 Not O and not S, Z1 is not O and not NR 11 , Z2 is not NR 21 , and the residues of the STING agonist are expressed by X 12 Connected to the rest of the molecule; When X 22 When it is O or S, then X 12 Not O and not S, Z1 is not O and not NR 11 , Z2 is not NR 21 , and the residues of the STING agonist are expressed by X 22 Connected to the rest of the molecule; When Z1 is O or NR 11 When X 12 Not O and not S, X 22 Not O and not S, Z2 is not NR 21 , and the residues of the STING agonist are connected to the rest of the molecule via Z1; When Z2 is NR 21 When X 12 Not O and not S, X 22 Not O and not S, Z1 is not O and not NR 11 , and the residues of the STING agonist are connected to the rest of the molecule via Z2.

17. The linker-drug conjugate of claim 16, wherein c is 1.

18. The linker-drug conjugate of claim 16, wherein w is 0 or 2.

19. The linker-drug conjugate according to claim 16, wherein: It has the following formula (IIa): or a salt thereof, in: - X1, X2, L1, W, Y, Q, c and w are as defined in claim 16, and - When w is 0, y is 0, and when w is an integer from 1 to 2, y is 0 or 1.

20. The linker-drug conjugate of claim 16, wherein at least X1 or X2 represents a halogen atom.

21. The linker-drug conjugate of claim 20, wherein at least X1 or X2 represents Br or Cl.

22. The linker-drug conjugate of claim 21, wherein one of X1 and X2 represents Br or Cl, and the other group represents H, Cl or Br.

23. The linker-drug conjugate of claim 16, wherein L1' represents -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, -cycloalkanediyl-, -(CH2) n -Arylene-, -Arylene-(CH2) p -、-(CH2) n -cycloalkanediyl-, or -cycloalkanediyl-(CH2) p -.

24. The linker-drug conjugate of claim 23, wherein L1' is -(CH2) n -or-(CH2CH2O) m -CH2-CH2-.

25. The linker-drug conjugate of claim 24, wherein L1' is -(CH2)5-.

26. The linker-drug conjugate of claim 16, wherein each W is selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, lysine protected by acetyl or formyl, arginine, arginine protected by tosyl or nitro, histidine, ornithine, ornithine protected by acetyl or formyl, and citrulline.

27. The linker-drug conjugate of claim 16, wherein: -w = 0 and (W) w is a key, or -w = 2 and (W) w is Val-Cit or Val-Ala.

28. The linker-drug conjugate of claim 16, wherein: -X1 and X2 are the same and are selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and phenoxy which is unsubstituted or substituted by one or more halogen atoms, or - One of X1 and X2 is H, and the other is selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and phenoxy that is unsubstituted or substituted by one or more halogen atoms.

29. The linker-drug conjugate of claim 28, wherein: -X1 and X2 are the same and are selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and unsubstituted or substituted phenoxy with one or more fluorine atoms, or - One of X1 and X2 is H, and the other is selected from Cl, Br, (C1-C6) alkoxy, and unsubstituted or substituted phenoxy, wherein the substituted phenoxy is substituted by one or more groups selected from halogen, CN, NO2 and phenoxy that is unsubstituted or substituted by one or more fluorine atoms.

30. The linker-drug conjugate of claim 16, wherein Q is a residue of maytansine, DM1 or DM4, or has: - The following formula (A): - The following formula (B): - The following formula (C): in: -R1 is H or OH, -R2 is (C1-C6) alkyl, COOH, COO-((C1-C6) alkyl) or thiazolyl, -R3 is H or (C1-C6) alkyl, -X4 is O or NR9, - R9 is H or (C1-C6) alkyl, and -t is an integer from 1 to 8.

31. A binding unit-drug conjugate of the following formula (III) or (IV): or a salt thereof, in: - the binding unit is an antibody or an antigen-binding fragment thereof; -L1 expression L1'-(CO-Z') Z’ A group wherein L1' is -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, cycloalkanediyl, -(CH2) n -Arylene-, -(CH2) n -cycloalkanediyl-, -arylene-(CH2) p -, -cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-(CH2) p -、-(CH2) n -cycloalkanediyl-(CH2) p -、-(CH2CH2O) m -CH2-CH2-arylene-(CH2) p -、-(CH2CH2O) m -CH2-CH2-cycloalkanediyl-(CH2) p -、-(CH2) n -Arylene-CH2-CH2-(CH2CH2O) m -, or -(CH2) n -cycloalkanediyl-CH2-CH2-(CH2CH2O) m -; - each W independently represents an amino acid unit; -Y is PAB-CO-(Z) z -, where PAB is The oxygen of the PAB unit is connected to CO-(Z) z ; -Z is -NR4-(CH2) u -NR5-, -NR4-(CH2) u -NR5-CO-, -NR4-(CH2) u -NR5-CO-(CH2) v -, or -NR4-(CH2) u -NR5-CO-(CH2) v -CO-, wherein the NR4 group is attached to the CO group of PAB-CO; -Z' is -NR4-(CH2) u -NR5- or -NR4-(CH2) u -NR5-CO-(CH2) v -, wherein the NR4 group is connected to the CO group of CO-Z'; - R4 and R5 are independently H or (C1-C6) alkyl; -Q represents the drug moiety; -c is 0 or 1; -m is an integer from 1 to 15; -n is an integer from 1 to 6; -p is an integer from 1 to 6; -s is an integer from 1 to 8; -u is an integer from 1 to 6; -v is an integer from 1 to 6; -w is an integer from 0 to 2; -y is 0 or 1; -z is 0 or 1; and -z' is 0 or 1, in: -Arylene means phenylene or naphthylene, -Cycloalkanediyl means a divalent saturated hydrocarbon ring having 4 to 10 carbon atoms, and - The drug moiety is selected from: - a residue of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE) or monomethyl dolastatin-10, or a residue of a derivative thereof having the following formula (C): in: -R1 is H or OH, -R2 is (C1-C6) alkyl, COOH, COO-((C1-C6) alkyl) or thiazolyl, -R3 is H or (C1-C6) alkyl, -X4 is O or NR9, - R9 is H or (C1-C6) alkyl, and -t is an integer from 1 to 8; - a residue of daunorubicin, doxorubicin, epirubicin, idarubicin, 2-pyrrolidoxorubicin, pre-2-pyrrolidoxorubicin or PNU-159682, or a residue of the following formula (A) or (B): - Residues of camptothecin or SN-38; - a residue of tubulysin A, tubulysin B, tubulysin C or tubulysin D; - the residue of esperamicin, calicheamicin gamma 1 or N-acetyldimethylhydrazine calicheamicin; - residues of maytansine, DM1 or DM4; - residues of duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA or CC-1065; - residues of α-amanitin, β-amanitin, γ-amanitin or ε-amanitin; - residues of anthramycin or SGD-1882; - The residue of a STING agonist of the following formula (D): in: -X 11 and X 21 are independently O or S, -X 12 and X 22 are independently OH, SH, O or S, -A 11 and A 21 are independently groups of the formula: in: Z1 is OR 11 NR 11 R 12 , O or NR 11 , where R 11 and R 12 Independently of each other, H and R 13 or COR 13 , where R 13 is (C1-C6)alkyl, aryl or aryl(C1-C6)alkyl, Z2 is H, NR 21 R 22 or NR 21 , where R 21 and R 22 Independently of each other, H and R 23 or COR 23 , where R 23 is (C1-C6)alkyl, aryl or aryl(C1-C6)alkyl, Z3 is N or CR 33 , where R 33 is H or a halogen atom, and · Z4 is H or (C1-C6) alkyl, -A 12 and A 22 are independently H, OH or F, and -A2 is H, or A2 and A 22 connected to each other, wherein A2 is CH2 and A 22 For O, in: When X 12 When it is O or S, then X 22 Not O and not S, Z1 is not O and not NR 11 , Z2 is not NR 21 , and the residues of the STING agonist are expressed by X 12 Connected to the rest of the molecule; When X 22 When it is O or S, then X 12 Not O and not S, Z1 is not O and not NR 11 , Z2 is not NR 21 , and the residues of the STING agonist are expressed by X 22 Connected to the rest of the molecule; When Z1 is O or NR 11 When X 12 Not O and not S, X 22 Not O and not S, Z2 is not NR 21 , and the residues of the STING agonist are connected to the rest of the molecule via Z1; When Z2 is NR 21 When X 12 Not O and not S, X 22 Not O and not S, Z1 is not O and not NR 11 , and the residues of the STING agonist are connected to the rest of the molecule via Z2.

32. The binding unit-drug conjugate of claim 31, wherein c is 1.

33. The binding unit-drug conjugate of claim 31, wherein w is 0 or 2.

34. The binding unit-drug conjugate of claim 31, wherein L1' represents -(CH2) n -、-(CH2CH2O) m -CH2-CH2-, arylene, -cycloalkanediyl-, -(CH2) n -Arylene-, -Arylene-(CH2) p -、-(CH2) n -cycloalkanediyl-, or -cycloalkanediyl-(CH2) p -.

35. The binding unit-drug conjugate of claim 34, wherein L1' is -(CH2) n -or-(CH2CH2O) m -CH2-CH2-.

36. The Binding Unit-Drug Conjugate of claim 35, wherein L1' is -(CH2)5-.

37. The binding unit-drug conjugate of claim 31, wherein each W is selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, lysine protected by acetyl or formyl, arginine, arginine protected by tosyl or nitro, histidine, ornithine, ornithine protected by acetyl or formyl, and citrulline.

38. The binding unit-drug conjugate of claim 31, wherein: -w = 0 and (W) w is a key, or -w = 2 and (W) w is Val-Cit or Val-Ala.

39. The binding unit-drug conjugate of claim 31, wherein Q is a residue of maytansine, DM1 or DM4, or has: - The following formula (A): - The following formula (B): or - The following formula (C): in: -R1 is H or OH, -R2 is (C1-C6) alkyl, COOH, COO-((C1-C6) alkyl) or thiazolyl, -R3 is H or (C1-C6) alkyl, -X4 is O or NR9, - R9 is H or (C1-C6) alkyl, and t is an integer from 1 to 8.

40. The binding unit-drug conjugate of claim 31, wherein the antibody is an IGF-1R antibody or a HER2 antibody.

41. A pharmaceutical composition comprising the binding unit-drug conjugate according to any one of claims 31 to 40 and at least one pharmaceutically acceptable excipient.

42. The binding unit-drug conjugate according to any one of claims 31 to 40 or Use of the pharmaceutical composition according to claim 41, for the manufacture of a medicament intended for the treatment of cancer selected from lung cancer, breast cancer and ovarian cancer.

Images