GSPT1 degrading agent, antibody conjugate containing GSPT1 degrading agent, and use thereof

Abstract

Provided are a GSPT1 degrading agent (a domide molecular glue derivative) having an anti-tumor effect and an antibody-drug conjugate thereof, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopic marker thereof, use thereof, and a preparation method therefor.

Description

GSPT1 degraders and antibody conjugates containing gspt1 degraders and uses thereof

[0001] CROSS-REFERENCE

[0002] The present application claims priority to the prior application filed with the China National Intellectual Property Office on August 8, 2024, with the patent application number CN202411085670.8, and the title of the invention being “GSPT1 degraders and antibody conjugates containing GSPT1 degraders and uses thereof”. The entire contents of the prior application are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the technical field of medicine, and specifically relates to GSPT1 degraders (dexamine molecular glue derivatives) with anti-tumor effects and antibody conjugate drugs thereof. BACKGROUND

[0004] Dexamine compounds have been used as clinical therapeutic drugs for immunomodulation and anti-tumor fields for many years. For example, thalidomide has been used for a long time for leprosy and various skin diseases, such as discoid lupus erythematosus, subacute cutaneous lupus erythematosus, Behcet's syndrome, etc. Pomalidomide and lenalidomide are commonly used for the treatment of multiple myeloma. In recent years, studies have shown that the mechanism of action of this type of dexamine molecule is similar to “molecular glue”, which achieves therapeutic effects by degrading the corresponding target protein. The existing dexamine small molecules on the market mainly have problems such as high toxicity and unsatisfactory efficacy. For example, thalidomide is the culprit of the famous “reaction stop event”. More and more studies are being conducted on the structural modification of this type of dexamine compound, such as CC-885, CC-90009, CC-220, CC-92480, CC-99282 developed by BMS, DKY709 developed by Novartis, MRT-2359 developed by Monte Rosa Therapeutics, BTX-1188, BTX-306 developed by Biotherys, and CFT7455 developed by C4 Therapeutics.

[0005] Among them, the most advanced CC-885 was terminated due to toxicity, and then Orum Therapeutics company carried out structural modification on CC-885 to obtain Smol006, and combined with antibody conjugated delivery technology, the antibody conjugated drugs ORM-5029 and ORM-6151 showed very good anti-tumor activity and good safety. However, we found in the research process that the activity of Smol006 is only at a medium level, and its activity as an antibody conjugated drug is far from enough for tumors with low receptor expression, so it is urgent to develop compounds with better activity. The purpose of the present application is to solve the above problems, and to obtain molecules with higher activity based on the structural modification of Smol006 for the development of antibody conjugated drugs. SUMMARY

[0006] The present application relates to antibody conjugated drugs (ADC) represented by formula I and intermediates thereof, preparation methods and uses.

[0007] To this end, in a first aspect, the present application provides an antibody conjugated drug represented by formula (I),

[0008] or a stereoisomer of the antibody conjugated drug, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof or an isotopically labeled material thereof,

[0009] wherein,

[0010] Ab is a ligand unit selected from an antibody (such as a monoclonal antibody) or an antigen binding fragment, a small molecule ligand, a polypeptide;

[0011] L is a linker unit, one end of which is connected to the ligand unit Ab, and the other end of which is connected to the bioactive molecule unit D'; preferably, L is a linker unit comprising a hydrophilic segment; further preferably, L is a linker unit comprising a hydrophilic segment in a self-cleavable segment;

[0012] D' is a bioactive molecule unit, and the structure is as follows:

[0013] wherein, * represents covalent connection with the linker unit L;

[0014] W 1 and W 2 are each independently selected from methylene and carbonyl, and W 1 and W 2 are not both methylene;

[0015] R 1 is selected from hydrogen, deuterium, halogen, cyano, amino, nitro, C1-C4 alkoxy or C1-C4 alkyl;

[0016] each R2 , R 3 , R 4 , R 5 each independently is selected from hydrogen, deuterium, C1-C4alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocyclyl, each n1, n2is independently selected from any integer between 0-6 (such as 0, 1, 2, 3, 4, 5, 6);

[0017] W 3 is absent or is selected from O, S, or NR a , wherein R a is selected from hydrogen, C1-C4alkyl, or 3-6 membered cycloalkyl;

[0018] each n3, n4is independently selected from any integer between 1-3 (such as 1, 2, 3);

[0019] each R 6 , R 7 , R 8 , R 9 is independently selected from hydrogen, deuterium, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, 3-8 membered cycloalkyl, halogen, hydroxyl, C1-C6deuteroalkyl, C1-C6hydroxyalkyl; or, R 6 and R 7 together with the carbon atom to which they are attached form a 3-6 membered cycloalkylene; or R 6 and R 8 together with the carbon atom to which they are attached form a 4-6 membered cycloalkylene;

[0020] each n5, n6is independently selected from any integer between 0-3 (such as 0, 1, 2, 3), and n5, n6are not simultaneously 0;

[0021] m represents the molar ratio of the bioactive molecule unit to the ligand unit Ab (also known as DAR, i.e., drug antibody coupling ratio);

[0022] m is 1 to 12 (the value is an integer or a decimal number (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)).

[0023] In some embodiments, the linker unit -L- is represented by the following formula:

[0024] -L1-L2-L3-L4-,

[0025] wherein L1is selected from or wherein * represents the connection to the thiol group of Ab (such as a monoclonal antibody), and ** represents the connection to L2.

[0026] In some embodiments, L2is a spacer selected from -L 2a-C(O)-, -L 2a -NR b -C(O)-L 2b -C(O)-, wherein L 2a selected from -Ci-C8alkylene-, -Ci-C8alkylene-C3-C8cycloalkylene-, -C6-C 14 arylene-, -C6-C 14 arylene-Ci-C8alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-Ci-C8alkylene-, straight chain or branched chain heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, straight chain or branched chain heteroalkylene-3-8 membered heterocyclyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, each of said alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene, heteroalkyl, heteroaryl, heterocyclyl being optionally substituted with one or more substituents independently selected from Ci-C6alkyl, heteroalkyl of 1-6 carbon atoms, Ci-C6alkoxy, hydroxyl, amino, carboxyl, or C3-C8cycloalkyl, said heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more (such as two or three) of N, O, or S;

[0027] L 2bstraight or branched heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, R b selected from the group consisting of hydrogen, C1-C6alkyl, C3-C8cycloalkyl, 3-8 membered heterocyclyl, C1-C6haloalkyl, heteroalkyl of 2-8 carbon atoms, C6-C10aryl, 5-6 membered heteroaryl, each of said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl optionally substituted with one or more substituents independently selected from the group consisting of C1-C6alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6alkoxy, amino, or carboxyl, said heteroalkyl, heterocyclyl, heteroaryl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more (such as two or three) of N, O, or S; 14 selected from the group consisting of hydrogen, C1-C6alkyl, C3-C8cycloalkyl, 3-8 membered heterocyclyl, C1-C6haloalkyl, heteroalkyl of 2-8 carbon atoms, C6-C10aryl, 5-6 membered heteroaryl, each of said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl optionally substituted with one or more substituents independently selected from the group consisting of C1-C6alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6alkoxy, amino, or carboxyl, said heteroalkyl, heterocyclyl, heteroaryl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more (such as two or three) of N, O, or S;

[0028] L3 is a polypeptide sequence selected from a peptide residue consisting of 2-8 (such as 2, 3, 4, 5, 6, 7, 8) natural or non-natural amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from the group consisting of C1-C6alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6alkoxy, hydroxyl, amino, carboxyl, or C3-C8cycloalkyl;

[0029] L4 is a self-cleaving fragment selected from:

[0030] or

[0031] wherein * indicates attachment to the carbonyl of L3 to form an amide bond, and ** indicates attachment to the oxygen atom of D’; R ca straight or branched heteroalkyl comprising 1-50 (preferably 4-50, more preferably 4-24, even more preferably 8-24, most preferably 10-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) -OCH2CH2- structural units, a peptide chain comprising 4-50 (preferably 4-24, more preferably 8-24, most preferably 10-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) natural or non-natural amino acids, a straight or branched heteroalkyl comprising a monosaccharide, an oligosaccharide or a polysaccharide;

[0032] X is absent, -N(R d )CH2- or -L 4a -;

[0033] wherein R d is selected from hydrogen, C1-C3 alkyl, 3-6 membered cycloalkyl, a straight or branched heteroalkyl comprising 4-50 (preferably 4-24, more preferably 6-24, most preferably 8-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) -OCH2CH2- or -CH2CH2SO2- structural units;

[0034] -L 4a - structure is R e and R f are each independently selected from H, C1-C3 alkyl, or R e and R f together with the atom to which they are attached form a 5-6 membered heterocyclyl; R gH, C1-C3 alkyl, 3-6 membered cycloalkyl, heteroalkyl of 2-6 carbon atoms; the heterocyclyl contains 1-6 (preferably 1-4, such as 1, 2, 3, 4) heteroatoms, wherein the heteroatoms are selected from one or more (such as two or three) of N, O, or S;

[0035] Y is C1-C6 alkylene or -Z-C(O)-, wherein Z is C1-C6 alkylene or heteroalkylene containing 1-24 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) -OCH2CH2- structural units;

[0036] n7 is an integer from 0 to 6 (such as 0, 1, 2, 3, 4, 5, 6).

[0037] R c selected from a hydrophilic segment; in some embodiments, R c selected from the following structures:

[0038] wherein r, s are each independently selected from an integer from 1-50 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0039] in some embodiments, R c selected from the following structures:

[0040] in some embodiments, Y is C1-C6 alkylene or -Z-C(O)-, wherein Z is C1-C6 alkylene or -C1-C6 alkylene-(OCH2CH2) v wherein v is selected from an integer from 1-12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0041] in some embodiments, Y is C1-C3 alkylene or -Z-C(O)-, wherein Z is C1-C3 alkylene or -C1-C3 alkylene-(OCH2CH2) v wherein v is selected from an integer from 1-12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0042] in some embodiments, Y is methylene, ethylene, n-propylene, i-propylene, or -Z-C(O)-, wherein Z is methylene-(OCH2CH2)v - ethylene-(OCH2CH2) v - n-propylene-(OCH2CH2) v - isopropylene-(OCH2CH2) v - wherein v is selected from an integer from 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0043] In some embodiments, Y is selected from -CH2-(OCH2CH2)4C(O)-.

[0044] In some embodiments, L2is selected from -L 2a - C(O)- or -L 2a - NR b - C(O)-L 2b - C(O)-, wherein L 2aselected from the group consisting of optionally substituted: methylene, ethylene, n- propylene, n-butylene, n-pentylene, n-hexylene, -Ci-C3alkylene-cyclohexanediyl-, - phenylene-, -phenylene-Ci-C3alkylene-, -5-6 membered heteroarylene-Ci-C3alkylene-, straight or branched heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, straight or branched heteroalkylene-3-6 membered heterocyclyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, said optional substitution means optionally substituted with one or more substituents independently selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, heteroalkyl of 1-3 carbon atoms, methoxy, ethoxy, n-propoxy, i-propoxy, hydroxyl, amino, carboxyl, or cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, said heteroalkylene, heterocyclyl, heteroalkyl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroatoms of the heteroalkylene, heterocyclyl, heteroarylene, heteroalkyl being selected from one or more of N, O, or S;

[0045] L 2b selected from the group consisting of optionally substituted: methylene, ethylene, n- propylene, n-butylene, n-pentylene, n-hexylene, -Ci-C3alkylene-cyclohexanediyl-, - phenylene-, -phenylene-Ci-C3alkylene-, -5-6 membered heteroarylene-Ci-C3alkylene-, straight or branched heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, straight or branched heteroalkylene-3-6 membered heterocyclyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, said optional substitution means optionally substituted with one or more substituents independently selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, heteroalkyl of 1-3 carbon atoms, methoxy, ethoxy, n-propoxy, i-propoxy, hydroxyl, amino, carboxyl, or cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, said heteroalkylene, heterocyclyl, heteroalkyl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroatoms of the heteroalkylene, heterocyclyl, heteroarylene, heteroalkyl being selected from one or more of N, O, or S; bselected from the group consisting of hydrogen, optionally substituted: methyl, ethyl, n-propyl, i-propyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 3-6 membered heterocyclyl, -CF3, -CF2CF3, heteroalkyl of 2-3 carbon atoms, phenyl, 5-6 membered heteroaryl, said optional substitution means each of the aforementioned substituents is optionally substituted with one or more substituents independently selected from methyl, ethyl, n-propyl, i-propyl, heteroalkyl of 2-3 carbon atoms, methoxy, ethoxy, n-propoxy, i-propoxy, amino or carboxyl, said heteroalkyl, heterocyclyl, heteroaryl, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroatoms of the heteroalkyl, heterocyclyl, heteroaryl are selected from one or more of N, O or S.

[0046] In some embodiments, L 2a selected from the group consisting of -C1-C8alkylene-, -C1-C8alkylene-C3-C8cycloalkylene, -C6-C 14 arylene-, -C6-C 14 arylene-C1-C8alkylene, straight chain or branched heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms, each of the alkylene, cycloalkylene, arylene, heteroalkylene is optionally substituted with one or more substituents independently selected from C1-C6alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6alkoxy, hydroxyl, amino, carboxyl or 3-8 membered cycloalkyl, said heteroalkylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroatoms of the heteroalkylene, heteroalkyl are selected from one or more of N, O or S; L 2b selected from the group consisting of -C1-C8alkylene-, straight chain or branched heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms, R b selected from the group consisting of hydrogen, C1-C6alkyl, heteroalkyl of 2-8 carbon atoms, C6-C 14 aryl, each of the alkyl, heteroalkyl, aryl is optionally substituted with one or more substituents independently selected from C1-C6alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6alkoxy, amino or carboxyl, said heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroatoms of the heteroalkyl, heteroalkylene are selected from one or more of N, O or S.

[0047] In some embodiments, L 2a selected from the group consisting of -(CH2)5-, -CH2CH2(OCH2CH2) p -, wherein p is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); L 2b -CH2CH2(OCH2CH2) q -CH2(OCH2CH2) q wherein q is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0048] In some embodiments, L2 is selected from the following structures:

[0049] * indicates attachment to L1, and ** indicates attachment to L3.

[0050] In some embodiments, L3 is selected from a peptide residue formed from 2-8 amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), C1-C6 alkyl substituted lysine (such as C1-C3 alkyl substituted lysine, such as methyl substituted lysine, ethyl substituted lysine, n-propyl substituted lysine, iso-propyl substituted lysine), and the like.

[0051] In some embodiments, L3 is selected from the following structures:

[0052] wherein * indicates attachment to L2, and ** indicates attachment to L4.

[0053] In some embodiments, L4 is selected from:

[0054] wherein * indicates attachment to L3, and ** indicates attachment to D'; R c , R e , R f , R g are as defined in any of the above, and v is selected from an integer from 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0055] In some embodiments, L4 is selected from:

[0056] wherein R cis defined as any one of the above, * indicates the connection to L3, and ** indicates the connection to D'.

[0057] In some embodiments, L4is preferably selected from:

[0058] wherein r is selected from an integer from 1 to 50 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), * indicates the connection to L3, and ** indicates the connection to D'.

[0059] In some embodiments, L is preferably selected from the following structures:

[0060] wherein r is defined as any one of the above, * indicates the connection to the thiol group of the ligand unit Ab (such as an antibody), and ** indicates the connection to the biologically active molecule D'.

[0061] In some embodiments, L is preferably selected from the following structures:

[0062] wherein * indicates the connection to the thiol group of the ligand unit Ab (such as an antibody), and ** indicates the connection to the biologically active molecule D'.

[0063] In some embodiments, W 1 and W 2 are each independently selected from methylene and carbonyl, and W 1 and W 2 are not simultaneously methylene; R 1 is selected from hydrogen, deuterium, halogen, cyano, amino, nitro, C1-C2alkoxy, or C1-C2alkyl; each R 2 , R 3 , R 4 , R 5 is independently selected from hydrogen, C1-C4alkyl, and n1, n2 are each independently selected from any integer from 0 to 3 (such as 0, 1, 2, 3); W 3 is absent or selected from O or NR a , wherein R a is selected from hydrogen, C1-C4alkyl, or 3-6 membered cycloalkyl; n3, n4 are each independently selected from any integer from 1 to 3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9each independently selected from the group consisting of hydrogen, deuterium, C1-C4 haloalkyl, C1-C4 alkyl, 3-5 membered cycloalkyl; or R 6 and R 7 together with the carbon atom to which they are attached form a 3-4 membered cycloalkylene; or R 6 and R 8 together with the carbon atom to which they are attached form a 4-6 membered cycloalkylene, wherein n5, n6are each independently selected from any integer between 0-2 (such as 0, 1, 2), and n5, n6are not simultaneously 0.

[0064] In some embodiments, W 1 is carbonyl, W 2 is methylene; R 1 is selected from the group consisting of halogen; each R 2 , R 3 , R 4 , R 5 is selected from the group consisting of hydrogen, methyl, n1, n2are each independently selected from any integer between 0-3 (such as 0, 1, 2, 3); W 3 is absent or selected from O or NR a , wherein R a is selected from the group consisting of hydrogen, C1-C2 alkyl or 3-4 membered cycloalkyl; n3, n4are each independently selected from any integer between 1-3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9 is selected from the group consisting of hydrogen, C1-C4 haloalkyl, C1-C4 alkyl, 3-5 membered cycloalkyl; or R 6 and R 7 together with the carbon atom to which they are attached form a 3-4 membered cycloalkylene; or R 6 and R 8 together with the carbon atom to which they are attached form a 4-6 membered cycloalkylene, wherein n5, n6are each independently selected from any integer between 0-2 (such as 0, 1, 2), and n5, n6are not simultaneously 0.

[0065] In some embodiments, W 1 is carbonyl, W 2 is methylene; R 1 is selected from the group consisting of fluorine, chlorine, bromine; each R 2 , R 3 , R 4 , R 5 is selected from the group consisting of hydrogen, n1, n2are each independently selected from any integer between 0-3 (such as 0, 1, 2, 3); W 3 is absent or selected from O or NR a , wherein R aselected from hydrogen, methyl, ethyl or cyclopropyl, cyclobutyl; each of n3, n4 is independently selected from any integer between 1 and 3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9 is independently selected from hydrogen, trifluoromethyl, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, cyclopropanyl, cyclobutanyl, cyclopentanyl, or R 6 and R 7 together with the carbon atom to which they are attached form a cyclopropanediyl or cyclobutylidene group, or R 6 and R 8 together with the carbon atom to which they are attached form a cyclobutylidene, cyclohexylidene group, wherein each of n5, n6 is independently selected from any integer between 0 and 2 (such as 0, 1, 2), and n5, n6 are not simultaneously 0.

[0066] In some embodiments, W 1 is carbonyl, W 2 is methylene; R 1 is selected from chlorine; each R 2 , R 3 , R 4 , R 5 is independently selected from hydrogen; each of n1, n2 is independently selected from any integer between 0 and 3 (such as 0, 1, 2, 3); W 3 is absent or selected from O; each of n3, n4 is independently selected from any integer between 1 and 3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9 is independently selected from hydrogen, trifluoromethyl, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, cyclopropanyl, cyclobutanyl, cyclopentanyl, or R 6 and R 7 together with the carbon atom to which they are attached form a cyclopropanediyl group, or R 6 and R 8 together with the carbon atom to which they are attached form a cyclobutylidene, cyclohexylidene group, wherein each of n5, n6 is independently selected from any integer between 0 and 2 (such as 0, 1, 2), and n5, n6 are not simultaneously 0.

[0067] In some embodiments, W 1 is carbonyl, W 2 is methylene; R 1 is selected from chlorine, R 2 , R 3 is independently selected from hydrogen; n1 is selected from 3, n2 is selected from 0; W 3 is selected from O; each of n3, n4 is independently selected from 1 or 2; each R6 R 7 R 8 R 9 each independently selected from hydrogen, trifluoromethyl, methyl, cyclopropanesulfonyl, or R 6 and R 7 together with the carbon atom to which they are attached form a cyclopropanediyl group, or R 6 and R 8 together with the carbon atom to which they are attached form a cyclobutanediyl group, a cyclohexanediyl group, wherein each of n5, n6 is independently selected from any integer from 0 to 2 (e.g., 0, 1, 2), and n5, n6 are not simultaneously 0.

[0068] In some embodiments, the present application provides an antibody drug conjugate of Formula I, selected from the following structures:

[0069] wherein Ab is a Ligand Unit; m represents the molar ratio of the Bioactive Molecule Unit to the Ligand Unit Ab (also referred to as DAR, i.e., Drug to Antibody Ratio), which is from 1 to 12 (the value is an integer or a decimal, e.g., an integer or a decimal from 1 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; when m is a decimal, it represents the average molar ratio of the Bioactive Molecule Unit to the Ligand Unit Ab).

[0070] In some embodiments, Ab is selected from a mAb, a diabody, or an antigen binding fragment thereof that targets HER2 (ErbB2), CD33, or BCMA.

[0071] In some embodiments, Ab is modifiable, e.g., one or more amino acid changes, additions, or deletions; in some embodiments, one or more amino acids of Ab comprise a glycosyl group; preferably, the Bioactive Molecule Unit is introduced via the glycosyl group.

[0072] In some embodiments, Ab is a mAb, a diabody, or an antigen binding fragment thereof that targets HER2, comprising a HER2-targeting heavy chain variable region and a light chain variable region, each comprising 3 CDRs,

[0073] the heavy chain variable region comprises:

[0074] CDR1-H having the sequence as set forth in SEQ ID NO: 1 or a functional variant thereof;

[0075] CDR2-H having a sequence as set forth in SEQ ID NO: 2, or a functional variant thereof; and

[0076] CDR3-H having a sequence as set forth in SEQ ID NO: 3, or a functional variant thereof;

[0077] the heavy chain variable region comprises:

[0078] CDR1-H having a sequence as set forth in SEQ ID NO: 7, or a functional variant thereof;

[0079] CDR2-H having a sequence as set forth in SEQ ID NO: 8, or a functional variant thereof; and

[0080] CDR3-H having a sequence as set forth in SEQ ID NO: 9, or a functional variant thereof;

[0081] In some embodiments, the Ab is a mAb, a diabody, or an antigen binding fragment thereof targeting HER2, comprising a heavy chain targeting HER2 and a light chain, the heavy chain being a sequence as set forth in SEQ ID NO: 13 or comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; and / or the light chain being a sequence as set forth in SEQ ID NO: 14 or comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0082] In some embodiments, the Ab is a mAb, a diabody, or an antigen binding fragment thereof targeting CD33, comprising a heavy chain variable region and a light chain variable region targeting CD33, the heavy chain variable region and the light chain variable region each comprising 3 CDRs,

[0083] the heavy chain variable region comprises:

[0084] CDR1-H having a sequence as set forth in SEQ ID NO: 7, or a functional variant thereof;

[0085] CDR2-H having a sequence as set forth in SEQ ID NO: 8, or a functional variant thereof; and

[0086] CDR3-H having a sequence as set forth in SEQ ID NO: 9, or a functional variant thereof;

[0087] the heavy chain variable region comprises:

[0088] CDR1-H having a sequence as set forth in SEQ ID NO: 7, or a functional variant thereof;

[0089] CDR2-L, which has the sequence as set forth in SEQ ID NO: 11 or a functional variant thereof; and

[0090] CDR3-L, which has the sequence as set forth in SEQ ID NO: 12 or a functional variant thereof.

[0091] In some embodiments, Ab is a CD33-targeting mAb, diabody or antigen-binding fragment thereof comprising a CD33-targeting heavy chain that is a sequence as set forth in SEQ ID NO: 15 or comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; and / or a CD33-targeting light chain that is a sequence as set forth in SEQ ID NO: 14 or comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0092] In a second aspect, the present application provides a bioactive molecule (a duocarmycin compound) D represented by formula (II):

[0093] or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material thereof;

[0094] wherein, W 1 , W 2 , W 3 , R 1 -R 9 , n1-n6 are as described above for the compound of formula I.

[0095] In some embodiments, the duocarmycin compound D is selected from the following structural specific structures as follows:

[0096] In a third aspect, the present application provides a linker-bioactive molecule represented by formula (III) or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material thereof:

[0097] L1’-L2-L3-L4-D’ (III)

[0098] wherein L1' is a linker unit precursor that can be converted to L1 of Formula I, thereby forming a covalent bond to Ab of Formula I;

[0099] L2, L3, L4, D' are defined as in Formula I.

[0100] In some embodiments, the linker unit precursor L1' is selected from or

[0101] In some embodiments, the Linker-Bioactive Molecule of Formula (III) is selected from the following structures:

[0102] In a fourth aspect, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate as previously described, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material of the antibody-drug conjugate; or a bioactive molecule unit as previously described, or a stereoisomer of the bioactive molecule unit, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material of the bioactive molecule unit; or a Linker-Bioactive Molecule as previously described, or a stereoisomer of the Linker-Bioactive Molecule, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material of the Linker-Bioactive Molecule; and optionally one or more pharmaceutically acceptable excipients.

[0103] In a fifth aspect, the present application provides use of an antibody-drug conjugate as previously described, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material of the antibody-drug conjugate; or a bioactive molecule unit as previously described, or a stereoisomer of the bioactive molecule unit, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material of the bioactive molecule unit; or a Linker-Bioactive Molecule as previously described, or a stereoisomer of the Linker-Bioactive Molecule, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled material of the Linker-Bioactive Molecule; or a pharmaceutical composition as previously described, in the manufacture of a medicament for the treatment and / or prevention of a cell proliferative disease (e.g., cancer).

[0104] In some embodiments, the present application provides an antibody drug conjugate as previously described, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or an isotopically-labeled material of the antibody drug conjugate; or a bioactive molecule unit as previously described, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or an isotopically-labeled material of the bioactive molecule unit; or a linker-bioactive molecule as previously described, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or an isotopically-labeled material of the linker-bioactive molecule; or a pharmaceutical composition as previously described, for use in the treatment and / or prevention of a cell proliferative disease (e.g., cancer).

[0105] In some embodiments, the present application provides a method of preventing and / or treating a cell proliferative disease (e.g., a cancer disease), comprising: administering to an individual in need thereof a prophylactically and / or therapeutically effective amount of an antibody drug conjugate as previously described, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or an isotopically-labeled material of the antibody drug conjugate; or a bioactive molecule unit as previously described, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or an isotopically-labeled material of the bioactive molecule unit; or a linker-bioactive molecule as previously described, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or an isotopically-labeled material of the linker-bioactive molecule; or a pharmaceutical composition as previously described.

[0106] In some embodiments, the cancer is selected from a solid tumor or a hematological tumor.

[0107] In some embodiments, the cancer comprises a liver cancer, a kidney cancer, a lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), a stomach cancer, an esophageal cancer, a urethral cancer, a bladder cancer, a colon cancer, a rectal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a pancreatic cancer, a melanoma, a hematological tumor or glioblastoma multiforme, a lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, or relapsed anaplastic large cell lymphoma), a cervical cancer, a uterine cancer, an endometrial cancer, a salivary gland cancer, a glioma, a neuroblastoma, a sarcoma, a colorectal cancer, a leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia, acute monocytic leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), a bone cancer, a skin cancer, a thyroid cancer, etc.

[0108] In a sixth aspect, the present application provides a method of preparing an antibody drug conjugate as previously described, or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or an isotopically-labeled material of the antibody drug conjugate.

[0109] including but not limited to the following methods:

[0110] General preparation method one:

[0111] D+L3'-L4'-protecting group L3'-L4'-D'+L1'-L2'L1'-L2-L3-L4'-D'L1'-L2-L3-L4-D'

[0112] wherein L3' is L3-protecting group, L2' is L2-protecting group, L1', L2, L3, L4, D' and D are as described in any preceding technical solution;

[0113] L4' is selected from:

[0114] or X is as described in any preceding technical solution;

[0115] Preferably:

[0116] when L4' is selected from , the step L1'-L2-L3-L4'-D'→L1'-L2-L3-L4-D' is absent;

[0117] when L4' is selected from or , the step L1'-L2-L3-L4'-D'→L1'-L2-L3-L4-D' is:

[0118] L1'-L2-L3-L4'-D'+Rc-N3→L1'-L2-L3-L4-D'.

[0119] General preparation method two:

[0120] Preferably,

[0121] wherein L1', L2, L3, L4 and D' are as described in any preceding technical solution;

[0122] D" is:

[0123] wherein R 1 -R 9 , W1-W3, n1-n6 are as described in any preceding technical solution.

[0124] Abbreviations and definitions

[0125] The following terms used in the present application have the following meanings unless otherwise stated.

[0126] When a trademarked name is used in this application, unless otherwise indicated, the trademarked name includes the product formulation, generic drug, and active pharmaceutical ingredient of the product of the trademarked name.

[0127] The term "antibody drug conjugate" or "antibody conjugated drug" (ADC) refers to the linkage of a targeting ligand such as an antibody (e.g., a monoclonal antibody), an antibody fragment to a biologically active molecule with biological activity through a stable chemical linker compound.

[0128] The term "linker-drug compound" or "linker-payload compound" refers to the moiety consisting of a linker compound and a biologically active compound in an "antibody-drug conjugate".

[0129] The linker-drug compound in the present application is linked to the antibody by a coupling method conventional in the art, including: lysine coupling, inter-heavy chain disulfide bond coupling, and directed coupling (Beck A, Reichert JM. Antibody-drug conjugates: Present and future; MAbs, 2014, 6: 15-17; McCombs J R, Owen S C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. The AAPS journal, 2015, 17: 339-351). The present application is preferably linked by inter-heavy chain disulfide bond coupling, i.e., the reaction of a thiol group (sulfur atom of a cysteine residue) formed after reduction of one or more of the inter-heavy chain disulfide bond sites (two sites between heavy chains, and two sites between heavy and light chains).

[0130] The term "solvate" means a physical association between a compound of this application and one or more solvent molecules (either organic or inorganic). This physical association can, for example, involve hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. Solvate molecules can be present in a regular or an orderly arrangement and / or be present in a non-ordered arrangement. Solvates can include stoichiometric or non-stoichiometric amounts of the solvent molecules. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods for solvation are known in the art.

[0131] The term "stereoisomers" refers to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like. Mixtures of any of the stereoisomers obtained can be separated, if desired, into the individual or substantially pure geometric or optical isomers on the basis of their physical or chemical differences by techniques ordinary skillful worker will recognize.

[0132] The term "tautomers" refers to isomers which differ in the arrangement of atoms or groups in space, which can interconvert by low energy barriers. If tautomerism is possible (as in solution), a chemical equilibrium of the tautomers can be reached. For example, prototropic tautomers (also known as proton shift tautomers) include tautomers which interconvert by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include tautomers which interconvert by reorganization of bonding electrons.

[0133] The term "heteroatom" refers to nitrogen, oxygen, sulfur, halogen atoms.

[0134] The term "one or more" refers to 1 to 12 (preferably 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), preferably 2, 3, 4, 5, 6.

[0135] The term "C m The term "C n " refers to the moiety having an integer number of carbon atoms in the range of m to n. For example, "C1-C3" indicates that the group can have 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.

[0136] The term "-(CH2) n " refers to the moiety having n -CH2- linked groups. For example, when the number of linking groups is 0, such as in "-(CH)0-", the linking group is a covalent bond.

[0137] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group including a straight chain or branched chain of 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms (i.e., C 1-10 alkyl), further preferably 1 to 8 carbon atoms (i.e., C 1-8 alkyl), and more preferably 1 to 6 carbon atoms (i.e., C 1-6 alkyl). For example, "C 1-6"Alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5, or 6), examples include but are not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, and the like.

[0138] The term "alkoxy" means -O-alkyl, the alkyl group being as defined above, i.e. containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and even more preferably 1 to 6 carbon atoms (specifically 1, 2, 3, 4, 5, or 6). Representative examples include, but are not limited to, methoxy, ethoxy, propyloxy, isopropoxy, butoxy, 1-methylpropyloxy, 2-methylpropyloxy, t-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropyloxy, 1,2-dimethylpropyloxy, 2,2-dimethylpropyloxy, 1-ethylpropyloxy, and the like.

[0139] The term "halogen" or "halo" means F, CI, Br, I.

[0140] The term "haloalkyl" means one, two, or multiple hydrogen atoms or all hydrogen atoms in an alkyl group as defined above are replaced by halogen. Representative examples of haloalkyl include CCI3, CHCI2, CH2CI, CF3, CHF2, CH2F, CBr3, CHBr2, CH2Br, CI3, CHI2, CH2I, CH2CF3, CF2CF3, and the like.

[0141] The term "aryl" means a monocyclic, bicyclic and tricyclic, aromatic carbocyclic ring system containing 6 to 16 carbon atoms, or 6 to 14 carbon atoms, or 6 to 12 carbon atoms, or 6 to 10 carbon atoms, preferably 6 to 10 carbon atoms. Examples of aryl groups can include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, or pyrenyl, and the like.

[0142] The term "heteroaryl" means an aromatic monocyclic or polycyclic ring system containing a 5-14 membered structure, or preferably a 5-10 membered structure, or preferably a 5-8 membered structure, more preferably a 5-6 membered structure, wherein 1, 2, 3 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms being independently selected from O, N or S, the number of heteroatoms preferably being 1, 2 or 3. Examples of heteroaryl groups include, but are not limited to, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiadiazolyl, triazinyl, phtalazinyl, quinolinyl, isoquinolinyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothienyl, benzopyridyl, benzopyrimidyl, benzopyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridyl, imidazo[l,2-a]pyridyl, pyrazolo[l,5-a]pyridyl, pyrazolo[l,5-a]pyrimidyl, imidazo[l,2-b]pyridazinyl, [l,2,4]triazolo[4,3-b]pyridazinyl, [l,2,4]triazolo[l,5-a]pyrimidyl, [l,2,4]triazolo[l,5-a]pyridyl, and the like.

[0143] The term "cycloalkyl" means a carbocyclic ring which is fully saturated and which can exist as a monocyclic, bridged or spirocyclic ring. Preferably it contains 3-12 carbon atoms (i.e. C3-12 cycloalkyl), more preferably 3-10 carbon atoms (C3-10 cycloalkyl), further preferably 3-7 carbon atoms (C3-7 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), 5-6 carbon atoms (C5-6 cycloalkyl). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl and the like.

[0144] The term "-alkyl-" or "alkylene" means a saturated straight-chain or branched-chain divalent hydrocarbon radical. For example, C1-C8 alkylene means a straight-chain or branched-chain alkylene group having 1-8 number of carbon atoms.

[0145] The term "heterocyclyl" refers to a non-aromatic ring that is fully saturated or partially unsaturated (not fully unsaturated heteroaromatic) and can exist as a monocyclic, bridged, or spirocyclic ring. Unless otherwise indicated, the heterocycle typically is a 3- to 7-membered ring containing 1 to 3 heteroatoms independently selected from sulfur, oxygen, and / or nitrogen (preferably 1 or 2 heteroatoms, but not including -0-0-, -0-S-, or -S-S- moieties). Non-limiting examples of heterocyclyl groups include, but are not limited to, oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothiophenyl, and the like.

[0146] The term "heteroalkyl" refers to an alkyl group in which one or more of the carbon atoms are replaced by a heteroatom (e.g., N, O, S, P, halogen) and / or a heteroatomic group (e.g., NH, -C(=0)-, -S(=0)-, -S(=0)2-). Replacement.

[0147] The term "derivative" refers to a compound formed by the substitution of an atom or a group of atoms in a parent compound molecule with another atom or group of atoms, which is referred to as a derivative of the parent compound.

[0148] The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of a compound (e.g., a drug, a drug-linker, or an antibody-linker-drug conjugate). The compound can contain at least one amino, imino, hydroxy, or carboxyl group and thus be capable of forming a salt with a corresponding acid or base. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, hydrochloride, hydrobromide, hydroiodide, nitrate, bisulfate, phosphate, acid phosphate (-H2PO4), phosphite, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, ammonium salt, calcium salt, and the like. Additionally, pharmaceutically acceptable salts have more than one charged atom in the structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counterions.

[0149] The term "amino acid" means a naturally occurring amino acid or a non-naturally occurring amino acid, represented by NH2-C(R'R")-C(=0)OH, wherein each of R' and R" is independently hydrogen, optionally substituted straight, branched, or cyclic alkyl, alkenyl, or alkynyl having from 1 to 10 carbon atoms, aryl, heteroaryl, or heterocyclyl, or R" and the N-terminal nitrogen atom can be taken together to form a heterocyclic ring, e.g., phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), C1-C6 alkyl substituted lysine (e.g., C1-C3 alkyl substituted lysine, such as methyl substituted lysine, ethyl substituted lysine, n-propyl substituted lysine, isopropyl substituted lysine), and the like.

[0150] The term "amino acid residue" means the corresponding residue when one hydrogen atom is removed from the amino terminus and / or one hydroxyl group is removed from the carboxyl terminus of an amino acid, e.g., -NH-C(R'R")-C(O)-; a residue of an amino acid selected independently of one another from the group consisting of alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valine (Nva), norleucine (Nle), or an analog of the above amino acids.

[0151] The term "peptide" means a short chain of amino acid monomers linked by peptide (amide) bonds, selected from a divalent peptidyl group comprising from 2 to 8 optionally substituted natural or non-natural, L or D form amino acid residues, each of said amino acid residues being the same or different, selected from the above definition, wherein the optional amino acid residues are further substituted with one or more substituents selected from C1-C6 alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6 alkoxy, hydroxyl, amino, carboxyl, or C3-C8 cycloalkyl.

[0152] The term "cancer" means a neoplasm formed by the clonal expansion of a cell that has lost normal regulatory controls of its growth at the genetic level as a result of the action of various carcinogenic factors on a local tissue of the body.

[0153] The term "linker" means a chemical moiety fragment that links one end to an antibody and the other end to a cytotoxic drug, represented by L.

[0154] The term "self-cleaving segment" is well known in the art, such as the p- aminobenzyl alcohol carbonate segment or -NHCH2- as commonly used in the art. In the specific embodiments of the present application, the "self-cleaving segment" of the linker of the present application comprises a modification of the "hydrophilic segment".

[0155] The term "hydrophilic segment" is well known in the art, which can comprise a polyethylene glycol group, such as a polyethylene glycol group with a terminal methoxy group, or further linked to other hydrophilic segments via a polyethylene glycol group. The hydrophilic segment can also include a polyamino acid segment, such as a polyglycine or a polysarcosine, which can be combined with a polyethylene glycol group. The hydrophilic segment can also be a monosaccharide, disaccharide or oligosaccharide, which can be a linear or cyclic sugar molecule, and can comprise a sugar amine, a sugar acid or a phosphosugar. Preferably, the sugar group is combined with a polyethylene glycol segment or a polyamino acid segment; more preferably, at least two sugar groups are introduced via a polyvalent linker, such as an aspartic acid, glutamic acid or lysine based linker. A polycarboxylic acid group, a polysulfonic acid group or a chelating group can also be included

[0156] The term "antibody" broadly refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site within a variable region of the immunoglobulin molecule, represented by Ab. Encompassed are intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies (such as bispecific antibodies, biparatopic antibodies, etc.), multivalent antibodies (such as tri-valent, tetra-valent, etc. antibodies having three, four or more antigen binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified configuration of the immunoglobulin molecule that exhibits the desired biological activity. When "antibody" and "antigen binding fragment / antigen binding portion" appear in the same context, "antibody" can be understood as the complete entity in relation to "antigen binding fragment / antigen binding portion", both together corresponding to the broad concept of antibody.

[0157] The term "heavy chain variable region (VH)," as used herein, refers to the amino-terminal variable region domain of an immunoglobulin heavy chain.

[0158] The term "light chain variable region (VL)," as used herein, refers to the amino-terminal variable region domain of an immunoglobulin light chain.

[0159] As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequences that together define the binding affinity and specificity of the Fv region of an antibody. Because, the specificity of an antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. The antibody combining site is composed of residues from the so-called hypervariable or complementarity determining regions (CDRs), interspersed among less variable regions called framework regions (FRs). The light (L) and heavy (H) chains of an antibody each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen binding site of a conventional antibody includes six CDRs, comprising the CDR sets from each of the heavy and light variable regions. Each VH and VL is composed of three CDRs and four FRs, in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0160] As used herein, the term "bispecific antibody" refers to whole or intact antibody molecules (e.g., IgM, IgG (including IgGl, IgG2, IgG3, and IgG4), IgA, IgD, or IgE) and any fragments thereof that bind to two or more different proteins, at least two of which are C5a, C5b, or C5aR (see above). The term bispecific antibody includes, for example, chimerized or chimeric antibodies, humanized antibodies, de-immunized human antibodies, and fully human antibodies. Bispecific antibodies also include, for example, F(ab')2 fragments or conjugates of two or more monospecific antibody fragments (e.g., two or more scFv, Fab, Fab', or Fd immunoglobulin fragments). In addition, bispecific intrabodies, minibodies, triabodies, and diabodies (see, e.g., Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1): 177-189; Poljak (1994) Structure 2(12): 1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety) are also included in the definition of bispecific antibodies and are compatible for use in the methods described herein. Also encompassed by the term bispecific antibody are tandem single-chain antibodies, single-chain diabodies, tandem single-chain diabodies, and fusion proteins containing a single-chain diabody and at least a portion of an immunoglobulin heavy chain constant region (e.g., a CH1 or CH3 region of a heavy chain polypeptide), as described in, e.g., Kontermann (2005) Acta Pharmacologica Sinica 26(1): 1-9; Kufer et al. (2004) Trends Biotechnol 22:238-244; and Kriangkum et al. (2001) Biomol Eng 18:31-40.

[0161] As used herein, the term "functional variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein, polypeptide, CDR and that retains one or more desired activities of the parent protein. A functional variant can be a protein fragment (i.e., a variant with N- and / or C-terminal deletions) that retains one or more desired activities of the parent protein.

[0162] As used herein, a sequence having "at least 85% identity" to a reference sequence is a sequence having 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, etc.) sequence identity to the entire length of the reference sequence over its entire length. Advantages

[0163] The ADC drug of the present application combines the tumor targeting effect of the antibody and the high bioactivity of the compound, becoming a biological missile with better efficacy and safety advantages. The antibody guides the ADC to bind to the target cell, achieving tumor tissue enrichment, reducing non-target tissue exposure, and reducing the toxicity that may be caused by systemic administration of bioactive compounds. The main technology of the present application is to provide a new type of molecular glue degradable compound and a linker, which are coupled with the antibody through a specific chemical method, thereby obtaining a new type of antibody conjugated drug with high efficiency and low toxicity. Biological tests show that the molecules have one or more of good bioactivity, safety, and other drug-related properties compared with similar ADC drugs. Therefore, the ADC drug provided by the present application has improved anti-tumor activity of the drug and / or improved the therapeutic window of the whole drug, and has high clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0164] Figure 1 is the inhibition effect of antibody drug conjugate on MV-4-11-LUC cell transplanted tumor

[0165] Figure 2 is a fluorescence imaging diagram of the inhibition effect of antibody drug conjugate on MV-4-11-LUC cell transplanted tumor

[0166] Figure 3 is the inhibition effect of antibody drug conjugate on HL-60 cell transplanted tumor

[0167] EMBODIMENTS

[0168] The present application will be further illustrated by the following examples, but these examples will not limit the scope of the present application. The test methods in the following examples are generally carried out under conventional conditions or under the conditions recommended by the manufacturer, unless otherwise specified. All percentages, proportions, ratios, or parts are by weight, unless otherwise specified.

[0169] Explanation of Abbreviations

[0170] Table 1. Explanation of Abbreviations

[0171] Example 1: Synthesis of intermediate 6:

[0172] Compound 1 (28.5 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol) and ethanol (400 mL) were added into a reaction flask, stirred and dissolved at room temperature. After the addition of carbon disulfide (17 g, 283 mmol) slowly, the reaction was heated to 100 °C and refluxed for 5 hours. After the removal of solvent under reduced pressure, water (50 mL) was added, the pH was adjusted to 6 with dilute hydrochloric acid, extracted with dichloromethane (100 mL x 3), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 2 (32.8 g). MS (ESI): (M+H) + Calculated 194.0, found 194.1.

[0173] Compound 2 (3.86 g, 20 mmol), triethylamine (2.42 g, 24 mmol) and tetrahydrofuran (36 mL) were added into a reaction flask, and methyl iodide (3.12 g, 22 mmol) was added into the above reaction solution at 0 °C. After the reaction was stirred at 25 °C for 1.5 hours, the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to give compound 3 (2.73 g). MS (ESI): (M+H) + Calculated 208.0, found 208.2.

[0174] Compound 3 (2.20 g, 10.6 mmol), diglycolic anhydride 4 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol) and tetrahydrofuran (30 mL) were added into a reaction flask, and the reaction was carried out at 25 °C for 1.5 hours. The solvent was removed under reduced pressure, and the residue was added with diethyl ether, filtered, and washed with water and diethyl ether to give compound 5 (3.4 g). MS (ESI): (M+H) + Calculated 324.1, found 324.2.

[0175] Compound 5 (3.4 g, 10.5 mmol) and glacial acetic acid (20 mL) were added into a reaction flask, and after dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0 °C. The reaction was carried out at room temperature for 1 hour, saturated sodium sulfite solution was added until the solution became colorless, extracted with ethyl acetate (100 mL x 3), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 6 (2.4 g). MS (ESI): (M+H) + Calculated 356.0, found 356.0.

[0176] Example 2: Synthesis of intermediate 14:

[0177] Compound 7 (9.65 g, 31 mmol), compound 8 (5.00 g, 31 mmol), EEDQ (11.5 g, 46.5 mmol) and super dry dichloromethane (90 mL) were added to a reaction flask. After reaction at 0 °C for 3 hours, the solvent was removed under reduced pressure, and the residue was slurried with 500 mL methyl tert-butyl ether to obtain compound 9 (9.01 g). MS (ESI): (M+H) + Calculated 455.2, found 455.2.

[0178] Chiral resolution of compound 9: Compound 9 (9 g) was resolved by SFC to obtain 9a (3.55 g, retention time 16.29 min) and 9b (3.63 g, retention time 13.55 min). SFC resolution method: Column type: DAICEL CHIRALCEL OD (250 mm-50 mm, 10 um); Mobile phase: A: CO2, B: CO2-ACN / i-PrOH (0.1% NH3H2O); Isocratic elution: B in A for 50%; Flow rate: 200 mL / min; Detector: PDA; Column temperature: 25 °C; Back pressure: 100 Bar;

[0179] Compound 9b (3.63 g, 8 mmol), super dry tetrahydrofuran (90 mL) were added to a reaction flask, stirred at 0 °C and DBU (1.21 g, 8 mmol) was added slowly, the reaction was removed to room temperature after half an hour. After TLC monitoring the disappearance of raw materials, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 10 (1.71 g). MS (ESI): (M+H) + Calculated 233.1, found 233.2.

[0180] Compound 10 (1.62 g, 7 mmol), compound 11 (302 mg, 7 mmol), EEDQ (2.60 g, 10.5 mmol) and super dry tetrahydrofuran (20 mL) were added to a reaction flask. After reaction at 0 °C for 3 hours, the solvent was removed under reduced pressure, and the residue was slurried with 50 mL ethyl acetate and 50 mL petroleum ether to precipitate white solid, which was repeated three times to obtain compound 12. MS (ESI): (M+H) + Calculated 554.3, found 554.4.

[0181] Compound 12 (1.66 g, 3 mmol), compound 13 (1.21 g, 6 mmol) and super dry tetrahydrofuran (150 mL) were added to a reaction flask, stirred and pyridine (474 uL, 6 mmol) was added dropwise, reacted at 65 °C for 1 hour, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 14 (1.83 g). MS (ESI): (M+H) +Calcd 335.5, found 335.5.

[0182] Example 3: Synthesis of intermediate 20:

[0183] Compound 15 (5.0 g, 25 mmol) was added to a single-necked flask and dissolved in 100 mL of dichloromethane. After the temperature was lowered to 0°C, triethylamine (5.3 mL, 37.5 mmol) was added, followed by dropwise addition of benzyl chloroformate (5.1 g, 30 mmol). After the dropwise addition was completed, the reaction was allowed to proceed to room temperature. After overnight, the reaction was complete. The reaction was diluted with water and extracted with dichloromethane (100 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the product 16 (6.6 g) as a colorless oil, MS (ESI): (M+H) + Calcd 335.5, found 335.5.

[0184] Compound 16 (6.5 g, 19.46 mmol) was added to a two-necked flask and dissolved in 100 mL of tetrahydrofuran. After being vacuumed three times, sodium hydride (1.168 g, 29.19 mmol) was added. After being stirred for 5 min, iodomethane (5.5 g, 38.92 mmol) was added under ice bath. The reaction was allowed to proceed to room temperature. After 1 h, the reaction was complete. The reaction was poured into 100 mL of saturated NH4Cl solution. The reaction was extracted with dichloromethane (100 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the product 17 (3.3 g) as a light yellow oil, MS (ESI): (M+H) + Calcd 349.2, found 349.2.

[0185] Compound 17 (3.3 g, 9.48 mmol) was added to a flask and dissolved in 50 mL of methanol. Palladium on carbon (330 mg) was added. The flask was vacuumed, replaced with hydrogen, and the reaction was allowed to proceed under hydrogen atmosphere. After 2 h, the reaction was complete. The reaction was filtered through celite. The reaction was concentrated under vacuum to give intermediate 18 (1.9 g), MS (ESI): (M+H) + Calcd 215.2, found 215.2.

[0186] Compound 18 (894 mg, 4.178 mmol), 14 (2.5 g, 3.48 mmol) were added into a reaction flask, dissolved in 20 mL of DMF, then DIEA (539 mg, 4.178 mmol) was added dropwise under ice-bath, after the addition was completed, the reaction was transferred to room temperature, after 3 hours, the reaction was completed, deionized water was added to the reaction solution, and the product was precipitated, extracted with dichloromethane (100 mL x 3), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain the product 19 (2.45 g) as a yellow-brown oil, MS (ESI): (M+H) + Calculated 794.4, found 794.4.

[0187] Compound 19 (4.3 g, 5.4 mmol) was added into a reaction flask, dissolved in 50 mL of dichloromethane, then 25 mL of trifluoroacetic acid was added to start the reaction, after 2 hours, the reaction was completed, after removing the dichloromethane and a large amount of trifluoroacetic acid by vacuum concentration, the crude product was obtained, and after column chromatography, the product 20 (2.7 g) was obtained as a yellowish oil, MS (ESI): (M+H) + Calculated 694.4, found 694.4.

[0188] Example 4: Synthesis of compound D1

[0189] Compound 21 (3.15 g, 15 mmol) was dissolved in tetrahydrofuran (20 mL), and platinum dioxide (645 mg, 3 mmol) was added under nitrogen protection, and the reaction was stirred after hydrogen replacement. After the reaction was completed, the solid was removed by filtration with the addition of diatomite, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column to obtain compound 22 (3 g). MS (ESI): (M+H) + Calculated 216.1, found 216.1.

[0190] Compound 22 (3 g, 14 mmol) was dissolved in dichloromethane (20 mL), and phosphorus tribromide (28 mmol) was added under nitrogen, and the reaction was stirred. After 2 hours, the reaction was completed, the reaction was quenched with water, and ethyl acetate and saturated sodium chloride solution were added for extraction, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column to obtain compound 23 (2.77 g). MS (ESI): (M+H) + Calculated 277.9, found 278.0.

[0191] Compound 24 (3.46 g, 20 mmol) was dissolved in DMF (10 mL), and sodium hydride (240 mg, 10 mmol) was added under nitrogen protection in an ice bath, and the reaction was stirred for half an hour. Compound 23 (2.77 g, 10 mmol) dissolved in DMF (10 mL) was added to the reaction system, and the reaction was stirred for 3 hours until completion. Saturated ammonium chloride solution was added to quench the reaction, and ethyl acetate and saturated sodium chloride solution were added for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 25 (2.89 g). MS (ESI): (M+H) + Calculated value 371.1, experimental value 371.2.

[0192] Compound 25 (2.89 g, 7.8 mmol) was dispersed in water (5 mL), and B2(OH)4 (1.4 g, 15.6 mmol) was added. The reaction was stirred at 100°C under nitrogen protection overnight. After the reaction was completed, ethyl acetate and saturated sodium chloride solution were added for extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 26 (2.38 g). MS (ESI): (M+H) + Calculated value 341.2, experimental value 341.2.

[0193] Compound 26 (2.38 g, 7 mmol) was dissolved in dichloromethane (100 mL), and DIEA (1.75 g, 14 mmol) and compound 27 (1.33 g, 8.4 mmol) were sequentially added to the solution, and the reaction was stirred at room temperature. After 30 minutes, the reaction was completed, and the reaction liquid was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 28 (2.76 g). MS (ESI): (M+H) + Calculated value 461.2, actual value 461.2.

[0194] Compound 28 (2.76 g, 6 mmol) was dissolved in DMF (80 mL), and then compound 29 (2.05 g, 6.6 mmol) was added, followed by the addition of DIEA (3.23 g, 25 mmol). The reaction was carried out by heating to 50°C, and after 3 hours, the reaction was completed. Ethyl acetate and saturated sodium chloride solution were added for extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 30 (3.65 g). MS (ESI): (M+H) + Calculated value 640.3, experimental value 640.4.

[0195] Compound 30 (3.65 g, 5.7 mmol) was dissolved in dichloromethane (19 mL), and hydrogen chloride in ethyl acetate (84 mmol, 21 mL, 4 M) was added dropwise to the solution under ice bath. A large amount of solid was observed as the solution was added dropwise, and the reaction was complete after two hours. The solvent was removed under reduced pressure at low temperature, and the compound 31 (2.77 g) was obtained after drying in vacuum. MS (ESI): (M+H) + Calculated 540.2, found 540.3.

[0196] Compound 31 (100 mg, 0.185 mmol), compound 32 (13 mg, 0.185 mmol), HATU (84 mg, 0.222 mmol) were added to a reaction bottle, and then dissolved in 2 mL of DMF. DIEA (48 mg, 0.37 mmol) was added dropwise under ice bath, and the reaction was carried out after being warmed to room temperature. The reaction was complete after one hour. The reaction solution was directly purified by reverse phase preparative chromatography, and D1 (84 mg) was obtained as a white solid after freeze-drying. MS (ESI): (M+H) + Calculated 598.2, found 598.2.

[0197] Example 5: Synthesis of compound D14

[0198] Compound 33 (4.2 g, 20 mmol) was dissolved in DMF (10 mL), and sodium hydride (240 mg, 10 mmol) was added under nitrogen protection under ice bath. The reaction was stirred for half an hour. Compound 23 (2.77 g, 10 mmol) in DMF (10 mL) was added to the reaction system, and the reaction was stirred for 3 hours. The reaction was complete after quenching with saturated ammonium chloride solution, and extraction was performed with ethyl acetate and saturated sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column to obtain compound 34 (3.4 g). MS (ESI): (M+H) + Calculated 399.2, found 399.2.

[0199] Compound 34 (3.4 g, 8.6 mmol) was dispersed in water (5 mL), and B2(OH)4 (1.55 g, 17.2 mmol) was added. The reaction was stirred overnight under nitrogen protection at 100°C. After the reaction was complete, extraction was performed with ethyl acetate and saturated sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column to obtain compound 35 (2.76 g). MS (ESI): (M+H) + Calculated 369.2, found 369.2.

[0200] Compound 35 (2.76 g, 7.5 mmol) was dissolved in dichloromethane (100 mL), to the solution was added DIEA (1.89 g, 15 mmol) and compound 27 (1.43 g, 9 mmol) sequentially, the reaction was stirred at room temperature. After 30 minutes, the reaction was complete, the reaction was concentrated under reduced pressure, the residue was purified by silica gel column to obtain compound 36 (3.3 g). MS (ESI): (M+H) + Calculated 489.2, found 489.2.

[0201] Compound 36 (3.3 g, 6.7 mmol) was dissolved in DMF (80 mL), then compound 29 (2.3 g, 7.4 mmol) was added, DIEA (3.87 g, 30 mmol) was added, the reaction was carried out by increasing the temperature to 50 °C, after 3 hours the reaction was complete, ethyl acetate and saturated sodium chloride solution were added for extraction, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, the residue was purified by silica gel column to obtain compound 37 (4.2 g). MS (ESI): (M+H) + Calculated 668.3, found 668.4.

[0202] Compound 37 (2.00 g, 3 mmol) was dissolved in dichloromethane (19 mL), hydrogen chloride ethyl acetate solution (44 mmol, 11 mL, 4M) was added dropwise to the solution under ice bath, a large amount of solid was observed as the solution was added, after 2 hours the reaction was complete, the solvent was removed under reduced pressure at low temperature, vacuum drying to obtain compound 38 (1.7 g). MS (ESI): (M+H) + Calculated 568.2, found 568.3.

[0203] Compound 38 (100 mg, 0.176 mmol), compound 32 (12.33 mg, 0.176 mmol), HATU (80.26 mg, 0.211 mmol) were added to the reaction bottle, dissolved in 2 mL of DMF, then DIEA (45.4 mg, 0.352 mmol) was added dropwise under ice bath, after the dropwise addition was completed, the reaction was carried out at room temperature. After 1 hour the reaction was complete. The reaction was directly purified by reverse phase preparative chromatography, and after freeze-drying, white solid D14 (73 mg) was obtained, MS (ESI): (M+H) + Calculated 626.2, found 626.2.

[0204] Example 6: Synthesis of compound D27

[0205] Compound 39 (4.2 g, 20 mmol) was dissolved in DMF (10 mL), and sodium hydride (240 mg, 10 mmol) was added under nitrogen protection in an ice bath, and the reaction was stirred for half an hour. Compound 23 (2.77 g, 10 mmol) dissolved in DMF (10 mL) was added to the reaction system, and the reaction was stirred for 3 hours until completion. Saturated ammonium chloride solution was added to quench the reaction, and ethyl acetate and saturated sodium chloride solution were added for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 40 (3.2 g). MS (ESI): (M+H) + Calculated value 399.2, experimental value 399.2.

[0206] Compound 40 (3.2 g, 8 mmol) was dispersed in water (5 mL), and B2(OH)4(1.44 g, 16 mmol) was added. The reaction was stirred at 100°C under nitrogen protection overnight. After the reaction was completed, ethyl acetate and saturated sodium chloride solution were added for extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 41 (2.72 g). MS (ESI): (M+H) + Calculated value 369.2, experimental value 369.2.

[0207] Compound 41 (2.72 g, 7.4 mmol) was dissolved in dichloromethane (100 mL), and DIEA (1.89 g, 15 mmol) and compound 27 (1.43 g, 9 mmol) were added in sequence to the solution, and the reaction was stirred at room temperature. After 30 minutes, the reaction was completed, and the reaction liquid was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 42 (3.1 g). MS (ESI): (M+H) + Calculated value 489.2, actual value 489.2.

[0208] Compound 42 (3.1 g, 6.3 mmol) was dissolved in DMF (80 mL), and then compound 29 (2.14 g, 6.9 mmol) was added, followed by the addition of DIEA (3.6 g, 28 mmol). The reaction was carried out by heating to 50°C, and after 3 hours the reaction was completed. Ethyl acetate and saturated sodium chloride solution were added for extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 43 (3.8 g). MS (ESI): (M+H) + Calculated value 668.3, experimental value 668.4.

[0209] Compound 43 (2.00 g, 3 mmol) was dissolved in dichloromethane (19 mL), and hydrogen chloride in ethyl acetate (44 mmol, 11 mL, 4 M) was added dropwise to the solution under ice bath. A large amount of solid was observed as the solution was added dropwise. The reaction was complete after two hours. The solvent was removed under reduced pressure at low temperature, and vacuum drying was performed to obtain compound 44 (1.75 g). MS (ESI): (M+H) + Calculated 568.2, found 568.3.

[0210] Compound 44 (100 mg, 0.176 mmol), compound 32 (12.33 mg, 0.176 mmol), HATU (80.26 mg, 0.211 mmol) were added to a reaction bottle, and then dissolved in 2 mL of DMF. DIEA (45.4 mg, 0.352 mmol) was added dropwise under ice bath. After the dropwise addition was complete, the reaction was allowed to proceed at room temperature. The reaction was complete after 1 hour. The reaction solution was directly purified by reverse phase preparative chromatography to obtain white solid D27 (66 mg), MS (ESI): (M+H) + Calculated 626.2, found 626.2.

[0211] Example 7: Synthesis of compounds D1-D13, D15-D26, D28-D52

[0212] The synthesis route of compounds D1-D13, D15-D26, D28-D52 is referenced to that of compound D1.

[0213] Example 8: Synthesis of compound LD1

[0214] Compound D1 (100 mg, 0.17 mmol), di(p-nitrophenyl) carbonate (73 mg, 0.24 mmol), and 3 mL of tetrahydrofuran were added to a reaction bottle, and then dissolved in 3 mL of DMF. DIEA (41 mg, 0.32 mmol) was added dropwise under ice bath. After the dropwise addition was complete, the reaction was allowed to proceed at room temperature. The reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 45 (80 mg), MS (ESI): (M+H) + Calculated 763.3, found 763.3.

[0215] Compound 45 (80 mg, 0.105 mmol), compound 20 (73 mg, 0.105 mmol) were added to a reaction bottle, and then dissolved in 3 mL of DMF. DIEA (13 mg, 0.21 mmol) was added dropwise under ice bath. After the dropwise addition was complete, the reaction was allowed to proceed at room temperature. After the reaction was complete, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 46 (100 mg), MS (ESI): (M+H)+ Calcd 1317.6, Found 1317.6.

[0216] Compound 46 (100 mg, 0.076 mmol) was added to the reaction bottle, dissolved with 3 mL of THF, and DBU (11.2 mg, 0.076 mmol) was added dropwise under ice bath. After the dropwise addition was completed, the reaction was allowed to proceed to room temperature. After the reaction was completed, compound 47 (83 mg) was obtained by purification, MS (ESI): (M+H) + Calcd 1095.5, Found 1095.6.

[0217] Compound 47 (83 mg, 0.076 mmol), compound 6 (27 mg, 0.076 mmol), EDCI (29 mg, 0.15 mmol), and HOBT (3 mg, 0.015 mmol) were added to the reaction bottle, dissolved with 3 mL of DMF, and the reaction was allowed to proceed at room temperature. After the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 48 (93 mg), MS (ESI): (M+H) + Calcd 1432.5, Found 1432.5.

[0218] Compound 48 (93 mg, 0.065 mmol), compound 49 (45 mg, 0.075 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (3 mg, 0.006 mmol), and cuprous bromide (17.1 mg, 0.006 mmol) were added to the reaction bottle, and nitrogen was replaced three times. THF / DMF / H2O (2 mL:0.5 mL:0.2 mL) was added, and the reaction was allowed to proceed at 25°C for 1 hour. After the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound LD1 (52 mg). MS (ESI): (M+H) + Calcd 2017.9, Found 2017.9.

[0219] Example 9: Synthesis of compound LD14

[0220] Compound D14 (100 mg, 0.16 mmol), di(p-nitrophenyl) carbonate (73 mg, 0.24 mmol), and 3 mL of tetrahydrofuran were added to the reaction bottle, dissolved with 3 mL of DMF, and DIEA (41 mg, 0.32 mmol) was added dropwise under ice bath. After the dropwise addition was completed, the reaction was allowed to proceed to room temperature. The reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 50 (83 mg), MS (ESI): (M+H) + Calcd 791.3, Found 791.3.

[0221] Compound 50 (80 mg, 0.101 mmol), compound 20 (70 mg, 0.101 mmol) were added into a reaction vial, dissolved in 3 mL of DMF, then DIEA (13 mg, 0.202 mmol) was added dropwise under ice-bath, after the addition was completed, the reaction was transferred to room temperature. After the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 51 (103 mg), MS (ESI): (M+H) + Calcd 1345.6, Found 1345.6.

[0222] Compound 51 (100 mg, 0.075 mmol) was added into a reaction vial, dissolved in 3 mL of THF, then DBU (11.2 mg, 0.075 mmol) was added dropwise under ice-bath, after the addition was completed, the reaction was transferred to room temperature. After the reaction was completed, compound 52 (83 mg) was obtained after purification, MS (ESI): (M+H) + Calcd 1123.5, Found 1123.6.

[0223] Compound 52 (83 mg, 0.075 mmol), compound 6 (29 mg, 0.075 mmol), EDCI (29 mg, 0.15 mmol), HOBT (3 mg, 0.015 mmol) were added into a reaction vial, dissolved in 3 mL of DMF, then the reaction was carried out at room temperature. After the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 53 (89 mg), MS (ESI): (M+H) + Calcd 1460.5, Found 1460.5.

[0224] Compound 53 (89 mg, 0.061 mmol, 1.0 eq), compound 49 (43 mg, 0.073 mmol), tris (3-hydroxypropyl triazole methyl) amine (3 mg, 0.006 mmol) and cuprous bromide (17.1 mg, 0.006 mmol) were added into a reaction vial, replaced with nitrogen three times, added THF / DMF / H2O (2 mL:0.5 mL:0.2 mL), reacted at 25°C for 1 hour, after the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound LD14 (56 mg). MS (ESI): (M+H) + Calcd 2045.9, Found 2045.9.

[0225] Example 10: Synthesis of compound LD27

[0226] Compound D27 (100 mg, 0.16 mmol), di(p-nitrophenyl) carbonate (73 mg, 0.24 mmol) and 3 mL of tetrahydrofuran were added into a reaction bottle, then dissolved in 3 mL of DMF, DIEA (41 mg, 0.32 mmol) was added dropwise under ice-bath, after the addition was completed, it was transferred to room temperature for reaction. The reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 54 (95 mg), MS (ESI): (M+H) + Calcd 791.3, Found 791.3.

[0227] Compound 54 (80 mg, 0.101 mmol), compound 20 (70 mg, 0.101 mmol) were added into a reaction bottle, then dissolved in 3 mL of DMF, DIEA (13 mg, 0.202 mmol) was added dropwise under ice-bath, after the addition was completed, it was transferred to room temperature for reaction. After the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 55 (108 mg), MS (ESI): (M+H) + Calcd 1345.6, Found 1345.6.

[0228] Compound 55 (100 mg, 0.075 mmol) was added into a reaction bottle, then dissolved in 3 mL of THF, DBU (11.2 mg, 0.075 mmol) was added dropwise under ice-bath, after the addition was completed, it was transferred to room temperature for reaction. After the reaction was completed, compound 56 (82 mg) was obtained after purification, MS (ESI): (M+H) + Calcd 1123.5, Found 1123.6.

[0229] Compound 56 (82 mg, 0.074 mmol), compound 6 (26 mg, 0.074 mmol), EDCI (29 mg, 0.15 mmol), HOBT (3 mg, 0.015 mmol) were added into a reaction bottle, then dissolved in 3 mL of DMF, and reacted at room temperature. After the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound 57 (84 mg), MS (ESI): (M+H) + Calcd 1460.5, Found 1460.5.

[0230] Compound 57 (84 mg, 0.058 mmol), compound 49 (38 mg, 0.064 mmol), tris(3- hydroxypropyltriazolylmethyl)amine (3 mg, 0.006 mmol) and cuprous bromide (17.1 mg, 0.006 mmol) were added into a reaction vial, purged with nitrogen for three times, THF / DMF / H2O (2 mL:0.5 mL:0.2 mL) was added, and the reaction was carried out at 25 °C for 1 hour. After the reaction was completed, the reaction solution was directly purified by reverse phase preparative chromatography to obtain compound LD27 (45 mg). MS (ESI): (M+H) + Calcd 2045.9, Found 2045.9.

[0231] Example 11: Synthesis of compounds LD2-LD13, LD15-LD26, LD28-LD52

[0232] The synthesis route of compounds LD2-LD13, LD15-LD26, LD28-LD52 refers to the synthesis of LD1.

[0233] Example 12: Synthesis of compound LD54

[0234] Compound 58 (100 mg, 0.16 mmol), compound 38 (96.5 mg, 0.16 mmol), HATU (72.5 mg, 0.19 mmol) were added into a reaction vial, dissolved in 2 mL of DMF, and then DIEA (41 mg, 0.32 mmol) was added dropwise under ice bath. After the addition was completed, the reaction was carried out at room temperature. After 1 hour, the reaction was completed. The reaction solution was directly purified by reverse phase preparative chromatography to obtain compound LD54 (112 mg). MS (ESI): (M+H) + Calcd 1166.4, Found 1166.5.

[0235] Example 12: Synthesis of compound LD55

[0236] Compound 58 (100 mg, 0.16 mmol), compound 44 (96.5 mg, 0.16 mmol), HATU (72.5 mg, 0.19 mmol) were added into a reaction vial, dissolved in 2 mL of DMF, and then DIEA (41 mg, 0.32 mmol) was added dropwise under ice bath. After the addition was completed, the reaction was carried out at room temperature. After 1 hour, the reaction was completed. The reaction solution was directly purified by reverse phase preparative chromatography to obtain compound LD55 (97 mg). MS (ESI): (M+H) + Calcd 1166.4, Found 1166.5.

[0237] Example 13: Synthesis of compounds LD53, LD56

[0238] The synthesis route of compounds LD53, LD56 is referenced to the synthesis of LD54.

[0239] Table 2. Linker-bioactive molecule number and structure

[0240] Example 14: General method for ADC sample preparation and DAR determination

[0241] The antibody of known sequence can be obtained by fermentation broth after protein expression method which is well known to those skilled in the art, and then high purity sample can be obtained by affinity chromatography, ion chromatography and other steps.

[0242] The antibody sample is diluted to about 10 mg / mL with a suitable buffer (consistent with the sample buffer), and an appropriate amount of reducing agent TCEP is added, and the number of equivalents is adjusted according to the target DAR, for example, when the target DAR is 2, 2-3 equivalents are added, when the target DAR is 4, 4-5 equivalents are added, and when the target DAR is 8, 8-10 equivalents are added, then the pH is adjusted to 7-7.4 with Tris buffer, and the reduction is carried out at room temperature for 1-1.5 hours. The antibody reduction intermediate can be monitored by CE-SDS, and after the antibody reduction is sufficient, an appropriate amount of saturated citric acid solution is first added to adjust the pH to about 6.5, then an excess of linker-bioactive molecule DMSO solution is added, so that the equivalent of linker-bioactive molecule is 3-4 times (target DAR is 2) / 4-6 times (target DAR is 4) / 10-15 times (target DAR is 8) of the equivalent of antibody, and the coupling reaction is carried out at room temperature for about 30 min. After the coupling is completed, the reaction solution is first filtered, and then ultrafiltration is carried out with a centrifugal concentration tube to remove excess linker-bioactive molecules and other small molecular impurities. After purification, the obtained sample is subjected to DAR determination by mass spectrometry.

[0243] The statistical data of all ADC samples are shown in Table 3:

[0244] Table 3. ADC drug number and structure

[0245] Test Example 1: In vitro cell activity test of payload

[0246] MV-4-11 (human acute monocytic leukemia cells) (medium: RPMI1640 + 10% FBS + 0.05 Mm β-me), THP-1 (human monocytic leukemia cells) (medium: RPMI1640 + 10% FBS + 0.05 Mm β-me), HL-60 (human acute promyelocytic leukemia cells) (medium: RPMI1640 + 20% FBS), MOLM-13 (human acute myeloid leukemia cells) (medium: RPMI1640 + 10% FBS), NCI-H929 (human myeloma cells) (medium: RPMI1640 + 15% FBS), SK-BR-3 (human breast cancer cells) (medium: McCoy's 5a + 15% FBS + 1% P / S) were purchased from Nanjing Kebai Biological Technology Co., Ltd. and were derived from the American Type Culture Collection (ATCC);

[0247] The experimental method is as follows:

[0248] Cell plating: when the tumor cells grow to a density of 80%-90%, the cells are digested with trypsin (0.25% Trypsin-EDTA, Gibco, item number: 25200072), centrifuged at 1000 rpm for 5 min, resuspended in complete medium and counted; adjust the cell density, take 75ul of cell suspension and inoculate in a black transparent bottom 96-well cell culture plate (Corning, item number: 3603), and adjust the cell density of MV-411, THP-1, HL-60, MOLM-13, NCI-H929 and SK-BR-3 cells to 3000, 3000, 4000, 3000, 3000 and 3000 cells per well respectively, and incubate in a carbon dioxide cell incubator overnight.

[0249] Sample preparation: add complete medium and different test samples (payload) corresponding to the cells in the first column of the U-shaped bottom 96-well plate, 200ul / well; the sample concentration is 2000nM, and the DMSO concentration is 2‰; add complete medium to the second column to the eleventh column, 200ul / well; take 50ul from the first column of samples and add to the second column, mix well, take 50ul from the second column of samples and add to the third column, and dilute by 5 times to the ninth column in this way.

[0250] Sample addition: the next day, after the cells adhere and grow, add the prepared payload samples of different concentrations to the 96-well cell culture plate, 75ul / well, 2 replicates for each sample, incubate in a 37°C, 5% carbon dioxide incubator for 4 days.

[0251] CellTiter-Lumi color development and plate reading: CellTiter-Lumi... TM The luminescent cell viability assay reagent was equilibrated to room temperature in the dark, and then 50 μL of CellTiter-Lumi was added to each well of a 96-well plate. TM (Item No.: C0065XL) Cell viability assay reagent. Place the sample on a shaker at 300 rpm and shake in the dark for 10 minutes to allow the cells to fully lyse. After the reaction is complete, measure the chemiluminescence intensity on a multi-functional microplate reader.

[0252] Data analysis: The data were processed and analyzed using Graphpad Prism 8.0 to generate cell viability curves and calculate IC50 values. The results are shown in Table 4.

[0253] Table 4. In vitro cell viability test of payload

[0254] Positive reference:

[0255] The same compound was obtained by referring to the synthesis method of the same compound in patent WO2021198965A1.

[0256] Conclusion: As can be seen from the statistical data in the table, the GSPT1 small molecule degrader (payload) studied in this invention has better half-cell killing activity than the positive reference molecule in a variety of cell types.

[0257] Test Example 2: Bioactive Molecular Proteomics Testing

[0258] (a) DC50 assay of GSPT1 protein degradation at the cellular level using payload:

[0259] Reagent source: The MV-4-11 cells (RPMI1640+10% FBS) used in the in vitro cellular protein degradation experiment of the payload of this invention were obtained from Nanjing Kebai Biotechnology and all cells were obtained from ATCC (American Type Culture Collection).

[0260] The experimental method is as follows:

[0261] (1). Cell collection and seeding: When the tumor cells MV-4-11 grow to a density of 80%-90%, centrifuge at 1000 rpm for 5 min, resuspend the cells in complete culture medium (RPMI 1640 + 10% FBS) and count them, and seed them into plates at 5E5 / 0.5 ml per well;

[0262] (2). Payload sample dilution: dilute Payload sample according to 1 uM final concentration, 5-fold gradient dilution;

[0263] (3). Add sample: add the diluted Payload sample and medium according to 1:1 (500ul+500ul) sample volume to the MV-4-11 cells in the plate, so that the final concentration of payload is 500nM, and the concentration of DMSO is 1 ‰;

[0264] (4). Incubate and collect cells: after the payload sample is incubated with the cells for 4 hours, collect the cells and label them;

[0265] (5). Centrifuge to remove supernatant: collect the cells of each sample at different concentrations in a 1.5ml EP tube, and wash the cells with PBS three times, 1000g, 5min centrifugation to remove the supernatant;

[0266] (6). Lysis of cells: add 500ul PBS to the EP tube containing the collected cells, and use the method of repeated freezing and thawing in liquid nitrogen and 37 degree water bath respectively, repeat freezing and thawing 3 times to lyse the cells;

[0267] (7). Centrifugation to remove supernatant: centrifuge the lysed cells at 10000xg, 4℃ for 10min, take the supernatant to a new EP tube, and label it;

[0268] (8). Protein quantification: BSA protein quantification, adjust the total protein concentration of cell lysate to 2mg / ml;

[0269] (9). Preheat the instrument: turn on JESS (Proteinsimple) 30min in advance;

[0270] (10). Sample and run: sample and run the digital Western test according to the operation steps of JESS instrument;

[0271] (11). Collect and process data: after the instrument runs, collect the data and use the obtained data to calculate the DC50 value, and the test data is shown in the following table 5:

[0272] Table 5. Payload in vitro inhibition activity

[0273] Positive reference:

[0274] Synthesized according to the synthesis method of the same compound in the reference patent WO2021198965A1.

[0275] (b) proteomic test of payload cellular level GSPT1 protein degradation

[0276] Reagent source: cells MV-4-11 (RPMI1640+10%FBS) for payload in vitro cellular level GSPT1 protein degradation experiment of the present application are from Nanjing Kebai Biological and cells are all from ATCC (American Type Culture Collection);

[0277] Test method:

[0278] (1). Cell collection and plating: when tumor cells MV-4-11 grow to a density of 80%-90%, centrifuge at 1000g for 5 min, resuspend the cells in complete culture medium (RPMI1640+10%FBS) and count, plate according to 2E7 cells in each T75 Flask resuspended in 15ml medium, three repeats for each sample;

[0279] (2). Payload dilution: according to the DC50 of each bioactive molecule measured by protein degradation experiment and combined with cell activity data, dilute the payload according to the final concentration of 100nM for positive reference, D130nM, D14 (3nM and 100nM) respectively, that is, the positive reference is 5*DC50, D1 is 10*DC50, D14 is 10*DC50 and 300*DC50, and the final concentration of DMSO is 1‰;

[0280] (3). Payload and cell incubation: add the above diluted payload to the culture bottle according to the ratio of cell suspension to payload diluent 1:1, and mark it, and treat it at 37℃ for 4 hours;

[0281] (4). Sample collection: after the incubation time reaches the designated incubation time, collect the sample in each Flask with a 50ml centrifuge tube, centrifuge at 1000g for 5 min, remove the supernatant, collect the cells, and wash the cells with PBS for 3 times;

[0282] (5). Cryopreservation of samples: remove the supernatant of the cell plate and freeze it at -80℃ for proteomic analysis;

[0283] (6). Proteomic analysis: after sample treatment before proteomic analysis, use DIA proteomic analysis method for data analysis, and the analysis and statistical results are shown in Table 6.

[0284] Table 6. Payload cellular level GSPT1 protein degradation results

[0285] Conclusion: (a) Under the same treatment time (4h), the DC 50 The activity is much higher than that of the positive control group; (b) Under the treatment condition of D1 and D14, the total down-regulated proteins are increased compared with the positive reference, but according to the condition of FC<0.5&P<0.05, the ranking of GSPT1 in D1 and D14 treatment groups is 3rd and 6th respectively, while the positive reference is 5 times of DC 50 Under the treatment condition of D1 and D14, the total down-regulated proteins are increased compared with the positive reference, but according to the condition of FC<0.5&P<0.05, the ranking of GSPT1 in D1 and D14 treatment groups is 3rd and 6th respectively, while the positive reference is 5 times of DC 50 Under the treatment condition of D1 and D14, the total down-regulated proteins are increased compared with the positive reference, but according to the condition of FC<0.5&P<0.05, the ranking of GSPT1 in D1 and D14 treatment groups is 3rd and 6th respectively, while the positive reference is 5 times of DC 50 Under the treatment condition of D1 and D14, the total down-regulated proteins are increased compared with the positive reference, but according to the condition of FC<0.5&P<0.05, the ranking of GSPT1 in D1 and D14 treatment groups is 3rd and 6th respectively, while the positive reference is 5 times of DC

[0286] Test Example 3: Cell activity test of ADC sample

[0287] MV-4-11 (RPMI1640+10%FBS), HL-60, purchased from Nanjing Kebai Biological and the cells are all from ATCC (American Type Culture Collection), NCI-N87 (human gastric cancer cells) (RPMI 1640 medium+15%FBS+1%P / S), NCI-N87-R (human gastric cancer DS8201 drug-resistant strain cells) (Huiyuan Biological, RPMI 1640 medium+10%FBS+2nM DS-8201) are used to evaluate the in vitro cell proliferation inhibition activity of the ADC of the present application.

[0288] The experimental method is as follows:

[0289] Cell plating: when the tumor cells grow to 80%-90% of the density, the cells are digested with trypsin (0.25% Trypsin-EDTA, Gibco, Cat No: 25200072), centrifuged at 1000 rpm for 5 min, and the cells are resuspended in complete medium and counted; adjust the cell density, take 75ul of cell suspension to inoculate in black transparent bottom 96-well cell culture plates (Corning, Cat No: 3603), and adjust the density of MV-4-11 and HL-60 cells to 3000 and 5000 / well, respectively, and incubate in a carbon dioxide cell incubator overnight.

[0290] ADC sample preparation: add complete medium and different samples to be tested in the first column of the U-shaped bottom 96-well plate, 250ul / well; the sample concentration is 200nM; add complete medium to the second to eleventh columns, 200ul / well; take 50ul from the first column of samples and add to the second column, mix well, take 50ul from the second column of samples and add to the third column, and so on, 5-fold dilution to the ninth column.

[0291] Sample addition: the next day, after the cells adhere and grow, add the prepared Payload samples of different concentrations to the 96-well cell culture plate, 75ul / well, 2 replicates for each sample, incubate in a 37°C, 5% carbon dioxide incubator for 4 days.

[0292] CellTiter-Lumi color development and plate reading: add 50ul of CellTiter-Lumi TM CellTiter-Lumi cell viability assay reagent is equilibrated to room temperature in the dark, then 50uL of CellTiter-Lumi (Cat No: C0065XL) cell viability assay reagent is added to each well of the 96-well plate and placed on a shaker at 300rpm in the dark for 10min to fully lyse the cells, and after sufficient reaction, the chemiluminescence intensity is measured on a multifunctional enzyme labeler.

[0293] Data analysis: the data is processed and analyzed by Graphpad Prism 8.0, the cell viability curve is drawn, and the IC50 value is calculated. The results are shown in Table 7:

[0294] Table 7. Cell activity test of ADC samples

[0295] From the results of Table 7, it can be seen that the activity of the ADC DAR4 samples was overall superior to that of DAR2 in both blood tumor MV-4-11 and HL-60 cells; the overall activity of the DAR2 / DAR4 samples was superior in MV-4-11 than in HL-60 in both blood tumor cells; the activity of ADC DAR8 was superior in NCI-N87 than in drug-resistant strain NCI-N87-R.

[0296] Test Example 4: ADC bystander effect test

[0297] The cell lines MV-4-11 (RPMI1640+10%FBS) and MDA-MB-468-LUC (DMEM+10%FBS+2ug / ml Puromycin) for the bystander effect test of the ADC of the present application were purchased from Nanjing Kebai Biological and the cell sources were all from ATCC (American Type Culture Collection).

[0298] Experimental procedure:

[0299] 1. Cell plating: When the tumor cells grow to a density of 80%-90%, the MDA-MB-468-LUC cells are digested with trypsin (0.25% Trypsin-EDTA, Gibco, Cat No: 25200072), centrifuged at 1000 rpm for 5 min, the cells are resuspended in complete medium and counted; the cell density is adjusted to 8E4 / mL, 37.5 μL of MDA-MB-468-LUC and 37.5 μL of MV4-11 cell suspension are inoculated in a black transparent bottom 96-well cell culture plate (Corning, Cat No: 3603), and the cell density of MDA-MB-468-luc is adjusted to 4E4 / mL, 75 μL of MDA-MB-468-LUC cells are inoculated in another black transparent bottom 96-well cell culture plate (Corning, Cat No: 3603), and incubated in a 37°C, 5% carbon dioxide cell incubator overnight.

[0300] 2. ADC sample preparation: Add complete medium and different samples to be tested in the first column of the U-shaped bottom 96-well plate, 250ul / well; the sample concentration is 200nM; add complete medium to the second to eleventh columns, 200ul / well; take 50ul from the first column of samples and add to the second column, mix well, take 50ul from the second column of samples and add to the third column, and so on, 5-fold dilution to the ninth column.

[0301] 3. Add sample: The next day after the cells adhered and grew, add the prepared Payload samples of different concentrations into the 96-well cell culture plate, 75ul / well, 2 repeats for each sample, incubate in a 37°C, 5% carbon dioxide incubator for 6 days.

[0302] 4. Read plate: CellTiter-Lumi and Bright-Glo TM Luciferase color development and plate reading: add CellTiter-Lumi TM and Bright-Glo TM Luciferase luminescence cell viability detection reagent to the room temperature in the dark, then add 50ul CellTiter-Lumi (item number: C0065XL) cell viability detection reagent to each well of the 96-well plate seeded with MDA-MB-468-Luc, and add 100ul Bright-Glo TM Luciferase detection reagent to each well of the 96-well plate seeded with MDA-MB-468-luc and MV4-1 mixed cells, and place it on a shaker at 300rpm in the dark for 10 minutes to fully lyse the cells. After sufficient reaction, measure the chemiluminescence intensity on a multifunctional microplate reader.

[0303] Table 8: ADC bystander effect test

[0304] Positive reference: The antibody is the same as ADC 10.

[0305] As can be seen from the results in Table 8, the cell killing activity of the ADC 10 sample of the present application on negative cells MDA-MB-468-Luc cells is 7.651nM, while the cell killing activity of the positive reference on MDA-MB-468-Luc is 0.9722nM, and the bystander effect is much higher than that of the ADC sample 10 of the present application. Starting from the main indication of the present application, in hematological tumors, bystander effect may bring safety toxicity to other blood cells and normal cells, therefore, from the perspective of bystander effect, the ADC 10 of the present application is superior to the positive reference.

[0306] Test Example 5: In vivo activity test of ADC sample

[0307] Select MV-4-11-luc cells to be inoculated into NCG mice by tail vein administration, fix the exposure time, and when the Total Flux [p / s] in the mice is about 3E7, randomly divide the mice into different groups, 5 mice in each group. Different drugs are injected by tail vein according to the pre-set dose. After administration, the mice are observed and measured regularly, and the Total Flux [p / s] in the mice is measured. The final fluorescence intensity and the body weight of the mice are measured at the end of the experiment, and the tumor inhibition rate is calculated. The test results are shown in Table 9 and Figure 1:

[0308] Table 9 In vivo activity test of ADC samples (MV-4-11-luc)

[0309] As can be seen from the in vivo efficacy results in Table 9, the TGI of the four candidate drug groups and the Xiazhi group is about 98-100% at a single needle 0.1 mpk dose. From the imaging data of the animals on D17 (Figure 2), the fluorescence imaging of the ADC14 group is the weakest, so the effect of ADC14 is the best in anti-tumor effect, and each experimental group has no significant effect on the body weight in the test period.

[0310] Select HL-60 cells to be inoculated into NOD SCID mice by subcutaneous injection, and after tumor formation, select mice with a tumor volume of about 120 cubic millimeters, and divide them into different groups, 5 mice in each group. Different drugs are injected by tail vein according to the pre-set dose. After administration, the mice are observed and measured regularly, and the body weight and tumor volume of the mice are measured. The tumor size and the body weight of the mice are measured at the end of the experiment, and the tumor inhibition rate is calculated. The test results are shown in Table 10 and Figure 3:

[0311] Table 10 In vivo activity test of ADC samples (HL-60)

[0312] As can be seen from the in vivo efficacy results in Table 10, except that the activity of the ADC57 DAR2 sample is poor, the in vivo efficacy activity TGI of the remaining samples is more than 95%, and each group has CR, the activity is better, and each experimental group has no significant effect on the body weight in the test period.

[0313] Sequence information related to the present application:

[0314] HER2

[0315] SYM001 (Trastuzumab) (WO03087131)

[0316] > Heavy chain (SEQ ID NO. 13)

[0317] Light chain (SEQ ID NO. 14)

[0318] CD33

[0319] SYM018 (CD33 AB) (Gemtuzumab-IgG1, see Orum patent WO2022254377)

[0320] Heavy Chain (SEQ ID NO. 15)

[0321] Light Chain (SEQ ID NO. 16)

Claims

1. An antibody-drug conjugate having the structure of formula (I) and its stereoisomers, prodrug, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, or isotopic label thereof: wherein, Ab is a ligand unit selected from an antibody (such as a monoclonal antibody) or an antigen-binding fragment, a small molecule ligand, a polypeptide; L is a linker unit, one end of which is connected to the ligand unit Ab, and the other end of which is connected to the bioactive molecule unit D’; D' is a bioactive molecule unit, the structure is as shown below: wherein, * represents covalent connection with the linker unit L; W 1 and W 2 are each independently selected from methylene and carbonyl, and W 1 and W 2 are not simultaneously methylene; R 1 selected from hydrogen, deuterium, halogen, cyano, amino, nitro, Ci-C4-alkoxy or Ci-C4-alkyl; each R 2 , R 3 , R 4 , R 5 is each independently selected from hydrogen, deuterium, C1-C4alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocyclyl, each n1, n2is independently selected from any integer between 0-6 (such as 0, 1, 2, 3, 4, 5, 6); W 3 is absent or selected from O, S or NR a wherein R a is selected from hydrogen, C1-C4alkyl or 3-6 membered cycloalkyl; n3, n4 are each independently selected from any integer from 1 to 3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9 is each independently selected from hydrogen, deuterium, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, 3-8 membered cycloalkyl, halogen, hydroxyl, C1-C6deuteroalkyl, C1-C6hydroxyalkyl; or, R 6 and R 7 together with the carbon atom to which they are attached form a 3-6 membered cycloalkylene; or R 6 and R 8 together with the carbon atom to which they are attached form a 4-6 membered cycloalkylene; n5, n6 are each independently selected from any integer from 0 to 3 (such as 0, 1, 2, 3), and n5, n6 are not simultaneously 0; m represents the molar ratio of the bioactive molecule unit to the ligand unit Ab (also known as DAR, i.e. drug antibody coupling ratio); m is 1 to 12 (the value is an integer or a decimal number (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)).

2. The antibody-drug conjugate having the structure of formula (I) according to claim 1, and stereoisomers, prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates or isotopically-labeled forms thereof, wherein the linker unit -L- is represented by the following formula: -L1-L2-L3-L4-, wherein * represents connection with the thiol group of Ab (such as a monoclonal antibody), and ** represents connection with L2; L3 is a polypeptide sequence selected from a peptide residue composed of 2-8 (such as 2, 3, 4, 5, 6, 7, 8) natural or unnatural amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from C1-C6 alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6 alkoxy, hydroxyl, amino, carboxyl, or C3-C8 cycloalkyl; wherein L1is selected from or Y is C1-C6 alkylene or -Z-C(O)-, wherein Z is C1-C6 alkylene or heteroalkylene containing 1-24 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) -OCH2CH2- structural units; In some embodiments, L2is a spacer selected from -L 2a -C(O)-, -L 2a -NR b -C(O)-L 2b -C(O)-, wherein L 2a selected from -C1-C8alkylene-, -C1-C8alkylene-C3-C8cycloalkylene-, -C6-C 14 arylene-, -C6-C 14 arylene-C1-C8alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C8alkylene-, a straight chain or branched chain heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, a straight chain or branched chain heteroalkylene-3-8 membered heterocyclyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, each of said alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene, heteroalkyl, heteroaryl, heterocyclyl being optionally substituted with one or more substituents independently selected from C1-C6alkyl, heteroalkyl of 1-6 carbon atoms, C1-C6alkoxy, hydroxyl, amino, carboxyl, or C3-C8cycloalkyl, said heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more (such as two or three) of N, O, or S; L 2b linear or branched heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) carbon atoms, R b hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, heteroalkyl of 2-8 carbon atoms, C6-C10 aryl, 5-6 membered heteroaryl, each of said alkyl, heteroalkyl, haloalkyl, aryl, heteroaryl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6 alkoxy, amino, or carboxyl, said heteroalkyl, heterocyclyl, heteroaryl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroatoms of said heteroalkyl, heterocyclyl, heteroaryl, heteroaryl being selected from one or more (such as two or three) of N, O, or S; 14 hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, heteroalkyl of 2-8 carbon atoms, C6-C10 aryl, 5-6 membered heteroaryl, each of said alkyl, heteroalkyl, haloalkyl, aryl, heteroaryl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, heteroalkyl of 2-6 carbon atoms, C1-C6 alkoxy, amino, or carboxyl, said heteroalkyl, heterocyclyl, heteroaryl containing 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroatoms of said heteroalkyl, heterocyclyl, heteroaryl, heteroaryl being selected from one or more (such as two or three) of N, O, or S; n7 is an integer from 0 to 6 (such as 0, 1, 2, 3, 4, 5, 6). L4 is a self-cleaving fragment selected from the group consisting of: or wherein * represents attachment to the carbonyl of L3 to form an amide bond, ** represents attachment to the oxygen atom of D'; R c selected from the group consisting of a linear or branched heteroalkyl comprising 1-50 (preferably 4-50, more preferably 4-24, even more preferably 8-24, most preferably 10-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) -OCH2CH2- structural units, a peptide chain comprising 4-50 (preferably 4-24, more preferably 8-24, most preferably 10-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) natural or non-natural amino acids, a linear or branched heteroalkyl comprising a monosaccharide, an oligosaccharide, or a polysaccharide; X is absent, -N(R d )CH2- or -L 4a -; Among them, R d Selected from hydrogen, C1-C3 alkyl, 3-6 membered cycloalkyl, straight-chain or branched heteroalkyl containing 4-50 (preferably 4-24, more preferably 6-24, most preferably 8-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) -OCH2CH2- or -CH2CH2SO2- structural units; - L 4a - the structure is R e and R f each independently is selected from H, C1-C3 alkyl, or R e and R f together with the atom to which they are attached form a 5-6 membered heterocyclyl; R g is selected from H, C1-C3 alkyl, 3-6 membered cycloalkyl, heteroalkyl of 2-6 carbon atoms, 5-6 membered heterocyclyl; said heterocyclyl contains 1-6 (preferably 1-4, such as 1, 2, 3, 4) heteroatoms, wherein the heteroatoms are selected from one or more (such as two or three) of N, O, or S; * represents connection with L1, and ** represents connection with L3. * represents connection with L2, and ** represents connection with L4.

3. The antibody-drug conjugate having the structure of Formula (I) according to any one of claims 1-2, and stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof, or isotopically-labeled versions thereof, wherein L2 is selected from the following structures: r is defined as in any of the preceding technical solutions, * represents connection with the thiol group of the ligand unit Ab (such as an antibody), and ** represents connection with the bioactive molecule D’; 4. The antibody drug conjugate having the structure of formula (I) as claimed in any one of claims 1 to 3, and stereoisomers, prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates or isotopically-labeled forms thereof, wherein L3 is selected from a peptide residue formed from 2 to 8 amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), C1-C6 alkyl substituted lysine (such as C1-C3 alkyl substituted lysine, such as methyl substituted lysine, ethyl substituted lysine, n-propyl substituted lysine, isopropyl substituted lysine) and the like; preferably L3 is selected from the following structures: Preferably, * represents connection with the thiol group of the antibody A, and ** represents connection with the hydroxyl group of the bioactive molecule D.

5. The antibody-drug conjugate having the structure of Formula (I) according to any one of claims 1-4, and stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof, or isotopically-labeled versions thereof, wherein L4 is selected from the following structures: wherein, * indicates attachment to L3, ** indicates attachment to D'; R c , R e , R f , R g v is selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

6. The antibody drug conjugate having the structure of Formula (I) of any one of claims 1-5, and stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof, or isotopically-labeled versions thereof, wherein R c is selected from a hydrophilic segment; preferably, R c is selected from the following structures: wherein r, s are each independently selected from an integer from 1 to 50 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50); further preferably, R c is selected from the following structures:

7. An antibody-drug conjugate having the structure of Formula (I) according to any one of claims 1-6, and stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof, or isotopically-labeled versions thereof, wherein L is selected from the following structures: wherein 8. The antibody-drug conjugate having the structure of formula (I) according to any one of claims 1-7, and stereoisomers, prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates or isotopically-labeled forms thereof, wherein the bioactive molecule unit D’: ​ ​ W 1 and W 2 are each independently selected from methylene and carbonyl, and W 1 and W 2 are not simultaneously methylene; R 1 is selected from hydrogen, deuterium, halogen, cyano, amino, nitro, C1-C2alkoxy, or C1-C2alkyl; each R 2 , R 3 , R 4 , R 5 is independently selected from hydrogen, C1-C4alkyl, each n1, n2 is independently selected from any integer from 0 to 3 (such as 0, 1, 2, 3); W 3 is absent or selected from O or NR a , wherein R a is selected from hydrogen, C1-C4alkyl, or 3-6 membered cycloalkyl; each n3, n4 is independently selected from any integer from 1 to 3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9 is independently selected from hydrogen, deuterium, C1-C4haloalkyl, C1-C4alkyl, 3-5 membered cycloalkyl; or R 6 and R 7 together with the carbon atom to which they are attached form a 3-4 membered cycloalkylene; or R 6 and R 8 together with the carbon atom to which they are attached form a 4-6 membered cycloalkylene, wherein each n5, n6 is independently selected from any integer from 0 to 2 (such as 0, 1, 2), and n5, n6 are not simultaneously 0; Preferably, W 1 is carbonyl, W 2 is methylene; R 1 is selected from halogen; each R 2 , R 3 , R 4 , R 5 is independently selected from hydrogen, methyl, n1, n2 are each independently selected from any integer between 0 and 3 (e.g. 0, 1, 2, 3); W 3 is absent or selected from O or NR a , wherein R a is selected from hydrogen, C1-C2 alkyl or 3-4 membered cycloalkyl; n3, n4 are each independently selected from any integer between 1 and 3 (e.g. 1, 2, 3); each R 6 , R 7 , R 8 , R 9 is independently selected from hydrogen, C1-C4 haloalkyl, C1-C4 alkyl, 3-5 membered cycloalkyl; or R 6 and R 7 together with the carbon atom to which they are attached form a 3-4 membered cycloalkylene group; or R 6 and R 8 together with the carbon atom to which they are attached form a 4-6 membered cycloalkylene group, wherein n5, n6 are each independently selected from any integer between 0 and 2 (e.g. 0, 1, 2), and n5, n6 are not simultaneously 0. Further preferred, W 1 is carbonyl, W 2 is methylene; R 1 is selected from fluorine, chlorine, bromine; each R 2 , R 3 , R 4 , R 5 are each independently selected from hydrogen, n1, n2 are each independently selected from any integer from 0 to 3 (such as 0, 1, 2, 3); W 3 is absent or selected from O or NR a , wherein R a is selected from hydrogen, methyl, ethyl or cyclopropyl, cyclobutyl; n3, n4 are each independently selected from any integer from 1 to 3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9 are each independently selected from hydrogen, trifluoromethyl, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, cyclopropanyl, cyclobutanyl, cyclopentanyl, or R 6 and R 7 together with the carbon atom to which they are attached form a cyclopropanediyl or cyclobutandiyl, or R 6 and R 8 together with the carbon atom to which they are attached form a cyclobutandiyl, cyclohexandiyl, wherein n5, n6 are each independently selected from any integer from 0 to 2 (such as 0, 1, 2), and n5, n6 are not simultaneously 0; Further preferred, W 1 is carbonyl, W 2 is methylene; R 1 is selected from chlorine; each R 2 , R 3 , R 4 , R 5 is independently selected from hydrogen; n1, n2 are each independently selected from any integer from 0 to 3 (such as 0, 1, 2, 3); W 3 is absent or selected from O; n3, n4 are each independently selected from any integer from 1 to 3 (such as 1, 2, 3); each R 6 , R 7 , R 8 , R 9 is independently selected from hydrogen, trifluoromethyl, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, cyclopropanyl, cyclobutanyl, cyclopentan- yl, or R 6 and R 7 together with the carbon atoms to which they are attached form a cyclopropanediyl group, or R 6 and R 8 together with the carbon atoms to which they are attached form a cyclobutanediyl group, a cyclohexanediyl group, wherein n5, n6 are each independently selected from any integer from 0 to 2 (such as 0, 1, 2), and n5, n6 are not simultaneously 0; Further preferred, W 1 is carbonyl, W 2 is methylene; R 1 is selected from chloro, R 2 , R 3 are each independently selected from hydrogen; n1 is selected from 3, n2 is selected from 0; W 3 is selected from O; n3, n4 are each independently selected from 1 or 2; each R 6 , R 7 , R 8 , R 9 are each independently selected from hydrogen, trifluoromethyl, methyl, cyclopropanes, or, R 6 and R 7 together with the carbon atom to which they are attached form a cyclopropane group, or R 6 and R 8 together with the carbon atom to which they are attached form a cyclobutane group, a cyclohexane group, wherein n5, n6 are each independently selected from any integer from 0 to 2 (e.g. 0, 1, 2), and n5, n6 are not simultaneously 0.

9. An antibody drug conjugate having a structure of Formula (I) according to any one of claims 1-8, and stereoisomers, prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates, or isotopically-labeled forms thereof, selected from the following structures: wherein Ab is a ligand unit as described in claims 1-8; m represents the molar ratio of the bioactive molecule unit to the ligand unit Ab (also known as DAR, i.e. drug antibody coupling ratio), which is 1 to 12 (the value is an integer or a decimal, for example an integer or a decimal of 1-8, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; when m is a decimal, it represents the average molar ratio of the bioactive molecule unit to the ligand unit Ab).

10. An antibody conjugated drug having the structure of formula (I) as described in any one of claims 1-9, and stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof, or isotopically-labeled versions thereof, Ab is selected from a mAb, a diabody or an antigen binding fragment thereof targeting HER2 (ErbB2), CD33, or BCMA; Preferably, Ab is a mAb, a diabody or an antigen binding fragment thereof targeting HER2, which comprises a HER2 targeting heavy chain variable region and a light chain variable region, both of which comprise 3 CDRs, the heavy chain variable region comprises: CDR1-H having the sequence as set forth in SEQ ID NO: 1 or a functional variant thereof; CDR2-H having the sequence as set forth in SEQ ID NO: 2 or a functional variant thereof; and CDR3-H having the sequence as set forth in SEQ ID NO: 3 or a functional variant thereof; the light chain variable region comprises: CDR1-L having the sequence as set forth in SEQ ID NO: 4 or a functional variant thereof; CDR2-L having the sequence as set forth in SEQ ID NO: 5 or a functional variant thereof; and CDR3-L having the sequence as set forth in SEQ ID NO: 6 or a functional variant thereof; or preferably, Ab is a mAb, a diabody or an antigen binding fragment thereof targeting CD33, which comprises a CD33 targeting heavy chain variable region and a light chain variable region, both of which comprise 3 CDRs, the heavy chain variable region comprises: CDR1-H having the sequence as set forth in SEQ ID NO: 7 or a functional variant thereof; CDR2-H having the sequence as set forth in SEQ ID NO: 8 or a functional variant thereof; and CDR3-H having the sequence as set forth in SEQ ID NO: 9 or a functional variant thereof; the light chain variable region comprises: CDR1-L having the sequence as set forth in SEQ ID NO: 10 or a functional variant thereof; CDR2-L having the sequence as set forth in SEQ ID NO: 11 or a functional variant thereof; and CDR3-L having the sequence as set forth in SEQ ID NO: 12 or a functional variant thereof.

11. A biologically active molecule D represented by formula (II): ###00003### (II) or stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof, or isotopically-labeled versions thereof of the bioactive molecule; wherein W 1 , W 2 , W 3 , R 1 - R 9 , n1-n6 are as described above for the compound of the formula (I) according to claim 1 or 8.

12. The biologically active molecule of claim 11 of the formula (II) and stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof, or isotopically-labeled versions thereof, having the particular structure: ###00006### 13. A linker-bioactive molecule represented by formula (III): L1’-L2-L3-L4-D’ (III) or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled form thereof. wherein L1’ is a precursor of a linker unit capable of being converted to L1 of formula I, thereby forming a covalent bond to Ab of formula I; L2, L3, L4, D’ are defined as in formula I; Preferably, the linker unit precursor L1' is selected from or 14. The conjugate of claim 13, the bioactive molecule is selected from the following structures:

15. A pharmaceutical composition comprising an antibody drug conjugate having the structure of formula (I) as described in any one of claims 1-10, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled form thereof; or a bioactive molecule unit having the structure of formula (II) as described in any one of claims 11-12, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled form thereof; or a linker-bioactive molecule having the structure of formula (III) as described in any one of claims 13-14, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled form thereof, and optionally one or more pharmaceutically acceptable excipients.

16. Use of an antibody drug conjugate having the structure of formula (I) as described in any one of claims 1-10, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled form thereof; or a bioactive molecule unit having the structure of formula (II) as described in any one of claims 11-12, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled form thereof; or a linker-bioactive molecule having the structure of formula (III) as described in any one of claims 13-14, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, or an isotopically-labeled form thereof; or a pharmaceutical composition as described in claim 15, in the manufacture of a medicament for the treatment and / or prevention of a cell proliferative disorder (e.g., cancer).

17. The use of claim 16, wherein the cancer comprises a liver cancer, a kidney cancer, a lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), a stomach cancer, an esophageal cancer, a urethral cancer, a bladder cancer, a colon cancer, a rectal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a pancreatic cancer, a melanoma, a hematological tumor or glioblastoma multiforme, a lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, or relapsed anaplastic large cell lymphoma), a cervical cancer, a uterine cancer, an endometrial cancer, a salivary gland cancer, a glioma, a neuroblastoma, a sarcoma, a colorectal cancer, a leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia, acute monocytic leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), a bone cancer, a skin cancer, a thyroid cancer, and the like.

Images