Drug conjugate of eribulin derivative, its preparation method and medical application

Abstract

The present disclosure relates to eribulin derivative drug conjugates, their preparation methods, and their pharmaceutical applications. Specifically, the present disclosure provides antibody-drug conjugates containing an eribulin derivative drug moiety. The present disclosure also relates to methods for treating cancer by administering the antibody-drug conjugates provided herein.

Description

[Technical Field]

[0001] The present disclosure relates to a drug conjugate of an eribulin derivative, its preparation method and its pharmaceutical application. [Background technology]

[0002] Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active drugs via stable chemical linker compounds, taking advantage of the specificity of antibodies in binding to surface antigens on normal and tumor cells and the high performance of drugs, while avoiding the relatively low therapeutic efficacy of the former and the excessive toxicity and side effects of the latter. This means that, compared with traditional chemotherapy drugs, antibody drug conjugates can bind precisely to tumor cells and reduce their impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

[0003] In 2000, the first antibody-drug conjugate, Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), was approved for marketing by the US FDA for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25(7):686; US4970198; US5079233; US5585089; US5606040; US5693762; US5739116; US5767285; US5773001).

[0004] In August 2011, Adcetris (brentuximab vedotin, Seattle Genetics) was approved under the US FDA Fast Track program for the treatment of Hodgkin's lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7):778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). Adcetris® is a novel targeted ADC drug that directly targets CD30 on lymphoma cells, causing endocytosis and inducing apoptosis of tumor cells.

[0005] Both Mylotarg and Adcetris are targeted therapies aimed at hematologic tumors, which have relatively simple histological structures compared to solid tumors. In February 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the U.S. FDA for the treatment of patients with advanced or metastatic breast cancer that is HER2-positive and resistant to trastuzumab (Herceptin) and paclitaxel (WO2005037992; US8088387). Kadcyla is the first drug approved by the U.S. FDA for the treatment of solid tumors.

[0006] Microtubules are powerful filamentous cytoskeletal proteins involved in various cellular functions, including intracellular movement and transport, cell signaling, and maintaining cell shape. Microtubules also play a crucial role in mitotic cell division by forming the mitotic spindle, which is necessary for the division of chromosomes into two daughter cells. Most of the biological functions of microtubules in all cells are regulated by polymerization dynamics, which are carried out by the reversible, noncovalent binding of α- and β-tubulin dimers to both ends of the microtubule. This dynamic behavior and the resulting control over microtubule length are essential for the proper function of the mitotic spindle. Consequently, even small changes in microtubule dynamics can engage the axial checkpoint, disrupt cell cycle progression during mitosis, and subsequently cause cell death (Mukhtar et al. (2014) Mol. Cancer Ther. 13:275-84). Because cancer cells divide rapidly, they are typically more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. Therefore, tubulin inhibitors and other microtubule-targeting agents are expected to be therapeutic agents for cancer (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790-803). Summary of the Invention

[0007] The present disclosure provides an antibody-drug conjugate (ADC) having the structure shown in formula (I) or a pharmaceutically acceptable salt or solvate thereof: [ka] where Ab is an antibody or an antigen-binding fragment thereof; L is a linker covalently linking Ab to D, and k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any number between any two numbers); -D is as shown in the following formula: [ka] Among them, R1a is hydrogen, alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; cycloalkyl groups (e.g., C 3-8 a cycloalkyl group, including but not limited to a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group, an aryl group, and a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the aryl group, and the heteroaryl group are each independently optionally substituted with an alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 an alkoxy group, substituted with one or more substituents selected from a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a halogen (e.g., fluorine, chlorine, bromine), a deuterium, an amino group, a cyano group, a nitro group, a hydroxy group, a hydroxyalkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, and a heteroaryl group, and preferably a methyl group; R 1b is hydrogen, alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups; cycloalkyl groups (e.g., C 3-8 a cycloalkyl group, including but not limited to a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group, an aryl group, and a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the aryl group, and the heteroaryl group are each independently optionally substituted with an alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6an alkoxy group, substituted with one or more substituents selected from methoxy, ethoxy, propoxy, and isopropoxy groups, halogen (e.g., fluorine, chlorine, and bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, and preferably hydrogen; Or, R 1a and R 1b together with the atom to which they are connected form a 5- to 8-membered heterocycloalkyl group, said heterocycloalkyl group optionally being an alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 Alkoxy groups, including, but not limited to, methoxy, ethoxy, propoxy, and isopropoxy groups; halogens (e.g., fluorine, chlorine, and bromine); deuterium; amino groups; cyano groups; nitro groups; hydroxy groups; hydroxyalkyl groups; and cycloalkyl groups (e.g., C 3-8 cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, and cyclohexyl groups, substituted with one or more substituents selected from the group consisting of heterocycloalkyl groups, aryl groups, and heteroaryl groups; and R 1a and R 1b is not hydrogen at the same time.

[0008] In some embodiments, in the antibody-drug conjugate, R in -D 1a and R 1b together with the atom to which they are attached form a 6- to 8-membered heterocycloalkyl group. In some embodiments, in the antibody-drug conjugate, R in -D 1a is selected from methyl groups. In some embodiments, in the antibody-drug conjugate, R in -D 1a , R 1b are each independently selected from methyl groups.

[0009] In some embodiments, in the antibody-drug conjugate, -D is [ka] is.

[0010] In some embodiments, the antibody-drug conjugate Ab-(LD) k In the formula, k is selected from 1 to 10 and may be an integer or a decimal number.

[0011] In another aspect, the disclosure further provides an antibody-drug conjugate (ADC) having the structure shown in formula (I) or a pharmaceutically acceptable salt or solvate thereof: [ka] Among them, -D is [ka] Selected from.

[0012] In some embodiments, the linker is stable extracellularly and can remain intact when the ADC is in the extracellular environment, but can be cleaved upon internalization into a cell, such as a cancer cell. In some embodiments, when the ADC enters a cell that expresses an antigen specific for the antibody portion of the ADC, the drug moiety of the eribulin analog is cleaved from the antibody moiety, releasing the unmodified eribulin analog upon cleavage.

[0013] In some embodiments, the cleavable portion of the linker is a cleavable peptide moiety. In some embodiments, relative to ADCs containing other cleavable moieties, ADCs containing a cleavable peptide moiety exhibit lower aggregation levels, improved drug-antibody ratios, increased on-target killing of cancer cells, reduced off-target killing of non-cancer cells, and / or relatively high drug loading (p). In some embodiments, the addition of a cleavable moiety to a non-cleavable linker increases cytotoxicity and / or efficacy. In some embodiments, the increased efficacy and / or cytotoxicity is increased efficacy and / or cytotoxicity in cancers that express moderate levels of the antigen targeted by the antibody portion of the ADC (e.g., moderate levels of FRA expression). In some embodiments, the cleavable peptide moiety is enzymatically cleavable, and the linker is an enzymatically cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, compared to other cleavage mechanisms, an enzymatically cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties described above.

[0014] In some embodiments, the linker comprises an amino acid unit, and the amino acid unit preferably comprises a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0015] In some embodiments, the linker of the Amino Acid units of the present disclosure is: [ka] Selected from.

[0016] In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit). In some embodiments, ADCs comprising Val-Cit exhibit improved stability, reduced off-target killing of cells, increased on-target killing of cells, lower aggregation levels, and / or higher drug loading relative to ADCs comprising other amino acid units or other cleavable moieties. On the other hand, some embodiments provide linkers that contain a cleavable sulfonamide moiety, said linkers being cleavable under reducing conditions.

[0017] In some embodiments, the linker comprises a cleavable disulfide moiety, wherein the linker is cleavable under reducing conditions. In contrast, the linker of the antibody conjugate of the present disclosure comprises at least one spacer unit linking eribulin derivative D to the cleavable moiety. In some embodiments, the linker comprises a spacer unit bound to D.

[0018] In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB), [ka] Includes: In some embodiments, the spacer unit is p-aminobenzoyl, [ka] Includes:

[0019] In some embodiments, the spacer unit is [ka] Including, wherein Z1 to Z5 are each independently selected from carbon atoms or nitrogen atoms; R 14 is selected from alkyl groups, cycloalkyl groups, aryl groups, and heteroaryl groups, and the alkyl groups, cycloalkyl groups, aryl groups, and heteroaryl groups are each independently optionally substituted with one or more substituents selected from alkyl groups, alkoxy groups, halogen, deuterium, amino groups, cyano groups, nitro groups, hydroxy groups, hydroxyalkyl groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, and heteroaryl groups; R 11 and R 12 are independently hydrogen, deuterium, and C 1-6 Alkyl group, C 3-6 cycloalkyl groups, preferably hydrogen, or R 11 and R 12 form a C3-6 cycloalkyl group together with the carbon atom to which they are attached, X is selected from -O- or -NH-, and L is an integer selected from 1 to 4; Q is VE-, which provides a glycosidic bond that can be cleaved by intracellular glycosidases, and E is -O-, -S-, or -NR 13 -Selected from R 13 is selected from hydrogen or a methyl group, and V is [ka] Among them, R 15 is selected from —COOH or CH 2 OH. In some embodiments, V is selected from —COOH.

[0020] In some embodiments, the spacer unit is [ka] Including, Z1, Z3, Z4, X, Q, R 11 , R 12 , R 14 is as described above.

[0021] In some embodiments, the spacer unit is -(CR a R b ) m1 -O(CR a R b ) m2 -CR 8 R 9 -C(O)-, -(CR a R b ) m1 NH-(CR a R b ) m2 -CR 8 R 9 -C(O)-, -(CR a R b ) m1 OCR 8 R 9 (CR a R b ) m2 -, -(CR a R b ) m1 OCR 8 R 9 -C(O)-, -(CR a R b ) m1 -O-(CR a R b ) m2 C(O)- or -(CR a R b ) m1 -S-(CR a R b ) m2 -CR 8 R 9 -C(O)-; Among them, R a and R b are the same or different and are each independently selected from hydrogen, deuterium, halogen, or alkyl groups; R 8 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, R 9 is hydrogen, haloalkyl group or C 3-6cycloalkyl groups, preferably hydrogen, or R 8 and R 9 are C together with the carbon atoms connected to them. 3-6 m1 and m2 each independently form a cycloalkyl group; m1 and m2 each independently represent 0, 1, 2, or 3;

[0022] In some embodiments, the spacer unit is -(CH2)3-C(O)-, -CH2-O-CH2-C(O)-, -(CH2)2-O-CH2-C(O)-, [ka] The present invention includes a portion selected from the group consisting of:

[0023] Meanwhile, in the antibody-drug conjugates (ADCs) of the present disclosure, LD is a chemical moiety represented by the formula: -Str-(Pep)-Sp-D Str is the stretch unit covalently attached to Ab; Sp is a spacer unit, Pep is selected from an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety such as: [ka] wherein W is -NH-heterocycloalkyl- or heterocycloalkyl group, and Y is a heteroaryl group, an aryl group, or -C(O)C 1-6 Alkylene group, C 2-6 Alkenylene group, C 1-6 Alkylene group or -C 1-6 alkylene-NH-, Each R 2 is independently C 1-10 Alkyl group, C 2-10 Alkenyl group, C 1-6 Alkylene-NH2, -(C 1-10 alkylene)NHC(NH)NH2 or -(C 1-10 alkylene)NHC(O)NH2; R 3and R 4 are independently H, C 1-10 Alkyl group, C 2-10 alkenyl, arylalkyl, or heteroarylalkyl group, or R 3 R 4 With C 3-7 capable of forming a cycloalkyl group, R 5 and R 6 are each independently C 1-10 Alkyl group, C 2-10 Alkenyl groups, arylalkyl groups, heteroarylalkyl groups, (C 1-10 alkylene)OCH2-, or R 5 R 6 With C 3-7 A cycloalkyl ring can be formed.

[0024] In some embodiments, Y in the antibody-drug conjugate (ADC) is [ka] Selected from.

[0025] Meanwhile, Str in the antibody-drug conjugate (ADC) is selected from the chemical moieties shown in the following formula: [ka] Among them, R 7 -W1-C(O)-, -C(O)-W1-C(O)-, -(CH2CH2O) p1 C(O)-, -(CH2CH2O) p1 CH2C(O)-, -(CH2CH2O) p1 CH2CH2C(O)-, where W1 is C 1-8 Alkylene group, C 1-8 alkylene-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, said heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O or S, among which: 1-8The alkylene group, cycloalkyl group, and straight-chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, deuterium, hydroxyl, cyano, amino, alkyl, haloalkyl, deuterated alkyl, alkoxy, and cycloalkyl groups; L 1 Ha-NR 10 (CH2CH2O) p1 CH2CH2C(O)-, -NR 10 (CH2CH2O) p1 CH2C(O)-, -S(CH2) p1 C(O)-, -(CH2) p1 C(O)- or a chemical bond, preferably a chemical bond, wherein p1 is an integer of 1 to 20; R 10 is selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0026] In some embodiments, C 1-8 The alkylene-cycloalkyl group is a methylene-cyclohexyl group: [ka] , ethylene-cyclohexane group: [ka] , methylene-cyclopentyl group: [ka] Selected from. In some embodiments, the linker may include at least one polyethylene glycol (PEG) moiety. The PEG moiety may be, for example, -(PEG) p1 -, [ka] wherein p1 is an integer of 1 to 20, for example [ka] is.

[0027] In some embodiments, the stretch unit in the linker comprises (PEG). In some embodiments, despite the shorter linker length, ADCs comprising shorter stretch units (e.g., (PEG)) exhibit lower aggregation levels and / or higher drug loading relative to ADCs comprising longer stretch units (e.g., (PEG)).

[0028] In some embodiments, the Str of the antibody-drug conjugate [ka] R in 7 is C 1-6 It is selected from alkyleneC(O)-, -(CH2-CH2O)2C(O)-, -(CH2-CH2O)2CH2C(O)-, -(CH2-CH2O)2CH2CH2C(O)-, -(CH2-CH2O)2CH2CH2C(O)-, -(CH2-CH2O)3C(O)- and -(CH2-CH2O)4C(O)-.

[0029] In some embodiments, the Str of the antibody-drug conjugate [ka] R in 7 Ha-C 1-8 It is selected from alkylene-cycloalkyl-C(O)-, -(CH2-CH2O)4CH2C(O)-, and -(CH2-CH2O)6CH2C(O)-.

[0030] In some embodiments, in the antibody-drug conjugate, the linker L is selected from the group consisting of maleimide-(PEG)2-Val-Cit, maleimide-(PEG)6-Val-Cit, maleimide-(PEG)8-Val-Cit, maleimide-(PEG)4-CH2CH2C(O)-Val-lys, maleimide-(CH2)5-Val-Cit, maleimide-(CH2)5-Val-lys, maleimide-(CH2)5-Gly-Gly-Phe-Gly, maleimide-(PEG)2-Ala-Ala-Asn, maleimide-(PEG)6-Ala-Ala-Asn, maleimide-(PEG)8-Ala-Ala-Asn, maleimide-(PEG)4-triazole-(PEG)3-sulfonamide, maleimide-(PEG) 2- CH2CH2C(O)-Val-lys, maleimide-(PEG)4-triazole-(PEG)3-sulfonamide or Mal-(PEG)4-triazole-(PEG)3-disulfide. In some embodiments, the linker L in the antibody-drug conjugate is maleimide-(PEG)4-CH2C(O)-Gly-Gly-Phe-Gly, maleimide-(PEG)2-CH2CH2C(O)-Gly-Gly-Phe-Gly, maleimide-(PEG)6-CH2C(O)-Gly-Gly-Phe-Gly-, maleimide-(CH2)5C(O)-Gly-Gly-Phe-Gly-, maleimide-C 1-8 Includes alkylene-cycloalkyl-C(O)—NH(CHCHO)CHC(O)-Gly-Gly-Phe-Gly-, maleimido-(PEG)-CHC(O)-Gly-Gly-Phe-Gly-, maleimido-(PEG)-CHCHC(O)-Val-Cit-, maleimido-(PEG)-Gly-Gly-Phe-Gly-, maleimido-(PEG)-CHC(O)-Val-Cit-, maleimido-(PEG)-CHC(O)-Val-Cit-, maleimido-(PEG)-CHC(O)-Val-Cit-, and maleimido-(PEG)-CHC(O)-Val-Cit-.

[0031] Meanwhile, in some embodiments, the antibody-drug conjugate is provided as follows: Str is selected from the chemical moieties shown in the formula: [ka] , among which, R 8 is C 1-10 Alkylene group, C 2-10 Alkenylene groups, (C 1-10 alkylene)O-, N(R d )-(C 2-6 alkylene)-N(R d ) and N(R d )-(C 2-6 alkylene), and each R d are independently H or a C1-C6 alkyl group.

[0032] In some embodiments, in the antibody-drug conjugate, L D is represented by a formula selected from the following: [ka] , among which, R 2 is a C alkylene group, (C alkyl)NHC(NH)NH or (C alkylene)NHC(O)NH; [ka] , among which, R 2 is C 1-6 Alkyl groups, (C 1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, [ka] , among which, R 2 is C 1-6 Alkyl group, C 2-6 Alkenylene groups, (C 1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, [ka] , among which, R 2 is C 1-6 Alkyl group, C2-6 Alkenylene groups, (C 1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, [ka] , among which, R 2 is C 1-6 Alkyl groups, (C 1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, and R 5 R 6 With C 3-7 forming a cycloalkyl ring, [ka] , among which, R 2 is C 1-6 Alkyl groups, (C 1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, and R 5 R 6 With C 3-7 forming a cycloalkyl ring, W, Str, and D are as described above.

[0033] In some embodiments, the antibody-drug conjugate (ADC) of the present disclosure is represented by the formula: [ka] , among which, R 2 is C 1-6 alkylene-NH2, (C1-6 alkylene)NHC(NH)NH2, or (C1-6 alkylene)NHC(O)NH2, k is selected from 1 to 10 and may be an integer or a decimal point, and p2 is an integer selected from 2 to 6; [ka] , among which, R 2 is C 1-6 Alkylene-NH2, (C1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6, [ka] , among which, R 2 is C 1-6 Alkylene-NH2, (C 1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, and R 5 and R 6 C 3-7 forming a cycloalkyl ring, k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6; [ka] , among which, R 2 is C 1-6 Alkylene-NH2, (C 1-6 alkylene)NHC(NH)NH2 or (C 1-6 alkylene)NHC(O)NH2, and R 5 and R 6 C 3-7 forming a cycloalkyl ring, k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6; Y, R 3 , R 4 , Ab, and D are as defined above.

[0034] Meanwhile, some embodiments provide antibody-drug conjugates (ADCs) represented by the following formula: [ka] , among which, R 8 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 9 is hydrogen, haloalkyl group or C3-6 cycloalkyl groups, preferably hydrogen, or R 8 and R 9 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6; [ka] , among which, R 8 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 9 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 8 and R 9 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2; [ka] , k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6; [ka] , k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6; [ka] , k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, and p3 is selected from 0, 1, or 2; [ka] , k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, and p3 is selected from 0, 1, or 2; [ka] , k is selected from 1 to 10 and may be an integer or a decimal, p1 is selected from 2, 4, 6, or 8, [ka] , k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, and p3 is selected from 0, 1, or 2; [ka] , k is selected from 1 to 10 and may be an integer or a decimal, p1 is selected from 2, 4, 6, or 8, [ka] , k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, and p3 is selected from 0, 1, or 2; [ka] , k is selected from 1 to 10 and may be an integer or a decimal, p2 is selected from 2, 4, 6, or 8, [ka] , k is selected from 1 to 10 and may be an integer or a decimal, p2 is selected from 2, 4, 6, or 8, [ka] , k is selected from 1 to 10 and may be an integer or a decimal, p2 is selected from 2, 4, 6, or 8, [ka] , k is selected from 1 to 10 and may be an integer or a decimal, p2 is selected from 2, 4, 6, or 8, [ka] , k is selected from 1 to 10 and may be an integer or a decimal, p2 is selected from 2, 4, 6, or 8, [ka] , k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, and p3 is selected from 0, 1, or 2; [ka] , k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, and p3 is selected from 0, 1, or 2; [ka] , among which, R 8 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 9 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 8 and R 9 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6; [ka] , among which, R 8 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 9 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 8 and R 9 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, k is selected from 1 to 10 and may be an integer or a decimal, and p2 is an integer selected from 2 to 6; [ka] , among which, R 8 is hydrogen, haloalkyl group or C3-6 cycloalkyl groups, preferably hydrogen; R 9 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 8 and R 9 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2; [ka] , among which, R 8 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 9 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 8 and R 9 are C together with the carbon atoms connected to them. 3-6 It forms a cycloalkyl group, k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2.

[0035] In some such embodiments, the antibody-drug conjugate (ADC) has the formula: [ka] [ka] [ka] [ka] [ka] In this case, k is selected from 1 to 10 and may be an integer or a decimal. 1ais preferably selected from methyl groups, R 1b is preferably selected from hydrogen.

[0036] Meanwhile, the antibody in the antibody-drug conjugate (ADC) of the present disclosure is selected from a mouse antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. In some embodiments, the antibody or antigen-binding fragment thereof in the antibody-drug conjugate (ADC) is an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis antibody, ...HER4 (ErbB4) antibody, an anti-HER2 (ErbB2) antibody, an anti-HER2 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-HER2 (ErbB2) antibody, an anti-HER2 (ErbB3) antibody, an anti-HER2 ( The antibody is selected from an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, an anti-SLC44A4 antibody, an anti-CD79 antibody, an anti-TROP-2 antibody, an anti-CD79B antibody, an anti-mesothelin antibody, or an antigen-binding fragment thereof.

[0037] In some embodiments, the antibody in the antibody-drug conjugate (ADC) is a known antibody selected from, but not limited to, trastuzumab, pertuzumab, nimotuzumab, enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab vedotin, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, and glematumumab, or an antigen-binding fragment thereof.

[0038] In some embodiments, the antibody in the antibody-drug conjugate (ADC) is selected from an anti-CD79B antibody or an antigen-binding fragment thereof, and comprises an antibody heavy chain variable region and / or an antibody light chain variable region, wherein: The antibody heavy chain variable region is 1) HCDR1, HCDR2 and HCDR3 represented by SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; or 2) HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. and / or the antibody light chain variable region is 1) LCDR1, LCDR2, and LCDR3 represented by SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or 2) Contains LCDR1, LCDR2 and LCDR3 represented by SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

[0039] In some embodiments, the anti-CD79B antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, and includes any one of the following (I) to (II): 1) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 represented by SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; 2) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; and and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 represented by SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

[0040] [Table 1]

[0041] In some embodiments, the anti-CD79B antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region is 1) a sequence represented by SEQ ID NO:3 or having at least 90%, 95%, 98%, 99% identity to SEQ ID NO:3, or 2) comprising a sequence represented by SEQ ID NO: 5 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 5; and / or the light chain variable region is 1) a sequence represented by SEQ ID NO:4 or having at least 90%, 95%, 98%, 99% identity to SEQ ID NO:4, or 2) comprising a sequence represented by SEQ ID NO:6 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO:6; Preferably, the heavy chain variable region of the anti-CD79B antibody or antigen-binding fragment is represented by the sequence SEQ ID NO:3 and the light chain variable region is represented by the sequence SEQ ID NO:4, or alternatively, the heavy chain variable region is represented by the sequence SEQ ID NO:5 and the light chain variable region is represented by the sequence SEQ ID NO:6.

[0042] [Table 2]

[0043] In some embodiments, the anti-CD79B antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region is 1) a sequence represented by SEQ ID NO:19 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO:19; or 2) comprising a sequence represented by SEQ ID NO: 21 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 21; and / or the light chain variable region is 1) a sequence represented by SEQ ID NO:20 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO:20; or 2) comprising a sequence represented by SEQ ID NO: 22 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 22; Preferably, the heavy chain variable region of the anti-CD79B antibody or antigen-binding fragment is represented by the sequence SEQ ID NO:19 and the light chain variable region is represented by the sequence SEQ ID NO:20, or alternatively, the heavy chain variable region is represented by the sequence SEQ ID NO:21 and the light chain variable region is represented by the sequence SEQ ID NO:22.

[0044] [Table 3]

[0045] In another embodiment, the following: [Table 4]

[0046] In some embodiments, the antibody in the antibody-drug conjugate (ADC) is selected from an anti-TROP-2 antibody. In some embodiments, the antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the sequences of which are represented by SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the sequences of which are represented by SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively.

[0047] In some embodiments, the anti-TROP-2 antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the same sequences as those of the heavy chain variable region represented by SEQ ID NO:29, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the same sequences as those of the light chain variable region represented by SEQ ID NO:30.

[0048] In some embodiments, the anti-TROP-2 antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence represented by SEQ ID NO:29 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO:30 or has at least 90% identity thereto. In some other embodiments, the anti-TROP-2 antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region whose sequence is set forth in SEQ ID NO:29 and a light chain variable region whose sequence is set forth in SEQ ID NO:30.

[0049] In some embodiments, the anti-TROP-2 antibody in the antibody-drug conjugate (ADC) comprises an antibody heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and common variants thereof, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions and common variants thereof, more preferably the antibody comprises a heavy chain constant region whose sequence is set forth in SEQ ID NO:31 and a light chain constant region whose sequence is set forth in SEQ ID NO:32. In some embodiments, the anti-TROP-2 antibody in the antibody-drug conjugate (ADC) comprises a heavy chain having the sequence set forth in SEQ ID NO:33 and a light chain having the sequence set forth in SEQ ID NO:34.

[0050] [Table 5]

[0051] [Table 6]

[0052] The heavy chain constant region of the antibody (anti-TROP-2 antibody) can be selected from the constant regions of human IgG1, IgG2, IgG4, and variants thereof, and the light chain constant region can be selected from the light chain constant region of human kappa or lambda chains or variants thereof. Exemplarily, the antibody heavy chain constant region is selected from the human IgG1 chain whose sequence is represented by SEQ ID NO:31, and the light chain constant region is selected from the human kappa chain constant region whose sequence is represented by SEQ ID NO:32.

[0053] [Table 7]

[0054] Illustratively, the above light chain / heavy chain constant regions are combined with the variable regions of the PD3 antibody to form a complete antibody, whose light chain / heavy chain sequences are as follows: [Table 8]

[0055] Furthermore, the antibody-drug conjugate (ADC) according to the present disclosure may comprise: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] In this case, k is selected from 1 to 10 and may be an integer or a decimal number. 1a is selected from methyl groups, and R 1b is selected from hydrogen.

[0056] The present disclosure further provides a compound of formula D, or a tautomeric, meso, racemic, enantiomeric, diastereomeric, mixture thereof, or a pharmaceutically acceptable salt thereof: [ka] Among them, R 1a is hydrogen, alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; cycloalkyl groups (e.g., C 3-8 cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, and cyclohexyl groups, aryl groups, and heteroaryl groups, wherein the alkyl, cycloalkyl, aryl, and heteroaryl groups are each independently optionally selected from alkyl groups (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 R is substituted with one or more substituents selected from the group consisting of alkoxy, methoxy, ethoxy, propoxy, and isopropoxy groups, halogen (e.g., fluorine, chlorine, and bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, and is preferably a methyl group; 1b is hydrogen, alkyl group (e.g., C 1-6alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups; cycloalkyl groups (e.g., C 3-8 cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, and cyclohexyl groups, aryl groups, and heteroaryl groups, wherein the alkyl, cycloalkyl, aryl, and heteroaryl groups are each independently optionally selected from alkyl groups (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 substituted with one or more substituents selected from the group consisting of alkoxy, methoxy, ethoxy, propoxy, and isopropoxy groups, halogen (e.g., fluorine, chlorine, and bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, preferably hydrogen, or a group A; or 1a and R 1b together with the atom to which they are connected form a 5- to 8-membered heterocycloalkyl group, said heterocycloalkyl group optionally being an alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 alkoxy groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups), halogens (e.g., fluorine, chlorine, bromine), deuterium, amino groups, cyano groups, nitro groups, hydroxy groups, hydroxyalkyl groups, cycloalkyl groups (e.g., C 3-8 cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, and cyclohexyl groups, substituted with one or more substituents selected from the group consisting of heterocycloalkyl groups, aryl groups, and heteroaryl groups; and R 1a and R 1b is not hydrogen at the same time.

[0057] In some embodiments, in the compound of formula D, R1a and R 1b are each independently C 1-6 In some embodiments, in the compound of formula D, R 1a is C 1-6 R is selected from alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups. 1b is selected from hydrogen. In some embodiments, in the compound of formula D, R 1a and R 1b together with the atom to which they are attached form a 5- to 8-membered heterocycloalkyl group.

[0058] In some embodiments, the compound of formula D is [ka] is.

[0059] In some embodiments, the compound of formula D is [ka] is.

[0060] In some embodiments, the compound of formula D is [ka] is.

[0061] The present disclosure further provides a compound of formula DZ, or in the form of a tautomer, meso-isomer, racemate, enantiomer, diastereomer, mixture thereof, or a pharmaceutically acceptable salt thereof: [ka] Among them, R 1a is hydrogen, alkyl group (e.g., C 1-6alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; cycloalkyl groups (e.g., C 3-8 cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, and cyclohexyl groups, aryl groups, and heteroaryl groups, wherein the alkyl, cycloalkyl, aryl, and heteroaryl groups are each independently optionally selected from alkyl groups (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 substituted with one or more substituents selected from an alkoxy group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a halogen (e.g., fluorine, chlorine, bromine), a deuterium atom, an amino group, a cyano group, a nitro group, a hydroxy group, a hydroxyalkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, and a heteroaryl group, and preferably a methyl group; R 1b is hydrogen, alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups; cycloalkyl groups (e.g., C 3-8 a cycloalkyl group, including but not limited to a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group, an aryl group, and a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the aryl group, and the heteroaryl group are each independently optionally substituted with an alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 an alkoxy group, substituted with one or more substituents selected from methoxy, ethoxy, propoxy, and isopropoxy groups, halogen (e.g., fluorine, chlorine, and bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, and preferably hydrogen; Or, R 1a and R 1b together with the atom to which they are connected form a 5- to 8-membered heterocycloalkyl group, said heterocycloalkyl group optionally being an alkyl group (e.g., C 1-6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups; alkoxy groups (e.g., C 1-6 alkoxy groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups), halogens (e.g., fluorine, chlorine, bromine), deuterium, amino groups, cyano groups, nitro groups, hydroxy groups, hydroxyalkyl groups, cycloalkyl groups (e.g., C 3-8 cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, and cyclohexyl groups, substituted with one or more substituents selected from the group consisting of heterocycloalkyl groups, aryl groups, and heteroaryl groups; and R 1a and R 1b is not hydrogen at the same time, Y is -O(CR a R b ) m2 -CR 8 R 9 -C(O)-, -NH-(CR a R b ) m2 -CR 8 R 9 -C(O)-, -O-CR 8 R 9 (CR a R b ) m2 -,-OCR 8 R 9 -C(O)-, -O(CR a R b ) m2 C(O)- or -S-(CR a R b ) m2 -CR 8 R 9 -C(O)-, among which R a and R b are the same or different and are each independently selected from hydrogen, deuterium, halogen, or alkyl groups; R8 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, R 9 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; Or, R 8 and R 9 C together with the carbon atoms connected to them 3-6 forming a cycloalkyl group, m2 is selected from 0, 1, 2 or 3;

[0062] In some embodiments, in the compound of formula DZ, R 1a and R 1b are each independently C 1-6 The alkyl group may be selected from alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups. In some embodiments, in the compound of formula DZ, R 1a is C 1-6 R is selected from alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups. 1b is selected from hydrogen. In some embodiments, in the compound of formula DZ, R 1a and R 1b together with the atom to which they are attached form a 5- to 8-membered heterocycloalkyl group.

[0063] In some embodiments, the compound of formula DZ, or a tautomer, meso isomer, racemate, enantiomer, diastereomer, mixture thereof, or pharmaceutically acceptable salt thereof, is a compound of formula DZ-1, or a tautomer, meso isomer, racemate, enantiomer, diastereomer, mixture thereof, or pharmaceutically acceptable salt thereof, [ka] Among them, R 8 is hydrogen, C 3-6Cycloalkylalkyl group or C 3-6 cycloalkyl groups, R 9 is hydrogen, haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 8 and R 9 C together with the carbon atoms connected to them 3-6 forming a cycloalkyl group, and m2 is selected from 0, 1, 2 or 3;

[0064] In some embodiments, the compound designated DZ is [ka] Selected from.

[0065] On the other hand, compounds shown in DZ provided in some embodiments may contain one or more asymmetric centers, e.g. [ka] teeth [ka] It could be.

[0066] In another aspect, the present disclosure further provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate and a pharmaceutically acceptable pharmaceutical carrier, diluent, or excipient. The disclosure further provides a use of the antibody-drug conjugate or the pharmaceutical composition in the preparation of a medicament for treating or preventing a tumor. In some embodiments, the tumor is a cancer associated with expression of HER2, HER3, B7H3, or EGFR.

[0067] The present disclosure further provides use of the antibody-drug conjugate or the pharmaceutical composition in the preparation of a medicament for treating and / or preventing cancer. In some embodiments, the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma. The present disclosure further provides a method for treating or preventing a tumor, the method comprising administering a therapeutically effective amount of the antibody-drug conjugate or the pharmaceutical composition to a patient in need thereof, wherein the tumor is preferably a cancer associated with expression of HER2, HER3, B7H3 or EGFR.

[0068] The present disclosure further provides a method for treating and / or preventing a tumor, the method comprising administering a therapeutically effective amount of the antibody-drug conjugate or the pharmaceutical composition to a patient in need thereof, wherein the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma. The present disclosure further provides the antibody-drug conjugate or the pharmaceutical composition for treating or preventing tumors, wherein the tumors are preferably cancers associated with the expression of HER2, HER3, B7H3 or EGFR.

[0069] The present disclosure further provides the antibody-drug conjugate or the pharmaceutical composition for treating and / or preventing a tumor, wherein the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma. The active compound can be in a form suitable for administration by any suitable route, preferably in unit dosage form or in a form that the patient can self-administer as a single dose. The unit dosage form of the compounds or compositions of the present disclosure can be a tablet, capsule, cachet, bottled drug solution, drug powder, granules, tablet, suppository, reconstituted powder, or liquid formulation.

[0070] The dose of the compound or composition used in the therapeutic methods of the present disclosure will generally vary depending on the severity of the disease, the weight of the patient, and the relative efficacy of the compound, although as a general guide, a suitable unit dose may be 0.1 to 1000 mg. In addition to the active compound, the pharmaceutical composition according to the present disclosure may contain one or more additives selected from fillers (diluents), binders, wetting agents, disintegrants, excipients, etc. The composition may contain 0.1 to 99% by weight of the active compound, depending on the method of administration.

[0071] Detailed Description of the Invention Unless otherwise limited, all technical and scientific terms used in this disclosure are accorded the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described in this disclosure can be used in the practice or testing of this disclosure, this disclosure describes the preferred methods and materials. In describing and claiming this disclosure, the following terms will be used in accordance with the following definitions. When trade names are used in this disclosure, applicants intend to include formulations of products bearing that trade name, generic drugs and active drug portions of products bearing that trade name. Unless otherwise specified, terms used in the specification and claims have the following meanings.

[0072] The term "drug" refers to a cytotoxic drug or an immunomodulator. A cytotoxic drug refers to a chemical molecule that is potently disruptive to the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to a lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, resulting in severe side effects. The term also includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), toxic agents, chemotherapeutic agents, antibiotics, and nucleases. Immunomodulatory agents are inhibitors of immune checkpoint molecules. In some embodiments of the present disclosure, the agent is designated D and is an immunomodulatory agent, particularly a TLR8 agonist.

[0073] The terms "linker," "linking unit," "linker unit," or "linking fragment" refer to a chemical fragment or bond that is linked at one end to a ligand and at the other end to a drug, and may be linked to another linker and then to a drug. The linker may include one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and linker reagents derived from coupling with N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, also referred to herein as MCC), and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB). The linker may include a stretch unit, a spacer unit, an amino acid unit, and an elongation unit. It can be synthesized by methods known in the art, such as the method described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates release of the drug in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992), U.S. Patent No. 5,208,020) can be used.

[0074] The term "stretch unit" refers to a fragment of a chemical structure that is covalently attached at one end to an antibody via a carbon atom and at the other end to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety. The term "spacer unit" refers to a structural fragment of a bifunctional compound that can be used to couple an amino acid unit with a cytotoxic drug to form an antibody-drug conjugate, and this coupling scheme can selectively attach the cytotoxic drug to the amino acid unit.

[0075] The term "amino acid" refers to an organic compound whose molecular structure contains an amino group and a carboxyl group, both of which are directly bonded to a -CH- structure. The general formula is HNCHRCOOH, where R can be H, a substituted or unsubstituted alkyl group, or the like. Depending on the position where the amino group is attached to the carbon atom of the carboxylic acid, amino acids can be classified as α-, β-, γ-, δ-, ε-, etc. In the biological world, naturally occurring amino acids that make up proteins have specific structural characteristics: their amino groups are directly attached to the α-carbon atom, i.e., α-amino acids, and include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline, etc. An example of an unnatural amino acid is citrulline. As is well known to those skilled in the art, unnatural amino acids do not constitute natural proteins and are therefore not involved in the synthesis of antibodies in this disclosure. The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p. 3558 (1968).

[0076] [Table 9] [Table 10]

[0077] In the present disclosure, the spacer unit is PAB, the structure of which is shown as a p-aminobenzyloxycarbonyl fragment, the structure of which is shown in formula (VI) and which is connected to D: [ka]

[0078] The linker element is MC=6-maleimidocaproyl, which has the following structure: [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker); Citrulline = 2-amino-5-ureidopentanoic acid, Me-Val-Cit = N-methyl-valine-citrulline (in which the linker peptide bond is modified so as not to be cleaved by cathepsin B); MC(PEG)6-OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines); SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate, SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate, SMCC = succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; IT = iminothiolane; Including but not limited to PBS = phosphate buffered saline.

[0079] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active agent by a stable linking unit. As used herein, "antibody drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic agent by a stable linking unit. The term "drug loading" may be expressed as the ratio of the amount of drug to the amount of antibody. As for the range of drug loading, each antibody (Ab) may be linked to 1 to 20 cytotoxic drugs (D), preferably 1 to 10. In an embodiment of the present disclosure, the drug loading is represented by k, which may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an average value between any two numbers. It is preferably 1 to 10, and more preferably 1 to 8, 2 to 8, 2 to 7, 3 to 8, 3 to 7, 3 to 6, 4 to 7, 4 to 6, or 4 to 5. The average amount of drug per ADC molecule after the coupling reaction can be characterized by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, monoclonal antibody molecular size variant assay (CE-SDS), and HPLC.

[0080] The monoclonal antibody molecular size variant assay (CE-SDS) disclosed herein utilizes capillary electrophoresis sodium dodecyl sulfate (CE-SDS) with ultraviolet detection to quantitatively measure the purity of recombinant monoclonal antibody products according to their molecular weights under reducing and non-reducing conditions by capillary electrophoresis (Chinese Pharmacopoeia 2015 Edition 0542). In one embodiment of the present disclosure, a cytotoxic drug is coupled to the N-terminal amino group and / or the ε-amino group of a lysine residue of a ligand via a linker unit, and generally, the number of drug molecules that can be coupled to an antibody in a coupling reaction is less than the theoretical maximum.

[0081] The loading of antibody-drug conjugates can be controlled by non-limiting methods, including: (1) Controlling the molar ratio of the linking reagent and the monoclonal; (2) Control of reaction time and temperature; (3) Selection of different reaction reagents.

[0082] The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to differences in the amino acid composition and sequence of the heavy chain constant regions. Therefore, immunoglobulins can be divided into five types, or so-called immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig molecules of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa or lambda chains based on differences in the constant regions. Each of the five Ig classes can have either kappa or lambda chains. The antibodies of the present disclosure are preferably specific antibodies against cell surface antigens on target cells, and non-limiting examples include the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, and anti-Lewis antibody. Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody or antigen-binding fragment thereof.In some embodiments, the antibody is chosen from trastuzumab (trade name Herceptin), pertuzumab (also known as 2C4, trade name Perjeta), nimotuzumab (trade name Taixinsheng), enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab vedotin, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, and glematumumab. In antibody heavy and light chains, approximately 110 amino acids near the N-terminus are highly variable and form the variable region (Fv region), while the remaining amino acid sequences near the C-terminus are relatively stable and form the constant region. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0083] Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred. The term "murine antibody" as used in this disclosure refers to an antibody prepared in mice according to the knowledge and skill in the art, in which a specific antigen is injected into a test subject, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. The term "chimeric antibody" refers to an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and can reduce the immune response elicited by mouse antibodies. To prepare a chimeric antibody, first, hybridomas secreting mouse-specific monoclonal antibodies are prepared, and then the variable region genes are cloned from the mouse hybridoma cells. Furthermore, if necessary, the constant region genes of a human antibody are cloned, and the mouse variable region genes and human constant region genes are ligated to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell system.

[0084] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting mouse CDR sequences onto a human antibody variable region framework, i.e., a framework sequence of a different human germline antibody. This overcomes the heterologous reactivity induced by chimeric antibodies containing a large amount of mouse protein components. Such framework sequences can be obtained from consensus DNA databases containing germline antibody gene sequences or from published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available online at www.mrccpe.com.ac.uk / vbase) and Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity associated with reduced immunogenicity, minimal back mutations or reverse mutations can be made to the human antibody variable region framework sequences to maintain activity. The humanized antibodies of the present disclosure also include phage-displayed humanized antibodies in which affinity maturation of CDRs has been performed. Further references describing methods by which murine antibodies can be used for humanization include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and coworkers [Jones et al., Nature, 321, 522 (1986); Riechmann et al., Nature, 332, 323-327 (1988); Verhoeyen et al., Science, 239, 1534 (1988)].

[0085] The terms "fully humanized antibody," "fully human antibody," or "fully human antibody" are also referred to as "fully humanized monoclonal antibodies," in which both the variable and constant regions of the antibody are human-derived, eliminating immunogenicity, toxicity, and side effects. The development of monoclonal antibodies has gone through four stages: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully humanized monoclonal antibodies. The present disclosure is a fully humanized monoclonal antibody. The main technologies related to the preparation of fully human antibodies include human hybridoma technology, EBV-mediated B lymphocyte transformation technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0086] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments included in the term "antigen-binding fragment" include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single antibody arm; (v) a single domain or dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); and (vi) an isolated complementarity-determining region (CDR), or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using recombinant techniques to generate a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same way as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, such as, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0087] Fab is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by treating an IgG antibody molecule with the protease papain (amino acid residue 224, which cleaves the H chain). Approximately half of the N-terminal H chain and the entire L chain are bound together by disulfide bonds. F(ab')2 is an antibody fragment containing two Fab regions connected by a hinge, with a molecular weight of approximately 100,000, which has antigen-binding activity and is obtained by digesting the lower portion of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin. Fab' is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2. Alternatively, Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0088] The terms "single-chain antibody," "single-chain Fv," or "scFv" include molecules of an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) connected by a linker. Such scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof, for example, a 1-4 repeat variant (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers for use in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0089] The term "CDR" refers to one of the six major hypervariable regions in an antibody variable domain that mediate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of a light chain variable domain and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of a heavy chain variable domain. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of the CDRs can be determined by any one of a variety of known methods, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, in a typical format, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable region (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3), according to the Kabat rules.According to the Chothia rules, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and those in VL are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). According to the combined Kabat and Chothia CDR definitions, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to the IMGT rules, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). According to the IMGT rules, the CDR regions of an antibody can be determined by the program IMGT / DomainGapAlign.

[0090] The term "antibody framework" refers to a part of a variable domain VL or VH that serves as a support for the antigen binding loops (CDRs) of said variable domain. Essentially, it is the variable structure domain without the CDRs. The term "epitope" or "antigenic determinant" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0091] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to an epitope of about 10 -7 Smaller than M, e.g., about 10-8 M, 10 -9 M or 10 -10 binds with an affinity (KD) less than or equal to M. The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0092] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, a vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be ligated. In another embodiment, the vector is a viral vector, into which other DNA segments can be ligated into the viral genome. The vectors disclosed herein can either autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or can integrate into the genome of a host cell after introduction and thereby replicate along with the host genome (e.g., non-episomal mammalian vectors). Methods for producing and purifying antibodies and antigen-binding fragments well known in the art are described, for example, in Chapters 5-8 and 15 of Reisenko's Antibody Laboratory Techniques Manual. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in the invention have one or more human-derived FR regions added to non-human CDR regions by genetic engineering techniques. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) homepage at http: / / imgt.cines.fr by aligning the IMGT human antibody variable region germline gene database with MOE software, or from the Immunoglobulin Journal 2001, ISBN 012441351.

[0093] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Bacteria amenable to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

[0094] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. A preferred conventional technique is the mammalian expression system, which induces antibody glycosylation, particularly at the highly conserved N-terminal site of the Fc region. Positive clones are cultured in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibodies can be purified by conventional techniques, for example, on an A or G Sepharose FF column containing a conditioned buffer. Nonspecifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and the antibody fragments are detected and collected by SDS-PAGE. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and multimers may be removed by conventional methods, such as molecular sieves or ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0095] "Identity" of an amino acid sequence refers to the percentage of amino acid residues in a first sequence that are similar to those in a second sequence, with alignment and gaps, if necessary, of the amino acid sequences to maximize the percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. To measure the percentage of amino acid sequence identity, alignment can be achieved by several methods within the skill of the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0096] The term "peptide" refers to a compound fragment between an amino acid and a protein, consisting of two or more amino acid molecules linked together by peptide bonds, and is a structural and functional fragment of a protein; for example, hormones, enzymes, etc. are peptides in nature. The term "sugar" refers to a biopolymer made up of the three elements C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides. The term "fluorescent probe" refers to a fluorescent molecule that exhibits characteristic fluorescence in the UV-Vis-NIR region and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) change sensitively depending on the properties of the environment in which it is located, such as polarity, refractive index, viscosity, etc. It interacts non-covalently with nucleic acids (DNA or RNA), proteins, or other macromolecular structures, changing one or more of its fluorescence properties, and is used to study the properties and behavior of macromolecular substances.

[0097] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms, more preferably an alkyl group containing from 1 to 10 carbon atoms, and most preferably an alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, and the like. Silyl group, 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2 n-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available linkage site, and the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0098] The term "heteroalkyl group" is an alkyl group containing one or more heteroatoms selected from N, O, or S, as defined above. A "monovalent group" is an atom or group that "formally" removes one monovalent from a compound. A "subunit" is an atom or group of atoms that "formally" removes two monovalents or one divalent from a compound. For example, an "alkyl group" refers to the moiety that remains after removing one hydrogen atom from an alkane molecule and includes straight- and branched-chain monovalent groups of 1 to 20 carbon atoms. Non-limiting examples of alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, s-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and various branched-chain isomers thereof.

[0099] The term "alkylene group" refers to a saturated, straight- or branched-chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of an alkane parent, and is a straight- or branched-chain alkylene group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH-), 1,1-ethylidene (-CH(CH)-), 1,2-ethylidene (-CHCH)-, 1,1-propylidene (-CH(CHCH)-), 1,2-propylidene (-CHCH(CH)-), 1,3-propylidene (-CHCHCHCH-), 1,4-butylidene (-CHCHCHCHCH-), and 1,5-butylidene (-CHCHCHCHCHCH-). An alkylene group can be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available linkage site, and the substituents are preferably independently and optionally selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo groups. Similarly, "alkenylene" is as previously defined.

[0100] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group or cycloalkyl group is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy groups. An alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogen atoms, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0101] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, where the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, and polycyclic cycloalkyl groups include spirocyclic, fused, and bridged cycloalkyl groups.

[0102] The term "heterocycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m(wherein m is an integer of 0 to 2), but does not include the ring moiety -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocycloalkyl groups include spirocyclic, fused-ring, and bridged-ring heterocycloalkyl groups.

[0103] The term "spiroheterocycloalkyl group" refers to a polycyclic heterocycloalkyl group having 5 to 20 members that share one atom (called a spiroatom) between the rings, in which one or more ring atoms is nitrogen, oxygen, or S(O). m (wherein m is an integer of 0 to 2), with the remaining ring atoms being carbon. It may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. It is preferably 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of spiro atoms shared between the rings, spiroheterocycloalkyl groups are classified as monospiroheterocycloalkyl groups, bisspiroheterocycloalkyl groups, or polyspiroheterocycloalkyl groups, with monospiroheterocycloalkyl groups and bisspiroheterocycloalkyl groups being preferred. More preferred are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocycloalkyl groups. Non-limiting examples of spiroheterocycloalkyl groups include: [ka] Includes:

[0104] The term "fused heterocycloalkyl group" refers to a 5- to 20-membered polycyclic heterocycloalkyl group in which each ring in the system shares an adjacent pair of atoms with another ring in the system, one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and one or more ring atoms is nitrogen, oxygen, or S(O) m (wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. 6 to 14-membered rings are preferred, and 7 to 10-membered rings are more preferred. Depending on the number of constituent rings, fused heterocycloalkyl groups may be classified as bicyclic, tricyclic, tetracyclic or polycyclic fused heterocycloalkyl groups, preferably bicyclic or tricyclic, and more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocycloalkyl groups. Non-limiting examples of fused heterocycloalkyl groups are: [ka] Includes:

[0105] The term "bridged heterocycloalkyl group" refers to a 5- to 14-membered polycyclic heterocycloalkyl group in which any two rings share two non-directly connected atoms, and may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and in which one or more ring atoms is nitrogen, oxygen, or S(O) m (wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. Preferably, the ring is 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of constituent rings, the bridged heterocycloalkyl group may be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocycloalkyl group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged heterocycloalkyl groups include: [ka] Includes:

[0106] The heterocycloalkyl group ring may be fused to an aryl group, a heteroaryl group, or a cycloalkyl group ring, wherein the ring connected to the parent structure is a heterocycloalkyl group, non-limiting examples of which include: [ka] Includes:

[0107] The heterocycloalkyl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0108] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π-electron system, preferably 6- to 10-membered, such as a phenyl group or a naphthyl group, preferably a phenyl group. The aryl group ring may be fused to a heteroaryl group, heterocycloalkyl group, or cycloalkyl group ring, in which the ring connected to the parent structure is an aryl group ring, non-limiting examples of which are: [ka] Includes:

[0109] The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen atom, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, and a heterocycloalkylthio group.

[0110] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5 or 6-membered, and examples thereof include furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl. The heteroaryl ring may be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, where the ring connected to the parent structure is the heteroaryl ring, and non-limiting examples thereof include: [ka] Includes:

[0111] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0112] The term "amino-protecting group" refers to a group that protects an amino group with an easily removable group so that the amino group is not altered when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, t-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups can be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy, or nitro. The amino-protecting group is preferably 9-fluorenylmethyloxycarbonyl.

[0113] The term "aminoheterocycloalkyl group" refers to a heterocycloalkyl group substituted with one or more amino groups, preferably one amino group, wherein the heterocycloalkyl group is defined above and "amino group" refers to -NH. Representative examples of the present disclosure are as follows: [ka]

[0114] The term "heterocycloalkylamino group" refers to an amino group substituted with one or more heterocycloalkyl groups, preferably one heterocycloalkyl group, wherein the amino group is defined above and the heterocycloalkyl group is defined above. Representative examples of the present disclosure are as follows: [ka]

[0115] The term "cycloalkylamino group" refers to an amino group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein the amino group is defined above and the cycloalkyl group is defined above. Representative examples of the present disclosure are as follows: [ka]

[0116] The term "cycloalkylalkyl group" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein the alkyl group is defined above and the cycloalkyl group is defined above. The term "haloalkyl group" refers to an alkyl group substituted with one or more halogens, wherein alkyl group is defined above.

[0117] The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl group is defined above. The term "hydroxy group" refers to an --OH group. The term "halogen" refers to fluorine, chlorine, bromine or iodine. The term "amino group" refers to -NH2. The term "nitro group" refers to -NO2. In chemical formulas, it is abbreviated as "Me" and stands for the methyl group.

[0118] The present disclosure further includes various deuterated forms of the compound of formula (I). Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of formula (I) by referring to relevant literature. When preparing the deuterated form of the compound of formula (I), commercially available deuterated starting materials may be used, or the compound may be synthesized by conventional techniques using deuterated reagents, including, but not limited to, deuterated borane, tritiated borane in tetrahydrofuran, lithium aluminum deuterated hydride, deuterated iodoethane, and deuterated iodomethane.

[0119] On the other hand, hydrogen in the functional groups of the compounds described in the present disclosure can be deuterated to obtain the corresponding deuterated compounds, which retain the same selectivity and potential as the hydrogen derivatives, but the deuterium bonds are more stable, resulting in different "ADME" or "pharmacokinetics" and providing clinically beneficial effects. Pharmacokinetics is the process by which an organism absorbs, distributes, metabolizes, and excretes excretion of exogenous chemicals.

[0120] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the phrase includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocycloalkyl group optionally substituted with an alkyl group" means that the alkyl group may, but need not, be present, and the phrase includes instances where the heterocycloalkyl group is substituted with an alkyl group and instances where the heterocycloalkyl group is not substituted with an alkyl group.

[0121] The term "substituted" means that one or more hydrogen atoms in a group, preferably 5 or less, more preferably 1 to 3 hydrogen atoms, are independently replaced with a corresponding number of substituents. Of course, substituents are only located at chemically feasible positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino group or a hydroxy group having free hydrogen may be unstable when bonded to a carbon atom having an unsaturated (e.g., olefinic) bond. The term "pharmaceutical composition" is intended to include a mixture of one or more compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, and other components, such as physiologically / pharmaceutically acceptable carriers and excipients, intended to facilitate administration to the body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0122] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of an antibody-drug conjugate of the present disclosure or a salt of a compound described herein, which salt is safe and effective when used in a mammalian body and has the requisite biological activity. Because the antibody-drug conjugate of the present disclosure contains at least one amino group, it can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0123] The term "solvate" refers to a pharmaceutically acceptable solvate of a ligand-drug conjugate compound of the present disclosure with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate. The term "drug carrier" is used in conjunction with the drugs of the present disclosure and refers to a system that changes the drug's entry into the human body and its distribution within the body, controls the drug's release rate, and delivers the drug to target organs. Drug carrier release and targeting systems can reduce drug degradation and loss, lower side effects, and improve bioavailability. For example, polymer surfactants, which are used as carriers, can self-assemble to form aggregates of various forms due to their unique amphiphilic structure, preferred examples of which include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and have good membrane permeability, making them good drug carriers.

[0124] The term "excipient" refers to a substance added to a drug formulation other than the main drug, and may also be called an additive. For example, adhesives, fillers, disintegrants, and lubricants in troches, the matrix portion of semi-solid preparations such as ointments and creams, and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure adjusters, and coloring agents in liquid preparations can all be called excipients. The term "diluent," also known as a filler, is primarily used to increase the weight and volume of lozenges. The addition of a diluent not only ensures a consistent volume, but also reduces the dosage deviation of the active ingredient and improves the compressibility of the drug. When the drug in a lozenge contains an oily ingredient, an absorbent must be added to absorb the oily substance so that the lozenge can be prepared in a "dry" state.

[0125] The compounds of the present disclosure may contain one or more asymmetric centers and therefore can form enantiomers, diastereomers, and other stereoisomeric forms defined by absolute stereochemistry as (R)- or (S)- or, as with amino acids, (D)- or (L)-. The present disclosure includes all possible isomers and their racemic and optically pure forms. Optically active (+)- and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or by conventional means, such as chromatography and fractional crystallization. Conventional means for preparing / separating individual enantiomers include chiral synthesis from appropriate optically pure precursors or resolution of the racemate (or a salt or derivative) by, for example, chiral high-performance liquid chromatography (HPLC). When a compound described herein contains an olefinic double bond or other center of geometric asymmetricity, unless otherwise specified, the compound is meant to include both E and Z geometric isomers, and all tautomers are also meant to be included.

[0126] In the chemical structures of the compounds according to the present disclosure, [ka] means that the configuration is not specified, that is, when chiral isomers exist in a chemical structure, the bond [ka] teeth [ka] or [ka] or [ka] and [ka] It contains two types of configurations at the same time. "Stereoisomer" refers to a compound made up of the same atoms joined by the same bonds but with different, incompatible three-dimensional structures. Various stereoisomers and mixtures thereof are contemplated in this disclosure, and further includes "enantiomers," which are two stereoisomers whose molecules are non-superimposable mirror images of one another. "Tautomer" refers to a proton moving from one atom of a molecule to another atom of the same molecule. The present disclosure includes any tautomers of the compounds. [Brief explanation of the drawings]

[0127] [Figure 1] 1 is a graph showing the trend of changes in body weight of animals in a test administration group different from the blank solvent group in Test Example 7 (horizontal axis: number of days, vertical axis: body weight). [Figure 2] 1 shows a graph of the trend of changes in tumor volume in animals in groups administered with a test drug different from the vehicle (horizontal axis: number of days, vertical axis: tumor volume). DETAILED DESCRIPTION OF THE INVENTION

[0128] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. See Sambrook et al., Molecular Cloning Manual, Laboratory Manual, Reisenko Laboratory; Modern Methods in Molecular Biology, edited by Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are commercially available conventional reagents.

[0129] The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts (δ) are 10 -6 The NMR was measured using a Bruker AVANCE-400 nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD) as the solvents, and tetramethylsilane (TMS) as the internal standard. For MS measurements, liquid chromatograph mass spectrometers Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS (manufacturer: Agilent, MS model number: 6110 / 6120 Quadrupole MS), waters ACQuity UPLC-QD / SQD (manufacturer: waters, MS model number: waters ACQuity Qda Detector / waters SQ Detector), and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model number: THERMO Q Exactive) were used. For high performance liquid chromatography (HPLC) analysis, a high performance liquid chromatograph Agilent HPLC 1200DAD, Agilent HPLC 1200VWD or Waters HPLC e2695-2489 is used.

[0130] For chiral HPLC analysis, a high performance liquid chromatograph Agilent 1260 DAD is used. For preparative high performance liquid chromatography, preparative chromatographs Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 are used. For chiral separation, a preparative chromatograph Shimadzu LC-20AP is used. Combiflash Rf200 (TELEDYNE ISCO) is used as the CombiFlash high-speed preparative chromatograph. Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used for thin-layer chromatography. The silica gel plate specifications for thin-layer chromatography (TLC) are 0.15 mm to 0.2 mm, and those for separating and purifying products by thin-layer chromatography are 0.4 mm to 0.5 mm. Silica gel column chromatography generally uses 200-300 mesh silica gel manufactured by Yantai Yellow Sea Silica Gel Co., Ltd. as a carrier. Known starting materials according to the present disclosure may be synthesized by adopting or following methods known in the art, or may be purchased commercially from commercial sources such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., and Darui Chemicals.

[0131] In the examples, unless otherwise stated, all reactions can be carried out under an argon or nitrogen atmosphere. An argon or nitrogen atmosphere refers to an argon or nitrogen balloon with a volume of approximately 1 L connected to the reaction flask. A hydrogen atmosphere refers to a hydrogen balloon with a volume of approximately 1 L attached to the reaction flask. For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenation apparatus are used. The hydrogenation reaction is generally carried out after three cycles of evacuation and hydrogen injection. For microwave reactions, a CEM Discover-S 908860 microwave reactor is used.

[0132] In the examples, unless otherwise specified, the term "solution" refers to an aqueous solution. Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C. Thin layer chromatography (TLC) is used to monitor the progress of the reactions in the examples. The volume ratio of the developing solvent used in the reaction, the eluent system of column chromatography used to purify the compound, and the developing solvent system of thin layer chromatography may be adjusted depending on the polarity of the compound, or may be adjusted by adding small amounts of basic or acidic reagents such as triethylamine and acetic acid.

[0133] The antibody-drug conjugates of the present disclosure are described in WO2020063676A, the entire text of which is incorporated herein by reference for the synthesis and testing of related compounds, and the synthesis of non-limiting examples thereof is as follows: 1. Preparation of antibodies Example 1-1. Cloning and expression of antigen protein The antibody (including light and heavy chains) and antigen were constructed by overlap extension PCR, a method known in the art. The DNA fragments obtained by overlap extension PCR were inserted into the expression vector pEE6.4 (Lonza Biologics) via the HindIII / BstBI enzyme cleavage sites and expressed in 293F cells (Invitrogen, Cat# R790-07). The resulting recombinant proteins were used for immunization or screening. The human CD79B gene sequence was derived from NCBI (NP_000617.1), and its extracellular domain (ECD) contains 159 amino acids (Met1-Asp159).

[0134] The amino acid sequence of the human CD79B extracellular domain (ECD) and human Fc region fusion protein (human CD79B ECD-hFc) is shown in SEQ ID NO:1: [Table 11] The amino acid sequence of the human CD79B extracellular domain (ECD) and His-tag fusion protein (human CD79B ECD-His) is represented in SEQ ID NO:2: [Table 12]

[0135] Example 1-2. Preparation of mouse monoclonal antibodies 1. Immunization of mice and detection of serum titers Balb / c and SJL mice were immunized intraperitoneally with the human CD79B extracellular domain (ECD) and human Fc region fusion protein (human CD79B ECD-hFc) and the human CD79B extracellular domain (ECD) and His-tag fusion protein (human CD79B ECD-His), respectively, to stimulate antibody formation against the human CD79B extracellular domain (ECD). Concurrently, SJL mice were immunized intraperitoneally with the monkey CD79B extracellular domain (ECD) and His-tag fusion protein (cyno CD79B ECD-His) to stimulate antibody formation against the monkey CD79B extracellular domain (ECD).

[0136] The experimental procedure is as follows: 1) Intraperitoneal immunization: The amount of antigen required for this immunization was calculated based on the immunization program. Protein antigens were diluted to the appropriate concentration with PBS as needed, followed by antigen emulsification. The emulsified antigen and adjuvant mixture was transferred to a 2.0 mL sterile syringe, taking care to remove any air bubbles. The mouse's tail was grasped with the right hand, and the skin on the head and neck was gently pinched with the thumb and index finger of the left hand. The mouse's abdomen was turned upright, and the injection site on the right abdomen was wiped with a 75% alcohol swab. The needle of the syringe filled with antigen was pointed upward, and the mouse's head was turned down. The needle was then inserted parallel to the skin, and the syringe was then inserted into the abdominal cavity at a 45-degree angle. The antigen and adjuvant mixture was slowly injected. After immunization was complete, the mouse was observed for at least 2 hours. 2) Collection of mouse serum: Mark the serum tube number corresponding to each mouse, identify the mouse's earring number, grasp the mouse with one hand, and collect approximately 100 μL of whole blood from the submaxillary vein on the mouse's face. Allow the collected whole blood sample to stand at room temperature for approximately 2 hours, then centrifuge and collect the upper serum from the centrifuge tube. Serum can be stored in a 4°C refrigerator for up to one week for related experiments such as antibody titer measurements. For long-term storage, serum can be stored in a -80°C refrigerator, but repeated freezing and thawing should be avoided. 3) ELISA serum titer measurement of immunized mice: Prior to the start of the experiment, a 96-well plate was appropriately marked and coated overnight in a 4°C refrigerator with 50 μL of antigen at a concentration of 1 μg / mL. The next day, the antigen-coated plate was removed and washed once with a plate washer (washing solution: 1X PBST). After washing, the plate was blocked with 1% BSA blocking solution prepared in 1X PBST at 37°C for 1 hour. After washing the plate three times with 1X PBST washing solution, various dilutions of test sera were added and incubated in a 37°C incubator for 1 hour. After washing the plate three times with 1X PBST washing solution, 100 μL of goat anti-mouse secondary antibody diluted 1:5000 was added and incubated at 37°C for 0.5 hours. After washing the plate, TMB color development solution A and B were mixed at a 1:1 ratio to develop the color. After 15 minutes, the color reaction was stopped with 1 N hydrochloric acid, and the fluorescence was detected at 450 nm using a Spectra Max M5 multifunction plate reader. 4) FACS serum titer measurement of immunized mice: DoHH2 cell or monkey peripheral blood mononuclear cell suspensions were centrifuged, the cells were resuspended in PBS containing 0.1% BSA, and counted. Test serum from each group of immunized mice was added and incubated at room temperature for 60 minutes, after which the cells were washed three times. Anti-mouse IgG (Fc specific)-FITC secondary antibody was added and incubated at room temperature in the dark for 30 minutes, after which the cells were washed three times. The cells were gently resuspended in PBS containing 0.1% BSA, and detected by the instrument. The above test results in the production of specific antibodies against CD79B in immunized mice, which can be used for cell fusion to produce hybridoma cell lines capable of secreting specific antibodies against CD79B.

[0137] 2. Hybridoma preparation and antibody screening Cell fusion was performed spontaneously or under artificial induction between mouse lymphocytes and myeloma cells SP2 / 0 (ATCC, CCL-121 TM) promotes fusion to hybridoma cells, which are endowed with antibody secretion function and capable of unlimited proliferation. The hybridoma cells were fused with lymphocytes and myeloma cells from immunized mice using electrofusion, and were then used for antibody screening. 1) Electrofusion experiment: SP2 / 0 cells were expanded in 10% DMEM medium one week before fusion. Spleens and lymph nodes were removed from sacrificed mice in a safety cabinet, washed and polished in culture dishes, and lymphocytes were collected. SP2 / 0 cells and lymphocytes were mixed proportionally, and the electrofusion device was started and programmed to perform the fusion. After fusion, the cells were seeded into 96-well plates and cultured in an incubator at 37°C and 5% CO2. Cell status was observed daily, and the cell fusion rate was calculated 5 days after fusion. The fused hybridoma cells were screened 9 to 14 days after fusion, and positive wells were selected and expanded in 24-well plates. 2) Subcloning by limiting dilution: Cell lines requiring subcloning were resuspended in 24-well culture wells and counted. The cell concentration of each cell line was diluted to 5-10 cells / mL, and the diluted cell suspension was added to a 15 cm disposable culture dish. 0.2 mL of the diluted cell suspension was added to each well of a 96-well culture plate, with each well containing 1-2 cells. The 96-well plate containing the cells was cultured in an incubator at 37°C with 5% CO2. After 7-10 days, the subcloning plates were detected and screened according to the cell growth status. Positive clones were transferred to 24-well plates and further confirmed as positive. 3) ELISA Cleaning: Prior to the start of the experiment, a 96-well plate was appropriately marked and coated overnight in a 4°C refrigerator with 50 μL of antigen per well at a concentration of 1 μg / mL. The next day, the antigen-coated plate was removed and washed once with a plate washer (washing solution: 1X PBST). After washing, the plate was blocked with 1% BSA blocking solution prepared in 1X PBST at 37°C for 1 hour. After washing the plate three times with 1X PBST washing solution, 50 μL of test cell supernatant was added and incubated in a 37°C incubator for 1 hour. After washing the plate three times with 1X PBST washing solution, 100 μL of goat anti-mouse secondary antibody diluted 1:5000 was added and incubated at 37°C for 0.5 hours. After washing the plate, TMB color development solution A and B were mixed in a 1:1 ratio to develop the color. After 15 minutes, the color reaction was stopped with 1 N hydrochloric acid, and the fluorescence was detected at 450 nm using a Spectra Max M5 multifunction plate reader. 4) FACS screening: The DOHH2 cell suspension was centrifuged, resuspended in PBS containing 0.1% BSA, and counted. The test cell supernatant was added and incubated at room temperature for 60 minutes, after which the cells were washed three times. Anti-Mouse IgG (Fc specific)-FITC secondary antibody was added and incubated at room temperature in the dark for 30 minutes, after which the cells were washed three times. The cells were gently resuspended in PBS containing 0.1% BSA and detected by the instrument. 5) Identification of hybridoma-positive clones: A large number of antibodies specific to human CD79B antigen were obtained by fusion and subcloning screening of mouse spleen cells. Of these, 17 hybridomas with the highest ELISA and FACS binding abilities were used to produce and purify antibodies. Table 1 shows the ELISA detection results of the culture supernatant of anti-human CD79B hybridoma-positive clone cells. Table 2 shows the FACS detection results of the culture supernatant of anti-human CD79B hybridoma-positive clone cells. At the same time, specific antibodies to monkey CD79B antigen were obtained, and of these, 4 hybridomas with the highest ELISA and FACS binding abilities were used to produce and purify antibodies. mIgG was used as a negative control in all cases.

[0138] Table 1. ELISA detection results of anti-human CD79B hybridoma positive clones [Table 13]

[0139] Table 2. FACS detection results of anti-human CD79B hybridoma positive clones [Table 14]

[0140] 3. Production, purification and identification of mouse monoclonal antibodies 1) Production and purification of mouse monoclonal antibodies: Observe the hybridoma cells that require antibody production under a microscope. Once the cells have grown to over 70% in good condition, harvest the cells and count them using a Countstar IC1000 cell counter. The cell concentration in the prepared medium was adjusted to 1-5 x 10. 5 The cell culture medium was adjusted to 10-15 cells / mL and transferred to a roller bottle. The roller bottle containing the cells was placed in a roller bottle incubator at 37°C and cultured for 10-15 days. Cell growth was monitored daily, and when the medium became orange-yellow and clear, it was removed for purification. The cell supernatant was purified using a Protein A column according to conventional methods. 2) ELISA detection of anti-human CD79B mouse monoclonal antibody: Prior to the start of the experiment, a 96-well plate was appropriately marked and coated overnight in a 4°C refrigerator with 50 μL of 1 μg / mL antigen per well. The next day, the plate was removed and washed once with a plate washer (washing solution: 1X PBST). After washing, the plate was blocked with 1% BSA blocking solution prepared in 1X PBST at 37°C for 1 hour. After washing the plate three times with 1X PBST washing solution, 50 μL of antibody diluted 1:10 at 100 nM was added and incubated in a 37°C incubator for 1 hour. After washing the plate three times with 1X PBST washing solution, 100 μL of goat anti-mouse secondary antibody diluted 1:5000 was added and incubated in a 37°C incubator for 0.5 hours. After washing the plate, TMB color development solution A and B were mixed in a 1:1 ratio and then developed. After 15 minutes, the color development reaction was stopped with 1N hydrochloric acid. Fluorescence readings were measured at 450 nm using a Spectra Max M5 multifunction plate reader. Among them, four anti-human CD79B mouse monoclonal antibodies, including mAb015 and mAb017, showed the highest ELISA binding affinity. 3) FACS detection of anti-human CD79B mouse monoclonal antibodies: DOHH2 cell suspensions were centrifuged, resuspended in PBS containing 0.1% BSA, and counted. 100 μL of 100 nM antibody diluted 1:10 was added and incubated in an incubator at room temperature for 1 hour. After washing three times, anti-mouse IgG (Fc specific)-FITC secondary antibody was added and incubated in the dark at room temperature for 30 minutes. The cells were then washed three times and gently resuspended in PBS containing 0.1% BSA before being loaded onto the instrument for detection. Among these, four anti-human CD79B mouse monoclonal antibodies, including mAb015 and mAb017, showed the highest FACS binding affinity. 4) SPR detection of anti-human CD79B mouse monoclonal antibody: The affinity of anti-human CD79B antibody with its antigen, human CD79B-His, was detected by surface plasmon resonance (SPR). The antigen, human CD79B-His protein, was immobilized on a CM5 chip. The coupling level was set to 100 RU. The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). The diluted antibody was injected into the experimental and control channels at a flow rate of 30 μL / min for 3 minutes, followed by dissociation for 5 minutes. Then, regeneration buffer (10 mM glycine, pH 1.5) was injected at a flow rate of 30 μL / min for 30 seconds. Data were analyzed using Biacore 8K evaluation software.

[0141] Example 1-3. Amino acid sequence determination of the variable region of a mouse monoclonal antibody The amino acid sequence of the variable regions of the high-affinity hybridoma monoclonal cell line obtained in Examples 1-2 was determined. A human-mouse chimeric antibody (cAb) was then recombinantly expressed and further characterized. The heavy and light chain variable regions of the antibody gene were amplified by reverse transcription PCR, ligated into a vector, and sequenced to obtain the light and heavy chain sequences of the monoclonal antibody. First, total cellular RNA from the highly active single-cell line obtained in Example 2 was extracted using an RNA purification kit (Qiagen, product number 74134; see the manual for the procedure). Next, single-stranded cDNA (i.e., cDNA reverse transcription using Oligo-dT primers) was prepared using a cDNA synthesis kit (Invitrogen, product number 18080-051). Using this as a template, the antibody light and heavy chain variable region sequences were synthesized by PCR. The PCR product was cloned into the TA vector pMD-18T and used for sequence analysis. The light and heavy chain sequences of the obtained antibody were each cloned into an expression vector (see Example 1-1 for the method), and the recombinant monoclonal antibody was expressed and its activity was verified (see Example 1-2 for the method), after which it was humanized.

[0142] The amino acid residues of the VH / VL CDRs of anti-human CD79B antibodies are assigned and annotated according to the Chothia numbering system. Sequence of the monoclonal antibody mAb015 from mouse hybridoma cells: [Table 15] Sequence of the monoclonal antibody mAb017 from mouse hybridoma cells: [Table 16]

[0143] The mouse CDR sequences are shown in Table 3. Table 3. CDR sequences of mouse anti-human CD79B antibodies [Table 17]

[0144] Example 1-4. Humanization of anti-human CD79B antibody The light and heavy chain sequences of the mouse anti-CD79B monoclonal antibody obtained in Examples 1-3 were compared for homology with antibody databases, followed by establishing a humanized antibody model. The model was then used to select the best humanized anti-CD79B monoclonal antibody as the preferred molecule through backmutation screening. This method began by searching publicly available mouse Fab crystal structure model databases (e.g., the PDB database) for crystal structures similar to the mouse candidate molecule. High-resolution (e.g., <2.5 Å) Fab crystal structures were then selected to establish the mouse Fab model. The mouse antibody light and heavy chain sequences were aligned with the sequences in the model, and sequences matching the mouse antibody sequences in the model were retained to obtain a mouse antibody structural model. Mismatched amino acids were identified as potential backmutation sites. The mouse antibody structural model was then run using Swiss-pdb viewer software, and energy was optimized (minimized). Different amino acid sites, excluding the CDRs, in the model were backmutated, and the resulting mutated (humanized) antibodies were compared with the unhumanized antibody for activity detection. Humanized antibodies with good activity were retained. The CDR regions were then optimized to avoid glycosylation, deamidation, and oxidation sites, etc. The antibodies were cloned, expressed, and purified by gene cloning and recombinant expression, and finally, humanized antibodies hAb015-10 and hAb017-10 with the best activity retention were selected by ELISA, FACS, and SPR detection.

[0145] The sequences of the humanized antibodies hAb015-10 and hAb017-10 are as follows: [Table 18]

[0146] Example 1-5: Construction of a cell line with high TROP-2 expression The pCDH-hTROP-2 lentiviral expression vector plasmid and Lipofectamine 3000 transfection reagent for pVSV-G and pCMV-dR8.91 lentiviral packaging vectors were transfected into 293T virus packaging cells. The virus-containing culture supernatant was collected, filtered, and centrifuged at ultrahigh speed. The concentrated virus was then used to infect Chinese hamster ovary cells CHO-K1. After selection with puromycin for 2-3 weeks, FACS single-cell sorting was performed. The amount of TROP-2 expression on the surface of lentivirus-infected CHO-K1 cells was detected by FACS, and CHO-K1 / hTROP-2 monoclonal cell lines with high TROP-2 expression were selected.

[0147] The TROP-2 amino acid sequence (Genbank: NP_002344.2) is as follows: [Table 19] [Table 20]

[0148] Examples 1-6: Preparation of anti-human TROP-2 monoclonal antibody The anti-human TROP-2 monoclonal antibody disclosed herein was prepared according to the method disclosed in WO03074566. Using the hRS7 antibody variable region gene as a template, point mutations were designed in the CDRs using computer software. The antibody was inserted into the protein expression vector Phr-IgG (containing a signal peptide and constant region gene (CH1-Fc / CL) fragment) by molecular cloning and expressed in HEK293 cells and Expi-CHO-S cells. The antibody was purified according to conventional methods. Activity was verified using CHO-K1 cells overexpressing huTROP-2 protein and huTROP-2 protein (His27-Thr274 Accession # NP_002344.2). Antibodies with good target binding activity were selected, among which the PD3 variable region sequence is as follows: [Table 21] Note: The underlined regions are the CDR regions as determined by the Kabat numbering convention.

[0149] Table 4. CDR regions of PD3 antibodies [Table 22]

[0150] The antibody heavy chain constant region can be selected from the constant regions of human IgG1, IgG2, IgG4, and variants thereof, and the light chain constant region can be selected from the light chain constant region of human kappa or lambda chains or variants thereof. Exemplary antibody heavy chain constant regions are selected from human IgG1 whose sequence is represented by SEQ ID NO:11, and light chain constant regions are selected from the human kappa chain whose sequence is represented by SEQ ID NO:12. [Table 23]

[0151] Illustratively, the above light chain / heavy chain constant regions are combined with the variable regions of the PD3 antibody to form a complete antibody, whose light chain / heavy chain sequences are as follows: [Table 24]

[0152] II. Preparation of Compounds Example 2-1: Synthesis of Compound L-1 [ka] [ka] [ka]

[0153] Step 1: Preparation of Compound 2 In an ice-water bath, compound 1 (50 mg, 0.08 mmol, prepared according to the method described in WO2017151979) was dissolved in 1.5 mL of N,N-dimethylformamide, followed by the addition of DIPEA (N,N-diisopropylethylamine, 18 mg, 0.14 mmol) and bis(p-nitrophenyl)carbonate (49 mg, 0.16 mmol). The mixture was then stirred at room temperature, and 20 mL of methyl tert-butyl ether was added. The mixture was stirred for 20 minutes, filtered, and dried to obtain 36 mg of solid compound 2. LC / MS (ESI): m / z 784.1 [M+H] + .

[0154] Step 2: Preparation of Compound D-1b Compound D-1a (eribulin, prepared according to the method of ZL201010236637.2) (72.91 mg, 0.1 mmol) was dissolved in 10 mL of tetrahydrofuran in an ice-water bath, and Fmoc-OSu (9-fluorenylmethyl succinimidyl carbonate, 41 mg, 0.12 mmol) was added. The mixture was stirred at room temperature until the reaction was complete. The mixture was concentrated under reduced pressure to obtain the crude product, which was used directly in the next reaction.

[0155] Step 3: Preparation of compound D-1c The crude product of compound D-1b obtained in the previous step was dissolved in 10 mL of anhydrous ether, and silver oxide (34.8 mg, 0.15 mmol) and iodomethane (28.4 mg, 0.2 mmol) were added. The mixture was stirred at room temperature until the reaction was complete, filtered, and concentrated under reduced pressure to obtain the crude product, which was then used directly in the next reaction.

[0156] Step 4: Preparation of Compound D-1 The crude product of compound D-1c obtained in the previous step was dissolved in 10 mL of tetrahydrofuran, 2 mL of diethylamine was added, and the mixture was stirred at room temperature until the reaction was complete. The mixture was concentrated under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate / petroleum ether) to obtain 3 mg of the target product, compound D-1. LC / MS (ESI): m / z 744.2 [M+H] + .

[0157] Step 5: Preparation of compound L-1 Compound D-1 (13.5 mg, 0.018 mmol) was dissolved in 1.5 mL of DMF, DIPEA (7 mg, 0.054 mmol) was added, and compound 2 (18 mg, 1.3 mmol) was added in portions. The mixture was stirred until the reaction was almost complete and concentrated to give the crude product. Separation by preparative HPLC (chromatography column: Welch XTimate C18 (5.0 μm*30.0*150 mm); mobile phase: A-water (0.1% formic acid): B-acetonitrile, gradient elution = 70:30-5:95 (16 min, flow rate: 30.0 mL / min) gave 6.5 mg of compound L-1 with a purity of 96.95%. LC / MS (ESI): m / z 1388.3 [M+H] + . 1 HNMR (CDCl3, 400M) δ 0.85~0.90 (m, 3H), 0.93~1.00 (m, 3H), 1.08~1.10 (m, 3H), 1.20~1.50 (m, 15H), 1.75~2.04 (m, 6H), 2.13~2.55 (m, 16H), 2.70~2.77 (m, 1H), 2.80~2.96 (m, 2H), 3.16~3.97 (m, 20H), 3.99~4.39 (m, 8H), 4.60~4.80 (m, 6H), 4.88~5.10 (m, 5H), 5.24~5.37 (m, 4H), 6.71 (s, 2H), 7.03 (d, J = 6.8 Hz, 1H), 7.18~7.30 (m, 3H), 7.63 (d, J = 8.0 Hz, 2H), 8.92 (bs, 1H).

[0158] Example 2-2 Synthesis of Compound D-2 [ka]

[0159] A reaction flask was charged with (R)-2-cyclopropyl-2-hydroxyacetic acid (4.7 mg, 0.04 mmol, 1.5 eq), THF, and the mixture was stirred to dissolve. The mixture was then cooled in an ice-water bath. EDCI HCl (8.0 mg, 0.04 mmol, 1.5 eq, 1-ethyl-3(3-dimethylpropylamine)carbodiimide hydrochloride), HOBT (5.4 mg, 0.04 mmol, 1.50 eq, 1-hydroxybenzotriazole), compound D-1a (20 mg, 0.027 mmol, 1.0 eq), and finally DIPEA (10.5 mg, 0.08 mmol, 3.0 eq) were added. After the addition of the materials was completed, the mixture was allowed to warm to room temperature (20°C) and stirred until the reaction was almost complete. 2 mL of water was added to quench the reaction, and the mixture was extracted with ethyl acetate (2 x 5 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether) to obtain 6.0 mg of compound D-2 with a purity of 98%. MS:827.8[M+H] + . 1H NMR (400 MHz, CDCl3) δ 6.82 (s, 1H), 5.08 (s, 1H), 4.93 (s, 1H), 4.89 (s, 1H), 4.81 (s, 1H), 4.70 (t, J = 4.4 Hz, 1H), 4.61 (t, J = 4.4 Hz, 1H), 4.42 - 4.25 (m, 3H), 4.23 - 4.16 (m, 1H), 4.12 (s, 1H), 4.03 (d, J = 9.3 Hz, 3H), 4.02 - 3.87 (m, 3H), 3.82 (d, J = 9.4 Hz, 1H), 3.78 - 3.70 (m, 3H), 3.58 (dd, J = 50.7, 8.9 Hz, 4H), 3.43 (s, 3H), 3.32 - 3.23 (m, 2H), 2.88 (d, J = 9.5 Hz, 2H), 2.72 (dd, J = 16.0, 10.0 Hz, 1H), 2.46 (d, J = 13.9 Hz, 4H), 2.33 (d, J = 13.8 Hz, 3H), 2.19 (dd, J = 21.4, 14.3 Hz, 4H), 2.08 (s, 1H), 1.97 (ddd, J = 13.6, 9.4, 4.7 Hz, 5H), 1.44 (d, J = 11.5 Hz, 3H), 1.27 (d, J = 12.2 Hz, 4H), 1.10 (d, J = 6.2 Hz, 3H), 0.68 - 0.45 (m, 4H).

[0160] Example 2-3 Synthesis of Compound D-3

change

[0161] Compound D-1 (22 mg, 0.03 mmol) and (R)-2-cyclopropyl-2-hydroxyacetic acid (7.0 mg, 0.06 mmol) were dissolved in 2 mL of DCM under a nitrogen atmosphere at room temperature. EtN (21 μL, 0.15 mmol) and DMTMM (20.3 mg, 0.069 mmol, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride) were then added sequentially and stirred at room temperature overnight. Upon completion of the reaction, the mixture was quenched by the addition of HO (2 mL). The mixture was extracted with DCM (2 mL x 3), and the organic phase was concentrated under reduced pressure (bath temperature 30 °C). The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether) to give compound D-3 (18 mg, 95% purity). MS: 863.8 [M+Na] + .

[0162] Example 2-4 Synthesis of Compound D-4 [ka] Compound D-4 was prepared by following the method of Example 2-2, using hydroxyacetic acid instead of (R)-2-cyclopropyl 2-hydroxyacetic acid. MS:787.82[M+H] + . 1HNMR (CDCl3, 400M):0.86~0.90 (m, 1H), 1.04~1.13 (m, 4H), 1.22~1.41 (m, 4H), 1.71~1.74 (m, 3H), 1.94~2.00 (m, 5H), 2.15~2.22 (m, 8H), 2.48 (S, 3H), 2.71~2.75 (m, 2H), 2.87~2.89 (m, 2H), 3.26~3.31 (m, 2H), 3.43 (S, 3H), 3.53~3.55 (m, 1H), 3.64~3.70 (m, 2H), 3.74 (s, 1H), 3.80~3.84 (m, 1H), 3.90~4.05 (m, 4H),4.12 (s, 3H), 4.18~4.20 (m,1H), 4.26~4.39 (m, 3H), 4.61 (t, J = 4.8 Hz, 1H), 4.619(t, J = 4.8 Hz, 1H), 4.82 (s, 1H), 4.82 (s, 1H), 4.88 (s, 1H), 4.93 (s, 1H), 5.08 (s, 1H),6.89(m, 1H).

[0163] Example 2-5 Preparation of Compound D-5 [ka] Compound D-5 was prepared by following the method of Example 2-2, using 1-hydroxycyclopropane-1-carboxylic acid instead of (R)-2-cyclopropyl-2-hydroxyacetic acid. MS: 835.7 [M+Na] + .

[0164] Example 2-6 Synthesis of Compound D-6 [ka] Using starting materials (R)-2-cyclopropyl-2-hydroxyacetic acid and E1-30 (synthesized according to the literature Bioorg. Med. Chem. Lett. 14 (2004) 5551-5554), compound of formula D-6 was prepared according to the method of Example 2-2. MS:850.64 [M+Na] + .

[0165] Example 2-7 Synthesis of Compound D-7 [ka] Using the starting materials 1-hydroxycyclopropane-1-carboxylic acid and E1-30 (synthesized according to the literature Bioorg. Med. Chem. Lett. 14 (2004) 5551-5554), the compound of formula D-7 was prepared according to the method of Example 2-2. MS:836.73 [M+Na] + .

[0166] Example 2-8 Synthesis of Compound D-8 [ka] Using starting materials p-hydroxyethylbenzoic acid and E1-30, the compound of formula D-8 was prepared according to the method of Example 2-2. MS:886.75 [M+Na] + .

[0167] Test Example 1: In vitro cytotoxic activity screening 1.1. Experimental principles and methods In this experiment, CTG was used to detect ATP content, which reflected the viability of tumor cells. First, cells were seeded at different densities and cultured for 3 and 5 days to determine IC. 50 The final culture conditions were determined based on the maximum inhibition rate and the maximum inhibition rate, and the killing effect of the toxin molecules was detected under these conditions. 1.2. Cell line selection Two disease models, breast cancer and NSCLC, were selected based on the experimental objectives, and three cell lines were used for the screening experiments: SKBR3 tumor cells (HER2+, ATCC, Cat. No. HTB-30), MDA-MB-468 (HER2-, ATCC, Cat. No. HTB-132), and A549 (human non-small cell lung cancer cells, ATCC, Cat. No. CCL-185).

[0168] 1.3. Establishing cell culture conditions 1) Cell culture: A549, SK-BR-3, and MDA-MB-468 cells were cultured in Ham's F-12K (Kaighn's) medium (Gibco, 21127030), McCoy's 5A medium (ThermoFisher, product number 16600108), and Leibovitz's L-15 medium (ThermoFisher, product number 11415-114), respectively, containing 10% FBS (Gibco, 10099-141). 2) Cell seeding: A549 cells were digested with trypsin, then suspended in the above medium, counted, and seeded with 4.3 × 10 5 , 7.2 × 10 5 , 11.5 x 10 5 Cells were removed from the wells and culture medium was added to bring the final volume to 26 mL. 180 μL of cell suspension was added to each well in columns 2 through 11 of a 96-well plate (Corning, Product No. 3903) to achieve cell densities of 3K, 5K, and 8K, respectively. Column 12 was filled with 200 μL of culture medium, and the remaining wells were filled with PBS. The above procedure was repeated for SKBR3 and MDA-MB-468 cells. Two parallel samples were prepared. 3) Drug preparation: Stock solutions of the positive control eribulin and compounds of the present disclosure were prepared in DMSO in a round-bottom 96-well plate (Corning, product number 3788). Column 1 of Dispensing Plate 1 was prepared with 2 mM stock solution (10-fold dilution of the stock solution with DMSO), which was then diluted 10-fold with DMSO in a gradient up to column 10, with column 11 containing DMSO. 95 μL of the corresponding culture medium was added to each well in columns 2 to 11 of Dispensing Plate 2, and 5 μL of the solution from columns 2 to 11 of Dispensing Plate 1 was aspirated into Dispensing Plate 2, mixed well, and 20 μL was aspirated and added to the seeded cells, followed by incubation for 3 and 5 days. 4) CTG detection (Cell Titer-Glo™, luminescent cell viability assay, Promega): On days 3 and 5, the cell plate was removed and equilibrated to room temperature. 90 μL of CTG was added to each well, and the reaction was allowed to proceed in the dark at room temperature for 10 minutes. The luminescence was then read using a microplate reader, and the IC was calculated.50 was calculated.

[0169] 1.4. Data Results Table 5 [Table 25] Conclusion: Compound D-1 showed excellent killing effect in three tumor cell lines, and was significantly superior to the positive drug eribulin.

[0170] Example 2-9: Synthesis of Compound D-9 [ka] [ka]

[0171] Step 1: Preparation of compound D-9a At room temperature, 0.3 mL of 1,4-dioxane and 0.3 mL of compound E-305 (31 mg, 0.042 mmol, synthesized according to Bioorg. Med. Chem. Lett. 14 (2004) 5551-5554.) were taken, followed by the addition of 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu) (17 mg, 0.050 mmol) and solid sodium carbonate (18 mg, 0.168 mmol). The mixture was stirred at room temperature overnight, and it was confirmed that the starting materials were almost completely converted. During the reaction, water was added to quench the reaction, and the mixture was extracted with ethyl acetate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether) to obtain 15 mg of the product. LC / MS (ESI): m / z 965.64 [M+H] + .

[0172] Step 2: Preparation of compound D-9b Compound D-9a (7 mg, 0.007 mmol) was dissolved in dichloromethane (0.5 mL) at room temperature, followed by the addition of molecular sieves 4A (10 mg), trimethyloxonium tetrafluoroborate (11 mg, 0.07 mmol), and proton sponge (16 mg, 0.07 mmol) and stirring at room temperature for 1 hour. After confirming that the starting material was almost completely converted, the reaction was quenched by adding water, extracted with methyl tert-butyl ether, washed with 1 N dilute hydrochloric acid, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:1) to give 7 mg of the product. LC / MS (ESI): m / z 979.68 [M+H] + .

[0173] Step 3: Preparation of compound D-9 In an ice-water bath, 1 mL of tetrahydrofuran was taken, and compound D-9b (10 mg, 0.01 mmol) was dissolved. DBU (6 μL, 0.04 mmol) was added dropwise, and the mixture was stirred until the reaction was complete. The reaction was quenched by adding water, extracted with dichloromethane, and concentrated under reduced pressure. The residue was separated by preparative HPLC (chromatography column: Welch Xtimate C18 (10 × 150 mm × 5 μm); mobile phase: A - water (20 mM NH4HCO3): B - acetonitrile, gradient elution = 30% B to 95% B) to obtain 5 mg of compound D-9. LC / MS (ESI): m / z 757.85 [M+H] + .

[0174] Example 2-10 Synthesis of Compound D-10 [ka] [ka]

[0175] Step 1: Preparation of compound D-10b Compound D-10a (6 mg, 0.008 mmol, synthesized according to Bioorg. Med. Chem. Lett. 21 (2011) 1639-1643) was dissolved in 2 mL of tetrahydrofuran in an ice-water bath, and lithium aluminum hydride solution (80 μL, 1 M soln. in THF, 0.08 mmol) was added dropwise. The reaction temperature was slowly raised to 40 °C with stirring. After confirming that the starting materials were almost completely converted by LCMS, the reaction was quenched with sodium sulfate decahydrate, stirred in an ice-water bath for half an hour, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was used directly in the next reaction. LC / MS (ESI): m / z 758.4 [M+H] + .

[0176] Step 2: Preparation of compound D-10c Compound D-10b (obtained in the previous step) was dissolved in 0.5 mL of 1,4-dioxane and 0.5 mL of water at room temperature. 9-Fluorenylmethyl succinimidyl carbonate (6.5 mg, 0.019 mmol) and sodium carbonate (6.8 mg, 0.064 mmol) were added sequentially. The mixture was stirred at room temperature overnight to confirm that the starting material was almost completely converted. The reaction was quenched by adding water, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 3:2) to obtain 14 mg of compound D-10c. LC / MS (ESI): m / z 980.4 [M+H] + .

[0177] Step 3: Preparation of compound D-10d In an ice-water bath, 1 mL of dichloromethane was taken, and compound D-10c (14 mg, 0.014 mmol) obtained in the previous step was dissolved. Dess-Martin oxidant (18.2 mg, 0.042 mmol) was added. The mixture was slowly warmed to room temperature with stirring until almost all of the starting material was converted by LCMS. The mixture was quenched with aqueous sodium bicarbonate, extracted with dichloromethane, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 3:2) to give 8 mg of compound D-10d. LC / MS (ESI): m / z 978.4 [M+H] + .

[0178] Step 4: Preparation of compound D-10 In an ice-water bath, 1 mL of tetrahydrofuran was taken and compound D-10d (8 mg, 0.008 mmol) obtained in the previous step was dissolved. DBU (6 μL, 0.032 mmol) was further added dropwise and stirred for 1 hour. TLC confirmed that the starting material was almost completely converted. During the reaction, water was added to quench the reaction, extracted with dichloromethane, and concentrated under reduced pressure. The residue was separated by preparative HPLC (chromatography column: Welch Boltimate C18 Core-Shell (4.6*50 mm*2.7 μm); mobile phase: A-water (20 mM NH4HCO3)) to obtain the target product compound D-10 (1.3 mg). LC / MS (ESI): m / z 755.93 [M+H] + .

[0179] Test Example 2: In vitro cytotoxic activity screening 2.1. Experimental principles and methods In this experiment, CTG was used to detect the content of ATP, which reflected the survival status of tumor cells.

[0180] 2.2. Establishing cell culture conditions 1) Cell culture: A549, SK-BR-3, and MDA-MB-468 cells were cultured in Ham's F-12K (Kaighn's) medium (Gibco, 21127030), McCoy's 5A medium (ThermoFisher, product number 16600108), and Leibovitz's L-15 medium (ThermoFisher, product number 11415-114), respectively, containing 10% FBS (Gibco, 10099-141). A549, SKBR3, and MDA-MB-468 were digested with trypsin, resuspended in medium, and counted to determine a cell density of 2.2 × 10 4 The cell suspension was adjusted to cells / mL and 135 μL of the cell suspension was added to each well of columns 2 to 11 of a 96-well plate. Column 12 served as a blank control. The plate was then cultured in a 5% CO2 incubator at 37°C for 24 hours. 2) Drug preparation: a) Preparation of stock solution: The test compound and positive control drug were dissolved in DMSO to give a stock solution concentration of 5 mM. b) Dispensing Plate 1: Stock solution was diluted 40-fold in column 1, followed by a 3-fold gradient dilution in columns 2 to 11. Column 12 was DMSO. c) Dispensing Plate 2: Add 196 μL of the appropriate culture medium to columns 2 to 11, and then aspirate 4 μL from columns 3 to 12 of Dispensing Plate 1 to columns 2 to 11 of Dispensing Plate 2. Mix well.

[0181] 2.3. Cell treatment 15 μL was drawn up from Dispensing Plate 2 and added to the seeded cells, which were then cultured in a 37°C incubator with 5% CO for 5 days. 2.4) CTG detection (Cell Titer-Glo™, luminescent cell viability assay): The cell plate was removed and equilibrated to room temperature. 75 μL of CTG was added to each well and incubated at room temperature for 10 min in the dark. The luminescence was measured using a microplate reader and the IC was calculated. 50 was calculated.

[0182] 2.5. Data Results Table 6 [Table 26]

[0183] The structural formulas of the positive control drug compound E-305 and compound E1-30 are shown below, and their preparation methods refer to the method in Bioorganic & Medicinal Chemistry Letters 14 (2004) 5551-5554: [ka]

[0184] Example 2-11: Synthesis of Compound L-2 [ka] [ka]

[0185] In an ice-water bath, compound D-1a (9 mg, 0.012 mmol) was dissolved in 0.3 mL DMF, DIPEA (3.5 mg, 0.028 mmol) was added, and compound 2 (7.8 mg, 0.011 mmol) was added in portions. The mixture was stirred until the reaction was almost complete, concentrated under reduced pressure to give the crude product, which was separated by preparative HPLC (chromatography column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NHOAc): B-acetonitrile, gradient elution) to give 4.95 mg of compound L-2 with a purity of 97%. LC / MS (ESI): m / z 1374.3 [M+H] + .

[0186] Example 2-12: Synthesis of Compound L-3 [ka] [ka] [ka]

[0187] Step 1: Preparation of Compound 4 Compound 4a (1.3 g, prepared by the method disclosed in WO2013106717) was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.2 g), benzyl bromide (1.35 mL), and tetrabutylammonium iodide (415 mg) were added sequentially. The mixture was stirred at room temperature until the reaction was almost complete, filtered, concentrated, and purified by silica gel column chromatography using petroleum ether / ethyl acetate as a developing solvent to obtain compound 4b.

[0188] Compounds 4b (121 mg) and 4c (180 mg) were placed in a reaction flask and 4 mL of tetrahydrofuran was added. Under a nitrogen atmosphere, the mixture was cooled to approximately 0 °C in an ice-water bath, potassium t-butoxide (109 mg, 0.98 mmol) was added, and the mixture was warmed to room temperature and stirred for 40 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, and sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added. The mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate as a developing solvent to obtain compound 4d. MS m / z (ESI): 515.0 [M+1] + It was.

[0189] Compound 4d (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (12 mg, 10% content, dry) was added. The atmosphere was purged with hydrogen gas three times and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to give the crude product, title compound 4 (13 mg). This product was used directly in the next reaction without further purification. MS m / z (ESI): 424.9 [M+1].

[0190] Step 2: Preparation of compound DZ-1a Compound 4 (13.4 mg, 0.0316 mmol, 1.7 eq) and the methanesulfonate salt of compound D-1a (15 mg, 0.0182 mmol, 1 eq) were weighed and dissolved in DMF (0.5 mL). Triethylamine (10 mg, 0.0988 mmol, 5.4 eq) and DMTMM (9.8 mg, 0.0332 mmol, 1.8 eq) were added while cooling in an ice bath. The reaction mixture was allowed to warm to room temperature and stirred until nearly complete. Water (2 mL) and ethyl acetate (3 mL) were added, and the mixture was diluted and separated. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography (ethyl acetate / petroleum ether) to give 16 mg of compound DZ-1a in 86.7% yield. LC / MS (ESI): m / z 1136.3 [M+H] + .

[0191] Step 3: Preparation of compound DZ-1b Compound DZ-1a (16 mg, 0.0141 mmol, 1 eq) obtained in the previous step was weighed and dissolved in THF (0.4 ml). Triethylamine (4.2 mg, 0.057 mmol, 4 eq) was added and stirred in an ice bath until almost complete. Diluted with dichloromethane (5 ml), washed with water (2 ml × 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was used directly in the next step. LC / MS (ESI): m / z 914.3 [M+H] + .

[0192] Step 4: Preparation of compound L-3 The crude compound DZ-1b (16 mg, 0.0175 mmol, 1 eq) obtained in the previous step and compound 6 (11.6 mg, 0.0246 mmol, 1.4 eq, prepared by the method of EP2907824) were weighed and dissolved in DMF (0.5 mL). HATU (9.9 mg, 0.026 mmol, 1.5 eq) and N,N-diisopropylethylamine (DIPEA) (5.5 mg, 0.0426 mmol, 2.4 eq) were added, and the mixture was stirred in an ice bath until the reaction was almost complete. Water (2 mL) and ethyl acetate (3 mL) were added, and the mixture was diluted and separated. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (chromatography column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH4OAc): B-acetonitrile, gradient elution) to obtain 10 mg of compound L-3. LC / MS (ESI): m / z 1368.3 [M+H] + .

[0193] Example 2-13: Synthesis of Compound L-4 [ka] [ka]

[0194] Step 1: Preparation of compound DZ-2a Compound 4 (11.6 mg, 0.0273 mmol, 1.5 eq) and compound D-1 (13.5 mg, 0.0181 mmol, 1 eq) were weighed and dissolved in N,N-dimethylformamide (0.5 mL). While cooling in an ice bath, DMTMM (10.1 mg, 0.0343 mmol, 1.3 eq) was added and stirred until the reaction was nearly complete. Water (2 mL) and ethyl acetate (3 mL) were added to quench the reaction, dilute the mixture, and separate the layers. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography (ethyl acetate / petroleum ether) to give 10 mg of compound in 47.9% yield. LC / MS (ESI): m / z 1150.2 [M+H] + .

[0195] Step 2: Preparation of compound DZ-2b Compound DZ-2a (10 mg, 0.0087 mmol, 1 eq) obtained in the previous step was weighed and dissolved in THF (1 mL). DBU (1,8-diazabicycloundec-7-ene) (5.2 mg, 0.034 mmol, 4 eq) was added and stirred in an ice bath until the reaction was almost complete. Dilution was added with dichloromethane (5 mL), and the mixture was washed with water (2 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was used directly in the next step. LC / MS (ESI): m / z 928.2 [M+H] + .

[0196] Step 3: Preparation of compound L-4 Compound DZ-2b (16 mg, 0.0087 mmol, 1 eq) obtained in the previous step and compound 6 (7.8 mg, 0.0165 mmol, 1.9 eq) were weighed and dissolved in DMF (0.5 mL). HATU (6.2 mg, 0.0163 mmol, 1.9 eq) and DIEA (5.7 mg, 0.0441 mmol, 5 eq) were added, and the mixture was stirred in an ice bath until the reaction was almost complete. Water (2 mL) and ethyl acetate (3 mL) were added, and the mixture was diluted and separated. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (chromatography column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH4OAc): B-acetonitrile, gradient elution) to give 3.5 mg of compound L-4, with a two-step yield of 29.1%. LC / MS (ESI): m / z 1382.2 [M+H] + .

[0197] Example 2-14: Preparation of antibody-drug conjugate ADC-1 [ka]

[0198] A solution of antibody PD3 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 0.9 mL, 60.6 nmol) was added to an aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 15.2 μL, 152 nmol) at 37°C. The solution was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound L-3 (0.83 mg, 606 nmol) was dissolved in 50 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-1 in PBS buffer (0.76 mg / mL, 10 mL), which was then stored frozen at 4 °C. The mean value calculated by capillary electrophoresis sodium dodecyl sulfate (CE-SDS) ultraviolet detection was k = 3.87.

[0199] Example 2-15: Preparation of antibody-drug conjugate ADC-2 [ka]

[0200] A solution of antibody PD3 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 1.14 mL, 77.2 nmol) was added to a solution of tri(2-carboxyethyl)phosphine (TCEP) in water (10 mM, 19.3 μL, 193 nmol) at 37°C. The solution was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound L-2 (0.83 mg, 772 nmol) was dissolved in 50 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-2 in PBS buffer (0.71 mg / mL, 12 mL), which was then stored frozen at 4 °C. The mean value was calculated using CE-SDS, and k was 3.88.

[0201] Example 2-16: Preparation of antibody-drug conjugate ADC-3 [ka]

[0202] A solution of antibody PD3 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 0.9 mL, 60.6 nmol) was added to an aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 15.2 μL, 152 nmol) at 37°C. The solution was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound L-1 (0.84 mg, 605 nmol) was dissolved in 50 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-3 in PBS buffer (0.77 mg / mL, 10.2 mL), which was then stored frozen at 4 °C. The mean value was calculated using CE-SDS, and k was 3.81.

[0203] Example 2-17: Preparation of antibody-drug conjugate ADC-4 [ka]

[0204] A solution of antibody PD3 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 0.9 mL, 60.6 nmol) was added to an aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 15.2 μL, 152 nmol) at 37°C. The solution was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound L-4 (0.84 mg, 608 nmol) was dissolved in 50 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-4 in PBS buffer (0.64 mg / mL, 13.5 mL), which was then stored frozen at 4 °C. The mean value was calculated using CE-SDS, and k was 3.88.

[0205] Example 2-18: Preparation of antibody-drug conjugate ADC-5 [ka]

[0206] A 10 mM tri(2-carboxyethyl)phosphine (TCEP) solution (16.8 μL, 168 nmol) was added to a PBS buffer solution of CD79B antibody hAb015-10 (0.05 M PBS buffer solution, pH 6.5; 10.0 mg / mL, 1.0 mL, 67.3 nmol) at 37°C. The solution was placed in a water bath oscillator and allowed to oscillate at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath. Compound L-1 (0.93 mg, 673 nmol) was dissolved in 50 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and oscillated at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-5 in PBS buffer (0.68 mg / mL, 9.6 mL), which was then stored frozen at 4 °C. The mean value was calculated using CE-SDS, and k was 4.07.

[0207] Test Example 3: The therapeutic effects of ADC-5 and Polivy on human diffuse large B-cell lymphoma WSU-DLCL2 subcutaneously transplanted tumors in nude mice were evaluated and compared. 3.1 Drug Information Blank group: hIgG1; ADC-5: a colorless, transparent liquid with a concentration of 0.68 mg / mL and a purity of 98.00%, sealed and stored in the dark at 2-8°C; Polivy (polatuzumab): A colorless, transparent liquid with a concentration of 5.83 mg / mL and a purity of 97.69%. Store sealed in the dark at 2-8°C.

[0208] 3.2 Drug Preparation All were diluted with saline to the desired concentration. Physiological saline solution was purchased from Otsuka Pharmaceutical Co., Ltd., China, with specifications of 10 mL:0.09 g. 3.3 Cells Human diffuse large B-cell lymphoma WSU-DLCL2 cells were purchased from DSMZ. WSU-DLCL2 cells were cultured in 10-cm culture dishes in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum and penicillin and streptomycin (GIBCO, product number 15070-063) in a 5% CO2 incubator at 37°C. Cells were passaged two to three times a week, and when they were in the exponential growth phase, they were harvested, counted, and seeded. 3.4 Experimental animals BALB / c-nu nude mice, female, 28-35 days old, were purchased from Beijing Huafukang Biotechnology Co., Ltd. Production license number: SCXK(Kyoto)2019-0008, Animal Certification No. 1103221911012510. Breeding environment: SPF grade.

[0209] 3.5 Experimental steps 2.0 × 10 for each nude mouse 7 WSU-DLCL2 cells were inoculated subcutaneously, and tumors grew to -100 mm 3 Once tumor volume reached 1000 mg / kg, the animals were divided into groups based on tumor volume (D0). Mice were administered intravenously (IV) at a dose of 10 mL / kg. The specific dose and administration schedule are listed in Table 3. Tumor volume was measured twice a week, and the mice were weighed and recorded. The use and welfare of the experimental animals were carried out in accordance with the regulations of the "Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)". The health status and mortality of the animals were monitored daily. Routine examinations included observation of the effects of the test substance and drugs on the daily behavior of the animals, such as behavioral activities, body weight changes, and appearance symptoms.

[0210] 3.6 Experimental indicators The experimental indicators were for examining the effect of the drug on tumor growth. The specific indicators were T / C% or tumor growth inhibition rate TGI (%). The diameter of the tumor was measured with a caliper twice a week. The calculation formula for the tumor volume (V) is as follows: V = 1 / 2 × a × b 2 Among them, a and b represent the length and width respectively. T / C(%) = (T - T0) / (C - C0) × 100. Among them, T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Tumor growth inhibition rate (TGI)(%) = 100 - T / C(%). When the tumor regressed, the tumor growth inhibition rate (TGI)(%) = 100 - (T - T0) / T0 × 100. When the tumor became smaller than the initial volume, that is, when T < T0 or C < C0, it was defined as partial response (PR), and when the tumor completely disappeared, it was defined as complete response (CR). When the experiment ended and the end point of the experiment was reached, or when the average tumor volume of the solvent group reached 1500 mm 3 the animals were sacrificed by CO2 anesthesia, and then dissected to remove the tumors and take pictures.

[0211] 3.7 Statistical analysis Unless otherwise specified, the comparison of tumor volumes between two groups used a two-sided Student's t-test, and a statistically significant difference was defined when P < 0.05.

[0212] 3.8 Results ADC-5 and Polivy (IV; D0, 1 mg / kg) inhibited tumors subcutaneously implanted in nude mice with human diffuse large B-cell lymphoma WSU-DLCL2 by 53% and 37%, respectively. Both tumor-bearing mice tolerated the drugs well and showed no obvious symptoms such as weight loss. Table 7 [Table 27] NOTE: IV intravenous injection

[0213] conclusion The antibody-drug conjugates ADC-5 and Polivy (positive control group) demonstrated significant antitumor activity against subcutaneously implanted tumors of human diffuse large B-cell lymphoma WSU-DLCL2 in nude mice, with tumor inhibition rates of 53% and 37%, respectively, and both drugs were well tolerated by tumor-bearing mice.

[0214] Test Example 4: Cell killing experiment 4.1 Experimental Objectives The purpose of this experiment was to measure the growth inhibitory activity of anti-TROP-2 antibody (PD3)-drug conjugates according to the present disclosure against different tumor cell lines: Miapaca2 tumor cells (human pancreatic cancer cells, Nanjing Kebai, Product No. CBP60544), Fadu tumor cells (human squamous cell carcinoma, ATCC, Product No. HTB-43), SK-OV-3 (human ovarian cancer cells, ATCC, Product No. HTB-77), K562 (human chronic myeloid leukemia cells, ATCC, Product No. CCL-243), HCC827 (human lung cancer cells, ATCC, Product No. CRL-2868), and BXPC3 (human pancreatic cancer cells, ATCC, Product No. CRL-1687). Cells were treated in vitro with different concentrations of antibody-drug conjugates and cultured for 6 days. After that, cell proliferation was detected using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Product No. G7573) reagent, and IC 50 The in vitro activity of the antibody-drug conjugate is evaluated based on the values.

[0215] 4.2 Experimental Method 1) Cell culture: MiaPaCa2, Fadu, SK-OV-3, K562, HCC827, and BXPC3 cells were cultured in DMEM / high glucose medium (GE, SH30243.01), MEM medium (Gibco, 11095080), McCoy's 5a medium (Gibco, 16600108), IMDM medium (ThermoFisher, 12440061), and RPIM1640 medium (Gibco, 11875119) containing 10% FBS (Gibco, 10099-141), respectively. 2) Cell seeding: On the day of the experiment, cells were digested with trypsin (0.25% Trypsin-EDTA (1x), Life Technologies, Product No. 25200-072), and then MiaPaCa2, Fadu, SK-OV-3, K562, HCC827, and BXPC3 were each prepared as a cell suspension in the appropriate medium to a density of 3.7 × 10 3 The suspension was adjusted to 500 cells / mL, and 135 μL of the suspension was added to each well of a 96-well plate (Corning, product number 3903). The cells in each well were then cultured at 37°C for 24 hours. 3) Drug preparation: The initial concentration of the test ADC stock solution was adjusted to 4 μM and added to the first column of a dispensing plate (Corning, product number 3599). Columns 2 to 9 of the dispensing plate were diluted 5-fold, and column 10 was filled with PBS. 15 μL of each well was added to a cell culture plate. 4) CTG detection: After culturing at 37°C for 6 days, the cell culture plate was removed and equilibrated to room temperature. 75 μL of CTG was added to each well, and the reaction was allowed to proceed in the dark at room temperature for 10 minutes. Luminescence was then read using a microplate reader (BMG Labtech, PHERAstar FS).

[0216] 4.3 Data analysis Data was processed and analyzed using Graphpad Prism 5. Results are shown in Table 8 below. Table 8 [Table 28]

[0217] Test Example 5: Bystander killing experiment 5.1 Experimental Purpose The antibody-drug conjugates disclosed herein were measured to determine whether they have a bystander killing effect on the TROP-2-negative MiaPaCa2 cells (human pancreatic adenocarcinoma in situ, Procell) when co-cultured with TROP-2-positive BXPC3 cells (human pancreatic adenocarcinoma cells, Procell). In a killing experiment, a 5 nM concentration of the antibody-drug conjugate was selected that had killing activity against the TROP-2-positive BXPC3 cells but not the TROP-2-negative MiaPaCa2 cells, and whether the antibody-drug conjugate has a bystander killing effect on the TROP-2-negative MiaPaCa2 cells was examined in a system in which both cells were co-cultured. 5.2 Experimental Method 1) Cell culture: MiaPaCa2 and BXPC3 cells were cultured in DMEM / high glucose medium (GE, SH30243.01) and RPIM1640 medium (Gibco, 11875119), respectively, containing 10% FBS (Gibco, 10099-141). 2) Cell seeding: On the day of the experiment, cells were digested with trypsin (0.25% Trypsin-EDTA (1x), Life Technologies, Product No. 25200-072), neutralized with fresh RPMI1640 medium (containing 10% FBS), centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in RPMI1640 + 10% FBS. After counting the cells, the BXPC3 cell density was adjusted to 6 x 10 4 The MiaPaCa2 cell density was adjusted to 1.5 x 10 cells / mL. 4 The concentration was adjusted to cells / mL. 500 μL of BXPC3 cells and 500 μL of MiaPaCa2 cells were added to each well of plate 1 of a 12-well plate. 500 μL of MiaPaCa2 cells and 500 μL of RPMI1640 + 10% FBS culture medium were added to plate 2 of a 12-well plate. The cells were cultured at 37°C under 5% carbon dioxide for 24 hours. 3) Preparation of antibody-drug conjugates: Antibody-drug conjugates ADC-1, ADC-2, ADC-3, and ADC-4 were each diluted to a concentration of 600 nM with RPMI 1640. 50 μL of this was then added to 100 μL of culture medium and diluted to 200 nM (40X, final concentration 5 nM). 25 μL was then added to a cell culture plate. A separate PBS control group was also cultured for six days. 4) Flow analysis: Cells from 12-well plates (Plates 1 and 2) were digested with trypsin, neutralized with fresh medium, centrifuged at 1000 rpm for 3 minutes, discarded the supernatant, and resuspended in 1 mL of FACS buffer (PBS + 2.5% FBS). 20 μL of cells were taken, 20 μL of trypan blue (Sigma, T8154-100ML) was added, and counted. Cells from Plate 1 were centrifuged at 1000 rpm for 3 minutes, discarded the supernatant, resuspended in 100 μL of FACS buffer, and 2 μL of TROP-2 (EGP-1) monoclonal antibody (MR54) (ThermoFisher, product number 12-6024-42) was added and incubated on ice for 30 minutes. Centrifuged at 2000 rpm for 1 minute at 4°C, resuspended in 150 μL of FACS buffer, and this procedure was repeated twice. Measurement was performed using a flow cytometer (BD, FACS Verse).

[0218] 5.3 Data analysis Data were processed and analyzed with Flowjo 10.0. CONCLUSIONS: All antibody conjugates demonstrated a bystander effect in the study.

[0219] Test Example 6: Pharmacokinetics test 1. Overview Non-naive beagle dogs were used as test animals, and the plasma drug concentrations were measured at various time points after intravenous injection of compound D-1 and eribulin using LC / MS / MS. The pharmacokinetic behavior of the compounds of the present disclosure in dogs was studied, and the pharmacokinetic characteristics were evaluated.

[0220] 2. Test plan 2.1 Test Drugs Compound D-1 and eribulin 2.2 Test animals Six male beagle dogs purchased from Medicilon Preclinical Research (Shanghai) LLC. were divided into two groups on average and used in the animal administration experiment. 2.3 Drug Preparation Compound D-1 was weighed and dissolved in 5% by volume of DMSO, 20% by volume of PG and 20% by volume of PEG400, and 55% by volume of saline was added to prepare a 0.25 mg / mL colorless and transparent solution. Eribulin was weighed and dissolved in 5% by volume of DMSO, 20% by volume of PG, and 20% by volume of PEG400, and then 55% by volume of saline was added to prepare a 0.25 mg / mL clear and colorless solution. 2.4 Administration Dogs in one group were intravenously administered with Compound D-1, all at a dose of 0.5 mg / kg and in a volume of 2 mL / kg. Dogs in the other group received intravenous administration of eribulin, all at a dose of 0.5 mg / kg and in a volume of 2 mL / kg.

[0221] 3, operation Dogs were injected with Compound D-1, and 1 mL of blood was collected before administration, 5 minutes after administration, and 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours later. The collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice. Plasma was centrifuged within 1 hour (2200 g, 2-8°C for 10 minutes). Plasma samples were stored in a -80°C freezer until testing. Dogs were administered an eribulin compound by injection, and 1 mL of blood was collected before administration, 5 minutes after administration, and 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours later. The collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice. Plasma was centrifuged within 1 hour (centrifugation at 2200 g for 10 minutes at 2-8°C). Plasma samples were stored in a -80°C freezer until testing. After injection, the content of the test compound in dog plasma was measured as follows: 25 μL of dog plasma was collected at each post-administration time point, and 50 μL (100 ng / mL) of camptothecin (China Institute for Biological Products Testing) as an internal standard solution and 200 μL of acetonitrile were added. The plasma was mixed by vortexing for 5 minutes and centrifuged for 10 minutes (3700 rpm). 3-4 μL of the supernatant was removed from the plasma sample and analyzed by LC / MS / MS (API4000 triple quadrupole tandem mass spectrometer (No. 2), Applied Biosystems; Shimadzu LC-30AD ultra-high performance liquid chromatography, Shimadzu, Japan).

[0222] 4. Pharmacokinetic parameter results The pharmacokinetic parameters of compounds according to the present disclosure are shown in Table 9 below. Table 9 [Table 29]

[0223] Test Example 7: Pharmacodynamic effects of ADC-2 and ADC-3 on human pharyngeal squamous cell carcinoma FaDu cell line subcutaneously transplanted tumors in BALB / c nude mice (1)Cell culture The human pharyngeal squamous cell carcinoma Fadu cell line (ScienCell Research Laboratories, ml-cs-0374) used in this experiment was cultured in MEM medium (supplemented with 10% (v / v) fetal bovine serum (FBS) (GIBCO, product number 10099-141) and 0.1% phosphate buffer) at 37°C in a 5% CO2 incubator. Mice were anesthetized with 3-4% isoflurane before inoculation. Cells were serially subcultured for 10 generations at 5 × 10 6 100 μL of Fadu cell culture medium was thoroughly mixed with an equal volume of Matrigel (solarbio) and inoculated subcutaneously into the right side of the back of each mouse near the armpit.

[0224] (2) Animal grouping and administration schedule The average tumor size is approximately 100 to 150 mm3 When the mice reached the age of 18, they were randomly assigned to groups of 8 mice per group based on tumor volume and body weight. The day of administration was defined as day 0. The group assignments and administration schedules are shown in Table 10 below: Table 10. Grouping and administration schedule [Table 30] Dosage: Animal dose was prepared at 10 μL / g body weight Tumor volume: After group allocation, tumor volume was measured twice a week for 4 consecutive weeks. Tumor volume (V) was calculated as follows: V = (length × width) 2 ) / 2. The relative tumor volume (RTV) of each mouse was calculated as RTV = Vt / V0, where Vt is the volume measured each time, and V0 is the volume at the start of treatment. Animal weight: After group allocation, mice were weighed and recorded twice a week. Observation of animal condition: In this experiment, animals given the solvent or test drug showed no abnormalities. The observation of ulcers in some animals was used as the endpoint.

[0225] (3) Criteria for discontinuing and resuming study drug administration If a mouse loses more than 15% of its body weight during the experiment, treatment is discontinued. The withdrawal period must be long enough for the mouse to regain its weight. Only one mouse is withdrawn from treatment, while the remaining mice are treated normally. The mouse's weight is then restored if its weight loss is less than 10% during the withdrawal period. The experiment is continued once the mouse regains its weight.

[0226] (4) Endpoint After the in vivo experiments were completed, all animals were sacrificed by CO2 asphyxiation and cervical dislocation. Tumors were harvested, weighed, and photographed. No samples were collected from tumor-bearing animals that died before the end of the in vivo experiments.

[0227] (5) Statistical analysis The results were expressed as mean ± SEM. Comparisons between the two groups were performed using Dunnett's multi-comparison test. A statistically significant difference was indicated by * if p < 0.05, ** if p < 0.01, and *** if p < 0.001.

[0228] (6) Results Body weight: The changes in body weight of animals in the vehicle and different test drug groups are shown in Figure 1. The body weight of the animals increased normally as the experiment progressed. Tumor volume: During the experiment, tumors grew slowly after inoculation of the animals in the blank solvent group (Group G1), and then rapidly increased in volume on Day 14. On Day 0 of the experiment, the mean tumor volume in Group G1 was 125.05 ± 3.66 mm. 3 On day 25, the mean tumor volume was 1854.48 ± 99.50 mm 3 According to the experimental data of tumor volume and relative tumor volume, a subcutaneous tumor model of human pharyngeal squamous cell carcinoma Fadu cell line was successfully established in BALB / c nude mice. On day 0 of the experiment, the mean tumor volume in the high-dose ADC-3 (3 mg / kg) group (G2 group) was 121.40 ± 3.18 mm 3 On day 25, the mean tumor volume was 721.56 ± 169.15 mm 3 During the experimental period, the high-dose ADC-3 group was able to significantly inhibit tumor growth compared with the blank vehicle group. Relative tumor growth rate and tumor weight inhibition rate: Relative tumor growth rate (T / C%): Used to evaluate the antitumor activity of drugs, calculated by the following formula: Relative tumor growth rate T / C (%) = treatment group (T) mean RTV / negative control group (C) mean RTV × 100%. The relative tumor growth rates of each group at each time point are shown in Table 11, and the trend chart is shown in Figure 2.

[0229] Table 11. Relative tumor growth rates in each group at each time point [Table 31]

[0230] Conclusion: After administration to the ADC-3 group, tumor volume in the high-dose group was significantly lower than that in the model group, while tumor volume in the low-dose group tended to decrease compared to the model group, but there was no statistical difference. This drug demonstrated a dose-dependent tumor growth inhibitory effect. At the same time, at a dose of 3 mg / kg, the in vivo efficacy of the ADC-3 group was superior to that of the ADC-2 group on day 25, with a significant difference between the two groups.

[0231] While specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative and that variations or modifications to these embodiments may be made without departing from the principles and essence of the present invention. Accordingly, the scope of the present disclosure is limited by the appended claims.

Claims

1. An antibody-drug conjugate represented by the following formula: 【Chemistry 1】 or 【Chemistry 2】 Among them, Ab is an antibody or antigen-binding fragment thereof; k is 1 to 10, -D is as shown in the following formula: 【Transformation 3】 Among them, R 1a is C 1-6 and R 1b is hydrogen, or a pharmaceutically acceptable salt or solvate thereof.

2. R 1a is methyl.

3. The antibody-drug conjugate of claim 1 , wherein the antibody is selected from a mouse antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

4. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, an anti-CD79 antibody, an anti-TROP-2 antibody, an anti-CD79B antibody, an anti-Mesothelin antibody, or an antigen-binding fragment thereof.

5. The antibody-drug conjugate of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is selected from trastuzumab, pertuzumab, nimotuzumab, enoblitzumab, emibetuzumab, inotuzumab, pinatuzumab vedotin, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, and glematumamab, or an antigen-binding fragment thereof.

6. the antibody is selected from an anti-CD79B antibody or an antigen-binding fragment thereof, and comprises an antibody heavy chain variable region and / or an antibody light chain variable region; The antibody heavy chain variable region is 1) HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; or 2) HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. and / or the antibody light chain variable region is 1) LCDR1, LCDR2, and LCDR3 represented by SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; or 2) The antibody-drug conjugate of any one of claims 1 to 4, comprising LCDR1, LCDR2 and LCDR3 represented by SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

7. The anti-CD79B antibody comprises a heavy chain variable region and a light chain variable region, and includes any one of the following 1) to 2): 1) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 represented by SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; 2) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; and 7. The antibody-drug conjugate of claim 6, comprising any one of a light chain variable region comprising LCDR1, LCDR2 and LCDR3 represented by SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

8. The anti-CD79B antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region is 1) a sequence represented by SEQ ID NO: 3 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 3; or 2) comprising a sequence represented by SEQ ID NO: 5 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 5; and / or the light chain variable region is 1) a sequence represented by SEQ ID NO: 4 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 4; or 2) The antibody-drug conjugate of claim 6 or 7, comprising a sequence represented by SEQ ID NO: 6 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO:

6.

9. The antibody-drug conjugate of claim 8, wherein the heavy chain variable region of the anti-CD79B antibody or antigen-binding fragment is represented by the sequence SEQ ID NO:3 and the light chain variable region is represented by the sequence SEQ ID NO:4, or the heavy chain variable region is represented by the sequence SEQ ID NO:5 and the light chain variable region is represented by the sequence SEQ ID NO:

6.

10. The anti-CD79B antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region is 1) a sequence represented by SEQ ID NO: 19 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 19; or 2) comprising a sequence represented by SEQ ID NO: 21 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 21; and / or the light chain variable region is 1) a sequence represented by SEQ ID NO: 20 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 20; or 2) The antibody-drug conjugate of any one of claims 6 to 9, comprising a sequence represented by SEQ ID NO: 22 or having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO:

22.

11. The antibody-drug conjugate of claim 10, wherein the heavy chain variable region of the anti-CD79B antibody or antigen-binding fragment is represented by the sequence SEQ ID NO: 19 and the light chain variable region is represented by the sequence SEQ ID NO: 20; or the heavy chain variable region is represented by the sequence SEQ ID NO: 21 and the light chain variable region is represented by the sequence SEQ ID NO:

22.

12. The antibody-drug conjugate of any one of claims 1 to 4, wherein the anti-TROP-2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the same sequences as those of the heavy chain variable region represented by SEQ ID NO: 29, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the same sequences as those of the light chain variable region represented by SEQ ID NO:

30.

13. The antibody-drug conjugate of claim 12, wherein the antibody is selected from anti-TROP-2 antibodies and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose sequences are represented by SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose sequences are represented by SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

14. The antibody-drug conjugate of claim 12 or 13, wherein the anti-TROP-2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 29 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 30 or has at least 90% identity thereto.

15. The antibody-drug conjugate of any one of claims 12 to 14, wherein the anti-TROP-2 antibody comprises a heavy chain variable region whose sequence is represented by SEQ ID NO: 29 and a light chain variable region whose sequence is represented by SEQ ID NO:

30.

16. The antibody-drug conjugate of any one of claims 12 to 15, wherein the anti-TROP-2 antibody comprises an antibody heavy chain constant region and a light chain constant region.

17. 17. The antibody-drug conjugate of claim 16, wherein the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region is selected from human antibody κ and λ chain constant regions.

18. 17. The antibody-drug conjugate of claim 16, wherein the antibody comprises a heavy chain constant region whose sequence is represented by SEQ ID NO: 31 and a light chain constant region whose sequence is represented by SEQ ID NO:

32.

19. The antibody-drug conjugate of any one of claims 12 to 16, wherein the anti-TROP-2 antibody comprises a heavy chain having the sequence represented by SEQ ID NO: 33 and a light chain having the sequence represented by SEQ ID NO:

34.

20. Selected from the following structural formulas: 【Chemistry 4】 2. The antibody-drug conjugate of claim 1, wherein k is selected from the group consisting of 1 to 10 and may be an integer or a decimal number.

21. R in D 1a is a methyl group, and R 1b 21. The antibody-drug conjugate of claim 20, wherein is hydrogen.

22. 22. A pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 21, and a pharmaceutically acceptable pharmaceutical carrier, diluent or excipient.

23. Use of the antibody-drug conjugate of any one of claims 1 to 21 or the pharmaceutical composition of claim 22 in the preparation of a medicament for treating or preventing tumors.

24. The method of claim 23, wherein the tumor is a cancer associated with expression of HER2, HER3, B7H3 or EGFR.

25. Use of the antibody-drug conjugate of any one of claims 1 to 21 or the pharmaceutical composition of claim 22 in the preparation of a medicament for treating and / or preventing cancer.

26. 26. The use of claim 25, wherein the cancer is breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.

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