Antibody drug conjugates containing glycosyl pyridine linkers

Abstract

The invention relates to an antibody drug conjugate containing a glycosyl pyridine linker. Specifically, the invention relates to a glycosyl pyridine connexon, an antibody drug conjugate containing the glycosyl pyridine connexon, and a preparation method and medical application of the glycosyl pyridine connexon and the antibody drug conjugate, in particular to application of the glycosyl pyridine connexon in preparation of drugs for treating cancers, autoimmune diseases and inflammatory diseases. The hydrophilicity of the antibody drug conjugate is enhanced through the specific glycosyl pyridine linker, and the antibody drug conjugate is controlled to release active drugs under the enzymatic action in cells.

Description

Technical Field

[0001] The present invention relates to a novel glycosyl pyridine type linker and an antibody drug conjugate containing the same, as well as a preparation method and use of the linker in preparing drugs for treating cancer, autoimmune diseases and inflammatory diseases. Background Art

[0002] Antibody-Drug Conjugates (ADCs) are a new type of targeted therapeutic drugs, mainly used in the treatment of cancer, autoimmunity and other fields. The basic design concept originated from the "magic bullet" and drug targeting concept first proposed by Paul Ehrlich in 1913, that is, the targeted delivery of drugs to the disease site through appropriate carriers. However, due to the limitations of antibody and highly active cytotoxic drug technology, it was not until 2000 that the first antibody-drug conjugate (Mylotarg) for the treatment of acute myeloid leukemia (AML) was developed. TM ) was approved by the FDA for marketing. So far, 12 ADC drugs have been approved for marketing, indicating that the application of antibody-drug conjugates in the field of tumor treatment has entered a rapid development stage.

[0003] Antibody-drug conjugates usually consist of three parts: antibodies or other types of ligands, highly active cytotoxic drugs, and linkers that couple the antibodies to the cytotoxic drugs. The mechanism of action is as follows: the antibody specifically recognizes and binds to cell surface antigens; the formed conjugate enters the cell by endocytosis, bringing in highly active cytotoxic drugs at the same time; the antibody is enzymatically hydrolyzed or the linker itself is broken, and the highly active cytotoxic drugs are released in an appropriate form to kill the target cells.

[0004] Based on the release mechanism of cytotoxic drugs in cells, linkers can be divided into non-cleavable linkers and cleavable linkers. Most of the ADC drugs currently on the market use cleavable linkers. According to the cleavage mechanism, cleavable linkers can be divided into the following main types: acid cleavage mechanism (such as hydrazones), cysteine / glutathione cleavage mechanism (such as disulfide bonds) and enzyme cleavage mechanism (such as peptides). Among them, enzyme cleavage mechanism linkers are favored for their stability and specificity of cleavage mechanism.

[0005] Cytotoxic drugs have a certain degree of hydrophobicity, and coupling them to antibodies usually leads to an increase in the hydrophobicity of the antibody-drug conjugate (relative to the antibody). This increase in hydrophobicity not only easily leads to the aggregation of ADC molecules (producing polymers), but also leads to an accelerated clearance rate of ADC in the body. In order to reduce the hydrophobicity of ADC drugs, hydrophilic linkers are usually used to reduce or offset the adverse effects of the hydrophobicity of cytotoxic drugs on ADC drugs.

[0006] In order to meet the development needs of the ADC drug field, this field urgently needs to develop linkers with new structures, cleavage mechanisms and hydrophilicity, so as to construct ADC drugs with better drugability. Summary of the invention

[0007] The present invention aims to provide a novel glycosyl pyridine linker and construct an antibody-drug conjugate based on the linker.

[0008] After years of in-depth research, the inventors have developed a new type of glycosyl pyridine linker. After the ADC drug constructed based on this linker binds to the cell surface antigen and is internalized into the cell, it can be degraded and released under the action of β-glucuronidase to release cytotoxic drugs, thereby achieving the effect of killing cells. This linker has good chemical stability and will not break during the in vivo circulation process, thereby increasing off-target toxicity. At the same time, this linker has good hydrophilicity, which can greatly improve the hydrophilicity of the ADC drug and the resulting in vivo metabolic stability. This linker technology has universality and can be applied to most ADC drugs, so it has broad application prospects.

[0009] Specifically, the present invention provides an antibody drug conjugate containing a "glycosyl pyridine type linker", wherein the glycosyl pyridine type linker structure contains a glycosyl (Su) substituted pyridine group. The pyridine group has two coupling groups, one of which can be coupled to an antibody or other type of ligand through other groups or directly, and the other coupling group can be connected to a cytotoxic drug through other groups or directly (as shown in the following formula).

[0010]

[0011] The antibody-drug conjugate produced can be used to deliver drugs to target cell populations, such as tumor cells. Antibody-drug conjugates can specifically bind to cell surface proteins, and the resulting conjugate is then internalized by the cell. In the cell, glycosidase (Glycoside Hydrolases, GH) selectively hydrolyzes the Su-O group to lose the sugar molecule, and the residual oxygen anion on the pyridine ring undergoes an electronic replay with the pyridine ring, releasing the drug connected to the para position as an active drug to produce efficacy (see the reaction formula below).

[0012]

[0013] Antibodies for constructing ADC drugs include chimeric antibodies, humanized antibodies, human antibodies; antibody fragments that can bind to antigens; antibody Fc fusion proteins; proteins; or other types of ligands. Drugs are highly active drugs, including but not limited to Maytansinoids, Auristatins, Calichemicins, Doxorubicins, Duocarmycins and CC-1065, PBD dimers, Camptothecins, Tubulysins, Amanitins.

[0014] Compared with traditional antibody-drug conjugates, the hydrophilicity of the antibody-drug conjugate prepared by the method of the present invention is increased, and the in vivo circulation stability is greatly improved, thereby greatly improving the drugability of the product.

[0015] Therefore, an object of the present invention is to provide an antibody drug conjugate as shown in Formula I,

[0016]

[0017] or a pharmaceutically acceptable salt or hydrate thereof,

[0018] in,

[0019] L is an antibody, antibody fragment, or other type of ligand;

[0020] A is a linker fragment, connected to both L and G;

[0021] G is a glycosyl-substituted pyridine structure, as shown in Formula II:

[0022]

[0023] in,

[0024] Su is a sugar molecule;

[0025] Each R is independently selected from hydrogen, CN, halogen, NO2 and C1-C4 alkyl;

[0026] X and Y are each independently selected from -CR 2 R 3 -、-C(=O)NR 4 -、-NR 5 C(=O)-, -O-;

[0027] Q is selected from -NR 6 -or-O-;

[0028] R 1 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton;

[0029] R 2 , R 3 , R 4 , R 5 and R 6 Each is independently selected from hydrogen or C1-C4 alkyl;

[0030] x, y, z are each independently selected from 0 or 1;

[0031] g is selected from 1 or 2;

[0032] W is an optional suicide linker;

[0033] w is selected from 0 or 1;

[0034] D is for medicine;

[0035] p is an integer selected from 1 to 4;

[0036] q is selected from an integer of 1-8.

[0037] In one embodiment, the antibody-drug conjugate of formula I according to the present invention is an antibody-drug conjugate of formula III:

[0038]

[0039] Among them, L, A, Q, R 1 , R, X, Y, W, D, Su, x, y, z, and w are as defined in Formula I.

[0040] In another embodiment, the antibody-drug conjugate of formula I according to the present invention is an antibody-drug conjugate of formula IV or formula V:

[0041]

[0042]

[0043] Among them, L, A, Q, R 1 , R, X, Y, W, D, x, y, z, and w are as defined in Formula I.

[0044] In another embodiment, the antibody-drug conjugate of formula I according to the present invention is an antibody-drug conjugate of formula VI or formula VII:

[0045]

[0046]

[0047] Among them, L, A, Q, R 1 , R, X, Y, D, x, y, and z are as defined in Formula I.

[0048] In a preferred embodiment, in Formula I, III, IV, V, VI, VII according to the present invention, x is 0;

[0049] y is 1;

[0050] Y is selected from -C(=O)NR 4 -、-NR 5 C(=O)-, -O-;

[0051] R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl.

[0052] In another preferred embodiment, in Formula I, III, IV, V, VI, VII according to the present invention, x is 1;

[0053] y is 1;

[0054] X is selected from -CR 2 R 3 -;

[0055] Y is selected from -C(=O)NR 4 -、-NR 5 C(=O)-, -O-;

[0056] R 2 and R 3 Each is independently selected from hydrogen or C1-C4 alkyl, preferably hydrogen;

[0057] R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl.

[0058] In another preferred embodiment, in Formula I, III, IV, V, VI, VII according to the present invention, R 1 is selected from C1-C6 alkylene, preferably C1-C6 straight-chain alkylene, more preferably C2-C4 straight-chain alkylene; z is 1.

[0059] In another preferred embodiment, according to Formula I, III, IV, V, VI, VII of the present invention, Q is -NR 6 -; R 6Each is independently selected from hydrogen or C1-C4 alkyl.

[0060] In another preferred embodiment, in Formula I, III, IV, V, VI, VII according to the present invention, each R is independently selected from hydrogen, halogen, C1-C4 alkyl, preferably hydrogen.

[0061] In another preferred embodiment, in Formula I, III, IV, V, VI, and VII according to the present invention, G has the structure shown below:

[0062]

[0063] Among them, R 7 Selected from hydrogen, C 1-4 Alkyl groups such as methyl, and water-soluble groups such as polyethylene glycol.

[0064] In another preferred embodiment, in Formula I, III, IV, V, VI, VII according to the present invention, g is 1.

[0065] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, A is a linker covalently linked to both the antibody and G.

[0066] In another preferred embodiment, according to Formula I, III, IV, V, VI, VII of the present invention, A is selected from the following structures:

[0067]

[0068]

[0069] Among them, R 8 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton;

[0070] R 9 is selected from H or halogen;

[0071] R 10 Selected from C1-C4 alkyl;

[0072] R 11 and R 12 Each is independently selected from C1-C6 alkylene.

[0073] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, L is a humanized, chimeric, or fully humanized antibody or antibody fragment, antibody Fc fusion protein, or other ligand.

[0074] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, L is an antibody or antibody fragment, antibody Fc fusion protein or other type of ligand against a cell surface receptor or tumor-associated antigen.

[0075] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, L is a humanized anti-CD30 monoclonal antibody or a humanized CD79b monoclonal antibody.

[0076] In another preferred embodiment, according to Formula I, III, IV, V, VI, VII of the present invention, L is Ab1, which has a heavy chain shown in SEQ ID NO: 1 and a light chain shown in SEQ ID NO: 2.

[0077] In another preferred embodiment, according to Formula I, III, IV, V, VI, VII of the present invention, L is Ab2, which has a heavy chain shown in SEQ ID NO: 3 and a light chain shown in SEQ ID NO: 4.

[0078] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, D is an anti-tumor drug, a drug for treating autoimmune diseases, or an anti-inflammatory drug, such as a microtubule protein inhibitor, a DNA alkylating agent, a DNA minor groove binder, or a DNA topoisomerase inhibitor.

[0079] In another preferred embodiment, in Formula I, III, IV, V, VI, and VII according to the present invention, D is monomethyl auristatin E (MMAE), SN-38, or exitecan.

[0080] Another object of the present invention is to provide a compound represented by formula (A) or a pharmaceutically acceptable salt thereof,

[0081]

[0082]

[0083] in,

[0084] A' is the linker fragment;

[0085] Each R is independently selected from hydrogen, CN, halogen, NO2 and C1-C4 alkyl; preferably hydrogen, halogen, C1-C4 alkyl, more preferably hydrogen;

[0086] X and Y are each independently selected from -CR 2 R 3 -、-C(=O)NR 4-、-NR 5 C(=O)-, -O-;

[0087] Q is selected from -NR 6 - or -O-, preferably -NR 6 -;

[0088] R 1 Selected from C1-C6 alkylene or C1-C6 alkylene containing O in the skeleton, preferably C1-C6 alkylene;

[0089] R 2 and R 3 Each is independently selected from hydrogen or C1-C4 alkyl, preferably hydrogen;

[0090] R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl;

[0091] R 6 Selected from hydrogen or C1-C4 alkyl;

[0092] x is 0 or 1;

[0093] y is 0 or 1, preferably 1;

[0094] z is 0 or 1, preferably 1;

[0095] D is the drug molecule;

[0096] p is selected from integers of 1 to 4, preferably 1 or 2.

[0097] In a preferred embodiment, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof according to the present invention, wherein A' is selected from the following structures:

[0098]

[0099]

[0100] in,

[0101] R 8 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton;

[0102] R 9 is selected from H or halogen;

[0103] R 10 Selected from C1-C4 alkyl;

[0104] R 11 and R 12 Each is independently selected from C1-C6 alkylene.

[0105] In another preferred embodiment, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof according to the present invention is selected from the following structures:

[0106]

[0107]

[0108] Among them, R 5 , R 6 and D are as defined in formula (A).

[0109] In another preferred embodiment, according to the compound of formula (A) of the present invention or a pharmaceutically acceptable salt thereof, wherein D is an anti-tumor drug, a drug for treating autoimmune diseases or an anti-inflammatory drug, such as a microtubule inhibitor, a DNA alkylating agent, a DNA minor groove binder, a DNA topoisomerase inhibitor; preferably, D is monomethyl auristatin E (MMAE), SN-38 or exitecan.

[0110] In another preferred embodiment, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof according to the present invention is selected from the following structures:

[0111]

[0112]

[0113]

[0114]

[0115] The present invention also provides a pharmaceutical composition, which contains the antibody-drug conjugate represented by Formula I, III, IV, V, VI or VII according to the present invention and a pharmaceutically acceptable carrier.

[0116] The present invention also provides the use of the antibody-drug conjugate represented by Formula I, III, IV, V, VI or VII according to the present invention or a pharmaceutical composition containing the same in the preparation of drugs for treating cancer, autoimmune diseases and inflammatory diseases.

[0117] The present invention also provides an antibody-drug conjugate represented by Formula I, III, IV, V, VI or VII according to the present invention or a pharmaceutical composition containing the same, which is used for treating cancer, autoimmune diseases and inflammatory diseases.

[0118] The present invention also provides a method for treating cancer, autoimmune diseases and inflammatory diseases, which comprises administering to a subject an effective amount of the antibody-drug conjugate represented by Formula I, III, IV, V, VI or VII according to the present invention or a pharmaceutical composition containing the same.

[0119] Terminology

[0120] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, an alkyl group containing 1 to 4 carbon atoms or an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0121] The term "alkylene" refers to a divalent alkyl group, wherein alkyl is as defined above, having from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C 1-20The alkylene group is preferably an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 alkylene), more preferably an alkylene group containing 1 to 6 carbon atoms (i.e., C 1-6 Alkylene), preferably an alkylene containing 1 to 4 carbon atoms (i.e., C 1-6 Alkylene). Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), and 1,4-butylene (-CH2CH2CH2CH2-). Alkylene may be substituted or unsubstituted, and when substituted, it may be substituted at any available point of attachment, and the substituent may be selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0122] The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, preferably an alkenyl group containing 2 to 4 carbon atoms, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2- or 3-butenyl, etc. The alkenyl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0123] The term "alkynyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon triple bond, preferably an alkynyl group containing 2 to 4 carbon atoms or preferably an alkynyl group containing 3 to 4 carbon atoms, such as ethynyl, propynyl, butynyl, etc. Alkynyl groups may be substituted or unsubstituted, and when substituted, the substituents may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0124] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include cycloalkyls of spiro rings, fused rings and bridged rings. The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclic ring, wherein the ring connected to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, tetrahydrobenzofuranyl, tetrahydrobenzoxazolyl, tetrahydrobenzisoxazolyl, cyclopentathienyl, tetrahydrobenzothiazolyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0125] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O). m (wherein m is an integer from 0 to 2) heteroatoms, but does not include -OO-, -OS- or -SS- ring parts, and the remaining ring atoms are carbon. Preferably, it contains 4 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 7 to 12 ring atoms, of which 1 to 4 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, etc., preferably 1, 2, 5-oxadiazolyl, pyranyl or morpholinyl. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups. The heterocyclic ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group. The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0126] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. More preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:

[0127]

[0128] The aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0129] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10 members, containing 1 to 3 heteroatoms; more preferably 5 or 6 members, containing 1 to 2 heteroatoms; preferably, for example, imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, thiazolyl, pyrazolyl or pyrimidyl, thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0130]

[0131] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0132] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0133] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.

[0134] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0135] "Pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, which are safe and effective when used in mammals and have the desired biological activity.

[0136] "Carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0137] The term "Drug / Antibody Ratio (DAR)" as used herein refers to the number of drugs conjugated to each antibody molecule. Because the antibody drug conjugate sample contains components with multiple different DAR values, the concepts of "average DAR value" and "DAR value distribution" are more suitable for describing the composition of antibody drug conjugates. The average DAR value is the ratio of the total number of drug molecules to the total number of antibodies in a sample, while the DAR value distribution refers to the content distribution of components with different DAR values ​​in the sample.

[0138] As used herein, the term "antibody" or "antibody unit" includes within its scope any portion of the antibody structure. This unit can bind, reactively associate, or complex a receptor, antigen, or other receptor unit possessed by a targeted cell population. An antibody can be any protein or protein-like molecule that can bind, complex, or react with a portion of a cell population to be treated or bioengineered.

[0139] The antibodies constituting the antibody-drug conjugates of the present invention preferably retain their antigen binding ability in their original wild state. Therefore, the antibodies of the present invention are able to bind to antigens (preferably specifically). The antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules related to tissue growth and differentiation (such as known or predicted functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known or predicted functional). Tumor-related factors can be cluster differentiation factors (such as CD proteins). The antigens bound to the antibodies described in the present invention can be one or a subset of the above classifications, while other subsets contain other molecules / antigens with special properties (compared to the target antigen).

[0140] Antibodies used in antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the industry and can be prepared by antibody preparation methods and information well known in the industry. In order to develop effective cellular level targets that can be used for cancer diagnosis and treatment, researchers strive to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, and rarely or not expressed on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancerous cells. Confirming such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer treatment.

[0141] Tumor-associated antigens include, but are not limited to, the tumor-associated antigens listed below (1)-(36). For convenience, antigen-related information known in the industry is indicated below, including name, other names, and gene bank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases, such as Genbank. Antibodies targeting corresponding tumor-associated antigens include all amino acid sequence variants and isotypes, which have at least 70%, 80%, 85%, 90%, or 95% homology with the sequences confirmed in the references, or have biological properties and characteristics that are completely consistent with the tumor-associated antigen sequences in the cited references.

[0142] Tumor-associated antigens (1)-(36):

[0143] (1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM_001203);

[0144] (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

[0145] (3) STEAP1 (six-transmembrane prostate epithelial antigen, Genbank accession number NM_012449);

[0146] (4) 0772P (CA125, MUC16, Genbank accession number AF361486);

[0147] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823);

[0148] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM_006424);

[0149] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878);

[0150] (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

[0151] (9) ETBR (endothelin type B receptor, Genbank accession number AY275463);

[0152] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763);

[0153] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane prostatic epithelial antigen 2, six-transmembrane prostate protein, Genbank accession number AF455138);

[0154] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636);

[0155] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP_003203 or NM_003212);

[0156] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Genbank accession number M26004);

[0157] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29, Genbank accession number NM_000626);

[0158] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764);

[0159] (17) HER2 (ErbB2, Genbank accession number M11730);

[0160] (18) NCA (CEACAM6, Genbank accession number M18728);

[0161] (19) MDP (DPEP1, Genbank accession number BC017023);

[0162] (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971);

[0163] (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053);

[0164] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442);

[0165] (23) ASLG659 (B7h, Genbank accession number AX092328);

[0166] (24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436);

[0167] (25) GEDA (Genbank accession number AY260763);

[0168] (26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456);

[0169] (27) CD22 (B cell receptor CD22-B isoform, Genbank accession number AK026467);

[0170] (28) CD79a (CD79A, CD79α, immunoglobulin-associated α, can covalently interact with Igβ (CD79B) and form a complex with Ig M molecules on the surface, transducing B cell-specific proteins involved in B cell differentiation signals, Genbank accession number NP_001774.1);

[0171] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, plays a role in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly in AIDS, lymphoma, myeloma, and leukemia, Genbank accession number NP_001701.1);

[0172] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen), which binds peptides and presents them to CD4 + T lymphocytes, Genbank accession number NP_002111.1);

[0173] (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, its defect may lead to the pathophysiological condition of idiopathic detrusor instability, Genbank accession number NP_002552.2);

[0174] (32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

[0175] (33) LY64 (lymphocyte antigen 64 (RP105), a member of the leucine-rich repeat-rich (LRR) family of type I membrane proteins, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, Genbank accession number NP_005573.1);

[0176] (34) FcRH1 (Fc receptor-like protein 1, a putative immunoglobulin Fc domain receptor containing C2-type Ig-like and ITAM domains, which may play a role in B lymphocyte differentiation, Genbank accession number NP_443170.1);

[0177] (35) IRTA2 (translocation-associated immunoglobulin superfamily receptor 2, a putative immune receptor that may play a role in B cell development and lymphomagenesis; gene dysregulation caused by translocation occurs in some B cell malignancies, Genbank accession number NP_112571.1);

[0178] (36) TENB2 (putative transmembrane proteoglycan related to the EGF / heregulin family of growth factors and follistatin, Genbank accession number AF179274).

[0179] As used herein, "drug" or code "D" refers to any compound having a desired biological activity and a reactive functional group for preparing the conjugate of the present invention. The desired biological activity includes diagnosis, cure, relief, treatment, and prevention of diseases in humans or other animals. Therefore, as long as the necessary reactive functional groups are present, the compounds referred to by the term "drug" include drugs confirmed in the official national pharmacopoeia, as well as the official U.S. Homeopathic Pharmacopoeia, the official National Formulary, or any supplements thereof. Typical drugs are listed in the Physician's Desk Drug Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). As new drugs are constantly discovered and developed, this patent stipulates that these drugs should also be included in the prodrugs of the conjugate drugs of the present invention.

[0180] Preferably, the drug refers to: a cytotoxic drug for cancer treatment; a protein or polypeptide with desired biological activity, for example, a toxin, such as abrin, ricin A, Pseudomonas exotoxin and diphtheria toxin; other suitable proteins include tumor necrosis factor, α-interferon, β-interferon, neurogenic growth factor, platelet-derived growth factor, tissue-type plasminogen activator growth factor, and biological response modifiers, such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor, or other growth factors.

[0181] On the one hand, the drug is maytansine or maytansinoids. Maytansine compounds inhibit cell proliferation by inhibiting the microtubule formation of tubulin (Science 1975, 189, 1002-1005; US5208020). Maytansinoids are derivatives of maytansine. Maytansine and maytansinoids have efficient cytotoxicity, but they have great limitations in the clinical application of cancer treatment, which is mainly due to the low selectivity of such molecules to tumors. However, this high cytotoxicity prompts them to become the preferred drug part of antibody drug conjugates. The following lists maytansine, maytansinoids and three maytansinoid molecular structures that are often used in antibody drug conjugate applications.

[0182]

[0183] The main raw material for synthesizing maytansinoids is maytansinol, which is mainly obtained by hydrolysis of ansamitocins. Ansamitocins can be prepared by fermentation. Ansamitocin derivatives (WO 2012 / 061590) and alaninyl maytansinol (US2012 / 0121615) are reported to be also used as drug "warheads" for antibody drug conjugates (these two types of molecular structures are shown below).

[0184]

[0185] In another aspect, the drug is auristatin. Auristatins are analogs of dolastatin 10, a biologically active peptide isolated from the marine mollusk Aplysia (US 7498298). Dolastatin 10 inhibits tubulin polymerization by binding to tubulin (the same binding region as vincristine). Dolastatin 10, auristatin PE, and auristatin E are all linear peptides containing four amino acids (three of which are unique to dolastatin compounds) and a C-terminal amide group. Two representative auristatins, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), are both preferred drug moieties for antibody drug conjugates.

[0186]

[0187] On the other hand, the drug is Tubulysins. Tubulysins are a class of natural products extracted from myxobacteria that can effectively inhibit the polymerization of tubulin and therefore have anti-mitotic activity, among which Tubulysin D has the best activity. Tubulysin D is a complex tetrapeptide compound that contains O-acyl / N,O-acetal functional groups in its structure, and is therefore unstable under both acidic and alkaline conditions. US2011 / 0021568 and US2013 / 0224228 respectively disclose a series of tubulysin analogs, which have the above unstable functional groups removed from their structures and have high cell activity.

[0188]

[0189] On the other hand, the drug is a calicheamicin. The calicheamicins are anti-tumor antibiotics that bind to the minor groove of DNA and cause double helix DNA breaks at specific sites, leading to cell apoptosis. The calicheamicins have high in vitro sub-picomolar activity, but their low therapeutic index excludes clinical application prospects. However, this high activity makes them ideal candidates for antibody-drug conjugates (such as Gemtuzumab and Inotuzumab Ozogamicin).

[0190]

[0191] On the other hand, the drug is doxorubicin. Doxorubicin can be inserted into the DNA double helix structure to block DNA replication, so it is used as a chemotherapy drug. However, due to the low cytotoxicity of doxorubicin (the half inhibitory concentration is 0.1-0.2 micromolar for human cancer cell lines, while the cytotoxic drug activity used for antibody drug conjugates is usually sub-nanomolar), its application in antibody drug conjugates is not common.

[0192]

[0193] On the other hand, the drugs are benzodipyrrole antibiotics (duocarmycins, CC-1065, etc.) and other cyclopropapyrroloind-4-one (CPI) derivatives. These compounds are effective DNA minor groove binding-alkylating agents. Cyclopropabenzindol-4-one (CBI) analogs have more stable chemical structures, higher biological activities, and are easier to synthesize than their parent compounds containing natural CPI alkylating subunits. A representative CBI derivative is a phenolic hydroxyl protected derivative CBI, which has weakened prodrug toxicity and enhanced water solubility.

[0194]

[0195] On the other hand, the drug is pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) or PBD dimers. PBD is a natural product produced by Streptomyces, and its unique property is that it can form non-twisted covalent adducts in the minor groove of DNA, specifically at the purine-guanine-purine sequence. The use of PBD as part of a small molecule strategy to target DNA sequences and as a new type of anticancer and antibacterial drug has attracted increasing interest (Biochemistry 2008, 47, 11818-11829). A flexible carbon chain is used to connect the C8 / C8' hydroxyl groups of two PBD units, and the resulting dimer has enhanced biological activity (WO 2011 / 130616). PBD dimers are believed to produce sequence-selective DNA damage, such as inverted 5′-Pu-GATC-Py-3′ interchain crosslinks, resulting in their biological activity. These compounds have been demonstrated to be highly potent cytotoxic drugs and could serve as candidate drugs for antibody-drug conjugates.

[0196]

[0197] On the other hand, the drug is camptothecin (CPT). Camptothecin and its derivatives, as topoisomerase (TOP1) inhibitors, selectively capture the TOP1 cleavage complex and become highly promising anticancer drugs. Camptothecin derivatives such as topotecan, irinotecan (CPT-11), and belotecan have been approved for the treatment of non-small cell lung cancer, ovarian cancer, and colorectal cancer. Such compounds have also been successfully applied to ADC drug design as warheads, such as SN-38, Exatecan (DX-8951f), and DXd.

[0198]

[0199] On the other hand, the drug is amanitin. Amanitin is an octapeptide toxin with a bicyclic structure produced by a poisonous fungus, Amanita phalloides. The toxin can bind specifically to RNA polymerase II (or B) and inhibit it non-competitively, thereby inhibiting the formation of phosphodiester bonds (the initiation and extension of RNA chain synthesis, and then inhibiting the catalytic synthesis of mRNA, leading to cell apoptosis. RNA polymerase II-catalyzed transcription is essential for cell function, so α-amanitin can kill both dividing cells and quiescent cells by a unique mechanism. This feature makes it an attractive class of ADC drug candidate cytotoxic drugs.

[0200]

[0201] On the other hand, drugs are not limited to the above-mentioned categories, but also include all drugs that can be used for antibody-drug conjugates, including but not limited to immune agonists (such as TLR7 / 8, Sting agonists), antibiotics, hormones, etc.

[0202] The term "linker" or "linker of antibody drug conjugate" as used herein refers to a molecule with bifunctional or multifunctional groups that can react with protein / antibody molecules and drug molecules, respectively, thus serving as a "bridge" to connect protein / antibody and drug molecules. According to the mechanism of drug release in cells, "linkers" or "linkers of antibody drug conjugates" can be divided into two categories: non-cleavable linkers and cleavable linkers.

[0203] A non-cleavable linker is a relatively stable linker, and its structure is difficult to degrade and break in the in vivo environment. For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing the active molecule composed of small molecule drugs, linkers, and antibody amino acid residues. The resulting changes in the drug molecular structure do not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Therefore, this type of active drug cannot kill neighboring tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect, bystander effect) (Bioconjugate Chem. 2010, 21, 5-13).

[0204] Common linkers include MC linker and MCC linker, as shown below.

[0205]

[0206] Cleavable linkers, as the name implies, can be cleaved within the target cell and release the active drug (the small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically labile linkers and enzyme-labile linkers.

[0207] Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties, including pH, glutathione concentration, etc.

[0208] Linkers that are sensitive to pH are often referred to as acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). The first generation of antibody-drug conjugates mostly use this type of linker, such as hydrazone, carbonate, acetal, and ketal. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on this type of linker usually have a shorter half-life (2-3 days). This shorter half-life limits the application of pH-sensitive linkers in the new generation of antibody-drug conjugates to a certain extent.

[0209] Glutathione-sensitive linkers are also called disulfide linkers. Drug release is based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low glutathione concentration in the blood (micromolar range). This is especially true for tumor cells, whose low oxygen content leads to increased reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have good stability in plasma.

[0210] Enzyme-labile linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cut by intralysosomal proteases, such as cathepsin B or plasmin (the content of such enzymes is increased in some tumor tissues). This peptide connection is considered to be very stable in the plasma circulation because the unfavorable pH value outside the cell and serum protease inhibitors cause the protease to be generally inactive outside the cell. In view of the high plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme-labile linkers include vc linkers.

[0211]

[0212] Suicide linkers are usually inserted between cleavable linkers and active drugs, or are part of cleavable linkers. The mechanism of action of suicide linkers is that when the cleavable linker is cleaved under appropriate conditions, the suicide linker can spontaneously rearrange its structure and release the active drug connected to it. Common suicide linkers include p-aminobenzyl alcohol (PAB).

[0213]

[0214] The antibody-drug conjugate provided by the present invention is composed of an antibody, a linker containing a glycosyl pyridine-type fragment, and a drug, wherein the glycosyl pyridine-type linker is an enzyme-cleavable linker. On the one hand, the antibody-drug conjugate provided by the present invention targets a specific cell population, binds to a specific protein (antigen) on the cell surface, the conjugate is internalized into the cell, and the drug is released into the cell in an active form.

[0215] On the other hand, the antibody-drug conjugate provided by the present invention targets a specific cell population, binds to a specific protein (antigen) on the cell surface, and produces efficacy; or releases the drug outside the cell, and the drug penetrates into the cell to produce efficacy.

[0216] The pharmaceutical composition of the present invention can be in the form of a sterile aqueous solution for injection. Acceptable vehicles and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution.

[0217] It is well known to those skilled in the art that the dosage of a drug depends on a variety of factors, including but not limited to the following factors: the activity of the specific compound used, the age of the patient, the weight of the patient, the health of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc. In addition, the best treatment method, such as the mode of treatment, the daily dosage of the general compound or the type of pharmaceutically acceptable salt can be verified according to traditional treatment plans.

[0218] The main advantages of the present invention are:

[0219] 1. The present invention provides an antibody-drug conjugate containing a glycosyl pyridine-type linker fragment. The glycosyl pyridine-type linker fragment has good hydrophilicity, thereby greatly improving the hydrophilicity of the antibody-drug conjugate, reducing the aggregate formation of the ligand-drug conjugate, and improving the PK properties of the antibody-drug conjugate in vivo.

[0220] 2. The glycosyl pyridine-type linker fragment provided by the present invention is suitable for the construction of most antibody-drug conjugates and has universal applicability, and therefore has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0221] Figure 1A-Figure 1KThe hydrophobic interaction chromatography (HIC) diagram of the antibody-drug conjugate of the present invention. Figure 1A-1E They correspond to antibody-drug conjugates Ab1-LD-02, Ab1-LD-10, Ab1-LD-11, Ab1-LD-12, and Ab1-LD-14, respectively; Figure 1F-1K They correspond to antibody-drug conjugates Ab2-LD-02, Ab2-LD-04, Ab2-LD-14, Ab2-LD-15, Ab2-LD-16 and Ab2-LD-17 respectively. DETAILED DESCRIPTION

[0222] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. All reactions were carried out under nitrogen protection (except for hydrogenation).

[0223] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The implementation methods and materials described herein are for demonstration purposes only.

[0224] Abbreviations

[0225] Ab Antibody

[0226] ACN Acetonitrile

[0227] ADC Antibody Drug Conjugate

[0228] BOC(Boc) tert-Butyloxycarbonyl

[0229] DIPEA Diisopropylethylamine

[0230] ELISA enzyme-linked immunosorbent assay

[0231] Eq(eq) equivalent

[0232] HIC Hydrophobic Interaction Chromatography

[0233] HPLC High Performance Liquid Chromatography

[0234] LCMS Liquid chromatography-mass spectrometry

[0235] mAb monoclonal antibody

[0236] MS

[0237] Prep-RP-HPLC Preparative High Performance Liquid Chromatography

[0238] Rt retention time

[0239] SDS-PAGE polyacrylamide gel electrophoresis

[0240] SEC Size Exclusion Chromatography

[0241] TEA Triethylamine

[0242] TFA Trifluoroacetic acid

[0243] Unless otherwise stated, all anhydrous reagents were purchased directly from suppliers and stored under nitrogen. All other reagents and solvents purchased were of high purity and were not further purified before use. The commercial reagents mentioned in the examples were used according to the manufacturer's instructions.

[0244] NMR spectra were collected on a Bruker Avance III 400 MHz NMR spectrometer. Chemical shifts (δ) are in ppm and are referenced to tetramethylsilane (chemical shift 0).

[0245] In the LC-MS method, low-resolution mass spectrometric data were collected on an Agilent 6110 (acid method) or 6120B (base method) mass spectrometer interfaced with a Hewlett-Packard Agilent 1200 HPLC:

[0246] Method 1: Acid HPLC method A Waters Sunfire C18 reverse phase column (4.60 × 50 mm, 3.5 μm) was used for separation with an eluent gradient of 5% to 95% phase B (acetonitrile, containing 0.01% TFA) in phase A (aqueous phase, containing 0.01% TFA) over 1.3 min at a flow rate of 2.0 mL / min and a column temperature of 50 °C;

[0247] Method 2: Acid HPLC method A Waters Sunfire C18 reverse phase column (4.6 × 30 mm, 2.7 μm) was used for separation with an eluent gradient of 5% to 95% phase B (acetonitrile, containing 0.01% TFA) in phase A (aqueous phase, containing 0.01% TFA) over 1.4 min at a flow rate of 2 mL / min and a column temperature of 50 °C;

[0248] Method 3: Alkaline HPLC method: A Waters Xbridge C18 reverse phase column (4.60×50 mm, 3.5 μm) was used for separation, and the eluent gradient was 5%-95% B phase (acetonitrile) in A phase (aqueous phase, containing 10 mM ammonium bicarbonate) within 1.5 minutes, the flow rate was 1.8 mL / min, and the column temperature was 50°C.

[0249] Preparative reverse phase-high performance liquid chromatography purification (prep-RP-HPLC) was performed on a Gilson instrument using a Waters Sunfire C18 reverse phase column (250×19 mm, 10 μm):

[0250] Method 4: Acid preparation, mobile phase: A: aqueous phase containing 0.1% TFA; B: ACN, flow rate: 15 mL / min;

[0251] Method 5: Alkaline preparation, mobile phase: A: aqueous phase containing 10 mM ammonium bicarbonate; B: ACN. Flow rate: 15 mL / min.

[0252] The cell lines Ramos (CRL-1596, ATCC) and BJAB (ACC757, DSMZ) used are human non-Hodgkin's lymphoma cells. The cell lines HH (CRL-2105, ATCC), L540 (CSC-C0233, Creative Bioarray) and SU-DHL-1 (CRL-2955, ATCC) used are human Hodgkin's lymphoma cells. Ab1 (humanized anti-CD79b monoclonal antibody, IgG1) and Ab2 (humanized anti-CD30 monoclonal antibody, IgG1) are antibodies developed by our company. Enzyme-labeled anti-antibody was purchased from Sigma (Shanghai). Substrate solution was purchased from Decent Biotechnology (Shanghai). Cell Counting Kit-8 (CCK-8) cell proliferation-toxicity detection kit was purchased from Dojindo (Shanghai).

[0253] The following is the sequence of Ab1 (humanized anti-CD79b monoclonal antibody, IgG1):

[0254] Ab1 heavy chain

[0255] QVQLVQSGAEVKKPGSSVKVSCKTSGNTFTSYGINWVKQAPGQGLEWIGEIFPRSGNIYYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKGGTGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0256] SEQ ID NO: 1

[0257] Ab1 light chain

[0258] DVVMTQSPLSLPVTLGQPASISCRSSQNIVHSDGNTYLEWYQQRPGQSPRLLIYKVSFRLSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0259] SEQ ID NO: 2

[0260] The following is the sequence of Ab2 (humanized anti-CD30 monoclonal antibody, IgG1):

[0261] Ab2 heavy chain

[0262] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWIKQAPGQGLEWIGYINPSSSYTDYNQNFKDKATLTADKSSSTAYMELSSLRSEDTAVYYCARREDYVPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0263] SEQ ID NO: 3

[0264] Ab2 light chain

[0265] DIVMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSLQPEDFATYYCQQHYRIPHTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0266] SEQ ID NO: 4

[0267] Synthesis of linker intermediate KL-1 in Example 1

[0268]

[0269] Step 1: Preparation of Compound 1

[0270] 2-Bromo-3-hydroxypyridine (15 g, 86.2 mmol), potassium hydroxide (KOH, 4.83 g, 86.2 mmol), and paraformaldehyde ((CH2O)n , 7.76g, 258.6mmol) was added to 135ml of water, and then the reaction solution was stirred at 80°C for 17 hours. The reaction solution was cooled to room temperature, acetic acid (5.17g, 86.2mmol) was added thereto, and the solvent was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (2 / 1)) to obtain yellow solid compound 1 (6.8g).

[0271] LCMS (method 1): Rt = 1.18 min, m / z (ES + )204.1[M+H] + .

[0272] Step 2: Preparation of compound 2

[0273] Compound 1 (6.8 g, 33.33 mmol), 4-methoxybenzyl chloride (5.7 g, 36.66 mmol) and potassium carbonate (K2CO3, 6.9 g, 50 mmol) were added to N,N-dimethylformamide (30 mL), and the reaction solution was stirred at room temperature for 17 hours. The mixed solution was poured into ice water (150 mL) and extracted with ethyl acetate (30 mL×4). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was washed with dichloromethane (15 mL×2) and dried to obtain white solid compound 2 (6.2 g).

[0274] LCMS (Method 3): Rt = 1.88 min, m / z (ES + )324.0[M+H] + .

[0275] Step 3: Preparation of compound 3

[0276] Compound 2 (6.2 g, 19.14 mmol) was dissolved in dimethyl sulfoxide (40 ml), and then 2-iodobenzoic acid (IBX, 6.4 g, 22.97 mmol) was added thereto under stirring. The reaction solution was stirred at room temperature for 17 hours, then filtered, and the filtrate was poured into an ice-water mixture (200 ml), and the mixture was extracted with ethyl acetate (30 mL×2). The combined organic phase was washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a crude white solid compound 3 (6.0 g).

[0277] LCMS (Method 3): Rt = 2.02 min, m / z (ES + )322.0[M+H] + .

[0278] Step 4: Preparation of compound 4

[0279] Compound 3 (6.0 g, 18.63 mmol), ethylene glycol (2.3 g, 37.26 mmol) and p-toluenesulfonic acid monohydrate (TsOH·H2O, 177 mg, 0.93 mmol) were added to toluene (50 mL) in sequence, and the reaction solution was stirred at 100°C for 17 hours. After the reaction solution was cooled to room temperature, water (100 mL) was added thereto for extraction, and the separated aqueous phase was extracted with ethyl acetate (30 mL×2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 1)) to obtain yellow solid compound 4 (1.8 g).

[0280] LCMS (method 1): Rt = 1.59 min, m / z (ES + )246.1[M+H] + .

[0281] Step 5: Preparation of Compound 5

[0282] Compound 4 (1.8 g, 7.32 mmol), 4-methoxybenzyl chloride (1.26 g, 8.05 mmol) and potassium carbonate (1.52 g, 10.98 mmol) were added to N,N-dimethylformamide (25 mL) in sequence, and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was poured into ice water (120 mL), and then extracted with ethyl acetate (20 mL × 2). The combined organic phase was washed with saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 4)) to obtain white solid compound 5 (2.0 g).

[0283] LCMS (Method 3): Rt = 2.01 min, m / z (ES + )366.0[M+H] + .

[0284] Step 6: Preparation of compound 6

[0285] Azide ethanol (4.75 g, 54.6 mmol) was added to a tetrahydrofuran solution of potassium tert-butoxide (KOtBu, 1N, 54.6 mL), and the reaction solution was stirred at room temperature for half an hour. Compound 5 (10.0 g, 27.3 mmol) was added to the above solution, and the reaction solution was stirred at 80°C for 2 hours. After the reaction solution was cooled to room temperature, water (300 mL) was added thereto, and then extracted with ethyl acetate (200 mL×3). The combined organic phase was washed with saturated brine (300 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain yellow oily compound 6 (8.5 g).

[0286] LCMS (Method 3): Rt = 2.06 min, m / z (ES + )373.1[M+H] + .

[0287] Step 7: Preparation of Compound 7

[0288] Compound 6 (54 g, 145 mmol) was dissolved in tetrahydrofuran (1000 mL) solution, and triphenylphosphine (PPh3, 57.1 g, 218 mmol) and water (10 mL) were added in sequence under stirring. The reaction solution was stirred at room temperature overnight, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 1) → methanol / dichloromethane (1 / 10)) to obtain white solid compound 7 (28.0 g).

[0289] LCMS (Method 3): Rt = 1.79 min, m / z (ES + )347.1[M+H] + .

[0290] Step 8: Preparation of Compound 8

[0291] Compound 7 (28 g, 145 mmol) and triethylamine (16.36 g, 162 mmol) were added to tetrahydrofuran (300 mL) in sequence, and then the solution was cooled to 0°C and methyloxycarbonyl chloride (FmocCl, 25.1 g, 97.2 mmol) was added. The reaction solution was stirred at room temperature for 1 hour, and then filtered. The filtrate was concentrated under reduced pressure to obtain a yellow oil. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 4)) to obtain a yellow solid compound 8 (25.0 g).

[0292] LCMS (Method 3): Rt = 2.21 min, m / z (ES + )569.1[M+H] + .

[0293] Step 9: Preparation of compound 9

[0294] Compound 8 (1.1 g, 1.93 mmol) was dissolved in a mixed solution of dichloromethane (8 mL) and water (0.8 mL), and trifluoroacetic acid (4 mL) was added thereto under ice bath cooling. The reaction solution was stirred for 1 hour under ice water bath cooling, and then a saturated sodium bicarbonate solution (100 mL) was added thereto to quench the reaction, and the mixed solution was extracted with dichloromethane (15 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 3)) to obtain yellow solid compound 9 (690 mg).

[0295] LCMS (Method 3): Rt = 1.67 min, m / z (ES + )405.1[M+H] + .

[0296] Step 10: Preparation of compound 11

[0297] Compound 9 (690 mg, 1.7 mmol), compound 10 (1.0 g, 2.55 mmol, prepared according to the method disclosed in WO2017 / 51249) and silver oxide (Ag2O, 1.97 g, 8.5 mmol) were added to acetonitrile (150 mL) in sequence, and the reaction solution was stirred at room temperature in the dark for 17 hours. The reaction solution was filtered, the filter cake was washed with tetrahydrofuran (15 mL × 3), and the filtrate was combined and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 3)) to obtain white solid compound 11 (800 mg).

[0298] LCMS (Method 3): Rt = 1.81 min, m / z (ES + )721.1[M+H] + .

[0299] Step 11: Preparation of compound 12

[0300] Compound 11 (800 mg, 1.11 mmol) was dissolved in a mixed solution of isopropanol (1 mL) and dichloromethane (5 mL), and then sodium borohydride (NaBH4, 63 mg, 1.67 mmol) was added under ice-water bath cooling. The reaction solution was stirred under ice-water bath for 1.5 hours, and then 25 ml of water was added thereto, and the mixed solution was extracted with dichloromethane (15 mL×3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 1)) to obtain white solid compound 12 (310 mg).

[0301] LCMS (method 1): Rt = 1.59 min, m / z (ES + )723.2[M+H] + .

[0302] Step 13: Preparation of compound KL-1

[0303] Compound 12 (270 mg, 0.373 mmol), bis(p-nitrophenyl) carbonate (227 mg, 0.746 mmol) and diisopropylethylamine (96 mg, 0.746 mmol) were added to a dry N,N'-dimethylformamide solution (5 mL) in sequence, and the reaction solution was stirred at room temperature for 17 hours. Water (30 mL) was added to the reaction solution, and then extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 10→1 / 1)) to obtain white solid compound KL-1 (310 mg).

[0304] LCMS (method 1): Rt = 2.27 min, m / z (ES + )888.2[M+H] + .

[0305] Example 2 Synthesis of Linker Intermediate KL-2

[0306]

[0307] Step 1: Preparation of compound 13

[0308] 2-Nitro-3-hydroxypyridine (7g, 50mmol) was dissolved in 37% formaldehyde aqueous solution (100mL), and then potassium hydroxide (2.8g, 50mmol) aqueous solution was added dropwise thereto. The reaction solution was stirred under heating reflux for 4 days. The reaction solution was cooled to room temperature, and then 1N hydrochloric acid solution (50mL) was added dropwise thereto. The resulting solution was extracted with ethyl acetate (100mL×3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a brown oily residue. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate (1 / 10→1 / 1)) to obtain yellow solid compound 13 (2.0g).

[0309] LCMS (method 1): Rt = 1.18 min, m / z (ES + )171.1[M+H] + .

[0310] 1H NMR (500MHz, CDCl3) δ10.20(s,1H),7.66-7.71(m,2H),4.80(s,2H),3.10(s,1H).

[0311] Step 2: Preparation of compound 14

[0312] Compound 13 (800 mg, 4.7 mmol) was dissolved in acetonitrile (50 mL), and 10 (1.5 g, 3.76 mmol) and silver oxide (2.17 g, 9.4 mmol) were added in turn under ice-water cooling, and the reaction solution was stirred at room temperature for 1 hour. The mixed solution was filtered, and the filter cake was washed with acetonitrile (50 mL × 2). The filtrate was combined and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 2→3 / 1)) to obtain yellow solid compound 14 (400 mg).

[0313] LCMS (Method 3): Rt = 1.72 min, m / z (ES+) 509.0 [M+Na] + .

[0314] 1 H NMR(500MHz, CDCl3)δ7.87(d,1H),7.64(d,1H),5.30-5.38(m,2H),5.21-5. 26(m,2H),4.79(s,2H),4.23(d,1H),3.74(s,3H),2.14(s,3H),2.07(d,6H).

[0315] Step 3: Preparation of compound 15

[0316] Compound 14 (520 mg, 1.02 mmol) was dissolved in ethyl acetate (100 ml), and then 10% palladium on carbon (Pd / C, 52 mg) was added, and the reaction solution was stirred and hydrogenated overnight at room temperature. The reaction solution was filtered to remove palladium on carbon, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain yellow oily compound 15 (450 mg).

[0317] LCMS (method 3): Rt = 1.64 min, m / z (ES + )457.1[M+H] + .

[0318] Step 4: Preparation of compound 16

[0319] Compound 15 (450 mg, 1 mmol) was dissolved in dichloromethane (20 mL), and then imidazole (102 mg, 1.5 mmol) and tert-butyldimethylsilyl chloride (TBSCl, 225 mg, 1.5 mmol) were added in sequence, and the reaction solution was stirred at room temperature for 2 hours. The mixed solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 10→1 / 2)) to obtain yellow solid compound 16 (350 mg).

[0320] LCMS (Method 3): Rt = 2.16 min, m / z (ES + )571.2[M+H] + .

[0321] Step 5: Preparation of compound 17

[0322] Fmoc-sarcosine (286 mg, 0.921 mmol) was dissolved in dichloromethane (10 ml), and then thionyl chloride (367 mg, 1.84 mmol) and a trace amount of N, N-dimethylformamide (4.5 mg, 0.0614 mmol) were added in sequence, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and dichloromethane (10 mL) was added to the residue, and then a dichloromethane solution (10 mL) of compound 16 (350 mg, 0.614 mmol) and DIPEA (158 mg, 1.23 mmol) were added thereto, and the resulting reaction solution was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 10→1 / 1)) to obtain yellow solid compound 17 (200 mg).

[0323] LCMS (method 1): Rt = 2.23 min, m / z (ES + )864.3[M+H] + .

[0324] Step 6: Preparation of compound 18

[0325] Compound 17 (190 mg, 0.22 mmol) was dissolved in tetrahydrofuran (6 mL), and then glacial acetic acid (AcOH, 2 mL) and water (2 mL) were added thereto, and the reaction solution was stirred at room temperature for 2 days. Saturated sodium bicarbonate solution (30 mL) was added to the reaction solution, and then extracted with ethyl acetate (30 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a yellow oil. The residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane (0 / 1→1 / 20)) to obtain a yellow solid compound 18 (100 mg). LCMS (Method 1): Rt=1.86 min, m / z (ES+ )750.2[M+H] + .

[0326] Step 7: Preparation of compound KL-2

[0327] Compound 18 (87 mg, 0.116 mmol), p-nitrophenyl chloroformate (35 mg, 0.174 mmol) and pyridine (13.7 mg, 0.174 mmol) were added to dichloromethane (4 mL) in sequence, and the reaction solution was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 10→1 / 2)) to obtain compound KL-2 (45 mg) as a white solid.

[0328] LCMS (method 1): Rt = 2.08 min, m / z (ES + )915.2[M+H] + .

[0329] Example 3 Synthesis of Linker Intermediate KL-3

[0330]

[0331] Step 1: Preparation of compound 19

[0332] Tert-butyl methyl (2-(methylamino)ethyl) carbamate (6.00 g, 31.6 mmol), 2-carboxy-3-hydroxypyridine (4.00 g, 28.8 mmol), 1-hydroxybenzotriazole (HOBt, 4.30 g, 31.6 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 6.10 g, 31.6 mmol) and diisopropylethylamine (5.60 g, 43.2 mmol) were added to N,N'-dimethylformamide (60 mL) in sequence, and the reaction solution was stirred at room temperature for 16 hours. Water (300 mL) was added to the reaction solution, and then extracted with ethyl acetate (150 mL × 3), and the combined organic phase was washed with saturated brine (200 mL × 2) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 5)) to give off-white solid compound 19 (6.96 g).

[0333] LCMS (Method 3): Rt = 1.77 min, m / z (ES + )310.2(M+H) + .

[0334] Step 2: Preparation of compound 20

[0335] Compound 19 (4.80 g, 15.5 mmol), triethylamine (3.13 g, 31 mmol) and 37% formaldehyde (HCHO, 3.77 g, 46.5 mmol) were added to water (50 mL) in sequence, and the reaction solution was stirred at 90°C for 16 hours. The reaction solution was cooled to room temperature, neutralized by adding glacial acetic acid, and then extracted with ethyl acetate (100 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 2)) to obtain an off-white solid compound 20 (1.4 g).

[0336] LCMS (Method 3): Rt = 1.63 min, m / z (ES + )340.2(M+H) + .

[0337] Step 3: Preparation of compound 21

[0338] Compound 20 (339 mg, 1.0 mmol), compound 10 (397 mg, 1.0 mmol) and silver oxide (462 mg, 2.0 mmol) were added to acetonitrile (50 mL) in sequence, and the reaction solution was stirred at 40°C in the dark for 16 hours. The reaction solution was cooled to room temperature and filtered, the filter cake was washed with acetonitrile, and the combined filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 5: 40%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 21 (120 mg).

[0339] LCMS (Method 3): Rt = 1.84 min, m / z (ES + )656.3(M+H) + .

[0340] Step 4: Preparation of compound KL-3

[0341] Compound 21 (100 mg, 0.15 mmol), bis(p-nitrophenyl) carbonate (48 mg, 0.24 mmol) and pyridine (20 mg, 0.25 mmol) were added to dichloromethane (3 mL) in sequence under ice bath cooling, and the reaction solution was stirred at room temperature for 16 hours. Water (20 mL) was added to the reaction solution, and then extracted with dichloromethane (30 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 10)) to obtain an off-white solid compound KL-3 (100 mg).

[0342] LCMS (Method 3): Rt = 2.06 min, m / z (ES+ )821.3(M+H) + .

[0343] Example 4 Synthesis of Linker Intermediate KL-4

[0344]

[0345] Step 1: Preparation of compound 22

[0346] 3-Hydroxypyridine (9.50 g, 100 mmol), 37% formaldehyde solution (12.20 g, 150 mmol), N-methylbenzylamine (11.5 g, 95 mmol), and concentrated hydrochloric acid (8.5 mL, 100 mmol) were added to ethanol (120 mL) in sequence, and the reaction solution was stirred at 85°C overnight. 10% sodium hydroxide solution was added to adjust the pH value to neutral, and the ethanol was removed by concentration under reduced pressure, and then extracted with ethyl acetate (80 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol (150 / 1)) to obtain a light yellow solid compound 22 (11.50 g).

[0347] LCMS (Method 3): Rt = 1.87 min, m / z (ES + )229.1(M+H) + .

[0348] Step 2: Preparation of compound 23

[0349] Compound 22 (12.5 g, 55 mmol) was dissolved in methanol (100 mL), and 10% palladium carbon (1.0 g) was added thereto. The reaction solution was stirred for 16 hours under hydrogenation conditions at room temperature. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol (10 / 1)) to obtain pink solid compound 23 (6.6 g).

[0350] LCMS (Method 3): Rt = 0.38 min, m / z (ES + )139.2(M+H) + .

[0351] Step 3: Preparation of compound 24

[0352] Boc-sarcosine (6.24 g, 33 mmol), compound 23 (5.10 g, 37 mmol), 1-hydroxybenzotriazole (5.00 g, 37 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.10 g, 37 mmol) and diisopropylethylamine (7.22 g, 56 mmol) were added to DMF (80 mL) in sequence, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was poured into 300 ml of water and extracted with ethyl acetate (200 mL × 3). The combined organic phase was washed with saturated brine (300 mL × 2) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol (80 / 1→30 / 1)) to obtain a light yellow solid compound 24 (7.8 g).

[0353] LCMS (Method 3): Rt = 1.75 min, m / z (ES + )310.1(M+H) + .

[0354] 1 HNMR(400MHz,DMSO-d6)δ7.98-7.97(m,1H),7.19-7.13(m,2H),4.56-4.51(m,2H),4.15-4.04(m,2H ), 2.99-2.98 (m, 1H), 2.84 (s, 1H), 2.79-2.76 (m, 3H), 2.72 (s, 1H), 1.40-1.38 (m, 4H), 1.28 (s, 5H).

[0355] Step 4: Preparation of compound 25

[0356] Compound 24 (7.72 g, 25 mmol), triethylamine (5.05 g, 50 mmol) and 37% formaldehyde solution (6.08 g, 75 mmol) were added to water (60 mL) in sequence, and the reaction solution was stirred at 90 ° C for 16 hours. The reaction solution was cooled to room temperature, acetic acid was added thereto to adjust the pH to neutral, and then extracted with ethyl acetate (60 mL × 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol (50 / 1→20 / 1)) to obtain an off-white solid compound 25 (1.32 g).

[0357] LCMS (Method 3): Rt = 1.67 min, m / z (ES + )340.1(M+H) + .

[0358] Step 5: Preparation of compound 26

[0359] Compound 25 (1.32 g, 3.9 mmol) was dissolved in dichloromethane (25 mL), and then active manganese dioxide (MnO2, 1.35 g, 15.6 mmol) was added thereto, and the reaction solution was stirred under reflux for 16 hours. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol (100 / 1→80 / 1)) to obtain off-white solid compound 26 (0.86 g).

[0360] LCMS (Method 3): Rt = 1.55 min, m / z (ES + )338.1(M+H) + .

[0361] Step 6: Preparation of compound 27

[0362] Compound 26 (438 mg, 1.3 mmol), compound 10 (560 mg, 1.4 mmol) and silver oxide (534 mg, 2.3 mmol) were added to acetonitrile (8 mL) in sequence, and the reaction solution was stirred at room temperature in the dark for 16 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 5: 40%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 27 (112 mg).

[0363] LCMS (Method 3): Rt = 1.96 min, m / z (ES + )654.2(M+H) + .

[0364] Step 7: Preparation of compound 28

[0365] Compound 27 (112 mg, 0.17 mmol) was dissolved in a mixed solution of isopropanol (1 mL) and dichloromethane (2 mL). Sodium borohydride (10 mg, 0.25 mmol) was added thereto under ice bath cooling, and the reaction solution was stirred at room temperature for 2 hours. 20 ml of water was added to the reaction solution, and then extracted with dichloromethane (20 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol (60 / 1)) to obtain an off-white solid compound 28 (100 mg).

[0366] LCMS (Method 3): Rt = 1.81 min, m / z (ES + )656.0(M+H) + .

[0367] Step 8: Preparation of compound KL-4

[0368] Compound 28 (92 mg, 0.14 mmol) and p-nitrochlorophenylformate (56 mg, 0.28 mmol) were added to dichloromethane (4 mL), and triethylamine (43 mg, 0.42 mmol) was added thereto under ice bath cooling, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate (10 / 1→3 / 1)) to obtain off-white solid compound KL-4 (41 mg).

[0369] LCMS (Method 3): Rt = 2.03 min, m / z (ES + )820.9(M+H) + .

[0370] Example 5 Synthesis of Linker Intermediate KL-5

[0371]

[0372] Step 1: Preparation of compound 29

[0373] Compound 6 (4.5 g, 12 mmol) was dissolved in a mixed solution of dichloromethane (16 mL) and water (0.4 mL), and trifluoroacetic acid (8 mL) was added thereto under ice bath cooling, and the reaction solution was stirred at room temperature for 2 hours. Saturated sodium bicarbonate solution (40 mL) was added to the reaction solution to quench the reaction, and then extracted with dichloromethane (50 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 3)) to obtain a colorless oily compound 29 (1.64 g).

[0374] LCMS (Method 3): Rt = 0.67 min, m / z (ES + )209.1(M+H) + .

[0375] Step 2: Preparation of compound 31

[0376] Compound 29 (422 mg, 2.03 mmol), compound 30 (1.0 g, 2.43 mmol, synthesized according to the method disclosed in US2019 / 367488) and silver oxide (938 mg, 4.06 mmol) were added to acetonitrile (50 mL) in sequence, and the reaction solution was stirred at room temperature in the dark for 17 hours. The reaction solution was filtered, the filter cake was washed with tetrahydrofuran (15 mL × 3), and then the combined filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 4→1 / 1 / 2)) to obtain a colorless oily compound 31 (874 mg).

[0377] LCMS (Method 3): Rt = 1.89 min, m / z (ES + )539.1(M+H) + .

[0378] Step 3: Preparation of compound 32

[0379] Compound 31 (874 mg, 1.62 mmol) was dissolved in a mixed solution of isopropanol (10 mL) and dichloromethane (20 mL), and then sodium borohydride (92 mg, 2.43 mmol) was added thereto under ice-water bath cooling conditions. The reaction solution was stirred under ice-water bath for 1 hour. Water (50 mL) was added to the reaction solution, and then extracted with ethyl acetate (50 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain colorless oily compound 32 (850 mg).

[0380] LCMS (method 1): Rt = 1.63 min, m / z (ES + )541.2(M+H) + .

[0381] Step 4: Preparation of compound KL-5

[0382] Compound 32 (850 mg, 1.57 mmol) was dissolved in dry dichloromethane (10 mL), and then p-nitrophenyl chloroformate (476 mg, 2.36 mmol) and pyridine (373 mg, 4.72 mmol) were added in sequence, and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 3→1 / 1)) to obtain colorless oily compound KL-5 (950 mg).

[0383] LCMS (method 1): Rt = 2.10 min, m / z (ES + )706.1(M+H) + .

[0384] Example 6 Synthesis of Linker-Drug LD-01

[0385]

[0386] Step 1: Preparation of compound 33

[0387] Compound KL-1 (240 mg, 0271 mmol), 1-hydroxybenzotriazole (18.4 mg, 0.136 mmol), pyridine (0.5 mL) and MMAE (195 mg, 0.271 mmol, Haoyuan Pharmaceutical) were added to dry N,N-dimethylformamide (2 mL) in sequence, and the reaction solution was stirred at room temperature for 17 hours. Acetic acid was added to the reaction solution to quench the reaction, and then purified by prep-RP-HPLC (method 4: 60%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 33 (200 mg).

[0388] LCMS (method 1): Rt = 1.98 min, m / z (ES + )1467.5(M+H) + .

[0389] Step 2: Preparation of compound 34

[0390] Compound 33 (200 mg, 0.136 mmol) was dissolved in methanol (10 mL), and then lithium hydroxide aqueous solution (1 M, 1.36 mL, 1.36 mmol) was added thereto. The reaction solution was stirred at room temperature for 3 hours, and then glacial acetic acid (150 mg) was added to quench the reaction, and purified by prep-RP-HPLC (method 4: 45%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 34 (100 mg).

[0391] LCMS (method 2): Rt = 1.21 min, m / z (ES + )1104.4(M+H) + .

[0392] Step 3: Preparation of compound LD-01

[0393] Compound 34 (20 mg, 0.018 mmol) was dissolved in N,N-dimethylformamide (1 mL), and compound 35 (6.7 mg, 0.022 mmol, Haoyuan Pharmaceutical) and diisopropylethylamine (9.3 mg, 0.072 mmol) were added thereto in sequence. The reaction solution was stirred at room temperature for 2 hours, and then glacial acetic acid (24 mg) was added to quench the reaction. The resulting solution was purified by prep-RP-HPLC (method 4: 50%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-01 (13.4 mg).

[0394] LCMS (method 1): Rt = 1.70 min, m / z (ES + )649.5[1 / 2(M+2H)] + .

[0395] Example 6 Synthesis of Linker-Drug LD-02

[0396]

[0397] Compound 34 (55 mg, 0.05 mmol) was dissolved in N,N-dimethylformamide (2 mL), and compound 36 (23 mg, 0.06 mmol, synthesized according to the method disclosed in EP2949343, 2015, A1) and diisopropylethylamine (25.8 mg, 0.2 mmol) were added thereto in sequence. The reaction solution was stirred at room temperature for 2 hours, and then glacial acetic acid (24 mg) was added to quench the reaction. The resulting solution was purified by prep-RP-HPLC (method 4: 50%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-02 (50 mg).

[0398] LCMS (method 2): Rt = 1.36 min, m / z (ES + )1378.4(M+H) + .

[0399] Example 7 Synthesis of Linker-Drug LD-03

[0400]

[0401] Step 1: Preparation of compound 33

[0402] Compound 37 (100 mg, 0.225 mmol, synthesized by referring to the method disclosed in WO2016 / 192528) was dissolved in tetrahydrofuran, and then N-hydroxysuccinimide (NHS, 39 mg, 0.34 mmol) and dicyclohexylcarbodiimide (DCC, 70 mg, 0.34 mmol) were added thereto in sequence, and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane (0 / 1→1 / 30)) to obtain yellow solid compound 37 (60 mg).

[0403] Step 2: Preparation of compound LD-03

[0404] Compound 34 (20 mg, 0.018 mmol) was dissolved in N,N-dimethylformamide (2 mL), and then compound 38 (15 mg, 0.027 mmol) and diisopropylethylamine (10 mg, 0.076 mmol) were added thereto in sequence. The reaction solution was stirred at room temperature for 2 hours, and then quenched by adding glacial acetic acid (24 mg). The resulting solution was purified by prep-RP-HPLC (method 4: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-03 (9.9 mg).

[0405] LCMS (method 1): Rt = 1.75 min, m / z (ES + )766.5[1 / 2(M+2H)] + .

[0406] Example 8 Synthesis of Linker-Drug LD-04

[0407]

[0408] Step 1: Preparation of compound 39

[0409] Piperidine (0.35 g, 4.1 mmol) was added to a solution of compound 33 (300 mg, 0.2 mmol) in tetrahydrofuran (30 mL), and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was purified by prep-RP-HPLC (method 4: 15%-85% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 39 (200 mg).

[0410] LCMS (method 1): Rt = 1.63 min, m / z (ES+) 623.0 [1 / 2 (M+2H)] + .

[0411] Step 2: Preparation of compound 40

[0412] Compound 39 (98 mg, 0.079 mmol), compound 40 (41 mg, 0.095 mmol, synthesized by referring to the method disclosed in CN107652219B), diisopropylethylamine (49 mg, 0.38 mmol) and HATU (60 mg, 0.098 mmol) were added to N,N-dimethylformamide (5 mL) in sequence, and the resulting reaction solution was stirred at room temperature for 17 hours. 30 ml of water was added to the reaction solution to quench the reaction, and the resulting solution was extracted with ethyl acetate (25 mL × 3). The combined organic phase was washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude yellow solid compound 41 (120 mg).

[0413] LCMS (method 1): Rt = 2.01 min, m / z (ES+) 829.6 [1 / 2 (M+2H)] + .

[0414] Step 3: Preparation of compound 42

[0415] Compound 41 (120 mg, 0.079 mmol) was added to a mixed solution of dioxane hydrochloride (4 M, 5 mL) and ethyl acetate (5 mL), and the resulting reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a crude yellow solid compound 42 (100 mg).

[0416] LCMS (method 1): Rt = 1.48 min, m / z (ES + )729.5[1 / 2(M+2H)] + .

[0417] Step 4: Preparation of compound 43

[0418] Compound 42 (100 mg, 0.069 mmol) was added to a mixed solution of methanol (1 mL) and water (2 mL), and then lithium hydroxide monohydrate (LiOH·H2O, 84 mg, 2 mmol) was added thereto. The reaction solution was stirred at room temperature for 2 hours, and then glacial acetic acid (120 mg) was added to quench the reaction. The resulting solution was purified by prep-RP-HPLC (method 4: 15%-85% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 43 (14 mg).

[0419] LCMS (method 1): Rt = 1.38 min, m / z (ES + )659.5[1 / 2(M+2H)] + .

[0420] Step 4: Preparation of compound LD-04

[0421] Compound 43 (14 mg, 0.010 mmol), compound 36 (8.2 mg, 21.2 μmol) and diisopropylethylamine (6 mg, 70.41 μmol) were added to dry N,N-dimethylformamide (1 mL) in sequence, and the resulting reaction solution was stirred at room temperature for 2 hours, and then glacial acetic acid (10 mg) was added to quench the reaction. The reaction solution was purified by prep-RP-HPLC (method 4: 15%-85% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-04 (14 mg).

[0422] LCMS (method 1): Rt = 1.64 min, m / z (ES + )933.6[1 / 2(M+2H)] + .

[0423] Example 9 Synthesis of Linker-Drug LD-05

[0424]

[0425] Step 1: Preparation of compound 44

[0426] Compound KL-1 (500 mg, 0.564 mmol), HOBt (38 mg, 0.282 mmol), pyridine (2 mL), diisopropylethylamine (291 mg, 2.256 mmol) and exotecan (300 mg, 0.564 mmol, Haoyuan Pharmaceutical) were added to dry N, N-dimethylformamide (20 mL) in sequence, and the resulting reaction solution was stirred at room temperature for 17 hours. 150 ml of water was added to the reaction solution, and then extracted with ethyl acetate (500 mL×4). The combined organic phase was washed with saturated brine (600 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The resulting brown oil was purified by silica gel column chromatography (eluent: methanol / dichloromethane (1 / 60→1 / 20)) to obtain brown solid compound 44 (1.74 g).

[0427] LCMS (method 1): Rt = 2.04 min, m / z (ES + )593.1[1 / 2(M+2H)] + .

[0428] Step 2: Preparation of compound 45

[0429] Compound 44 (400 mg, 0.338 mmol) was dissolved in methanol (80 mL), and the resulting solution was cooled in an ice-water bath, and then lithium hydroxide aqueous solution (1 N, 13.5 mL, 13.5 mmol) was added thereto. After the reaction solution was reacted at 0°C for 2 hours, glacial acetic acid (1 g) was added to quench. The mixed solution was concentrated to one third under reduced pressure, and then purified by prep-RP-HPLC (method 4: 20%-90% B in 8 minutes → 95% B in 4 minutes) to obtain yellow solid compound 45 (80 mg).

[0430] LCMS (method 1): Rt = 1.55 min, m / z (ES + )411.8[1 / 2(M+2H)] + .

[0431] Step 3: Preparation of compound LD-05

[0432] Compound 45 (40 mg, 0.0487 mmol) was dissolved in anhydrous N,N-dimethylformamide solution (2 mL), and then compound 35 (18 mg, 0.0585 mmol) was added thereto. The reaction solution was cooled to 0°C, and then diisopropylethylamine (25 mg, 0.195 mmol) was added, and the reaction solution was stirred at room temperature for 4 hours. Glacial acetic acid (25 mg) was added to the reaction solution to quench the reaction, and the resulting mixture was purified by prep-RP-HPLC (method 4: 30%-90% B in 8 minutes → 95% B in 4 minutes) to obtain 13.7 mg of yellow solid compound LD-05 (13.7 mg).

[0433] LCMS (method 1): Rt = 1.81 min, m / z (ES + )1015.4(M+H) + .

[0434] Example 10 Synthesis of Linker-Drug LD-06

[0435]

[0436] Compound 45 (40 mg, 0.0487 mmol) was dissolved in anhydrous N,N-dimethylformamide solution (2 mL), and then compound 36 (18 mg, 0.0585 mmol) was added. The solution was cooled to 0°C, diisopropylethylamine (25 mg, 0.195 mmol) was added thereto, and the reaction solution was stirred at room temperature for 4 hours. Glacial acetic acid (25 mg) was added to quench the reaction, and the resulting solution was purified by prep-RP-HPLC (method 4: 30%-90% B in 8 minutes → 95% B in 4 minutes) to obtain yellow solid compound LD-06 (9.4 mg).

[0437] LCMS (method 1): Rt = 1.81 min, m / z (ES + )1096.4(M+H) + .

[0438] Example 11 Synthesis of Linker-Drug LD-07

[0439]

[0440] Step 1: Preparation of compound 46

[0441] Tert-butyl methyl (2-(methylamino)ethyl) carbamate (550 mg, 2.93 mmol) and triethylamine (325 mg, 3.22 mmol) were added to toluene (15 mL) in sequence, and triphosgene (348 mg, 1.17 mmol) was then added under ice bath cooling. The reaction solution was reacted for 2 hours under ice bath cooling, then warmed to room temperature and continued to react for 16 hours. The reaction solution was filtered, the resulting filtrate was concentrated to remove the solvent, and then pyridine (15 mL) and SN-38 (918 mg, 2.34 mmol, Titan Technology) were added thereto in sequence, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane (1 / 40→1 / 20)) to obtain yellow solid compound 46 (670 mg).

[0442] LCMS (Method 3): Rt = 1.89 min, m / z (ES + )606.1(M+H) + .

[0443] Step 2: Preparation of compound 47

[0444] Compound 46 (310 mg, 0.51 mmol) was dissolved in dichloromethane (4 mL), and then hydrochloric acid-dioxane solution (4 M, 4 mL, 16 mmol) was added, and the reaction solution was stirred at room temperature for 4 hours. The reaction solution was concentrated to remove the solvent to obtain yellow solid compound 47 (277 mg).

[0445] LCMS (method 1): Rt = 1.41 min, m / z (ES + )506.2(M+H) + .

[0446] Step 3: Preparation of compound 48

[0447] Compound 47 (174 mg, 0.32 mmol), compound KL-1 (284 mg, 0.32 mmol), HOBt (43 mg, 0.32 mmol) and pyridine (0.5 mL) were added to anhydrous N, N-dimethylformamide (5 mL) in sequence, and the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, water (30 mL) was added, and the mixed solution was extracted with ethyl acetate (50 mL × 4). The combined organic phase was washed with saturated brine (60 mL × 2) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane (1 / 40→1 / 20)) to obtain yellow solid compound 48 (324 mg).

[0448] LCMS (Method 3): Rt = 1.98 min, m / z (ES + )628.3[1 / 2(M+2H)] + .

[0449] Step 4: Preparation of compound 49

[0450] Compound 48 (324 mg, 0.26 mmol) was dissolved in a mixed solution of methanol (2 mL) and water (2 mL), and then lithium hydroxide monohydrate (109 mg, 2.6 mmol) was added thereto, and the reaction solution was stirred at room temperature for 2 hours. Glacial acetic acid (160 mg) was added to quench the reaction, and the resulting solution was purified by prep-RP-HPLC (method 4: 20%-80% B in 8 minutes → 95% B in 4 minutes) to obtain yellow solid compound 49 (52 mg).

[0451] LCMS (method 1): Rt = 1.36 min, m / z (ES + )893.3(M+H) + .

[0452] Step 5: Preparation of compound LD-07

[0453] Compound 49 (30 mg, 0.034 mmol), compound 35 (9 mg, 0.034 mmol) and diisopropylethylamine (17 mg, 0.136 mmol) were added to anhydrous N,N-dimethylformamide (1 mL) in sequence, and the reaction solution was stirred at room temperature for 3 hours. Glacial acetic acid (20 mg) was added to quench the reaction, and the resulting solution was purified by prep-RP-HPLC (method 4: 30%-90% in 8 minutes → 95% in 4 minutes B) to obtain yellow solid compound LD-07 (16 mg).

[0454] LCMS (method 1): Rt = 1.56 min, m / z (ES + )1087.4(M+H)+ .

[0455] Example 12 Synthesis of Linker-Drug LD-08

[0456]

[0457] Compound 49 (21.8 mg, 0.024 mmol), compound 36 (8.5 mg, 0.024 mmol) and diisopropylethylamine (12.6 mg, 0.098 mmol) were added to anhydrous N,N-dimethylformamide (1 mL) in sequence, and the reaction solution was stirred at room temperature for 3 hours. Glacial acetic acid (20 mg) was added to quench the reaction, and the resulting solution was purified by prep-RP-HPLC (method 4: 35%-90% B in 8 minutes → 95% B in 4 minutes) to obtain yellow solid compound LD-08 (6.7 mg).

[0458] LCMS (method 1): Rt = 1.74 min, m / z (ES + )584.3[1 / 2(M+2H)] + .

[0459] Example 13 Synthesis of Linker-Drug LD-09

[0460]

[0461]

[0462] Step 1: Preparation of compound 51

[0463] Compound 50 (6.4 mg, 0.026 mmol, Titan Technology), HATU (48.6 mg, 0.128 mmol), compound 38 (80 mg, 0.064 mmol) and diisopropylethylamine (33 mg, 0.256 mmol) were added to DMF (5 mL) in sequence, and the reaction solution was stirred at room temperature for 5 hours. Glacial acetic acid (20 mg) was added to quench the reaction, and the mixture was purified by prep-RP-HPLC (method 4: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 51 (30 mg).

[0464] LCMS (method 2): Rt = 1.92 min, m / z (ES + )900.9[1 / 3(M+3H)] + .

[0465] Step 2: Preparation of compound 52

[0466] Compound 51 (30 mg, 0.011 mmol) was dissolved in ethyl acetate (0.8 mL), and then 4N hydrochloric acid dioxane solution (0.2 mL) was added, and the reaction solution was stirred at room temperature for 5 hours. The solvent was removed by concentration under reduced pressure to obtain compound 52 (20 mg) as a white solid.

[0467] LCMS (method 2): Rt = 2.26 min, m / z (ES + )867.5[1 / 3(M+3H)] + .

[0468] Step 3: Preparation of compound 53

[0469] Compound 52 (20 mg, 0.0077 mmol) was dissolved in methanol (1 mL), and 1 M aqueous lithium hydroxide solution (77 μL, 0.077 mmol) was added under ice bath cooling, and the reaction solution was stirred at room temperature for 3 hours. Glacial acetic acid (2 mg) was added to quench the reaction, and the mixture was purified by prep-RP-HPLC (method 4: 20%-95% B in 8 minutes → 95% B in 4 minutes) to obtain compound 53 (4 mg) as a white solid.

[0470] LCMS (method 2): Rt = 2.04 min, m / z (ES + )744.0[1 / 3(M+3H)] + .

[0471] Step 4: Preparation of compound LD-09

[0472] Compound 53 (4 mg, 0.0017 mmol) was dissolved in DMF (0.5 mL), compound 36 (0.8 mg, 0.002 mmol) was added thereto, and then diisopropylethylamine (0.88 mg, 0.0068 mmol) was added under ice bath cooling, and the reaction solution was stirred at room temperature for 3 hours. Glacial acetic acid (2 mg) was added to quench, and purified by prep-RP-HPLC (method 4: 35%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-09 (0.8 mg).

[0473] LCMS (method 2): Rt = 2.17 min, m / z (ES + )865.2[1 / 3(M+3H)] + .

[0474] Example 14 Synthesis of Linker-Drug LD-10

[0475]

[0476] Step 1: Preparation of compound 54

[0477] Compound KL-2 (30 mg, 0.033 mmol) was dissolved in dry N,N-dimethylformamide (0.8 mL), and then 1-hydroxybenzotriazole (2.3 mg, 0.017 mmol), pyridine (0.2 mL) and MMAE (23.7 mg, 0.033 mmol) were added thereto in sequence. The reaction solution was stirred at room temperature overnight, and then purified by prep-RP-HPLC (method 4: 65%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 54 (20 mg).

[0478] LCMS (method 1): Rt = 2.14 min, m / z (ES + )747.3[1 / 2(M+2H)] + .

[0479] Step 2: Preparation of compound 55

[0480] Compound 54 (20 mg, 0.0134 mmol) was dissolved in methanol (1 mL), and then 1N aqueous lithium hydroxide solution (0.135 mL, 0.134 mmol) was added. The reaction solution was stirred at room temperature for 5 hours, and glacial acetic acid (0.1 mL) was added to quench the reaction, and purified by prep-RP-HPLC (method 4: 50%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 55 (10 mg).

[0481] LCMS (method 1): Rt = 1.47 min, m / z (ES + )566.4[1 / 2(M+2H)] + .

[0482] Step 3: Preparation of compound LD-10

[0483] Compound 55 (13 mg, 0.0115 mmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), and then compound 32 (5.4 mg, 0.0138 mmol) and diisopropylethylamine (5.9 mg, 0.046 mmol) were added thereto in sequence. The reaction solution was stirred at room temperature for 5 hours, glacial acetic acid (6 mg) was added to quench the reaction, and purified by prep-RP-HPLC (method 4: 55%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-10 (4 mg).

[0484] LCMS (method 1): Rt = 1.73 min, m / z (ES + )703.3[1 / 2(M+2H)] + .

[0485] Example 15 Synthesis of Linker-Drug LD-11

[0486]

[0487] Step 1: Preparation of compound 56

[0488] Compound KL-3 (100 mg, 0.12 mmol), MMAE (70 mg, 0.10 mmol), 1-hydroxybenzotriazole (7 mg, 0.05 mmol) and pyridine (0.05 mL) were added to anhydrous N,N'-dimethylformamide (1 mL) in sequence, and the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, it was purified by prep-RP-HPLC (method 5: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 52 (25 mg).

[0489] LCMS (method 1): Rt = 2.04 min, m / z (ES + )700.4[1 / 2(M+2H)] + .

[0490] Step 2: Preparation of compound 57

[0491] Compound 56 (25 mg, 0.0179 mmol) was dissolved in ethyl acetate (2 mL), and then 4 M hydrochloric acid dioxane solution (2 mL, 8 mmol) was added thereto, and the reaction solution was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain yellow solid compound 57 (20 mg).

[0492] LCMS (method 1): Rt = 1.53 min, m / z (ES + )650.5[1 / 2(M+2H)] + .

[0493] Step 3: Preparation of compound 58

[0494] Compound 57 (20 mg, 0.015 mmol) was dissolved in methanol (1 mL), and then 1 M aqueous lithium hydroxide solution (0.15 mL, 0.15 mmol) was added thereto, and the reaction solution was stirred at room temperature for 3 hours. Glacial acetic acid (0.1 mL) was added to quench the reaction, and the resulting solution was purified by prep-RP-HPLC (method 4: 50%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 58 (14 mg).

[0495] LCMS (method 1): Rt = 1.42 min, m / z (ES + )1160.8(M+H) +.

[0496] Step 4: Preparation of compound LD-11

[0497] Compound 58 (14 mg, 0.0121 mmol) was dissolved in N,N'-dimethylformamide (1 mL), and then compound 36 (5.6 mg, 0.0145 mmol) and diisopropylethylamine (9.4 mg, 0.0726 mmol) were added thereto in sequence, and the reaction solution was stirred at room temperature for 16 hours. Glacial acetic acid (10 mg) was added to quench the reaction, and the resulting solution was purified by prep-RP-HPLC (method 4: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-11 (2.7 mg).

[0498] LCMS (method 2): Rt = 1.78 min, m / z (ES + )717.4[1 / 2(M+2H)] + .

[0499] Example 16 Synthesis of Linker-Drug LD-12

[0500]

[0501] Step 1: Preparation of compound 59

[0502] Compound KL-4 (41 mg, 0.05 mmol), MMAE (43 mg, 0.06 mmol), 1-hydroxybenzotriazole (3 mg, 0.02 mmol) and pyridine (0.03 mL) were added to DMF (1 mL) in sequence, and the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, it was purified by prep-RP-HPLC (method 5: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 59 (50 mg).

[0503] LCMS (Method 3): Rt = 2.14 min, m / z (ES + )700.5[1 / 2(M+2H)] + .

[0504] Step 2: Preparation of compound 60

[0505] Compound 59 (50 mg, 0.04 mmol) was dissolved in ethyl acetate (1 mL), and 4 M hydrochloric acid dioxane solution (0.3 mL) was added thereto under ice bath cooling, and the reaction solution was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was purified by prep-RP-HPLC (method 4: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound 60 (30 mg).

[0506] LCMS (method 3): Rt = 1.53 min, m / z (ES + )650.6[1 / 2(M+2H)] + .

[0507] Step 3: Preparation of compound 61

[0508] Compound 60 (30 mg, 0.02 mmol) was dissolved in methanol (0.5 mL), and 1 M aqueous lithium hydroxide solution (0.5 mL) was added thereto under ice bath cooling, and the reaction solution was stirred at room temperature for 2 hours. Glacial acetic acid (30 mg) was added to quench the reaction, and the mixture was purified by prep-RP-HPLC (method 4: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain compound 61 (15 mg) as a white solid.

[0509] LCMS (method 2): Rt = 1.43 min, m / z (ES + )1160.7(M+H) + .

[0510] Step 4: Preparation of compound LD-12

[0511] Compound 61 (15 mg, 0.01 mmol) was dissolved in N,N-dimethylformamide (0.9 mL), and compound 36 (6.2 mg, 0.02 mmol) and diisopropylethylamine (6.7 mg, 0.05 mmol) were added sequentially under ice bath cooling, and the reaction solution was stirred at room temperature for 16 hours. Acetic acid was added to quench the reaction, and the mixture was purified by prep-RP-HPLC (method 4: 30%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-12 (2 mg).

[0512] LCMS (method 2): Rt = 1.68 min, m / z (ES + )717.5[1 / 2(M+2H)] + .

[0513] Example 17 Linker-Drugs LD-13, LD-14 and LD-15

[0514] Linker-drugs LD-13, LD-14 and LD-15 were synthesized according to the methods reported in the literature (LD-13: US7498298; LD-14: WO2014114207; LD-15: WO2019033773).

[0515]

[0516]

[0517] Example 18 Synthesis of Linker-Drug LD-16

[0518]

[0519] Step 1: Synthesis of compound 62

[0520] Compound KL-5 (197 mg, 0.28 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and then pyridine (0.5 mL), 1-hydroxybenzotriazole (19 mg, 0.14 mmol) and MMAE (201 mg, 0.28 mmol) were added thereto in sequence, and the reaction solution was stirred at room temperature for 17 hours. Acetic acid was added to the reaction solution to quench the reaction, and then water was added and extracted with ethyl acetate (30 mL×2). The combined organic phase was washed with saturated brine in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate (1 / 0→5 / 1→3 / 1), then dichloromethane / methanol (30 / 1)) to obtain colorless oil 62 (308 mg).

[0521] LCMS (Method 3): rt = 2.15 min; m / z (ES + )643.0[1 / 2(M+2H)] + .

[0522] Step 2: Synthesis of compound 63

[0523] Compound 62 (308 mg, 0.24 mmol) was dissolved in methanol (4.8 mL), and then 1M lithium hydroxide aqueous solution (2.4 mL, 2.4 mmol) was added thereto, and the reaction solution was stirred at room temperature for 3 hours. Glacial acetic acid (144 mg) was added to the reaction solution to quench the reaction, and then water was added and extracted with dichloromethane (30 mL×2). The combined organic phase was washed with saturated brine in turn, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol (40 / 1→15 / 1)) to obtain colorless gum compound 63 (240 mg).

[0524] LCMS (Method 3): rt = 1.91 min, m / z (ES + )1116.5(M+H) + .

[0525] Step 3: Synthesis of compound 64

[0526] Compound 63 (240 mg, 215 μmol) was dissolved in tetrahydrofuran (4 mL), and triphenylphosphine (67 mg, 258 μmol) and water (23 mg) were added thereto under stirring, and the reaction solution was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by prep-RP-HPLC (method 4: 15%-95% B in 8 minutes → 95% B in 4 minutes) to obtain compound 64 (150 mg) as a white solid.

[0527] LCMS (method 1): rt = 1.45 min, m / z (ES + )1091.6(M+H) + .

[0528] Step 4: Synthesis of compound LD-16

[0529] Compound 64 (21 mg, 19 μmol) was dissolved in N,N-dimethylformamide (1 mL), and compound 36 (8.5 mg, 22 μmol) and diisopropylethylamine (10 mg, 76 μmmol) were added thereto in sequence, and the reaction solution was stirred at room temperature for 4 hours. Glacial acetic acid (20 mg) was added to the reaction solution to quench the reaction, and then purified by prep-RP-HPLC (method 4: 15%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-16 (13.6 mg).

[0530] LCMS (Method 3): rt = 1.84 min, m / z (ES + )683.0[1 / 2(M+2H)] +2 .

[0531] Example 19 Synthesis of Linker-Drug LD-17

[0532]

[0533] Compound 64 (27.2 mg, 25 μmol) was dissolved in N,N-dimethylformamide (1 mL), and then compound 65 (25.3 mg, 32.5 μmol), HATU (19 mg, 50 μmol) and diisopropylethylamine (12.9 mg, 100 μmmol) were added thereto in sequence, and the reaction solution was stirred at room temperature for 2 hours. Glacial acetic acid (26 mg) was added to the reaction solution to quench the reaction, and the mixture was purified by prep-RP-HPLC (method 4: 20%-95% B in 8 minutes → 95% B in 4 minutes) to obtain white solid compound LD-17 (27 mg).

[0534] LCMS (method 2): rt = 1.09 min, m / z (ES +)926.8[1 / 2(M+2H)] + .

[0535] Example 20 Preparation of Antibody Drug Conjugates

[0536] Tris(2-carboxyethyl)phosphine hydrochloride (2-10 eq, stock solution concentration 10 mM) was added to the antibody solution (2-10 mg / mL, 67 mM PBS buffer, pH 7.0-7.2). The reaction solution was incubated in a 37°C constant temperature shaker for 2 hours. The reaction solution was cooled to about 10°C and ultrafiltered (Merck Millipore Ultra, 50000MWCO) or gel filtration exchange buffer (20mM citric acid-trisodium citrate, 50mM sodium chloride, 1mM diethylenetriaminepentaacetic acid, pH 5.8-6.2), add dimethyl sulfoxide and linker-drug (dimethyl sulfoxide stock solution, 3-15 equivalents), and ensure that the volume proportion of dimethyl sulfoxide in the reaction solution is about 10%. The coupling reaction is carried out at 10°C for 1.5 hours.

[0537] An excess of cysteine ​​solution was added to the reaction solution to quench the unreacted linker-drug. The quenching reaction was carried out at 10°C for 30 minutes. The reaction solution was first ultrafiltered (Merck Millipore The cysteine-linker-drug adduct and excess cysteine ​​were removed by filtration with a 100% elution (Ultrasonic A, 50000 MWCO) or gel filtration, and then sterilized by a 0.22 μm pore size filter (Merck Millex-GV Filter) to obtain the antibody drug conjugate, which was stored at 4°C.

[0538] The above preparation method was used to prepare antibody-drug conjugates (see Table 1) using Ab1 and Ab2 as antibodies and LD-01 to LD-17 as linker-drugs.

[0539] Table 1 Prepared Antibody Drug Conjugates

[0540]

[0541] NA: Not prepared.

[0542] 1) Determination of average DAR value

[0543] The average DAR value is determined by one of the following two methods depending on the actual situation.

[0544] 1a) Hydrophobic interaction chromatography (HIC)

[0545] It is calculated by the percentage of the relative peak area on the HIC spectrum and the number of drugs carried by the corresponding component (see Anal. Chem. 2013, 85, 1699-1704). The weighted peak share measures the contribution of a single DAR value component to the average DAR value, which is the relative peak area (%) multiplied by the number of drugs carried by the component. The average DAR value is obtained by summing the weighted peak shares of all components and then dividing by 100, as shown in the following formula:

[0546] DAR = Σ(relative peak area × the amount of drug carried by the component) / 100

[0547] 1b) Mass spectrometry (MS)

[0548] Take an appropriate amount of sample and dilute to 1 mg / mL with 150 mM ammonium acetate solution. Ultra-high performance liquid chromatography Acquity UPLC (Waters) and high-resolution Xevo G2-XS QTOF (Waters) mass spectrometer were used in the experiment, and 10 μL was injected for detection. Mass spectrometry data in positive ion mode were collected. The collected non-denaturing mass spectrometry data were analyzed and processed using UNIFI software.

[0549] The average DAR value measurement results of the antibody drug conjugates of the present invention are shown in Table 2 below.

[0550] Table 2 Average DAR values ​​of antibody drug conjugates

[0551] Antibody Drug Conjugates Average DAR value Antibody Drug Conjugates Average DAR value Ab1-LD-01 <![CDATA[8.0 a ]]> Ab2-LD-01 NA Ab1-LD-02 <![CDATA[4.1 a ]]> Ab2-LD-02 <![CDATA[4.0 a <!-- 56 -->]]> Ab1-LD-04 NA Ab2-LD-04 <![CDATA[2.0 a ]]> Ab1-LD-05 NA Ab2-LD-05 <![CDATA[7.5 b ]]> Ab1-LD-06 NA Ab2-LD-06 <![CDATA[4.0 b ]]> Ab1-LD-07 <![CDATA[8.0 b ]]> Ab2-LD-07 NA Ab1-LD-08 <![CDATA[4.0 b ]]> Ab2-LD-08 NA Ab1-LD-09 <![CDATA[8.1 a ]]> Ab2-LD-09 NA Ab1-LD-10 <![CDATA[4.1 a ]]> Ab2-LD-10 NA Ab1-LD-11 <![CDATA[4.3 a ]]> Ab2-LD-11 NA Ab1-LD-12 <![CDATA[4.4 a ]]> Ab2-LD-12 NA Ab1-LD-13 NA Ab2-LD-13 <![CDATA[4.1 a ]]> Ab1-LD-14 <![CDATA[4.2 a ]]> Ab2-LD-14 <![CDATA[4.2 a ]]> Ab1-LD-15 <![CDATA[2.2 a ]]> Ab2-LD-15 <![CDATA[2.1 a ]]> Ab1-LD-16 NA Ab2-LD-16 <![CDATA[4.0 a ]]> Ab1-LD-17 NA Ab2-LD-17 <![CDATA[2.2 a ]]>

[0552] NA: Not prepared.

[0553] a: HIC method

[0554] b: MS method

[0555] 2) Hydrophobic interaction chromatography (HIC) analysis

[0556] Hydrophobic interaction chromatography was measured on an Agilent 1100 chromatograph. The stationary phase was a TSKgel butyl-NPR column (4.6×35 mm, 2.5 μm, Tosoh (Shanghai) Biotechnology Co., Ltd.). The elution gradient was a linear gradient, replacing from 100% buffer A [50 mM potassium phosphate (pH 7.0) + 1.5 M ammonium sulfate] to 100% buffer B [80% v / v 50 mM potassium phosphate (pH 7.0), 20% v / v isopropanol] within 25 minutes. The flow rate was 0.8 mL / min, the column temperature was set at 30° C., and the detection wavelength was set at 280 nm.

[0557] The HIC spectrum of ADC in the present invention is shown in Figure 1A-Figure 1K The main peak retention times are shown in Tables 3 and 4.

[0558] Table 3 Main peak retention time of HIC spectrum based on Ab1 ADC

[0559]

[0560]

[0561] As shown in Table 3, compared with the ADC drug Ab1-LD-14 using the vc-PABC cleavable linker, Ab1-LD-02, Ab1-LD-10, Ab1-LD-11 and Ab1-LD-12 all use the glycosyl pyridine linker of the present invention. The comparison of the main peak retention time of the HIC spectrum shows that the latter's peak time is significantly lower than the former.

[0562] Table 4 Main peak retention time of HIC spectrum based on Ab2 ADC

[0563] Antibody Drug Conjugates Retention time (min) Average DAR value Ab2-LD-02 12.0 4.0 Ab2-LD-04 9.9 2.0 Ab2-LD-14 13.5 4.2 Ab2-LD-15 12.5 2.1 Ab2-LD-16 11.2 4.0 Ab2-LD-17 9.3 2.2

[0564] As shown in Table 4, when the average DAR value is about 4, the main peak retention time of the HIC spectra of antibody drug conjugates Ab2-LD-02 and Ab2-LD-16 using glycosyl pyridine type linkers is significantly shorter than that of antibody Ab2-LD-14 using vc-PABC linker. When the average DAR value is about 2, the main peak retention time of the HIC spectra of antibody drug conjugates Ab2-LD-04 and Ab2-LD-17 using glycosyl pyridine type linkers is significantly shorter than that of antibody Ab2-LD-15 using vc-PABC linker.

[0565] The above results show that compared with the commonly used vc-PABC linker, the use of glycosyl pyridine linkers can significantly increase the hydrophilicity of ADC products. The increase in the hydrophilicity of ADC products can reduce the aggregate content of ADC products and improve the in vivo PK properties of ADC products.

[0566] 3) Size Exclusion Chromatography (SEC) Analysis

[0567] Size exclusion chromatography was measured on an Agilent 1100 or 1260 chromatograph. The stationary phase was a TSKgel G3000SWXL column (7.8×300 mm, 5 μm, Tosoh (Shanghai) Biotechnology Co., Ltd.). The mobile phase was 62 mM disodium hydrogen phosphate, 55 mM potassium dihydrogen phosphate, 100 mM sodium sulfate, pH 6.7±0.1. The flow rate was 0.7 mL / min, the column temperature was set at 25°C, and the detection wavelength was set at 280 nm.

[0568] The monomer content of the ADC products in the embodiments of the present invention is above 99% as tested by SEC.

[0569] Experimental Example 1 Effect of the Antibody Drug Conjugate of the Present Invention on Inhibiting Cell Proliferation

[0570] Cell Proliferation Assay

[0571] The cell inhibitory activity of antibodies or antibody-drug conjugates was determined by the following method: mammalian cells expressing tumor-associated antigens or receptor proteins were inoculated on 96-well plates, 8,000 cells were inoculated per well, and suspended in 200 μL of DMEM high-glucose cell culture medium (GIBCO); the initial concentration of the ADC sample was 2 μg / mL, and it was diluted 3 times with DMEM high-glucose cell culture medium containing 2% FBS (GIBCO); the original culture medium was removed, 200 μL of ADC sample was added to each well, and incubation continued for 72 hours; the original culture medium was removed, 100 μL of CCK-8 colorimetric solution was added to each well, and incubation continued for 30 minutes; the absorbance value at 450nm / 630nm was read with an enzyme reader, the curve was drawn, and the IC 50 .

[0572] The results of the cell proliferation inhibition effect of the antibody drug conjugate of the present invention are shown in Tables 5 and 6.

[0573] Table 5 Results of the inhibitory effect of the antibody-drug conjugates of the present invention on cell proliferation

[0574]

[0575] The antibody drug conjugates prepared by the present invention all have good cell proliferation inhibition effect. As shown in Table 5, the cell proliferation inhibition effect of Ab1-LD-04 is slightly better than that of Ab1-LD-15, and the difference in structure between the two lies mainly in the cleavable linker part, the former adopts the glycosyl pyridine type linker of the present invention, and the latter adopts the vc-PABC type linker.

[0576] Table 6 Results of the inhibitory effect of the antibody-drug conjugates of the present invention on cell proliferation

[0577]

[0578] As shown in Table 6, when the average DAR values ​​are similar, the antibody drug conjugate using a glycosyl pyridine type linker has a comparable cell proliferation inhibition effect to the antibody drug conjugate using a vc-PABC type linker.

[0579] It can be seen that the ADC drug constructed based on the glycosyl pyridine linker of the present invention can effectively release cell-active drugs after entering the cells, thereby playing a role in killing cells.

[0580] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An antibody-drug conjugate of formula I, or a pharmaceutically acceptable salt or hydrate thereof, in, L is an antibody, antibody fragment, or other type of ligand; A is a linker fragment, connected to both L and G; G is a glycosyl-substituted pyridine structure, as shown in Formula II: in, Su is a sugar molecule; Each R is independently selected from hydrogen, CN, halogen, NO2 and C1-C4 alkyl; X and Y are each independently selected from -CR 2 R 3 -、-C(=O)NR 4 -、-NR 5 C(=O)-, -O-; Q is selected from -NR 6 -or-O-; R 1 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton; R 2 , R 3 , R 4 , R 5 and R 6 Each is independently selected from hydrogen or C1-C4 alkyl; x, y, z are each independently selected from 0 or 1; g is selected from 1 or 2; W is an optional suicide linker; w is selected from 0 or 1; D is for medicine; p is an integer selected from 1 to 4; q is selected from an integer of 1-8.

2. The antibody-drug conjugate of formula I according to claim 1, which is an antibody-drug conjugate of formula III: in, L, A, Q, R 1 , R, X, Y, W, D, Su, x, y, z, w as defined in claim 1.

3. The antibody-drug conjugate of formula I according to claim 1 or 2, which is an antibody-drug conjugate of formula IV or formula V: in, L, A, Q, R 1 , R, X, Y, W, D, x, y, z, w as defined in claim 1.

4. The antibody-drug conjugate of formula I according to any one of claims 1 to 3, which is an antibody-drug conjugate of formula VI or formula VII: in, L, A, Q, R 1 , R, X, Y, D, x, y, z as defined in claim 1.

5. The antibody-drug conjugate of formula I according to any one of claims 1 to 4, wherein: x is 0; y is 1; Y is selected from -C(=O)NR 4 -、-NR 5 C(=O)-, -O-; R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl.

6. The antibody-drug conjugate of formula I according to any one of claims 1 to 4, wherein: x is 1; y is 1; X is selected from -CR 2 R 3 -; Y is selected from -C(=O)NR 4 -、-NR 5 C(=O)-, -O-; R 2 and R 3 Each is independently selected from hydrogen or C1-C4 alkyl, preferably hydrogen; R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl.

7. The antibody-drug conjugate of formula I according to any one of claims 1 to 6, wherein: R 1 Selected from C1-C6 alkylene, preferably C1-C6 straight chain alkylene, more preferably C2-C4 straight chain alkylene; z is 1.

8. The antibody drug conjugate of formula I according to any one of claims 1 to 7, wherein: Q is -NR 6 -; R 6 Each is independently selected from hydrogen or C1-C4 alkyl.

9. The antibody drug conjugate of formula I according to any one of claims 1 to 8, wherein: Each R is independently selected from hydrogen, halogen, C1-C4 alkyl, preferably hydrogen.

10. The antibody-drug conjugate of formula I according to any one of claims 1 to 9, wherein: G has the following structure: Among them, R 7 Selected from hydrogen, C 1-4 Alkyl groups such as methyl, and water-soluble groups such as polyethylene glycol.

11. The antibody drug conjugate of formula I according to any one of claims 1 to 10, wherein g is 1.

12. The antibody drug conjugate of formula I according to any one of claims 1 to 11, wherein A is selected from the following structures: in, R 8 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton; R 9 is selected from H or halogen; R 10 Selected from C1-C4 alkyl; R 11 and R 12 Each is independently selected from C1-C6 alkylene.

13. The antibody-drug conjugate of formula I according to any one of claims 1 to 12, wherein L is a humanized, chimeric or fully humanized antibody or antibody fragment or antibody Fc fusion protein.

14. The antibody-drug conjugate of formula I according to any one of claims 1 to 13, wherein L is an antibody or antibody fragment or antibody Fc fusion protein against a cell surface receptor or a tumor-associated antigen.

15. The antibody-drug conjugate of formula I according to any one of claims 1 to 14, wherein L is a humanized anti-CD30 monoclonal antibody or a humanized CD79b monoclonal antibody. 16 . The antibody drug conjugate of formula I according to any one of claims 1 to 15 , wherein L is Ab1 having a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO:

2.

17. The antibody drug conjugate of formula I according to any one of claims 1 to 15, wherein L is Ab2 having a heavy chain as shown in SEQ ID NO: 3 and a light chain as shown in SEQ ID NO:

4.

18. The antibody-drug conjugate of formula I according to any one of claims 1 to 17, wherein D is an anti-tumor drug, a drug for treating an autoimmune disease, or an anti-inflammatory drug, such as a tubulin inhibitor, a DNA alkylating agent, a DNA minor groove binder, a DNA topoisomerase inhibitor, and the like.

19. The antibody drug conjugate of formula I according to any one of claims 1 to 18, wherein D is monomethyl auristatin E (MMAE), SN-38 or exotecan.

20. A compound represented by formula (A) or a pharmaceutically acceptable salt thereof, in, A' is the linker fragment; Each R is independently selected from hydrogen, CN, halogen, NO2 and C1-C4 alkyl; preferably hydrogen, halogen, C1-C4 alkyl, more preferably hydrogen; X and Y are each independently selected from -CR 2 R 3 -、-C(=O)NR 4 -、-NR 5 C(=O)-, -O-; Q is selected from -NR 6 - or -O-, preferably -NR 6 -; R 1 Selected from C1-C6 alkylene or C1-C6 alkylene containing O in the skeleton, preferably C1-C6 alkylene; R 2 and R 3 Each is independently selected from hydrogen or C1-C4 alkyl, preferably hydrogen; R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl; R 6 Selected from hydrogen or C1-C4 alkyl; x is 0 or 1; y is 0 or 1, preferably 1; z is 0 or 1, preferably 1; D is the drug molecule; p is selected from integers of 1 to 4, preferably 1 or 2.

21. The compound of formula (A) or a pharmaceutically acceptable salt thereof according to claim 20, wherein A' is selected from the following structures: in, R 8 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton; R 9 is selected from H or halogen; R 10 Selected from C1-C4 alkyl; R 11 and R 12 Each is independently selected from C1-C6 alkylene.

22. The compound of formula (A) according to claim 20 or 21 or a pharmaceutically acceptable salt thereof, which is selected from the following structures: in, R 5 , R 6 and D as defined in claim 17.

23. The compound represented by formula (A) or a pharmaceutically acceptable salt thereof according to any one of claims 20 to 22, wherein: D is an anti-tumor drug, a drug for treating autoimmune diseases or an anti-inflammatory drug, such as a microtubule inhibitor, a DNA alkylating agent, a DNA minor groove binder, or a DNA topoisomerase inhibitor; preferably, D is monomethyl auristatin E (MMAE), SN-38 or exitecan.

24. A compound of formula (A) or a pharmaceutically acceptable salt thereof according to any one of claims 20 to 23, selected from the following structures:

25. A pharmaceutical composition comprising the antibody drug conjugate of formula I according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

26. Use of the antibody drug conjugate of formula I according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 25 in the preparation of a medicament for treating cancer, autoimmune diseases and inflammatory diseases.

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