Antibody-drug conjugate containing glycosylpyridine linker

Abstract

An antibody-drug conjugate containing a glycosylpyridine linker. Specifically, the present invention relates to a glycosylpyridine linker, and an antibody-drug conjugate containing the glycosylpyridine linker, a preparation method therefor, and a medical use thereof, particularly a use in the preparation of a drug for treating cancers, autoimmune diseases, and inflammatory diseases. By means of the specific glycosylpyridine linker, the hydrophilicity of the antibody-drug conjugate is enhanced, and the antibody-drug conjugate is controlled to release an active drug under the intracellular enzymatic action.

Description

Antibody-drug conjugates containing glycosylpyridine linkers Technical Field

[0001] The present invention relates to a novel glycosyl pyridine 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 class of targeted therapeutics primarily used in the treatment of cancer and autoimmune diseases. Their fundamental design philosophy stems from the "magic bullet" and drug targeting concepts first proposed by Paul Ehrlich in 1913, which involves delivering drugs to the site of disease via an appropriate carrier. However, due to limitations in antibody and highly active cytotoxic drug technology, ADCs were not widely used until 2000, when the first ADC (Mylotarg) for the treatment of acute myeloid leukemia (AML) was developed. TM ) was approved for marketing by the FDA. So far, 12 ADC drugs have been approved for marketing, marking that the application of antibody-drug conjugates in the field of tumor treatment has entered a rapid development stage.

[0003] Antibody-drug conjugates typically consist of three components: an antibody or other type of ligand, a highly active cytotoxic drug, and a linker that couples the antibody and cytotoxic drug. Their mechanism of action is as follows: the antibody specifically recognizes and binds to cell surface antigens; the resulting conjugate is internalized into the cell, carrying the highly active cytotoxic drug with it; the antibody is enzymatically degraded or the linker itself is cleaved, releasing the highly active cytotoxic drug in an appropriate form to kill the target cells.

[0004] Based on the intracellular release mechanism of cytotoxic drugs, linkers can be divided into non-cleavable and cleavable linkers. The vast majority of currently marketed ADCs utilize cleavable linkers. Cleavable linkers can be further categorized by their cleavage mechanism into the following main types: acid cleavage (e.g., hydrazones), cysteine / glutathione cleavage (e.g., disulfide bonds), and enzymatic cleavage (e.g., peptides). Enzymatic cleavage linkers are favored for their stability and cleavage specificity.

[0005] Cytotoxic drugs have a certain degree of hydrophobicity. Conjugating them to antibodies often results in an increase in the hydrophobicity of the antibody-drug conjugate (relative to the antibody). This increased hydrophobicity not only easily leads to aggregation of ADC molecules (forming multimers) but also accelerates the ADC's clearance in the body. To reduce the hydrophobicity of ADC drugs, hydrophilic linkers are often used to reduce or offset the adverse effects of the cytotoxic drug's hydrophobicity on the ADC drug.

[0006] 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.

[0007] Summary of the Invention

[0008] The present invention aims to provide a novel glycosyl pyridine linker and construct an antibody-drug conjugate based on this linker.

[0009] After years of in-depth research, the inventors have developed a novel glycosylpyridine linker. ADC drugs constructed based on this linker bind to cell surface antigens and are internalized into cells. After being degraded by β-glucuronidase, they release cytotoxic drugs, achieving a cell-killing effect. This linker has excellent chemical stability and does not break during in vivo circulation, thereby increasing off-target toxicity. Furthermore, this linker exhibits excellent hydrophilicity, which can significantly improve the hydrophilicity of ADC drugs and the resulting metabolic stability in vivo. This linker technology is universal and can be applied to the vast majority of ADC drugs, thus possessing broad application prospects.

[0010] Specifically, the present invention provides an antibody-drug conjugate containing a glycosylpyridine linker. This glycosylpyridine linker structure contains a glycosyl (Su)-substituted pyridine group. The pyridine group has two conjugated groups, one of which can be coupled to an antibody or other type of ligand via another group or directly, and the other conjugated group can be linked to a cytotoxic drug via another group or directly (as shown in the formula below).

[0011] The resulting antibody-drug conjugate can be used to deliver drugs to target cell populations, such as tumor cells. The antibody-drug conjugate can specifically bind to cell surface proteins, and the resulting conjugate is then internalized by the cell. Inside the cell, glycosidases (GHs) selectively hydrolyze the Su-O group, releasing the sugar molecule. The residual oxygen anion on the pyridine ring undergoes an electron recombination reaction with the pyridine ring, releasing the para-linked drug as an active drug to produce its therapeutic effect (see the reaction formula below).

[0012] Antibodies used to construct ADCs include chimeric, humanized, and human antibodies; antigen-binding antibody fragments; antibody-Fc fusion proteins; proteins; or other types of ligands. Drugs are highly active, including but not limited to maytansinoids, auristatins, calicheamicins, doxorubicins, duocarmycins and CC-1065, PBD dimers, camptothecins, tubulysins, and amanitins.

[0013] Compared with traditional antibody-drug conjugates, the antibody-drug conjugates prepared by the method of the present invention have increased hydrophilicity and greatly improved in vivo circulation stability, thereby greatly enhancing the drugability of the product.

[0014] Therefore, an object of the present invention is to provide an antibody drug conjugate as shown in Formula I,

[0015] or a pharmaceutically acceptable salt or hydrate thereof,

[0016] in,

[0017] L is an antibody, antibody fragment, or other type of ligand;

[0018] A is a linker fragment, connected to both L and G;

[0019] G is a glycosyl-substituted pyridine structure, as shown in Formula II:

[0020] in,

[0021] Su is a sugar molecule;

[0022] Each R is independently selected from hydrogen, CN, halogen, NO2 and C1-C4 alkyl;

[0023] X and Y are each independently selected from -CR 2 R 3 -, -C(=O)NR 4 -、-NR 5 C(=O)-, -O-;

[0024] Q is selected from -NR 6 -or-O-;

[0025] R 1Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton;

[0026] R 2 、R 3 、R 4 、R 5 and R 6 Each independently selected from hydrogen or C1-C4 alkyl;

[0027] x, y, z are each independently selected from 0 or 1;

[0028] g is selected from 1 or 2;

[0029] W is an optional suicide linker;

[0030] w is selected from 0 or 1;

[0031] D is for medicine;

[0032] p is an integer selected from 1 to 4;

[0033] q is selected from an integer of 1 to 8.

[0034] In one embodiment, the antibody-drug conjugate of formula I according to the present invention is an antibody-drug conjugate of formula III:

[0035] Among them, L, A, Q, R 1 , R, X, Y, W, D, Su, x, y, z, w are as defined in Formula I.

[0036] 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:

[0037] Among them, L, A, Q, R 1 , R, X, Y, W, D, x, y, z, w are as defined in Formula I.

[0038] 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:

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

[0040] In a preferred embodiment, according to the formula I, III, IV, V, VI, VII of the present invention,

[0041] x is 0;

[0042] y is 1;

[0043] Y is selected from -C(=O)NR 4 -、-NR 5 C(=O)-, -O-;

[0044] R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl.

[0045] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, x is 1;

[0046] y is 1;

[0047] X is selected from -CR 2 R 3 -;

[0048] Y is selected from -C(=O)NR 4 -、-NR 5 C(=O)-, -O-;

[0049] R 2 and R 3 Each independently selected from hydrogen or C1-C4 alkyl, preferably hydrogen;

[0050] R 4 and R 5 Each is independently selected from hydrogen or C1-C4 alkyl.

[0051] In another preferred embodiment, in Formula I, III, IV, V, VI, and 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.

[0052] In another preferred embodiment, according to Formula I, III, IV, V, VI, VII of the present invention, Q is -NR 6 -;R 6 Each is independently selected from hydrogen or C1-C4 alkyl.

[0053] In another preferred embodiment, in Formulas I, III, IV, V, VI and VII according to the present invention, each R is independently selected from hydrogen, halogen, C1-C4 alkyl, preferably hydrogen.

[0054] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, G has the structure shown below:

[0055] Among them, R 7 Selected from hydrogen, C 1-4 Alkyl groups such as methyl, and water-solubilizing groups such as polyethylene glycol.

[0056] In another preferred embodiment, in Formula I, III, IV, V, VI, and VII according to the present invention, g is 1.

[0057] 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.

[0058] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, A is selected from the following structures:

[0059] Among them, R 8 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton;

[0060] R 9 is selected from H or halogen;

[0061] R 10 Selected from C1-C4 alkyl;

[0062] R 11 and R 12 Each is independently selected from C1-C6 alkylene.

[0063] In another preferred embodiment, according to Formula I, III, IV, V, VI, and VII of the present invention, L is a ligand such as a humanized, chimeric, or fully humanized antibody or antibody fragment, or an antibody Fc fusion protein.

[0064] 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 targeting a cell surface receptor or tumor-associated antigen.

[0065] 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.

[0066] In another preferred embodiment, according to Formula I, III, IV, V, VI, and 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.

[0067] In another preferred embodiment, according to Formula I, III, IV, V, VI, and 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.

[0068] 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 inhibitor, a DNA alkylating agent, a DNA minor groove binder, or a DNA topoisomerase inhibitor.

[0069] 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.

[0070] Another object of the present invention is to provide a compound represented by formula (A) or a pharmaceutically acceptable salt thereof,

[0071] in,

[0072] A' is the linker fragment;

[0073] Each R is independently selected from hydrogen, CN, halogen, NO2 and C1-C4 alkyl; preferably hydrogen, halogen, C1-C4 alkyl, more preferably hydrogen;

[0074] X and Y are each independently selected from -CR 2 R 3 -, -C(=O)NR 4 -、-NR 5 C(=O)-, -O-;

[0075] Q is selected from -NR 6 - or -O-, preferably -NR 6 -;

[0076] R 1 Selected from C1-C6 alkylene or C1-C6 alkylene containing O in the skeleton, preferably C1-C6 alkylene;

[0077] R 2 and R 3 Each independently selected from hydrogen or C1-C4 alkyl, preferably hydrogen;

[0078] R 4 and R 5 Each independently selected from hydrogen or C1-C4 alkyl;

[0079] R 6 Selected from hydrogen or C1-C4 alkyl;

[0080] x is 0 or 1;

[0081] y is 0 or 1, preferably 1;

[0082] z is 0 or 1, preferably 1;

[0083] D is the drug molecule;

[0084] p is selected from integers of 1 to 4, preferably 1 or 2.

[0085] 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:

[0086] in,

[0087] R 8 Selected from C1-C6 alkylene, or C1-C6 alkylene containing O in the skeleton;

[0088] R 9 is selected from H or halogen;

[0089] R 10 Selected from C1-C4 alkyl;

[0090] R 11 and R 12 Each is independently selected from C1-C6 alkylene.

[0091] In another preferred embodiment, the compound represented by formula (A) according to the present invention or a pharmaceutically acceptable salt thereof is selected from the following structures:

[0092] Among them, R 5 、R 6 and D are as defined in formula (A).

[0093] In another preferred embodiment, according to the compound represented by 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, or a DNA topoisomerase inhibitor; preferably, D is monomethyl auristatin E (MMAE), SN-38 or exitecan.

[0094] In another preferred embodiment, the compound represented by formula (A) according to the present invention or a pharmaceutically acceptable salt thereof is selected from the following structures:

[0095] The present invention also provides a pharmaceutical composition comprising the antibody-drug conjugate of Formula I, III, IV, V, VI or VII according to the present invention and a pharmaceutically acceptable carrier.

[0096] 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 a drug for treating cancer, autoimmune diseases and inflammatory diseases.

[0097] 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 to treat cancer, autoimmune diseases and inflammatory diseases.

[0098] 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.

[0099] Terminology

[0100] 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. When substituted, the substituent may be substituted at any available point of attachment and may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate.

[0101] 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 1-20 The alkylene group is preferably an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 alkylene), more preferably an alkylene containing 1 to 6 carbon atoms (i.e., C 1-6 Alkylene), further preferably an alkylene containing 1 to 4 carbon atoms (i.e., C 1-6Alkylene). Non-limiting examples of alkylene include, but are not limited to, methylene (—CH—), 1,1-ethylene (—CH(CH)—), 1,2-ethylene (—CHCH)—, 1,1-propylene (—CH(CHCH)—), 1,2-propylene (—CHCH(CH)—), 1,3-propylene (—CHCHCHCH—), and 1,4-butylene (—CHCHCHCHCH—). Alkylene may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment. The substituent may be selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0102] 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 ethenyl, 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, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0103] 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 groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0104] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls. The cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocyclyl ring, wherein the ring attached to the parent structure is a cycloalkyl. Non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptanyl, tetrahydrobenzofuranyl, tetrahydrobenzoxazolyl, tetrahydrobenzisoxazolyl, cyclopentathienyl, and tetrahydrobenzothiazolyl. The cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate.

[0105] 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 excluding the ring portion of -OO-, -OS- or -SS-, 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 groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate.

[0106] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing 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 attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0107] The aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0108] 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-membered, containing 1 to 3 heteroatoms; more preferably 5 or 6-membered, containing 1 to 2 heteroatoms; preferably, for example, imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, thiazolyl, pyrazolyl or pyrimidinyl, 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:

[0109] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylate.

[0110] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and that 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.

[0111] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0112] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with 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, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0113] "Pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that are safe and effective when used in mammals and have the desired biological activity.

[0114] "Carrier" refers to a vehicle or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0115] The term "Drug / Antibody Ratio (DAR)" as used herein refers to the number of drug-conjugated molecules per antibody molecule. Because ADC samples contain components with varying DAR values, the concepts of "average DAR" and "DAR distribution" are more appropriate for describing the composition of ADCs. The average DAR is the ratio of the total number of drug molecules to the total number of antibodies in a sample, while the DAR distribution refers to the distribution of the concentrations of components with varying DAR values ​​in the sample.

[0116] As used herein, the terms "antibody" or "antibody unit" include within their scope any portion of an antibody structure. This unit can bind, reactively associate with, 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.

[0117] 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 (TAAs), 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 those known or predicted to be functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as those known or predicted to be functional). Tumor-related factors can be cluster differentiation factors (such as CD proteins). The antigens bound to the antibodies of 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).

[0118] 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 using methods and information for antibody preparation that are well known in the industry. In order to develop effective cellular 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, while being 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 than on the surface of non-cancerous cells. Identifying such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer treatment.

[0119] Tumor-associated antigens include, but are not limited to, the tumor-associated antigens listed below (1)-(36). For convenience, information related to antigens well known in the art 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 that 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 references.

[0120] Tumor-associated antigens (1)-(36):

[0121] (1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM_001203);

[0122] (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

[0123] (3) STEAP1 (six-transmembrane prostate epithelial antigen, Genbank accession number NM_012449);

[0124] (4) 0772P (CA125, MUC16, Genbank accession number AF361486);

[0125] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823);

[0126] (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);

[0127] (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);

[0128] (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

[0129] (9) ETBR (endothelin type B receptor, Genbank accession number AY275463);

[0130] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763);

[0131] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane prostate epithelial antigen 2, six-transmembrane prostate protein, Genbank accession number AF455138);

[0132] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636);

[0133] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP_003203 or NM_003212);

[0134] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Genbank accession number M26004);

[0135] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29, Genbank accession number NM_000626);

[0136] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764);

[0137] (17) HER2 (ErbB2, Genbank accession number M11730);

[0138] (18) NCA (CEACAM6, Genbank accession number M18728);

[0139] (19) MDP (DPEP1, Genbank accession number BC017023);

[0140] (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971);

[0141] (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053);

[0142] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442);

[0143] (23) ASLG659 (B7h, Genbank accession number AX092328);

[0144] (24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436);

[0145] (25) GEDA (Genbank accession number AY260763);

[0146] (26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456);

[0147] (27) CD22 (B cell receptor CD22-B isoform, Genbank accession number AK026467);

[0148] (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);

[0149] (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);

[0150] (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);

[0151] (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);

[0152] (32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

[0153] (33) LY64 (lymphocyte antigen 64 (RP105), a member of the leucine-rich repeat-rich type I membrane protein (LRR) family, 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);

[0154] (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);

[0155] (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);

[0156] (36) TENB2 (putative transmembrane proteoglycan related to the EGF / heregulin family of growth factors and follistatin, Genbank accession number AF179274).

[0157] As used herein, "drug" or the code "D" refers to any compound having a desired biological activity and a reactive functional group for preparing the conjugates of the present invention. The desired biological activity includes diagnosing, curing, alleviating, treating, or preventing a disease 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 recognized in official national pharmacopeias, as well as drugs recognized in the official U.S. Homeopathic Pharmacopoeia, the official National Formulary, or any supplements thereof. Typical drugs are listed in the Physician's Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). As new drugs are continuously discovered and developed, this patent stipulates that these drugs should also be included in the prodrugs of the conjugate drugs of the present invention.

[0158] Preferably, the drug refers to: a cytotoxic drug for cancer treatment; a protein or polypeptide with desired biological activity, such as toxins, 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 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.

[0159] On the one hand, medicine is maytansine (maytansine) or maytansinoids (maytansinoids). Maytansine compounds inhibit cell proliferation by inhibiting the microtubule formation of tubulin (Science 1975,189,1002-1005; US 5208020). Maytansinoids are derivatives of maytansine. Maytansine and maytansinoids all have efficient cytotoxicity, but they have very large 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 first choice drug part of antibody drug conjugates. Listed below are three maytansine-like molecular structures that are often utilized in antibody drug conjugate applications, including maytansine, maytansinoids, and antibody drug conjugates.

[0160] The main raw material for synthetic maytansinoids is maytansinol, which is primarily obtained by hydrolysis of ansamitocins. Ansamitocins can be produced by fermentation. Ansamitocin derivatives (WO 2012 / 061590) and alaninyl maytansinol (US 2012 / 0121615) have also been reported as drug "warheads" in antibody-drug conjugates (the structures of these two types of molecules are shown below).

[0161] On the other hand, the drugs are auristatins. 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.

[0162] On the other hand, the drug is a tubulysin. Tubulysins are a class of natural products extracted from myxobacteria that effectively inhibit tubulin polymerization, thus exhibiting antimitotic activity. Tubulysin D exhibits the most activity. Tubulysin D is a complex tetrapeptide compound containing O-acyl / N,O-acetal functional groups, making it unstable under both acidic and alkaline conditions. US 2011 / 0021568 and US 2013 / 0224228, respectively, disclose a series of tubulysin analogs that have these unstable functional groups removed from their structures while still exhibiting high cellular activity.

[0163] On the other hand, the drug is a calicheamicin. Calcheamicins are antitumor antibiotics that bind to the minor groove of DNA and induce double-helix DNA breaks at specific sites, leading to cell apoptosis. While calicheamicins exhibit high in vitro activity at sub-picomolar levels, their low therapeutic index precludes their clinical application. However, this high activity makes them ideal candidates for antibody-drug conjugates (e.g., gemtuzumab ozogamicin and inotuzumab ozogamicin).

[0164] On the other hand, drugs are doxorubicins. Doxorubicins can intercalate into the DNA double helix, thereby blocking DNA replication, and are therefore used as chemotherapy drugs. However, due to the low cytotoxicity of doxorubicins (the half-maximal inhibitory concentration is 0.1-0.2 micromolar against human cancer cell lines, while the cytotoxic activity of drugs used in antibody-drug conjugates is generally sub-nanomolar), their application in antibody-drug conjugates is not widespread.

[0165] On the other hand, drugs include benzodipyrrole antibiotics (duocarmycins, CC-1065, etc.) and other cyclopropapyrroloind-4-one (CPI) derivatives. These compounds are potent DNA minor groove binding-alkylating agents. Cyclopropabenzindol-4-one (CBI) analogs have more stable chemical structures, higher biological activity, and are easier to synthesize than their parent compounds containing the natural CPI alkylating subunit. A representative CBI derivative is the phenolic hydroxyl-protected CBI derivative, which has weakened prodrug toxicity and enhanced water solubility.

[0166] 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 be able to produce sequence-selective DNA damage, such as the reversed 5'-Pu-GATC-Py-3' interchain crosslink, which leads to its biological activity. These compounds have been shown to be highly potent cytotoxic drugs and may serve as candidates for antibody-drug conjugates.

[0167] On the other hand, the drug is camptothecin (CPT). Camptothecin and its derivatives act as topoisomerase (TOP1) inhibitors, selectively capturing the TOP1 cleavage complex and becoming 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. These compounds have also been successfully used as warheads in ADC drug design, such as SN-38, exatecan (DX-8951f), and DXd.

[0168] On the other hand, the drug is an amanitin. Amanitin is an octapeptide toxin with a bicyclic structure produced by the poisonous fungus Amanita phalloides. This toxin can specifically bind to RNA polymerase II (or B) and non-competitively inhibit it, thereby inhibiting the formation of phosphodiester bonds (initiation and elongation 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 and quiescent cells through a unique mechanism. This property makes it an attractive cytotoxic drug candidate for ADC drugs.

[0169] On the other hand, drugs are not limited to the categories mentioned above, 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.

[0170] The term "linker" or "antibody drug conjugate linker" as used herein refers to a bifunctional or multifunctional molecule that can react with both a protein / antibody molecule and a drug molecule, thereby serving as a "bridge" to connect the protein / antibody and drug molecule. Based on the mechanism of intracellular drug release, "linkers" or "antibody drug conjugate linkers" can be divided into two categories: non-cleavable linkers and cleavable linkers.

[0171] A non-cleavable linker is a relatively stable linker whose 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 as follows: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing an active molecule composed of a small molecule drug, a linker, and antibody amino acid residues. The resulting change in the drug molecular structure does 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 target antigen (antigen-negative cells) (bystander effect) (Bioconjugate Chem. 2010, 21, 5-13). Common linkers such as MC linkers and MCC linkers are shown below.

[0172] Cleavable linkers, as the name suggests, can be cleaved within target cells to 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.

[0173] Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties, such as pH and glutathione concentration.

[0174] pH-sensitive linkers are also commonly 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 the 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 hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on this type of linker usually have a short half-life (2-3 days). This short half-life has, to a certain extent, limited the application of pH-sensitive linkers in the new generation of antibody-drug conjugates.

[0175] Glutathione-sensitive linkers, also known as disulfide linkers, trigger drug release based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low glutathione concentration (micromolar range) in blood. This is particularly true for tumor cells, where low oxygen levels lead to increased reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable, resulting in good stability in plasma.

[0176] Enzyme-labile linkers, such as peptide linkers, enable better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (the levels of such enzymes are increased in some tumor tissues). This peptide linkage is considered very stable in the plasma circulation because proteases are generally inactive outside the cell due to the unfavorable extracellular pH and serum protease inhibitors. Due to their high plasma stability and good intracellular cleavage selectivity and efficiency, enzyme-labile linkers are widely used as cleavable linkers in antibody-drug conjugates. Typical enzyme-labile linkers include the VC linker.

[0177] Suicide linkers are typically embedded between a cleavable linker and an active drug, or they themselves are part of the cleavable linker. The mechanism of action of a suicide linker is that upon cleavage of the cleavable linker under appropriate conditions, the suicide linker undergoes spontaneous structural rearrangement, releasing the attached active drug. Common suicide linkers include p-aminobenzyl alcohol (PAB).

[0178] The antibody-drug conjugate provided by the present invention is composed of an antibody, a linker containing a glycosylpyridine-type fragment, and a drug. The glycosylpyridine-type linker is an enzyme-cleavable linker. In one aspect, the antibody-drug conjugate provided by the present invention targets specific cell populations, binds to specific cell surface proteins (antigens), and the conjugate is internalized into the cell, where the drug is released into the cell in an active form.

[0179] On the other hand, the antibody-drug conjugates provided by the present invention target specific cell populations, bind to specific proteins (antigens) on the cell surface, and produce efficacy; or release drugs outside the cells, and the drugs penetrate into the cells to produce efficacy.

[0180] 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.

[0181] 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: the activity of the specific compound used, the patient's age, the patient's weight, the patient's health condition, the patient's behavior, the patient's diet, the time of administration, the route of administration, the rate of excretion, the combination of drugs, etc. In addition, the optimal treatment regimen, 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 protocols.

[0182] The main advantages of the present invention are:

[0183] 1. The present invention provides an antibody-drug conjugate containing a glycosylpyridine-type linker fragment. The glycosylpyridine-type linker fragment has good hydrophilicity, thereby significantly increasing the hydrophilicity of the antibody-drug conjugate, reducing aggregate formation in the ligand-drug conjugate, and improving the in vivo PK properties of the antibody-drug conjugate.

[0184] 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, thus having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0185] Figures 1A-1K are hydrophobic interaction chromatography (HIC) images of the antibody-drug conjugates of the present invention. Figures 1A-1E correspond to antibody-drug conjugates Abl-LD-02, Abl-LD-10, Abl-LD-11, Abl-LD-12, and Abl-LD-14, respectively; and Figures 1F-1K 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

[0186] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally performed under conventional conditions or as recommended by the manufacturer. All reactions were performed under nitrogen protection (except for the hydrogenation reaction).

[0187] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The methods and materials described herein are for illustrative purposes only.

[0188] Abbreviations Ab Antibody ACN Acetonitrile ADC Antibody Drug Conjugate BOC (Boc) Tert-Butyloxycarbonyl DIPEA Diisopropylethylamine ELISA Enzyme-Linked Immunosorbent Assay Eq (eq) Equivalent HIC Hydrophobic Interaction Chromatography HPLC High-Performance Liquid Chromatography LCMS Liquid Chromatography-Mass Spectrometry mAb Monoclonal Antibody MS Mass Spectrometry Prep-RP-HPLC Preparative High-Performance Liquid Chromatography Rt Retention Time SDS-PAGE Polyacrylamide Gel Electrophoresis SEC Size Exclusion Chromatography TEA Triethylamine TFA Trifluoroacetic Acid

[0189] Unless otherwise noted, 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.

[0190] 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).

[0191] In the LC-MS analysis method, low-resolution mass spectrometric data were acquired on an Agilent 6110 (acid method) or 6120B (base method) mass spectrometer interfaced with an Agilent 1200 HPLC:

[0192] Method 1: Acid-HPLC separation was performed using a Waters Sunfire C18 reversed-phase column (4.60 × 50 mm, 3.5 μm) 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.

[0193] Method 2: Acid-HPLC separation was performed using a Waters Sunfire C18 reversed-phase column (4.6 × 30 mm, 2.7 μm) 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.

[0194] Method 3: Alkaline HPLC method: Separation was performed using a Waters Xbridge C18 reversed-phase column (4.60×50 mm, 3.5 μm) with an eluent gradient of 5% to 95% phase B (acetonitrile) in phase A (aqueous phase containing 10 mM ammonium bicarbonate) over 1.5 minutes at a flow rate of 1.8 mL / min and a column temperature of 50°C.

[0195] Preparative reverse phase-high performance liquid chromatography (prep-RP-HPLC) purification was performed on a Gilson instrument using a Waters Sunfire C18 reverse phase column (250×19 mm, 10 μm):

[0196] Method 4: Acid preparation, mobile phase: A: aqueous phase containing 0.1% TFA; B: ACN, flow rate: 15 mL / min;

[0197] Method 5: Alkaline preparation, mobile phase: A: aqueous phase containing 10 mM ammonium bicarbonate; B: ACN. Flow rate: 15 mL / min.

[0198] The Ramos (CRL-1596, ATCC) and BJAB (ACC757, DSMZ) cell lines used are human non-Hodgkin's lymphoma cells. The HH (CRL-2105, ATCC), L540 (CSC-C0233, Creative Bioarray), and SU-DHL-1 (CRL-2955, ATCC) cell lines used are human Hodgkin's lymphoma cells. Ab1 (humanized anti-CD79b monoclonal antibody, IgG1) and Ab2 (humanized anti-CD30 monoclonal antibody, IgG1) were both developed in-house. The enzyme-labeled anti-antibody was purchased from Sigma (Shanghai). The substrate solution was purchased from Decent Biotechnology (Shanghai). The Cell Counting Kit-8 (CCK-8) cell proliferation-toxicity assay kit was purchased from Dojindo (Shanghai).

[0199] The following is the sequence of Ab1 (humanized anti-CD79b monoclonal antibody, IgG1):

[0200] Ab1 heavy chain

[0201] Ab1 light chain

[0202] The following is the sequence of Ab2 (humanized anti-CD30 monoclonal antibody, IgG1):

[0203] Ab2 heavy chain

[0204] Ab2 light chain

[0205] Example 1 Synthesis of Linker Intermediate KL-1

[0206] Step 1: Preparation of compound 1

[0207] 2-Bromo-3-hydroxypyridine (15 g, 86.2 mmol), potassium hydroxide (KOH, 4.83 g, 86.2 mmol) and paraformaldehyde ((CH2O) n , 7.76 g, 258.6 mmol) was added to 135 ml of water, and the reaction solution was stirred at 80°C for 17 hours. The reaction solution was cooled to room temperature, acetic acid (5.17 g, 86.2 mmol) was added, 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 Compound 1 (6.8 g) as a yellow solid.

[0208] LCMS (Method 1): Rt = 1.18 min, m / z (ES + )204.1[M+H] + .

[0209] Step 2: Preparation of compound 2

[0210] 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). The reaction mixture was stirred at room temperature for 17 hours. The mixture was poured into ice water (150 mL) and extracted with ethyl acetate (30 mL x 4). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was washed with dichloromethane (15 mL x 2) and dried to obtain Compound 2 (6.2 g) as a white solid.

[0211] LCMS (Method 3): Rt = 1.88 min, m / z (ES + )324.0[M+H] + .

[0212] Step 3: Preparation of compound 3

[0213] Compound 2 (6.2 g, 19.14 mmol) was dissolved in dimethyl sulfoxide (40 ml), and 2-iodobenzoic acid (IBX, 6.4 g, 22.97 mmol) was added 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). The mixture was extracted with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated brine, 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).

[0214] LCMS (Method 3): Rt = 2.02 min, m / z (ES + )322.0[M+H] + .

[0215] Step 4: Preparation of compound 4

[0216] 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 for extraction. The separated aqueous phase was extracted with ethyl acetate (30 mL x 2). 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 / petroleum ether (1 / 1)) to obtain compound 4 (1.8 g) as a yellow solid.

[0217] LCMS (Method 1): Rt = 1.59 min, m / z (ES + )246.1[M+H] + .

[0218] Step 5: Preparation of compound 5

[0219] 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 sequentially to N,N-dimethylformamide (25 mL), and the reaction mixture was stirred at room temperature for 17 hours. The reaction mixture was poured into ice water (120 mL), followed by extraction with ethyl acetate (20 mL x 2). The combined organic phases were washed sequentially with saturated brine, 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 compound 5 (2.0 g) as a white solid.

[0220] LCMS (Method 3): Rt = 2.01 min, m / z (ES + )366.0[M+H] + .

[0221] Step 6: Preparation of compound 6

[0222] Azidoethanol (4.75 g, 54.6 mmol) was added to a solution of potassium tert-butoxide in tetrahydrofuran (KOtBu, 1 N, 54.6 mL), and the reaction mixture 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 mixture was stirred at 80°C for 2 hours. After the reaction mixture was cooled to room temperature, water (300 mL) was added, and then extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated brine (300 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain Compound 6 (8.5 g) as a yellow oil.

[0223] LCMS (Method 3): Rt = 2.06 min, m / z (ES + )373.1[M+H] + .

[0224] Step 7: Preparation of Compound 7

[0225] Compound 6 (54 g, 145 mmol) was dissolved in tetrahydrofuran (1000 mL), and triphenylphosphine (PPh3, 57.1 g, 218 mmol) and water (10 mL) were added sequentially 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 compound 7 (28.0 g) as a white solid.

[0226] LCMS (Method 3): Rt = 1.79 min, m / z (ES + )347.1[M+H] + .

[0227] Step 8: Preparation of Compound 8

[0228] Compound 7 (28 g, 145 mmol) and triethylamine (16.36 g, 162 mmol) were added sequentially to tetrahydrofuran (300 mL). The solution was then cooled to 0°C and methyloxycarbonyl chloride (FmocCl, 25.1 g, 97.2 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour and then filtered. The filtrate was concentrated under reduced pressure to yield a yellow oil. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 4)) to yield Compound 8 (25.0 g) as a yellow solid.

[0229] LCMS (Method 3): Rt = 2.21 min, m / z (ES + )569.1[M+H] + .

[0230] Step 9: Preparation of compound 9

[0231] 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 under ice-cooling. The reaction solution was stirred under ice-water cooling for 1 hour, and then saturated sodium bicarbonate solution (100 mL) was added to quench the reaction. The mixture 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 compound 9 (690 mg) as a yellow solid.

[0232] LCMS (Method 3): Rt = 1.67 min, m / z (ES + )405.1[M+H] + .

[0233] Step 10: Preparation of compound 11

[0234] 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 sequentially to acetonitrile (150 mL). The reaction mixture was stirred in the dark at room temperature for 17 hours. The reaction mixture was filtered, and the filter cake was washed with tetrahydrofuran (15 mL x 3). The combined filtrates were 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 compound 11 (800 mg) as a white solid.

[0235] LCMS (Method 3): Rt = 1.81 min, m / z (ES +)721.1[M+H] + .

[0236] Step 11: Preparation of compound 12

[0237] Compound 11 (800 mg, 1.11 mmol) was dissolved in a mixture of isopropanol (1 mL) and dichloromethane (5 mL). Sodium borohydride (NaBH4, 63 mg, 1.67 mmol) was then added under ice-water cooling. The reaction mixture was stirred under ice-water cooling for 1.5 hours, and then 25 mL of water was added. The mixture was extracted with dichloromethane (15 mL x 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 Compound 12 (310 mg) as a white solid.

[0238] LCMS (Method 1): Rt = 1.59 min, m / z (ES + )723.2[M+H] + .

[0239] Step 13: Preparation of compound KL-1

[0240] 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 sequentially to a dry N,N'-dimethylformamide solution (5 mL). The reaction solution was stirred at room temperature for 17 hours. Water (30 mL) was added to the reaction solution, followed by extraction with ethyl acetate (30 mL x 3). The combined organic phases were washed sequentially with saturated brine (30 mL x 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 Compound KL-1 (310 mg) as a white solid.

[0241] LCMS (Method 1): Rt = 2.27 min, m / z (ES + )888.2[M+H] + .

[0242] Example 2 Synthesis of Linker Intermediate KL-2

[0243] Step 1: Preparation of compound 13

[0244] 2-Nitro-3-hydroxypyridine (7 g, 50 mmol) was dissolved in 37% formaldehyde solution (100 mL). Aqueous potassium hydroxide (2.8 g, 50 mmol) was then added dropwise. The reaction mixture was stirred under reflux for 4 days. The reaction mixture was cooled to room temperature, and 1N hydrochloric acid solution (50 mL) was added dropwise. The resulting solution was extracted with ethyl acetate (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding a tan oily residue. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate (1 / 10 → 1 / 1)) to yield Compound 13 (2.0 g) as a yellow solid.

[0245] LCMS (Method 1): Rt = 1.18 min, m / z (ES + )171.1[M+H] + .

[0246] 1 H NMR (500MHz, CDCl3) δ10.20(s,1H),7.66-7.71(m,2H),4.80(s,2H),3.10(s,1H).

[0247] Step 2: Preparation of compound 14

[0248] Compound 13 (800 mg, 4.7 mmol) was dissolved in acetonitrile (50 mL). Compound 10 (1.5 g, 3.76 mmol) and silver oxide (2.17 g, 9.4 mmol) were added sequentially under ice-water cooling. The reaction mixture was stirred at room temperature for 1 hour. The mixed solution was filtered, and the filter cake was washed with acetonitrile (50 mL x 2). The combined filtrates were 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 compound 14 (400 mg) as a yellow solid.

[0249] LCMS (Method 3): Rt=1.72 min, m / z (ES+) 509.0 [M+Na] + .

[0250] 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).

[0251] Step 3: Preparation of compound 15

[0252] Compound 14 (520 mg, 1.02 mmol) was dissolved in ethyl acetate (100 ml), and 10% palladium on carbon (Pd / C, 52 mg) was added. The reaction mixture was stirred and hydrogenated overnight at room temperature. The reaction mixture was filtered to remove the palladium on carbon, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding Compound 15 (450 mg) as a yellow oil.

[0253] LCMS (Method 3): Rt = 1.64 min, m / z (ES + )457.1[M+H] + .

[0254] Step 4: Preparation of compound 16

[0255] Compound 15 (450 mg, 1 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of imidazole (102 mg, 1.5 mmol) and tert-butyldimethylsilyl chloride (TBSCl, 225 mg, 1.5 mmol). The reaction mixture was stirred at room temperature for 2 hours. The mixture 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 afford Compound 16 (350 mg) as a yellow solid.

[0256] LCMS (Method 3): Rt = 2.16 min, m / z (ES + )571.2[M+H] + .

[0257] Step 5: Preparation of compound 17

[0258] Fmoc-sarcosine (286 mg, 0.921 mmol) was dissolved in dichloromethane (10 ml), followed by the addition of thionyl chloride (367 mg, 1.84 mmol) and a trace amount of N,N-dimethylformamide (4.5 mg, 0.0614 mmol). The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed by concentration under reduced pressure, and dichloromethane (10 mL) was added to the residue. Compound 16 (350 mg, 0.614 mmol) and a dichloromethane solution of DIPEA (158 mg, 1.23 mmol) (10 mL) were then added. The resulting reaction mixture was stirred at room temperature overnight. The solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 10 → 1 / 1)) to afford Compound 17 (200 mg) as a yellow solid.

[0259] LCMS (method 1): Rt = 2.23 min, m / z (ES + )864.3[M+H] + .

[0260] Step 6: Preparation of compound 18

[0261] Compound 17 (190 mg, 0.22 mmol) was dissolved in tetrahydrofuran (6 mL), and glacial acetic acid (AcOH, 2 mL) and water (2 mL) were added thereto. 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 phases were 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 yellow solid compound 18 (100 mg). LCMS (Method 1): Rt=1.86 min, m / z (ES + )750.2[M+H] + .

[0262] Step 7: Preparation of compound KL-2

[0263] 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 sequentially to dichloromethane (4 mL). The reaction mixture was stirred at room temperature overnight. The solvent was removed by concentration under reduced pressure, 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.

[0264] LCMS (method 1): Rt = 2.08 min, m / z (ES + )915.2[M+H] + .

[0265] Example 3 Synthesis of Linker Intermediate KL-3

[0266] Step 1: Preparation of compound 19

[0267] 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 sequentially to N,N'-dimethylformamide (60 mL). The reaction mixture was stirred at room temperature for 16 hours. Water (300 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (150 mL x 3). The combined organic phases were washed sequentially with saturated brine (200 mL x 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 / 5)) to give Compound 19 (6.96 g) as an off-white solid.

[0268] LCMS (Method 3): Rt = 1.77 min, m / z (ES + )310.2(M+H) + .

[0269] Step 2: Preparation of compound 20

[0270] 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 sequentially to water (50 mL), and the reaction mixture was stirred at 90°C for 16 hours. The reaction mixture was cooled to room temperature, neutralized by adding glacial acetic acid, and then extracted with ethyl acetate (100 mL x 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 / petroleum ether (1 / 2)) to obtain Compound 20 (1.4 g) as an off-white solid.

[0271] LCMS (Method 3): Rt = 1.63 min, m / z (ES + )340.2(M+H) + .

[0272] Step 3: Preparation of compound 21

[0273] Compound 20 (339 mg, 1.0 mmol), compound 10 (397 mg, 1.0 mmol), and silver oxide (462 mg, 2.0 mmol) were added sequentially to acetonitrile (50 mL). The reaction mixture was stirred in the dark at 40°C for 16 hours. The reaction mixture was cooled to room temperature and filtered. The filter cake was washed with acetonitrile, and the combined filtrates were concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (Method 5: 40% to 95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 21 (120 mg) as a white solid.

[0274] LCMS (Method 3): Rt = 1.84 min, m / z (ES + )656.3(M+H) + .

[0275] Step 4: Preparation of compound KL-3

[0276] 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 sequentially to dichloromethane (3 mL) under ice-cooling conditions. The reaction solution was stirred at room temperature for 16 hours. Water (20 mL) was added to the reaction solution, followed by extraction with dichloromethane (30 mL x 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 / petroleum ether (1 / 10)) to obtain Compound KL-3 (100 mg) as an off-white solid.

[0277] LCMS (Method 3): Rt = 2.06 min, m / z (ES + )821.3(M+H) + .

[0278] Example 4 Synthesis of Linker Intermediate KL-4

[0279] Step 1: Preparation of compound 22

[0280] 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 sequentially to ethanol (120 mL). The reaction mixture was stirred at 85°C overnight. 10% sodium hydroxide solution was added to adjust the pH to neutral. The ethanol was removed by concentration under reduced pressure, and the mixture was 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 yield Compound 22 (11.50 g) as a light yellow solid.

[0281] LCMS (Method 3): Rt = 1.87 min, m / z (ES + )229.1(M+H) + .

[0282] Step 2: Preparation of compound 23

[0283] Compound 22 (12.5 g, 55 mmol) was dissolved in methanol (100 mL), and 10% palladium on carbon (1.0 g) was added. The reaction mixture was stirred at room temperature under hydrogenation conditions for 16 hours. The reaction mixture 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 Compound 23 (6.6 g) as a pink solid.

[0284] LCMS (Method 3): Rt=0.38 min, m / z (ES + )139.2(M+H) + .

[0285] Step 3: Preparation of compound 24

[0286] 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 sequentially to DMF (80 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into 300 mL of water and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed sequentially with saturated brine (300 mL x 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: dichloromethane / methanol (80 / 1 → 30 / 1)) to obtain compound 24 (7.8 g) as a pale yellow solid.

[0287] LCMS (Method 3): Rt = 1.75 min, m / z (ES + )310.1(M+H) + .

[0288] 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).

[0289] Step 4: Preparation of compound 25

[0290] 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 to adjust the pH to neutral, and then extracted with ethyl acetate (60 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: dichloromethane / methanol (50 / 1→20 / 1)) to obtain compound 25 (1.32 g) as an off-white solid.

[0291] LCMS (Method 3): Rt = 1.67 min, m / z (ES + )340.1(M+H) + .

[0292] Step 5: Preparation of compound 26

[0293] Compound 25 (1.32 g, 3.9 mmol) was dissolved in dichloromethane (25 mL), and active manganese dioxide (MnO2, 1.35 g, 15.6 mmol) was added. The reaction mixture was stirred at reflux for 16 hours. The reaction mixture 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 Compound 26 (0.86 g) as an off-white solid.

[0294] LCMS (Method 3): Rt = 1.55 min, m / z (ES + )338.1(M+H) + .

[0295] Step 6: Preparation of compound 27

[0296] Compound 26 (438 mg, 1.3 mmol), compound 10 (560 mg, 1.4 mmol), and silver oxide (534 mg, 2.3 mmol) were added sequentially to acetonitrile (8 mL). The reaction mixture was stirred in the dark at room temperature for 16 hours. The reaction mixture 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% to 95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 27 (112 mg) as a white solid.

[0297] LCMS (Method 3): Rt = 1.96 min, m / z (ES + )654.2(M+H) + .

[0298] Step 7: Preparation of compound 28

[0299] Compound 27 (112 mg, 0.17 mmol) was dissolved in a mixture of isopropanol (1 mL) and dichloromethane (2 mL). Sodium borohydride (10 mg, 0.25 mmol) was added under ice-cooling, and the reaction mixture was stirred at room temperature for 2 hours. 20 mL of water was added to the reaction mixture, followed by extraction with dichloromethane (20 mL x 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: dichloromethane / methanol (60 / 1)) to yield Compound 28 (100 mg) as an off-white solid.

[0300] LCMS (Method 3): Rt = 1.81 min, m / z (ES + )656.0(M+H) + .

[0301] Step 8: Preparation of compound KL-4

[0302] Compound 28 (92 mg, 0.14 mmol) and p-nitrophenyl chloroformate (56 mg, 0.28 mmol) were added to dichloromethane (4 mL). Triethylamine (43 mg, 0.42 mmol) was added under ice-cooling conditions, 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 compound KL-4 (41 mg) as an off-white solid.

[0303] LCMS (Method 3): Rt = 2.03 min, m / z (ES + )820.9(M+H) + .

[0304] Example 5 Synthesis of Linker Intermediate KL-5

[0305] Step 1: Preparation of compound 29

[0306] Compound 6 (4.5 g, 12 mmol) was dissolved in a mixture of dichloromethane (16 mL) and water (0.4 mL). Trifluoroacetic acid (8 mL) was then added under ice-cooling conditions, and the reaction mixture was stirred at room temperature for 2 hours. Saturated sodium bicarbonate solution (40 mL) was added to the reaction mixture to quench the reaction, followed by extraction with dichloromethane (50 mL x 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 afford compound 29 (1.64 g) as a colorless oil.

[0307] LCMS (Method 3): Rt=0.67 min, m / z (ES + )209.1(M+H) + .

[0308] Step 2: Preparation of compound 31

[0309] 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 in the dark at room temperature for 17 hours. The reaction solution was filtered, the filter cake was washed with tetrahydrofuran (15 mL × 3), and 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 give compound 31 (874 mg) as a colorless oil.

[0310] LCMS (Method 3): Rt = 1.89 min, m / z (ES + )539.1(M+H) + .

[0311] Step 3: Preparation of compound 32

[0312] Compound 31 (874 mg, 1.62 mmol) was dissolved in a mixture of isopropanol (10 mL) and dichloromethane (20 mL). Sodium borohydride (92 mg, 2.43 mmol) was then added under ice-water cooling. The reaction mixture was stirred under ice-water bath for 1 hour. Water (50 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding compound 32 (850 mg) as a colorless oil.

[0313] LCMS (Method 1): Rt = 1.63 min, m / z (ES + )541.2(M+H) + .

[0314] Step 4: Preparation of compound KL-5

[0315] Compound 32 (850 mg, 1.57 mmol) was dissolved in dry dichloromethane (10 mL), and p-nitrophenyl chloroformate (476 mg, 2.36 mmol) and pyridine (373 mg, 4.72 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 17 hours. The solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether (1 / 3 → 1 / 1)) to obtain compound KL-5 (950 mg) as a colorless oil.

[0316] LCMS (Method 1): Rt = 2.10 min, m / z (ES + )706.1(M+H) + .

[0317] Example 6 Synthesis of Linker-Drug LD-01

[0318] Step 1: Preparation of compound 33

[0319] 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 sequentially to dry N,N-dimethylformamide (2 mL). The reaction 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 afford Compound 33 (200 mg) as a white solid.

[0320] LCMS (Method 1): Rt = 1.98 min, m / z (ES +)1467.5(M+H) + .

[0321] Step 2: Preparation of compound 34

[0322] Compound 33 (200 mg, 0.136 mmol) was dissolved in methanol (10 mL), and then aqueous lithium hydroxide solution (1 M, 1.36 mL, 1.36 mmol) was added. The reaction solution was stirred at room temperature for 3 hours, then quenched by the addition of glacial acetic acid (150 mg). The solution was purified by prep-RP-HPLC (Method 4: 45%-95% B in 8 minutes → 95% B in 4 minutes) to give compound 34 (100 mg) as a white solid.

[0323] LCMS (method 2): Rt = 1.21 min, m / z (ES + )1104.4(M+H) + .

[0324] Step 3: Preparation of compound LD-01

[0325] 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 sequentially. 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 compound LD-01 (13.4 mg) as a white solid.

[0326] LCMS (method 1): Rt = 1.70 min, m / z (ES + )649.5[1 / 2(M+2H)] + .

[0327] Example 6 Synthesis of Linker-Drug LD-02

[0328] 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 sequentially. The reaction solution was stirred at room temperature for 2 hours, then quenched by the addition of glacial acetic acid (24 mg). The resulting solution was purified by prep-RP-HPLC (Method 4: 50% to 95% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-02 (50 mg) as a white solid.

[0329] LCMS (Method 2): Rt = 1.36 min, m / z (ES + )1378.4(M+H) + .

[0330] Example 7 Synthesis of Linker-Drug LD-03

[0331] Step 1: Preparation of compound 33

[0332] Compound 37 (100 mg, 0.225 mmol, synthesized according to the method disclosed in WO2016 / 192528) was dissolved in tetrahydrofuran, and N-hydroxysuccinimide (NHS, 39 mg, 0.34 mmol) and dicyclohexylcarbodiimide (DCC, 70 mg, 0.34 mmol) were added sequentially. 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 Compound 37 (60 mg) as a yellow solid.

[0333] Step 2: Preparation of compound LD-03

[0334] Compound 34 (20 mg, 0.018 mmol) was dissolved in N,N-dimethylformamide (2 mL), and compound 38 (15 mg, 0.027 mmol) and diisopropylethylamine (10 mg, 0.076 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 hours and then quenched by the addition of glacial acetic acid (24 mg). The resulting solution was purified by prep-RP-HPLC (Method 4: 30% to 95% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-03 (9.9 mg) as a white solid.

[0335] LCMS (method 1): Rt = 1.75 min, m / z (ES + )766.5[1 / 2(M+2H)] + .

[0336] Example 8 Synthesis of Linker-Drug LD-04

[0337] Step 1: Preparation of compound 39

[0338] To a solution of compound 33 (300 mg, 0.2 mmol) in tetrahydrofuran (30 mL) was added piperidine (0.35 g, 4.1 mmol), and the reaction was stirred at room temperature for 17 hours. The reaction was purified by prep-RP-HPLC (Method 4: 15%-85% B in 8 minutes → 95% B in 4 minutes) to afford compound 39 (200 mg) as a white solid.

[0339] LCMS (method 1): Rt=1.63 min, m / z (ES+) 623.0 [1 / 2 (M+2H)] + .

[0340] Step 2: Preparation of compound 40

[0341] Compound 39 (98 mg, 0.079 mmol), compound 40 (41 mg, 0.095 mmol, synthesized according to the method disclosed in CN107652219B), diisopropylethylamine (49 mg, 0.38 mmol), and HATU (60 mg, 0.098 mmol) were sequentially added to N,N-dimethylformamide (5 mL). 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 x 3). The combined organic phases were washed sequentially with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude yellow solid of compound 41 (120 mg).

[0342] LCMS (method 1): Rt=2.01 min, m / z (ES+) 829.6 [1 / 2 (M+2H)] + .

[0343] Step 3: Preparation of compound 42

[0344] Compound 41 (120 mg, 0.079 mmol) was added to a mixture of dioxane hydrochloride (4 M, 5 mL) and ethyl acetate (5 mL), and the resulting reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude yellow solid compound 42 (100 mg).

[0345] LCMS (method 1): Rt = 1.48 min, m / z (ES + )729.5[1 / 2(M+2H)] + .

[0346] Step 4: Preparation of compound 43

[0347] Compound 42 (100 mg, 0.069 mmol) was added to a mixture of methanol (1 mL) and water (2 mL), followed by the addition of lithium hydroxide monohydrate (LiOH·H2O, 84 mg, 2 mmol). The reaction mixture was stirred at room temperature for 2 hours, then quenched by the addition of glacial acetic acid (120 mg). The resulting solution was purified by prep-RP-HPLC (Method 4: 15%-85% B in 8 minutes → 95% B in 4 minutes) to afford compound 43 (14 mg) as a white solid.

[0348] LCMS (Method 1): Rt = 1.38 min, m / z (ES + )659.5[1 / 2(M+2H)] + .

[0349] Step 4: Preparation of compound LD-04

[0350] Compound 43 (14 mg, 0.010 mmol), compound 36 (8.2 mg, 21.2 μmol), and diisopropylethylamine (6 mg, 70.41 μmol) were added sequentially to dry N,N-dimethylformamide (1 mL). The resulting reaction solution was stirred at room temperature for 2 hours, then quenched by the addition of glacial acetic acid (10 mg). The reaction solution was purified by prep-RP-HPLC (Method 4: 15%-85% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-04 (14 mg) as a white solid.

[0351] LCMS (method 1): Rt = 1.64 min, m / z (ES + )933.6[1 / 2(M+2H)] + .

[0352] Example 9 Synthesis of Linker-Drug LD-05

[0353] Step 1: Preparation of compound 44

[0354] Compound KL-1 (500 mg, 0.564 mmol), HOBt (38 mg, 0.282 mmol), pyridine (2 mL), diisopropylethylamine (291 mg, 2.256 mmol), and exitecan (300 mg, 0.564 mmol, Haoyuan Pharmaceutical) were added sequentially to dry N,N-dimethylformamide (20 mL). The resulting reaction solution was stirred at room temperature for 17 hours. 150 mL of water was added to the reaction solution, followed by extraction with ethyl acetate (500 mL x 4). The combined organic phases were washed with saturated brine (600 mL x 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 compound 44 (1.74 g) as a brown solid.

[0355] LCMS (Method 1): Rt = 2.04 min, m / z (ES + )593.1[1 / 2(M+2H)] + .

[0356] Step 2: Preparation of compound 45

[0357] Compound 44 (400 mg, 0.338 mmol) was dissolved in methanol (80 mL). The resulting solution was cooled in an ice-water bath, and then aqueous lithium hydroxide solution (1 N, 13.5 mL, 13.5 mmol) was added. The reaction mixture was reacted at 0°C for 2 hours, and then quenched by adding glacial acetic acid (1 g). 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 afford Compound 45 (80 mg) as a yellow solid.

[0358] LCMS (method 1): Rt = 1.55 min, m / z (ES + )411.8[1 / 2(M+2H)] + .

[0359] Step 3: Preparation of compound LD-05

[0360] Compound 45 (40 mg, 0.0487 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and compound 35 (18 mg, 0.0585 mmol) was added. The reaction solution was cooled to 0°C, and diisopropylethylamine (25 mg, 0.195 mmol) was added. 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. The resulting mixture was purified by prep-RP-HPLC (Method 4: 30% to 90% B in 8 minutes → 95% B in 4 minutes) to obtain 13.7 mg of compound LD-05 as a yellow solid (13.7 mg).

[0361] LCMS (Method 1): Rt = 1.81 min, m / z (ES + )1015.4(M+H) + .

[0362] Example 10 Synthesis of Linker-Drug LD-06

[0363] Compound 45 (40 mg, 0.0487 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), followed by the addition of compound 36 (18 mg, 0.0585 mmol). The solution was cooled to 0°C, and diisopropylethylamine (25 mg, 0.195 mmol) was added. The reaction was stirred at room temperature for 4 hours. The reaction was quenched by the addition of glacial acetic acid (25 mg). The resulting solution was purified by prep-RP-HPLC (Method 4: 30% to 90% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-06 (9.4 mg) as a yellow solid.

[0364] LCMS (method 1): Rt = 1.81 min, m / z (ES + )1096.4(M+H) + .

[0365] Example 11 Synthesis of Linker-Drug LD-07

[0366] Step 1: Preparation of compound 46

[0367] Tert-butyl methyl (2-(methylamino)ethyl)carbamate (550 mg, 2.93 mmol) and triethylamine (325 mg, 3.22 mmol) were added sequentially to toluene (15 mL), and triphosgene (348 mg, 1.17 mmol) was then added under ice-bath cooling. The reaction solution was reacted under ice-bath cooling for 2 hours, then warmed to room temperature and continued to react for 16 hours. The reaction solution was filtered, and the resulting filtrate was concentrated to remove the solvent. Pyridine (15 mL) and SN-38 (918 mg, 2.34 mmol, Titan Technology) were then added sequentially thereto, 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 Compound 46 (670 mg) as a yellow solid.

[0368] LCMS (Method 3): Rt = 1.89 min, m / z (ES + )606.1(M+H) + .

[0369] Step 2: Preparation of compound 47

[0370] 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. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated to remove the solvent to obtain compound 47 (277 mg) as a yellow solid.

[0371] LCMS (Method 1): Rt = 1.41 min, m / z (ES + )506.2(M+H) + .

[0372] Step 3: Preparation of compound 48

[0373] 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 sequentially to anhydrous N,N-dimethylformamide (5 mL). The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, water (30 mL) was added, and the mixed solution was extracted with ethyl acetate (50 mL x 4). The combined organic phases were washed sequentially with saturated brine (60 mL x 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: methanol / dichloromethane (1 / 40 → 1 / 20)) to obtain Compound 48 (324 mg) as a yellow solid.

[0374] LCMS (Method 3): Rt = 1.98 min, m / z (ES+ )628.3[1 / 2(M+2H)] + .

[0375] Step 4: Preparation of compound 49

[0376] Compound 48 (324 mg, 0.26 mmol) was dissolved in a mixture of methanol (2 mL) and water (2 mL), and lithium hydroxide monohydrate (109 mg, 2.6 mmol) was added. The reaction 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 afford Compound 49 (52 mg) as a yellow solid.

[0377] LCMS (Method 1): Rt = 1.36 min, m / z (ES + )893.3(M+H) + .

[0378] Step 5: Preparation of compound LD-07

[0379] Compound 49 (30 mg, 0.034 mmol), compound 35 (9 mg, 0.034 mmol), and diisopropylethylamine (17 mg, 0.136 mmol) were added sequentially to anhydrous N,N-dimethylformamide (1 mL). The reaction was stirred at room temperature for 3 hours. Glacial acetic acid (20 mg) was added to quench the reaction. The resulting solution was purified by prep-RP-HPLC (Method 4: 30%-90% in 8 minutes → 95% in 4 minutes B) to afford compound LD-07 (16 mg) as a yellow solid.

[0380] LCMS (Method 1): Rt = 1.56 min, m / z (ES + )1087.4(M+H) + .

[0381] Example 12 Synthesis of Linker-Drug LD-08

[0382] 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 sequentially to anhydrous N,N-dimethylformamide (1 mL). The reaction was stirred at room temperature for 3 hours. Glacial acetic acid (20 mg) was added to quench the reaction. The resulting solution was purified by prep-RP-HPLC (Method 4: 35% to 90% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-08 (6.7 mg) as a yellow solid.

[0383] LCMS (Method 1): Rt = 1.74 min, m / z (ES + )584.3[1 / 2(M+2H)] + .

[0384] Example 13 Synthesis of Linker-Drug LD-09

[0385] Step 1: Preparation of compound 51

[0386] 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 sequentially to DMF (5 mL). The reaction was stirred at room temperature for 5 hours. The reaction was quenched by the addition of glacial acetic acid (20 mg) and purified by prep-RP-HPLC (Method 4: 30% to 95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 51 (30 mg) as a white solid.

[0387] LCMS (Method 2): Rt = 1.92 min, m / z (ES + )900.9[1 / 3(M+3H)] + .

[0388] Step 2: Preparation of compound 52

[0389] Compound 51 (30 mg, 0.011 mmol) was dissolved in ethyl acetate (0.8 mL), and 4N hydrochloric acid in dioxane (0.2 mL) was added. The reaction mixture was stirred at room temperature for 5 hours and concentrated under reduced pressure to remove the solvent to obtain compound 52 (20 mg) as a white solid.

[0390] LCMS (Method 2): Rt = 2.26 min, m / z (ES + )867.5[1 / 3(M+3H)] + .

[0391] Step 3: Preparation of compound 53

[0392] Compound 52 (20 mg, 0.0077 mmol) was dissolved in methanol (1 mL) and cooled in an ice bath. 1M aqueous lithium hydroxide solution (77 μL, 0.077 mmol) was added, and the reaction was stirred at room temperature for 3 hours. The reaction was quenched by the addition of glacial acetic acid (2 mg) and purified by prep-RP-HPLC (Method 4: 20% to 95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 53 (4 mg) as a white solid.

[0393] LCMS (Method 2): Rt = 2.04 min, m / z (ES + )744.0[1 / 3(M+3H)] + .

[0394] Step 4: Preparation of compound LD-09

[0395] Compound 53 (4 mg, 0.0017 mmol) was dissolved in DMF (0.5 mL), and compound 36 (0.8 mg, 0.002 mmol) was added. Diisopropylethylamine (0.88 mg, 0.0068 mmol) was then added under ice-cooling. The reaction was stirred at room temperature for 3 hours. Glacial acetic acid (2 mg) was added for quenching, and the product was purified by prep-RP-HPLC (Method 4: 35% to 95% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-09 (0.8 mg) as a white solid.

[0396] LCMS (Method 2): Rt = 2.17 min, m / z (ES + )865.2[1 / 3(M+3H)] + .

[0397] Example 14 Synthesis of Linker-Drug LD-10

[0398] Step 1: Preparation of compound 54

[0399] Compound KL-2 (30 mg, 0.033 mmol) was dissolved in dry N,N-dimethylformamide (0.8 mL), and 1-hydroxybenzotriazole (2.3 mg, 0.017 mmol), pyridine (0.2 mL), and MMAE (23.7 mg, 0.033 mmol) were added sequentially. The reaction mixture 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 afford Compound 54 (20 mg) as a white solid.

[0400] LCMS (method 1): Rt = 2.14 min, m / z (ES +)747.3[1 / 2(M+2H)] + .

[0401] Step 2: Preparation of compound 55

[0402] Compound 54 (20 mg, 0.0134 mmol) was dissolved in methanol (1 mL), and 1N aqueous lithium hydroxide solution (0.135 mL, 0.134 mmol) was added. The reaction mixture was stirred at room temperature for 5 hours, quenched by the addition of glacial acetic acid (0.1 mL), and purified by prep-RP-HPLC (Method 4: 50%-95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 55 (10 mg) as a white solid.

[0403] LCMS (Method 1): Rt = 1.47 min, m / z (ES + )566.4[1 / 2(M+2H)] + .

[0404] Step 3: Preparation of compound LD-10

[0405] Compound 55 (13 mg, 0.0115 mmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), and compound 32 (5.4 mg, 0.0138 mmol) and diisopropylethylamine (5.9 mg, 0.046 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 5 hours, quenched by the addition of glacial acetic acid (6 mg), and purified by prep-RP-HPLC (Method 4: 55%-95% B in 8 minutes → 95% B in 4 minutes) to obtain compound LD-10 (4 mg) as a white solid.

[0406] LCMS (Method 1): Rt = 1.73 min, m / z (ES + )703.3[1 / 2(M+2H)] + .

[0407] Example 15 Synthesis of Linker-Drug LD-11

[0408] Step 1: Preparation of compound 56

[0409] 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 sequentially to anhydrous N,N'-dimethylformamide (1 mL). The reaction was stirred at room temperature for 16 hours. After completion of the reaction, the product was purified by prep-RP-HPLC (Method 5: 30% to 95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 52 (25 mg) as a white solid.

[0410] LCMS (Method 1): Rt = 2.04 min, m / z (ES + )700.4[1 / 2(M+2H)] + .

[0411] Step 2: Preparation of compound 57

[0412] Compound 56 (25 mg, 0.0179 mmol) was dissolved in ethyl acetate (2 mL), and a 4 M hydrochloric acid solution in dioxane (2 mL, 8 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction, the solvent was removed by vacuum concentration to obtain Compound 57 (20 mg) as a yellow solid.

[0413] LCMS (Method 1): Rt = 1.53 min, m / z (ES + )650.5[1 / 2(M+2H)] + .

[0414] Step 3: Preparation of compound 58

[0415] Compound 57 (20 mg, 0.015 mmol) was dissolved in methanol (1 mL), and 1 M aqueous lithium hydroxide solution (0.15 mL, 0.15 mmol) was added. The reaction 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 afford Compound 58 (14 mg) as a white solid.

[0416] LCMS (Method 1): Rt = 1.42 min, m / z (ES + )1160.8(M+H) + .

[0417] Step 4: Preparation of compound LD-11

[0418] Compound 58 (14 mg, 0.0121 mmol) was dissolved in N,N'-dimethylformamide (1 mL), and compound 36 (5.6 mg, 0.0145 mmol) and diisopropylethylamine (9.4 mg, 0.0726 mmol) were added sequentially. The reaction was stirred at room temperature for 16 hours. The reaction was quenched by the addition of glacial acetic acid (10 mg). The resulting solution was purified by prep-RP-HPLC (Method 4: 30% to 95% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-11 (2.7 mg) as a white solid.

[0419] LCMS (Method 2): Rt = 1.78 min, m / z (ES + )717.4[1 / 2(M+2H)] + .

[0420] Example 16 Synthesis of Linker-Drug LD-12

[0421] Step 1: Preparation of compound 59

[0422] 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 sequentially to DMF (1 mL). The reaction was stirred at room temperature for 16 hours. After completion of the reaction, the product was purified by prep-RP-HPLC (Method 5: 30% to 95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 59 (50 mg) as a white solid.

[0423] LCMS (Method 3): Rt = 2.14 min, m / z (ES + )700.5[1 / 2(M+2H)] + .

[0424] Step 2: Preparation of compound 60

[0425] Compound 59 (50 mg, 0.04 mmol) was dissolved in ethyl acetate (1 mL). 4 M hydrochloric acid in dioxane (0.3 mL) was added under ice-cooling. The reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The residue was purified by prep-RP-HPLC (Method 4: 30% to 95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 60 (30 mg) as a white solid.

[0426] LCMS (Method 3): Rt = 1.53 min, m / z (ES + )650.6[1 / 2(M+2H)] +.

[0427] Step 3: Preparation of compound 61

[0428] 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 under ice-cooling. The reaction was stirred at room temperature for 2 hours. The reaction was quenched by the addition of glacial acetic acid (30 mg) and purified by prep-RP-HPLC (Method 4: 30%-95% B in 8 minutes → 95% B in 4 minutes) to afford Compound 61 (15 mg) as a white solid.

[0429] LCMS (Method 2): Rt = 1.43 min, m / z (ES + )1160.7(M+H) + .

[0430] Step 4: Preparation of compound LD-12

[0431] Compound 61 (15 mg, 0.01 mmol) was dissolved in N,N-dimethylformamide (0.9 mL). Compound 36 (6.2 mg, 0.02 mmol) and diisopropylethylamine (6.7 mg, 0.05 mmol) were added sequentially under ice-cooling. The reaction was stirred at room temperature for 16 hours. Acetic acid was added to quench the reaction and the product was purified by prep-RP-HPLC (Method 4: 30% to 95% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-12 (2 mg) as a white solid.

[0432] LCMS (Method 2): Rt = 1.68 min, m / z (ES + )717.5[1 / 2(M+2H)] + .

[0433] Example 17 Linker-Drugs LD-13, LD-14 and LD-15

[0434] 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).

[0435] Example 18 Synthesis of Linker-Drug LD-16

[0436] Step 1: Synthesis of compound 62

[0437] Compound KL-5 (197 mg, 0.28 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL). Pyridine (0.5 mL), 1-hydroxybenzotriazole (19 mg, 0.14 mmol), and MMAE (201 mg, 0.28 mmol) were then added sequentially. The reaction mixture was stirred at room temperature for 17 hours. Acetic acid was added to quench the reaction, followed by water and extraction with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated brine, 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), followed by dichloromethane / methanol (30 / 1)) to afford 62 (308 mg) as a colorless oil.

[0438] LCMS (Method 3): rt = 2.15 min; m / z (ES + )643.0[1 / 2(M+2H)] + .

[0439] Step 2: Synthesis of compound 63

[0440] Compound 62 (308 mg, 0.24 mmol) was dissolved in methanol (4.8 mL), and a 1 M aqueous lithium hydroxide solution (2.4 mL, 2.4 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Glacial acetic acid (144 mg) was added to the reaction mixture to quench the reaction. Water was then added, and the mixture was extracted with dichloromethane (30 mL x 2). The combined organic phases were washed with saturated brine, 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 afford compound 63 (240 mg) as a colorless gum.

[0441] LCMS (Method 3): rt = 1.91 min, m / z (ES + )1116.5(M+H) + .

[0442] Step 3: Synthesis of compound 64

[0443] Compound 63 (240 mg, 215 μmol) was dissolved in tetrahydrofuran (4 mL), and triphenylphosphine (67 mg, 258 μmol) and water (23 mg) were added with stirring. 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 provide Compound 64 (150 mg) as a white solid.

[0444] LCMS (method 1): rt = 1.45 min, m / z (ES + )1091.6(M+H) + .

[0445] Step 4: Synthesis of compound LD-16

[0446] 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 sequentially. The reaction was stirred at room temperature for 4 hours. Glacial acetic acid (20 mg) was added to the reaction solution to quench the reaction. The product was then purified by prep-RP-HPLC (Method 4: 15% to 95% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-16 (13.6 mg) as a white solid.

[0447] LCMS (Method 3): rt = 1.84 min, m / z (ES + )683.0[1 / 2(M+2H)] +2 .

[0448] Example 19 Synthesis of Linker-Drug LD-17

[0449] Compound 64 (27.2 mg, 25 μmol) was dissolved in N,N-dimethylformamide (1 mL), and compound 65 (25.3 mg, 32.5 μmol), HATU (19 mg, 50 μmol), and diisopropylethylamine (12.9 mg, 100 μmmol) were added sequentially. The reaction was stirred at room temperature for 2 hours. Glacial acetic acid (26 mg) was added to the reaction solution to quench the reaction. The mixture was purified by prep-RP-HPLC (Method 4: 20% to 95% B in 8 minutes → 95% B in 4 minutes) to afford compound LD-17 (27 mg) as a white solid.

[0450] LCMS (method 2): rt = 1.09 min, m / z (ES + )926.8[1 / 2(M+2H)] + .

[0451] Example 20 Preparation of Antibody Drug Conjugates

[0452] 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, 50,000 MWCO) or gel filtration exchange buffer (20 mM citric acid-trisodium citrate, 50 mM sodium chloride, 1 mM diethylenetriaminepentaacetic acid, pH 5.8-6.2), add dimethyl sulfoxide and linker-drug (dimethyl sulfoxide stock solution, 3-15 equivalents), ensuring that the dimethyl sulfoxide content of the reaction solution is approximately 10% by volume. The coupling reaction is carried out at 10°C for 1.5 hours.

[0453] Excess 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 50000 MWCO Ultra or gel filtration, and then sterilized through a 0.22 μm pore size filter (Merck Millex-GV Filter) to obtain the antibody drug conjugate, which was stored at 4°C.

[0454] Using the above preparation method, Ab1 and Ab2 were used as antibodies, and LD-01 to LD-17 were used as linker-drugs to prepare antibody-drug conjugates (see Table 1).

[0455] Table 1 Prepared Antibody Drug Conjugates

[0456] NA: Not prepared.

[0457] 1) Determination of average DAR value

[0458] The average DAR value is determined by one of the following two methods depending on the actual situation.

[0459] 1a) Hydrophobic interaction chromatography (HIC)

[0460] The weighted peak contribution (WPC) is calculated by combining the relative peak area percentages of each component on the HIC spectrum with the drug quantity carried by the corresponding component (see Anal. Chem. 2013, 85, 1699-1704). The WPC measures the contribution of a single DAR component to the average DAR value and is calculated by multiplying the relative peak area (%) by the drug quantity carried by that component. The average DAR value is calculated by summing the weighted peak contributions of all components and dividing by 100, as shown below:

[0461] DAR = Σ(relative peak area × amount of drug carried by the component) / 100

[0462] 1b) Mass spectrometry (MS)

[0463] An appropriate amount of sample was diluted to 1 mg / mL with 150 mM ammonium acetate solution. The experiment was performed using an Acquity UPLC (Waters) and a high-resolution Xevo G2-XS QTOF (Waters) mass spectrometer, with a 10 μL injection. Mass spectrometric data were acquired in positive ion mode. The native mass spectrometric data were analyzed and processed using UNIFI software.

[0464] The average DAR value measurement results of the antibody drug conjugates of the present invention are shown in Table 2 below.

[0465] Table 2 Average DAR values ​​of antibody drug conjugates

[0466] NA: Not prepared.

[0467] a: HIC method

[0468] b: MS method

[0469] 2) Hydrophobic interaction chromatography (HIC) analysis

[0470] Hydrophobic interaction chromatography was performed 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 linear, shifting 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] over 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.

[0471] The HIC spectra of the ADC of the present invention are shown in Figures 1A to 1K, and the retention times of the main peaks are shown in Tables 3 and 4.

[0472] Table 3 Main peak retention time of HIC spectrum based on Ab1 ADC

[0473] 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 glycosylpyridine linker of the present invention. Comparison of the main peak retention times of the HIC spectra shows that the peak elution times of the latter are significantly lower than those of the former.

[0474] Table 4 Main peak retention time of HIC spectrum based on Ab2 ADC

[0475] As shown in Table 4, when the average DAR value is approximately 4, the HIC main peak retention times of Ab2-LD-02 and Ab2-LD-16, antibody drug conjugates using glycosylpyridine linkers, are significantly shorter than those of Ab2-LD-14, an antibody using a vc-PABC linker. When the average DAR value is approximately 2, the HIC main peak retention times of Ab2-LD-04 and Ab2-LD-17, antibody drug conjugates using glycosylpyridine linkers, are significantly shorter than those of Ab2-LD-15, an antibody using a vc-PABC linker.

[0476] These results demonstrate that, compared to the commonly used vc-PABC linker, the use of a glycosylpyridine linker can significantly increase the hydrophilicity of ADC products. This increased hydrophilicity can reduce the aggregate content of ADC products and improve their in vivo PK properties.

[0477] 3) Size Exclusion Chromatography (SEC) Analysis

[0478] Size exclusion chromatography was performed 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 consisted of 62 mM disodium hydrogen phosphate, 55 mM potassium dihydrogen phosphate, and 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.

[0479] The monomer content of the ADC products in the embodiments of the present invention is above 99% as tested by SEC.

[0480] Experimental Example 1: Effect of the Antibody Drug Conjugate of the Present Invention on Inhibiting Cell Proliferation

[0481] Cell Proliferation Assay

[0482] The cytostatic activity of antibodies or antibody-drug conjugates was determined by the following method: mammalian cells expressing tumor-associated antigens or receptor proteins were seeded in 96-well plates, with 8,000 cells per well, and suspended in 200 μL of DMEM high-glucose cell culture medium (GIBCO); the ADC sample had an initial concentration of 2 μg / mL and was diluted 3-fold in DMEM high-glucose cell culture medium containing 2% FBS (GIBCO); the original culture medium was removed, and 200 μL of ADC sample was added to each well, and the cells were incubated for a further 72 hours; the original culture medium was removed, and 100 μL of CCK-8 colorimetric solution was added to each well, and the cells were incubated for a further 30 minutes; the absorbance values ​​at 450 nm / 630 nm were read using a microplate reader, and the curve was plotted to calculate the IC 50 .

[0483] The results of the cell proliferation inhibition effect of the antibody-drug conjugates of the present invention are shown in Tables 5 and 6.

[0484] Table 5 Results of the Antibody Drug Conjugates of the Present Invention Inhibiting Cell Proliferation

[0485] The antibody-drug conjugates prepared according to the present invention all exhibited significant cell proliferation inhibition. As shown in Table 5, Ab1-LD-04 exhibited slightly superior cell proliferation inhibition compared to Ab1-LD-15. The structural difference between the two lies primarily in the cleavable linker: the former utilizes the glycosylpyridine linker of the present invention, while the latter utilizes a vc-PABC linker.

[0486] Table 6 Results of the Antibody Drug Conjugates of the Present Invention Inhibiting Cell Proliferation

[0487] As shown in Table 6, when the average DAR values ​​are similar, the antibody drug conjugate using a glycosylpyridine type linker has a comparable cell proliferation inhibitory effect to that of the antibody drug conjugate using a vc-PABC type linker.

[0488] 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.

[0489] All documents mentioned herein are incorporated by reference in this application as if each were individually incorporated by reference. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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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