Antibody-drug conjugates, pharmaceutical compositions thereof and uses thereof

Abstract

Provided are antibody-drug conjugates, pharmaceutical compositions thereof and uses thereof. In particular, provided are antibody-drug conjugates of formula (I), drug-linkers for preparing the antibody-drug conjugates, and uses of the antibody-drug conjugates in combating tumors. TIFF2025513163000280.tif39149

Description

[Technical field]

[0001] This application is based on and claims priority from Chinese Patent Application No. 202210465896.5, filed on April 29, 2022. The disclosure of the Chinese Patent Application is incorporated herein in its entirety.

[0002] The present application is in the field of medicine, and in particular relates to antibody-drug conjugates, pharmaceutical compositions thereof and uses thereof. [Background technology]

[0003] In antibody drug conjugates (ADCs), antibodies are coupled to small molecule cytotoxic drugs via specific linkers, and the antibodies act as carriers to transport small molecule cytotoxic drugs to target cells, thereby reducing systemic exposure and improving safety. Therefore, this is a hot topic in the research and development of tumor targeted therapy.

[0004] With the development of ADC technology, immunostimulatory antibody conjugates (ISACs) have been developed. ISACs are formed by coupling immune agonists or immune modulators, such as TLR7 and / or TLR8 agonists, to antibodies via cleavable / non-cleavable linkers. Immunostimulatory antibody conjugates combine the precise targeting of antibodies with the immune activation and long-lasting immune memory effects of immunostimulants, thereby realizing safe systemic administration and inducing anti-tumor immune responses.

[0005] In the research of antitumor treatment, there is a clear trend of development from single-drug treatment to combination treatment. From the perspective of clinical needs, the ideal combination of drugs should be able to improve clinical efficacy, reduce drug toxicity, and delay or avoid the development of drug resistance. On the one hand, drug combination refers to the use of two existing drugs, such as the combination of a cytotoxic drug and an immunostimulant, and on the other hand, refers to the combination of two active ingredients to form a new single drug.

[0006] Taking advantage of the targeting properties of antibodies, dual payload-type bifunctional immunomodulatory antibody-drug conjugates (iADCs) obtained by coupling a cytotoxic drug and an immunostimulant to an antibody can simultaneously invade tumor cells and the tumor immune microenvironment to exert dual effects, i.e., the tumoricidal effect of the cytotoxic drug and the local immune activation of the immunostimulant, thereby improving the efficacy of antibody-drug conjugates in the treatment of cancer.

[0007] In the present invention, a dual-payload type bifunctional immunoregulatory antibody-drug conjugate is obtained by coupling a cytotoxic drug and a TLR agonist to an antibody, which can simultaneously invade tumor cells and the tumor immune microenvironment to exert dual effects, i.e., the tumoricidal effect of the cytotoxic drug and the local immune activation of the TLR agonist, thereby improving the cancer treatment effect compared with the antibody-drug conjugate to which a cytotoxic drug is coupled alone.

[0008] In particular, the present invention relates to a compound of formula (I): [ka] [In the formula, Ab represents the targeting moiety; M represents a linking site connected to a targeting moiety; X represents a linker connecting M and Aa; Aa represents an amino acid fragment or a peptide fragment formed from two or more amino acids; L 1 Aa and D 1 represents a covalent bond or linker connecting L 2 Aa and D 2 represents a linker connecting D 1 is a cytotoxic drug fragment; D 2 is a TLR agonist fragment; and m is selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0009] In some embodiments, the Ab is a targeting moiety capable of targeting an antigen of interest, where the targeting moiety targets a cell surface receptor or a tumor surface antigen.

[0010] In some embodiments, an Ab is an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment comprises a monoclonal antibody, a polyclonal antibody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a murine antibody, a humanized antibody, a fully human antibody, or a fusion protein comprising the antigen-binding region of an antibody.

[0011] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof, and the antigen-binding fragment is a Fab, Fab', F(ab') 2 , Fv, disulfide bond linked Fv, and scFv.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of an anti-Her-2 antibody, an anti-EGFR antibody, an anti-VEGFR antibody, an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an anti-Trop-2 antibody.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof is trastuzumab.

[0014] In some embodiments, M is a covalent bond or has the following structure: [ka] selected from the group consisting of; Each a is independently an integer selected from 1 to 5, b is an integer selected from 1 to 3, position 1 of M is connected to Ab, and position 2 of M is connected to X.

[0015] In some embodiments, M is a covalent bond or has the following structure: [ka] selected from the group consisting of; Each a is independently an integer selected from 1 to 5, b is an integer selected from 1 to 3, position 1 of M is connected to Ab, and position 2 of M is connected to X.

[0016] In some embodiments, M is a covalent bond or has the following structure: [ka] is selected from the group consisting of:

[0017] In some embodiments, M is a covalent bond or has the following structure: [ka] is selected from the group consisting of:

[0018] In some embodiments, M is a covalent bond or has the following structure: [ka] is selected from the group consisting of:

[0019] In some embodiments, X is a covalent bond or C 1~6 Alkylene, -NH-(CH 2 ) c -C(O)-, [ka] selected from the group consisting of; Each c is an integer independently selected from 1 to 10, position 3 of X is connected to M, and position 4 of X is connected to Aa.

[0020] In some embodiments, X is a covalent bond or [ka] and; Each c is independently an integer selected from 1 to 10 (e.g., an integer from 5 to 10, e.g., an integer from 8 to 10), and position 3 of X is connected to M, and position 4 of X is connected to Aa.

[0021] In some embodiments, Aa is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Gly, Phe, Ala, Val, Ser, Thr, His, Trp, Cys, Asp, Glu, Lys, Tyr, and Arg, and optionally each amino acid is selected from the group consisting of C 1~6 In some embodiments, the polyethylene glycol hydrophilic structural unit is independently modified by an alkyl or polyethylene glycol hydrophilic structural unit. [ka] and each c is independently an integer selected from 1 to 10 (eg, an integer from 5 to 10, such as an integer from 8 to 10).

[0022] In some embodiments, Aa is selected from the group consisting of fragments of the following amino acids: Gly, Phe, Ala, Val, Cys, Asp, Glu, Lys, and Arg, and peptide fragments formed from any combination of two or more of these amino acids, wherein each amino acid is selected from the group consisting of C 1~6 Optionally, the alkyl group or the polyethylene glycol hydrophilic structural unit is independently modified. In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10 (eg, an integer from 5 to 10, such as an integer from 8 to 10).

[0023] In some embodiments, Aa is selected from the group consisting of fragments of the following amino acids: Val, Cys, Asp, Glu, and Lys, and peptide fragments formed from any combination of two or more of these amino acids, wherein the carboxy terminus of Glu and Lys is [ka] and each c is independently an integer selected from 1 to 10 (e.g., an integer from 5 to 10, e.g., an integer from 8 to 10).

[0024] In some embodiments, Aa has the structure: [ka] selected from the group consisting of; Each R 7 , H, C 1~6 independently selected from the group consisting of alkyl, alkylene, and polyethylene glycol hydrophilic structural units; 8 are independently selected from the group consisting of hydroxyl and polyethylene glycol hydrophilic structural units; position 5 is connected to X and position 6 is connected to L 1 and L 2 Position 7 is connected to one of the L 1 and L 2 is connected to the other of the two.

[0025] In some embodiments, position 5 is connected to X and position 6 is connected to L 1 Position 7 is connected to L 2 is connected to.

[0026] In some embodiments, position 5 is connected to X and position 6 is connected to L 2 Position 7 is connected to L 1 is connected to.

[0027] In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10 (eg, an integer from 5 to 10, such as an integer from 8 to 10).

[0028] In some embodiments, R 7 is H.

[0029] In some embodiments, Aa has the structure: [ka] selected from the group consisting of; Each R 7 , H, C 1~6 independently selected from the group consisting of alkyl, alkylene, and polyethylene glycol hydrophilic structural units; 8 are independently selected from the group consisting of hydroxyl and polyethylene glycol hydrophilic structural units; position 5 is connected to X and position 6 is connected to L 1 and L 2 Position 7 is connected to one of the L 1 and L 2 Preferably, position 5 is connected to X and position 6 is connected to L 1 Position 7 is connected to L 2 is connected to.

[0030] In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10 (eg, an integer from 5 to 10, such as an integer from 8 to 10).

[0031] In some embodiments, L 1 is a covalent bond or a non-cleavable or cleavable linker, the cleavable linker being capable of being cleaved by an enzyme present in the pathological environment, the enzyme being selected from the group consisting of proteases, phosphatases, pyrophosphatases, β-glucuronidase, β-galactosidase and sulfatase.

[0032] In some embodiments, L 1 is a covalent bond or -L a -L b -L c - and L a is a covalent bond or C 1~6 Alkylene, -NH-(CH 2 ) c -C(O)-, [ka] selected from the group consisting of; each c is independently an integer selected from 1 to 10; L b is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L c is a covalent bond, -NH-CH 2 or has the following structure: [ka] is selected from the group consisting of:

[0033] In some embodiments, L 1is a covalent bond or -L a -L b -L c - and L a is a covalent bond or C 1~6 Alkylene, -NH-(CH 2 ) c -C(O)-, [ka] each c is independently an integer selected from 1 to 10; L b is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L c is a covalent bond, -NH-CH 2 or has the following structure: [ka] is selected from the group consisting of:

[0034] In some embodiments, L a is a covalent bond, or [ka] each c is independently an integer selected from 1 to 10.

[0035] In some embodiments, L b is a covalent bond or has the structure: [ka] selected from the group consisting of; R 9is selected from the group consisting of H, acetyl, fluorenyloxycarbonyl, trityl, and a polyethylene glycol hydrophilic structural unit. In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10.

[0036] In some embodiments, L c is -NH-CH 2 -or [ka] It is.

[0037] In some embodiments, L 1 is a covalent bond or -L a -L b -L c - and L a is a covalent bond, or [ka] each c is independently an integer selected from 1 to 10; L b is a covalent bond, or [ka] selected from the group consisting of; L c is -NH-CH 2 -or [ka] It is.

[0038] In some embodiments, L 1is a covalent bond or has the structure: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is 、 D 1 is connected to.

[0039] In some embodiments, L 1 is a covalent bond or has the structure: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is 、 D 1 is connected to.

[0040] In some embodiments, L 1 teeth, [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0041] In some embodiments, L 1 teeth, [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0042] In some embodiments, L 1 teeth: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0043] In some embodiments, L 2 is a non-cleavable linker or a cleavable linker, the cleavable linker being capable of undergoing cleavage by an enzyme present in a pathological environment, the enzyme being selected from the group consisting of proteases, phosphatases, pyrophosphatases, β-glucuronidases, β-galactosidases and sulfatases.

[0044] In some embodiments, L 2 -L d -L e -L f and L d is a covalent bond or the following: 1~6 Alkylene, -C(O)-(CH 2 ) d -NH-, [ka] each d is independently an integer selected from 1 to 12; L e is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L f is a covalent bond, -CH 2 -NH- or the following structure: [ka] is selected from the group consisting of:

[0045] In some embodiments, L 2 -L d-L e -L f and L d is a covalent bond or the following: 1~6 Alkylene, -C(O)-(CH 2 ) d -NH-, [ka] each d is independently an integer selected from 1 to 12; L e is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L f is a covalent bond, -CH 2 -NH- or the following structure: [ka] is selected from the group consisting of:

[0046] In some embodiments, L e is a covalent bond or has the structure: [ka] selected from the group consisting of; Each R 10 are independently selected from the group consisting of H, acetyl, fluorenyloxycarbonyl, trityl and a polyethylene glycol hydrophilic structural unit; preferably, the polyethylene glycol hydrophilic structural unit is [ka] and each d is independently an integer selected from 1 to 10.

[0047] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0048] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0049] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0050] In some embodiments, L 2 teeth: [ka] and; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0051] In some embodiments, L 2 teeth: [ka] and; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0052] In some embodiments, D 1 is selected from the group consisting of cytotoxic drug fragments, the cytotoxic drugs being selected from the group consisting of tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, the tubulin inhibitors including, but not limited to, dolastatins, auristatins, maytansines, tubulysins and cryptomycins, the DNA damaging agents including, but not limited to, PBDs, duocarmycins and calicheamicins, and the topoisomerase inhibitors including, but not limited to, camptothecins and their derivatives.

[0053] In some embodiments, the tubulin inhibitor is selected from the group consisting of dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3 and DM4, and the topoisomerase inhibitor is selected from the group consisting of camptothecin, SN-38, exatecan, topotecan, belotecan, 10-hydroxy-camptothecin, 9-amino-camptothecin, doxorubicin, epirubicin and PNU-159682.

[0054] In some embodiments, D 1 has the following structure: [ka] is selected from the group consisting of:

[0055] In some embodiments, D 2 is a TLR agonist fragment.

[0056] In some embodiments, the TLR agonist is selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist, and a TLR9 agonist.

[0057] In some embodiments, D 2 is a TLR agonist fragment, wherein the TLR agonist is selected from the group consisting of a TLR7 agonist, a TLR8 agonist, and a TLR7 / 8 agonist.

[0058] In some embodiments, D 2 is a TLR agonist fragment, the TLR agonist being a compound represented by formula (II): [ka] [In the formula, X 1 is N or C; X 2 is N or C; X 1 and X 2 at least one of is N; X 3 is selected from the group consisting of O, S and C; X 4 are O, S, N and CR 4 selected from the group consisting of; R 1 is C 1~6 Alkyl and -C 1~6 Alkylene-OC 1~6 selected from the group consisting of alkyl; R 2 is hydrogen or a group of formula -L 3 -L 4 -L 5 -L 6 and; L 3 is a covalent bond or -C 1~6 is alkylene; L 4 is a covalent bond, C 3~10 Cycloalkyl, 3-12 membered heterocyclyl, C 6~10 aryl and 5-10 membered heteroaryl, wherein cycloalkyl, heterocyclyl, aryl and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~6 Alkyl, C1~6 Alkoxy and -C 1~6 Alkylene-NH 2 optionally substituted with one or more groups selected from the group consisting of: L 5 is a covalent bond, -O-, -S-, -C(O)-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -NR 5 -, -C(O)-NR 5 -, -NR 5 -C(O)-, -OC(O)-NR 5 -, -NR 5 -C(O)-O-, -NR 5 -C(O)-NR 5 -, -S(O) r -NR 5 -, -NR 5 -S(O) r - and -NR 5 -S(O) r -NR 5 - selected from the group consisting of; L 6 is hydrogen, C 1~6 Alkyl, -(O-CH 2 CH 2 ) n -OC 1~6 Alkyl and -C 1~6 Alkylene-(O-CH 2 CH 2 ) n -OC 1~6 alkyl, where alkyl is selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6 optionally substituted with one or more groups selected from the group consisting of alkyl; R 3 is hydrogen or a group of formula -L 7 -L 8 -L 9 -L 10 and; L 7 is a covalent bond and C 1~6 alkylene; L 8 is a covalent bond, C 3~10 Cycloalkyl, 3-12 membered heterocyclyl, C 6~10 aryl, and 5-10 membered heteroaryl, wherein cycloalkyl, heterocyclyl, aryl, and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~6 Alkyl, C 1~6 Alkoxy and -C 1~6 Alkylene-NH 2 optionally substituted with one or more groups selected from the group consisting of: L 9 is a covalent bond, -O-, -S-, -C(O)-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -NR 6 -, -C(O)-NR 6 -, -NR 6 -C(O)-, -OC(O)-NR 6 -, -NR 6 -C(O)-O-, -NR 6 -C(O)-NR 6 -, -S(O) p -NR 6 -, -NR 6 -S(O) p - and -NR 6 -S(O) p -NR 6 - selected from the group consisting of; L 10 is hydrogen, C 1~6 Alkyl, -(O-CH 2 CH 2 ) q -OC 1~6 Alkyl, and -C 1~6 Alkylene-(O-CH 2 CH 2 ) q -OC 1~6 alkyl, where alkyl is selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6optionally substituted with one or more groups selected from the group consisting of alkyl; R 4 is hydrogen, halogen, cyano, hydroxyl, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy and C 3~10 cycloalkyl; Each R 5 are independently hydrogen or C 1~6 is alkyl; Each R 6 are independently hydrogen or C 1~6 is alkyl; each r is independently 1 or 2; Each n is independently an integer selected from 1 to 25; each p is independently 1 or 2; Each q is independently an integer selected from 1 to 25; D 2 In formula (II), R 2 or R 3 Via L 2 connected to].

[0059] In some embodiments, each n is independently selected from 1 to 15, and preferably n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0060] In some embodiments, each q is independently selected from 1 to 15, and preferably, q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0061] In some embodiments, X 3 is selected from the group consisting of S and N.

[0062] In some embodiments, L 3 is a covalent bond and C 1~3 Alkylene is selected from the group consisting of:

[0063] In some embodiments, L 4 is a covalent bond, 3- to 12-membered heterocyclyl, C 6~10 aryl, and 5-10 membered heteroaryl, wherein heterocyclyl, aryl, and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~3 Alkyl, C 1~3 Alkoxy and -C 1~6 Alkylene-NH 2 and optionally substituted with one or more groups selected from the group consisting of:

[0064] In some embodiments, L 5 is a covalent bond, -NR 5 -, -C(O)-NR 5 -, -NR 5 -C(O)-, -OC(O)-NR 5 -, -NR 5 -C(O)-O-, -NR 5 -C(O)-NR 5 -, -S(O) r -NR 5 -, -NR 5 -S(O) r - and -NR 5 -S(O) r -NR 5 -, each R 5 is hydrogen and C 1~6 alkyl; preferably, L 5 is a covalent bond and -NR 5 -, each R 5 is hydrogen and C 1~6 alkyl.

[0065] In some embodiments, L 6 is hydrogen, C 1~6 Alkyl, and -C 1~6 Alkylene-(O-CH 2 CH 2 ) n -OC 1~6alkyl, where alkyl is selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6 and n is 1 to 10.

[0066] In some embodiments, L 8 is selected from the group consisting of a covalent bond and 3- to 12-membered heterocyclyl, where heterocyclyl is hydrogen, halogen, cyano, and C 1~6 and optionally substituted with one or more groups selected from the group consisting of alkyl.

[0067] In some embodiments, L 9 is a direct bond.

[0068] In some embodiments, L 10 is hydrogen, C 1~6 Alkyl, and -C 1~6 Alkylene-(O-CH 2 CH 2 ) q -OC 1~6 alkyl, and q is 1 to 10.

[0069] In some embodiments, R 3 is hydrogen and the formula -L 7 -L 8 -L 9 -L 10 selected from the group consisting of; L 8 is selected from the group consisting of a covalent bond and 3- to 12-membered heterocyclyl, where heterocyclyl is hydrogen, halogen, cyano, and C 1~6 optionally substituted with one or more groups selected from the group consisting of alkyl; L 9 is a covalent bond; L 10 is hydrogen, C 1~6 and -C1~6 Alkylene-(O-CH 2 CH 2 ) q -OC 1~6 selected from the group consisting of alkyl; q is selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0070] In some embodiments, X 1 is C; X 2 is N; X 3 is S; X 4 CR 4 Selected from; R 1 is C 1~6 alkyl; R 2 is the formula -L 3 -L 4 -L 5 -L 6 Selected from; L 3 is a covalent bond and C 1~6 alkylene; L 4 is a covalent bond, 3- to 12-membered heterocyclyl, C 1~6 aryl and 5-10 membered heteroaryl, wherein heterocyclyl, aryl and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~3 Alkyl, C 1~3 Alkoxy and -C 1~6 Alkylene-NH 2 optionally substituted with one or more groups selected from the group consisting of: L 5 is a covalent bond and -NR 5 -, each R 5 is hydrogen and C 1~6 independently selected from the group consisting of alkyl; L 6 is hydrogen, C 1~6 Alkyl and -C 1~6 Alkylene-(O-CH2 CH 2 ) n -OC 1~6 alkyl, the alkyl group being selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6 and optionally substituted with one or more groups selected from the group consisting of alkyl.

[0071] In some embodiments, D 2 has the following structure: [ka] is selected from the group consisting of:

[0072] In some embodiments, the antibody-drug conjugate has the following structure: [ka] TIFF2025513163000048.tif211149 TIFF2025513163000049.tif174149 TIFF2025513163000050.tif160149 TIFF2025513163000051.tif217149 TIFF2025513163000052.tif221149 TIFF2025513163000053.tif209149 TIFF2025513163000054.tif224149 TIFF2025513163000055.tif190149 TIFF2025513163000056.tif213149 TIFF2025513163000057.tif220149 TIFF2025513163000058.tif216149 TIFF2025513163000059.tif226149 TIFF2025513163000060.tif120149 selected from the group consisting of; Each m is independently selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0073] In some embodiments, the Ab is trastuzumab.

[0074] In some embodiments, the antibody-drug conjugate has a DAR value of 2.0 to 5.0, e.g., 2.0 to 2.5, 2.0 to 3.0, 2.0 to 3.5, 2.0 to 4.0, 2.0 to 4.5, 2.0 to 5.0, 2.5 to 3.0, 2.5 to 3.5, 2.5 to 4.0, 2.5 to 4.5, 2.5 to 5.0, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 4.0 to 4.5, 4.0 to 5.0, or 4.0 to 5.0.

[0075] Drug-Linker In another embodiment, the present invention provides a drug-linker simultaneously coupled to a cytotoxic drug and a TLR agonist, which can be used to prepare the above-mentioned antibody-drug conjugates.

[0076] In particular, the present invention relates to a compound of formula (III): [ka] [In the formula, M 1 is a precursor of a linking moiety connected to a targeting moiety, the targeting moiety being as defined in any one of the preceding paragraphs; X is a linker connecting M and Aa; Aa is an amino acid fragment or a peptide fragment formed from two or more amino acids; L 1 is a covalent bond or Aa and D 1 is a linker connecting L 2 Aa and D 2 is a linker connecting D 1 is a cytotoxic drug fragment; D 2 is a TLR agonist fragment], or a pharma- ceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof.

[0077] In some embodiments, M 1 has the following structure: [ka] selected from the group consisting of; Each a is independently an integer selected from 1 to 5, b is an integer selected from 1 to 3, and M a is a carboxyl active group (eg, a pentafluorophenoxy group).

[0078] In some embodiments, M 1 has the following structure: [ka] selected from the group consisting of; Each a is independently an integer selected from 1 to 5, b is an integer selected from 1 to 3, and M a is a carboxyl active group.

[0079] In some embodiments, M 1 is a covalent bond or has the structure: [ka] is selected from the group consisting of:

[0080] In some embodiments, M 1 is a covalent bond or has the structure: [ka] is selected from the group consisting of:

[0081] In some embodiments, X is a covalent bond or C 1~6 Alkylene, -NH-(CH 2 ) c -C(O)-, [ka] selected from the group consisting of; Each c is independently an integer selected from 1 to 10, position 3 of X is connected to M, and position 4 of X is connected to Aa.

[0082] In some embodiments, X is a covalent bond or [ka] and; Each c is independently an integer selected from 1 to 10 (e.g., an integer from 5 to 10, e.g., an integer from 8 to 10), and position 3 of X is connected to M, and position 4 of X is connected to Aa.

[0083] In some embodiments, Aa is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Gly, Phe, Ala, Val, Ser, Thr, His, Trp, Cys, Asp, Glu, Lys, Tyr, and Arg, and optionally each amino acid is selected from the group consisting of C 1~6 In some embodiments, the polyethylene glycol hydrophilic structural unit is independently modified with an alkyl or polyethylene glycol hydrophilic structural unit. [ka] and each c is independently an integer selected from 1 to 10 (eg, an integer from 5 to 10, such as an integer from 8 to 10).

[0084] In some embodiments, Aa is selected from the group consisting of fragments of the following amino acids: Gly, Phe, Ala, Val, Cys, Asp, Glu, Lys, and Arg, and peptide fragments formed from any combination of two or more of these amino acids, wherein each amino acid is selected from the group consisting of C 1~6 Optionally, the alkyl group or the polyethylene glycol hydrophilic structural unit is independently modified. In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10 (eg, an integer from 5 to 10, such as an integer from 8 to 10).

[0085] In some embodiments, Aa is selected from the group consisting of fragments of the following amino acids: Val, Cys, Asp, Glu, and Lys, and peptide fragments formed from any combination of two or more of these amino acids, wherein the carboxy terminus of Glu and Lys is [ka] and each c is independently an integer selected from 1 to 10 (e.g., an integer from 5 to 10, e.g., an integer from 8 to 10).

[0086] In some embodiments, Aa has the structure: [ka] selected from the group consisting of; Each R7 , H, C 1~6 independently selected from the group consisting of alkyl, alkylene, and polyethylene glycol hydrophilic structural units; 8 are independently selected from the group consisting of hydroxyl and polyethylene glycol hydrophilic structural units; position 5 is connected to X and position 6 is connected to L 1 and L 2 Position 7 is connected to one of the L 1 and L 2 In some embodiments, position 5 is connected to X and position 6 is connected to L 1 Position 7 is connected to L 2 In some embodiments, position 5 is connected to X and position 6 is connected to L 2 Position 7 is connected to L 1 In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10 (e.g., an integer from 5 to 10, e.g., an integer from 8 to 10). 7 is H.

[0087] In some embodiments, Aa has the structure: [ka] selected from the group consisting of; Each R 7 , H, C 1~6 independently selected from the group consisting of alkyl, alkylene, and polyethylene glycol hydrophilic structural units; 8 are independently selected from the group consisting of hydroxyl and polyethylene glycol hydrophilic structural units; position 5 is connected to X and position 6 is connected to L 1 and L 2 Position 7 is connected to one of the L 1 and L 2Preferably, position 5 is connected to X and position 6 is connected to L 1 Position 7 is connected to L 2 In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10 (eg, an integer from 5 to 10, such as an integer from 8 to 10).

[0088] In some embodiments, L 1 is a covalent bond or a non-cleavable or cleavable linker, the cleavable linker being cleaved by an enzyme present in the pathological environment, the enzyme being selected from the group consisting of proteases, phosphatases, pyrophosphatases, β-glucuronidase, β-galactosidase and sulfatase.

[0089] In some embodiments, L 1 is a covalent bond or -L a -L b -L c - and L a is a covalent bond or C 1~6 Alkylene, -NH-(CH 2 ) c -C(O)-, [ka] selected from the group consisting of; each c is independently an integer selected from 1 to 10; L b is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L c is a covalent bond, -NH-CH2 or has the following structure: [ka] is selected from the group consisting of:

[0090] In some embodiments, L 1 is a covalent bond or -L a -L b -L c - and L a is a covalent bond or C 1~6 Alkylene, -NH-(CH 2 ) c -C(O)-, [ka] each c is independently an integer selected from 1 to 10; L b is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L c is a covalent bond, -NH-CH 2 or has the following structure: [ka] is selected from the group consisting of:

[0091] In some embodiments, L a is a covalent bond, or [ka] and each c is independently an integer selected from 1 to 10. In some embodiments, L bis a covalent bond or has the structure: [ka] selected from the group consisting of; R 9 is selected from the group consisting of H, acetyl, fluorenyloxycarbonyl, trityl, and a polyethylene glycol hydrophilic structural unit. In some embodiments, the polyethylene glycol hydrophilic structural unit is [ka] and each c is independently an integer selected from 1 to 10.

[0092] In some embodiments, L c is -NH-CH 2 -or [ka] It is.

[0093] In some embodiments, L 1 is a covalent bond or -L a -L b -L c - and L a is a covalent bond, or [ka] each c is independently an integer selected from 1 to 10; L b is a covalent bond, or [ka] selected from the group consisting of; L c is -NH-CH 2 -or [ka] It is.

[0094] In some embodiments, L 1 is a covalent bond or has the structure: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0095] In some embodiments, L 1 is a covalent bond or has the structure: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0096] In some embodiments, L 1 has the following structure: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0097] In some embodiments, L 1 has the following structure: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0098] In some embodiments, L 1 has the following structure: [ka] selected from the group consisting of; Position 8 is connected to Aa and position 9 is D 1 is connected to.

[0099] In some embodiments, L 2 is a non-cleavable linker or a cleavable linker, the cleavable linker being cleaved by an enzyme present in the pathological environment, the enzyme being selected from the group consisting of proteases, phosphatases, pyrophosphatases, β-glucuronidases, β-galactosidases and sulfatases.

[0100] In some embodiments, L 2 -L d -L e -L f and L d is a covalent bond or the following: 1~6 Alkylene, -C(O)-(CH 2 ) d -NH-, [ka] each d is independently an integer selected from 1 to 12; L e is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L f is a covalent bond, -CH 2 -NH- or the following structure: [ka] is selected from the group consisting of:

[0101] In some embodiments, L 2 -L d -L e -L f and L d is a covalent bond or the following: 1~6 Alkylene, -C(O)-(CH 2 ) d -NH-, [ka] each d is independently an integer selected from 1 to 12; L e is a covalent bond or is selected from the group consisting of an amino acid fragment and a peptide fragment formed from two or more amino acids, the amino acids being selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; L f is a covalent bond, -CH 2 -NH- or the following structure: [ka] is selected from the group consisting of:

[0102] In some embodiments, L e is a covalent bond or has the structure: [ka] selected from the group consisting of; Each R 10 are independently selected from the group consisting of H, acetyl, fluorenyloxycarbonyl, trityl and a polyethylene glycol hydrophilic structural unit; preferably, the polyethylene glycol hydrophilic structural unit is [ka] and each d is independently an integer selected from 1 to 10.

[0103] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0104] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0105] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0106] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0107] In some embodiments, L 2 has the following structure: [ka] selected from the group consisting of; Position 10 is connected to Aa and position 11 is connected to D 2 is connected to.

[0108] In some embodiments, D 1 is selected from the group consisting of cytotoxic drug fragments, the cytotoxic drug is selected from the group consisting of tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, the tubulin inhibitors include, but are not limited to, dolastatins, auristatins, maytansines, tubulysins and cryptomycins, the DNA damaging agents include, but are not limited to, PBDs, duocarmycins and calicheamicins, and the topoisomerase inhibitors include, but are not limited to, camptothecin and its derivatives.

[0109] In some embodiments, the tubulin inhibitor is selected from the group consisting of dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3, and DM4, and the topoisomerase inhibitor is selected from the group consisting of camptothecin, SN-38, exatecan, topotecan, belotecan, 10-hydroxy-camptothecin, 9-amino-camptothecin, doxorubicin, epirubicin, and PNU-159682.

[0110] In some embodiments, D 1 has the following structure: [ka] is selected from the group consisting of:

[0111] In some embodiments, D 2 is a TLR agonist fragment.

[0112] In some embodiments, the TLR agonist is selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist, and a TLR9 agonist.

[0113] In some embodiments, D 2 is a TLR agonist fragment, wherein the TLR agonist is selected from the group consisting of a TLR7 agonist, a TLR8 agonist, and a TLR7 / 8 agonist.

[0114] In some embodiments, D 2 is a TLR agonist fragment, the TLR agonist having formula (II): [ka] [In the formula, X 1 is N or C; X 2 is N or C; X 1 and X 2 at least one of is N; X 3 is selected from the group consisting of O, S and C; X 4 are O, S, N and CR 4 selected from the group consisting of; R 1 is C 1~6 Alkyl and -C 1~6 Alkylene-OC 1~6 selected from the group consisting of alkyl; R 2 is hydrogen or a group of formula -L 3 -L 4 -L 5 -L 6 and; L 3 is a covalent bond or -C 1~6 is alkylene; L 4 is a covalent bond, C3~10 Cycloalkyl, 3-12 membered heterocyclyl, C 6~10 aryl and 5-10 membered heteroaryl, wherein cycloalkyl, heterocyclyl, aryl and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~6 Alkyl, C 1~6 Alkoxy and -C 1~6 Alkylene-NH 2 optionally substituted with one or more groups selected from the group consisting of: L 5 is a covalent bond, -O-, -S-, -C(O)-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -NR 5 -, -C(O)-NR 5 -, -NR 5 -C(O)-, -OC(O)-NR 5 -, -NR 5 -C(O)-O-, -NR 5 -C(O)-NR 5 -, -S(O) r -NR 5 -, -NR 5 -S(O) r - and -NR 5 -S(O) r -NR 5 - selected from the group consisting of; L 6 is hydrogen, C 1~6 Alkyl, -(O-CH 2 CH 2 ) n -OC 1~6 Alkyl and -C 1~6 Alkylene-(O-CH 2 CH 2 ) n -OC 1~6 alkyl, where alkyl is selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6 optionally substituted with one or more groups selected from the group consisting of alkyl; R 3is hydrogen or a group of formula -L 7 -L 8 -L 9 -L 10 and; L 7 is a covalent bond and C 1~6 alkylene; L 8 is a covalent bond, C 3~10 Cycloalkyl, 3-12 membered heterocyclyl, C 6~10 aryl, and 5-10 membered heteroaryl, wherein cycloalkyl, heterocyclyl, aryl, and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~6 Alkyl, C 1~6 Alkoxy and -C 1~6 Alkylene-NH 2 optionally substituted with one or more groups selected from the group consisting of: L 9 is a covalent bond, -O-, -S-, -C(O)-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -NR 6 -, -C(O)-NR 6 -, -NR 6 -C(O)-, -OC(O)-NR 6 -, -NR 6 -C(O)-O-, -NR 6 -C(O)-NR 6 -, -S(O) p -NR 6 -, -NR 6 -S(O) p - and -NR 6 -S(O) p -NR 6 - selected from the group consisting of; L 10 is hydrogen, C 1~6 Alkyl, -(O-CH 2 CH 2 ) q -OC 1~6 Alkyl, and -C 1~6 Alkylene-(O-CH 2 CH 2 ) q -OC 1~6alkyl, where alkyl is selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6 optionally substituted with one or more groups selected from the group consisting of alkyl; R 4 is hydrogen, halogen, cyano, hydroxyl, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy and C 3~10 cycloalkyl; Each R 5 are independently hydrogen or C 1~6 is alkyl; Each R 6 are independently hydrogen or C 1~6 is alkyl; each r is independently 1 or 2; Each n is independently an integer selected from 1 to 25; each p is independently 1 or 2; Each q is independently an integer selected from 1 to 25; D 2 In formula (II), R 2 or R 3 Via L 2 is a compound represented by the formula:

[0115] In some embodiments, each n is independently selected from 1 to 15, and preferably n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0116] In some embodiments, each q is independently selected from 1 to 15, and preferably, q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0117] In some embodiments, X 3is selected from the group consisting of S and N.

[0118] In some embodiments, L 3 is a covalent bond and C 1~3 Alkylene is selected from the group consisting of:

[0119] In some embodiments, L 4 is a covalent bond, 3- to 12-membered heterocyclyl, C 6~10 aryl, and 5-10 membered heteroaryl, wherein heterocyclyl, aryl, and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~3 Alkyl, C 1~3 Alkoxy and -C 1~6 Alkylene-NH 2 and optionally substituted with one or more groups selected from the group consisting of:

[0120] In some embodiments, L 5 is a covalent bond, -NR 5 -, -C(O)-NR 5 -, -NR 5 -C(O)-, -OC(O)-NR 5 -, -NR 5 -C(O)-O-, -NR 5 -C(O)-NR 5 -, -S(O) r -NR 5 -, -NR 5 -S(O) r - and -NR 5 -S(O) r -NR 5 -, each R 5 is hydrogen and C 1~6 alkyl; preferably, L 5 is a covalent bond and -NR 5 -, each R 5 is hydrogen and C 1~6 alkyl.

[0121] In some embodiments, L 6is hydrogen, C 1~6 Alkyl, and -C 1~6 Alkylene-(O-CH 2 CH 2 ) n -OC 1~6 alkyl, where alkyl is selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6 and n is 1 to 10.

[0122] In some embodiments, L 8 is selected from the group consisting of a covalent bond and 3- to 12-membered heterocyclyl, where heterocyclyl is hydrogen, halogen, cyano, and C 1~6 and optionally substituted with one or more groups selected from the group consisting of alkyl.

[0123] In some embodiments, L 9 is a direct bond.

[0124] In some embodiments, L 10 is hydrogen, C 1~6 Alkyl, and -C 1~6 Alkylene-(O-CH 2 CH 2 ) q -OC 1~6 alkyl, and q is 1 to 10.

[0125] In some embodiments, R 3 is hydrogen and the formula -L 7 -L 8 -L 9 -L 10 selected from the group consisting of; L 8 is selected from the group consisting of a covalent bond and 3- to 12-membered heterocyclyl, where heterocyclyl is hydrogen, halogen, cyano, and C 1~6optionally substituted with one or more groups selected from the group consisting of alkyl; L 9 is a covalent bond; L 10 is hydrogen, C 1~6 and -C 1~6 Alkylene-(O-CH 2 CH 2 ) q -OC 1~6 selected from the group consisting of alkyl; q is selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0126] In some embodiments, X 1 is C; X 2 is N; X 3 is S; X 4 CR 4 Selected from; R 1 is C 1~6 alkyl; R 2 is the formula -L 3 -L 4 -L 5 -L 6 Selected from; L 3 is a covalent bond and C 1~6 alkylene; L 4 is a covalent bond, 3- to 12-membered heterocyclyl, C 1~6 aryl and 5-10 membered heteroaryl, wherein heterocyclyl, aryl and heteroaryl are selected from the group consisting of hydrogen, halogen, cyano, C 1~3 Alkyl, C 1~3 Alkoxy and -C 1~6 Alkylene-NH 2 optionally substituted with one or more groups selected from the group consisting of: L 5 is a covalent bond and -NR 5 -, each R 5is hydrogen and C 1~6 independently selected from the group consisting of alkyl; L 6 is hydrogen, C 1~6 Alkyl and -C 1~6 Alkylene-(O-CH 2 CH 2 ) n -OC 1~6 alkyl, where alkyl is selected from the group consisting of hydrogen, halogen, hydroxyl, -NH 2 , -NH-C(O)-OC 1~6 Alkyl, C 1~6 Alkoxy, carboxyl and -C(O)-OC 1~6 and optionally substituted with one or more groups selected from the group consisting of alkyl.

[0127] In some embodiments, D 2 has the following structure: [ka] is selected from the group consisting of:

[0128] In some embodiments, the compound or a pharma- ceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof has the following structure: [ka] TIFF2025513163000106.tif212149 TIFF2025513163000107.tif185149 TIFF2025513163000108.tif163149 TIFF2025513163000109.tif185149 TIFF2025513163000110.tif226149 TIFF2025513163000111.tif226149 TIFF2025513163000112.tif226149 TIFF2025513163000113.tif193149 TIFF2025513163000114.tif226149 TIFF2025513163000115.tif226149 TIFF2025513163000116.tif226149 TIFF2025513163000117.tif196149 TIFF2025513163000118.tif184149 is selected from the group consisting of:

[0129] Pharmaceutical Compositions In another aspect, the invention provides a pharmaceutical composition comprising an antibody-drug conjugate, a compound (e.g., a drug linker, a TLR agonist or a cytotoxic drug) or a pharma- ceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof described in any one of the preceding paragraphs, and one or more pharmaceutical excipients.

[0130] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known or will be apparent to those skilled in the art in light of the present disclosure. According to REMINGTON'S PHARMACEUTICAL SCIENCES, edited by Martin, EW, Mack Publishing Company, 19th Edition (1995), a method for preparing a pharmaceutical composition includes adding a suitable pharmaceutical excipient.

[0131] use In another aspect, the present invention provides the use of an antibody-drug conjugate, a compound (e.g., a drug linker, a TLR agonist or a cytotoxic drug), or a pharma- ceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof according to any one of the foregoing in the manufacture of an antibody-drug conjugate, in particular in the manufacture of an antibody-drug conjugate according to any one of the foregoing.

[0132] In another aspect, the invention provides the use of an antibody-drug conjugate, a compound (e.g., a drug linker, a TLR agonist or a cytotoxic drug), or a pharma- ceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition according to any one of the foregoing, in the manufacture of a medicament for treating and / or preventing cancer (e.g., a HER2 positive cancer, such as, for example, a HER2 positive gastric cancer, breast cancer or non-small cell lung cancer).

[0133] In another aspect, the present invention provides an antibody-drug conjugate, a compound (e.g., a drug linker, a TLR agonist or a cytotoxic drug), or a pharma- ceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition according to any one of the foregoing, for use in the treatment and / or prevention of cancer (e.g., a HER2 positive cancer, such as, for example, a HER2 positive gastric cancer, breast cancer or non-small cell lung cancer).

[0134] In another aspect, the present invention provides a method of treating and / or preventing cancer (e.g., a HER2-positive cancer, such as, for example, a HER2-positive gastric cancer, breast cancer, or non-small cell lung cancer), comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of an antibody-drug conjugate, a compound (e.g., a drug linker, a TLR agonist, or a cytotoxic drug), or a pharma- ceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, or a pharmaceutical composition according to any one of the foregoing.

[0135] Preparation method In another aspect, the present invention provides a method for preparing a compound represented by formula (III) of the present invention.

[0136] X of the compound of formula (III) is a structural fragment [ka] If X contains [ka] and X a and X b is in X [ka] The present invention provides a method for preparing a compound of formula (III), comprising the steps of: [ka] [In the formula, LG 1 and L.G. 2 represents a leaving group including, but not limited to, halogen, hydroxyl, succinimide ester, active ester, etc., and PG 1 and P.G. 2represents a protecting group including, but not limited to, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenyloxycarbonyl (Fmoc), benzyl, acetyl, tert-butyl, and the like; L 1a , L 1 which preferably contains an unsaturated double bond and can be connected to Aa by an addition reaction occurring on the unsaturated double bond; D 1 , D 2 , L 1 , L 2 , X, Aa and M 1 is as defined above]; Step 1: subjecting compound III-1 and compound III-2 to a condensation reaction to obtain compound III-3; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, preferably a combination of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 2: subjecting compound III-3 to a deprotection reaction to obtain compound III-4; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable acid, which may be selected from the group consisting of hydrochloric acid and trifluoroacetic acid, preferably trifluoroacetic acid. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 3: subjecting compound III-4 and compound III-5 to a condensation reaction to obtain compound III-6; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, preferably a combination of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 4: subjecting compound III-6 to a deprotection reaction to obtain compound III-7; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable acid, preferably trifluoroacetic acid, which may be selected from the group consisting of acetic acid, hydrobromic acid, hydrochloric acid, and trifluoroacetic acid. The reaction may be carried out with the addition of triethylsilane as an ion trapping agent. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 5: subjecting compound III-7 and compound III-8 to an addition reaction to obtain compound III-9; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran, dimethylsulfoxide, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 6: subjecting compound III-9 and compound III-10 to a cyclization reaction to obtain compound III; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, water and any combination thereof, and may preferably be a mixed solvent of dimethyl sulfoxide and water. The reaction is preferably carried out in the presence of a metal catalyst. The metal catalyst may be selected from the group consisting of copper sulfate, copper iodide, copper bromide, and copper acetate, and may preferably be copper sulfate. The reaction is preferably carried out in the presence of ascorbic acid or sodium ascorbate. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, 1 to 12 hours.

[0137] The present invention provides another method for preparing a compound of formula (III) where X is a covalent bond, the method comprising the steps of: [ka] [In the formula, LG 1 , L.G. 2 and L.G. 3 represents a leaving group including, but not limited to, halogen, hydroxyl, succinimide ester, active ester, etc., and PG 1 and P.G. 2 represents a protecting group including, but not limited to, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenyloxycarbonyl (Fmoc), benzyl, acetyl, tert-butyl, and the like; D 1 , D 2 , L 1 , L 2 , Aa and M 1 is as defined above]; Step 1: subjecting compound III-11 and compound III-12 to a condensation reaction to obtain compound III-13; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 2: subjecting compound III-13 to a deprotection reaction to obtain compound III-14; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable acid, which may be selected from the group consisting of hydrochloric acid, trifluoroacetic acid and zinc bromide, preferably zinc bromide. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 3: subjecting compound III-14 and compound III-5 to a condensation reaction to obtain compound III-15; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 4: subjecting compound III-15 to a deprotection reaction to obtain compound III-16; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of N,N-dimethylformamide, dichloromethane, tetrahydrofuran and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a suitable base. The base may be selected from the group consisting of diethylamine and piperidine, preferably piperidine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 6 hours. Step 5: subjecting compound III-16 and compound III-17 to a substitution reaction to obtain compound III; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of dichloromethane, tetrahydrofuran, 1,2-dichloroethane, diethyl ether, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine and 4-dimethylaminopyridine, preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours.

[0138] The present invention provides another method for preparing a compound of formula (III) where X is a covalent bond, the method comprising the steps of: [ka] [In the formula, LG 1 , L.G. 2 and L.G. 3 represents a leaving group including, but not limited to, halogen, hydroxyl, succinimide ester, active ester, etc., and PG 1 and P.G. 2 represents a protecting group including, but not limited to, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenyloxycarbonyl (Fmoc), benzyl, acetyl, tert-butyl, and the like; D 1 , D 2 , L 1 , L 2 , Aa and M 1 is as defined above]; Step 1: subjecting compound III-18 and compound III-19 to a condensation reaction to obtain compound III-20; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 2: subjecting compound III-20 to a deprotection reaction to obtain compound III-21; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable acid, which may be selected from the group consisting of hydrochloric acid and trifluoroacetic acid, preferably trifluoroacetic acid. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 3: subjecting compound III-21 and compound III-22 to a condensation reaction to obtain compound III-23; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 4: subjecting compound III-23 to a deprotection reaction to obtain compound III-24; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of N,N-dimethylformamide, dichloromethane, tetrahydrofuran and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a suitable base. The base may be selected from the group consisting of diethylamine and piperidine, preferably piperidine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 6 hours. Step 5: subjecting compound III-24 and compound III-25 to a substitution reaction to obtain compound III; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of dichloromethane, tetrahydrofuran, 1,2-dichloroethane, diethyl ether, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine and 4-dimethylaminopyridine, preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours.

[0139] When Aa is a polypeptide fragment formed from two or more amino acids, Aa is -Aa 1 -Aa 2 - and Aa 1 and Aa 2 is an amino acid fragment constituting Aa; the present invention provides another method for preparing a compound of formula (III), the method comprising the steps of: [ka] [In the formula, LG 1 , L.G. 2 and L.G. 3 represents a leaving group including, but not limited to, halogen, hydroxyl, succinimide ester, active ester, etc., and PG 1 , P.G. 2 and P.G. 3 represents a protecting group including, but not limited to, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenyloxycarbonyl (Fmoc), benzyl, acetyl, tert-butyl, and the like; 1 and Aa 2 is the amino acid fragment that constitutes Aa; D 1 , D 2 , L 1 , L 2 , X, Aa and M 1 is as defined above]; Step 1: subjecting compound III-26 and compound III-27 to a condensation reaction to obtain compound III-28; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 2: subjecting compound III-28 to a deprotection reaction to obtain compound III-29; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran and any combination thereof, preferably 1,4-dioxane. The reaction is preferably carried out in the presence of a suitable acid. The acid may be selected from the group consisting of acetic acid, hydrobromic acid, hydrochloric acid and trifluoroacetic acid, preferably hydrochloric acid. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 3: subjecting compound III-29 and compound III-30 to a condensation reaction to obtain compound III-31; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 4: subjecting compound III-31 to a deprotection reaction to obtain compound III-32; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable acid, which may be selected from the group consisting of hydrochloric acid and trifluoroacetic acid, preferably trifluoroacetic acid. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 5: subjecting compound III-32 and compound III-33 to a condensation reaction to obtain compound III-34; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 6: subjecting compound III-34 to a deprotection reaction to obtain compound III-35; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of N,N-dimethylformamide, dichloromethane, tetrahydrofuran and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a suitable base. The base may be selected from the group consisting of diethylamine and piperidine, preferably piperidine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 6 hours. Step 7: subjecting compound III-35 and compound III-36 to a substitution reaction to obtain compound III; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of dichloromethane, tetrahydrofuran, 1,2-dichloroethane, diethyl ether, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours.

[0140] When Aa is a polypeptide fragment formed from two or more amino acids, Aa is -Aa 1 -Aa 2 The present invention provides another method for preparing a compound of formula (III), the method comprising the steps of: [ka] [In the formula, LG 1 , L.G.2 and L.G. 3 represents a leaving group including, but not limited to, halogen, hydroxyl, succinimide ester, active ester, etc., and PG 1 and P.G. 2 represents a protecting group including, but not limited to, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenyloxycarbonyl (Fmoc), benzyl, acetyl, tert-butyl, and the like; 1 and Aa 2 are, respectively, an amino acid fragment or a polypeptide fragment formed from two or more amino acids; D 1 , D 2 , L 1 , L 2 , X, Aa and M 1 is as defined above]; Step 1: subjecting compound III-37 and compound III-38 to a condensation reaction to obtain compound III-39; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. This reaction is preferably carried out in the presence of a base. The base can be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, with N,N-diisopropylethylamine being preferred. This reaction is preferably carried out at an appropriate temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for an appropriate time, for example, from 1 to 12 hours. Step 2: subjecting compound III-39 to a deprotection reaction to obtain compound III-40; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of N,N-dimethylformamide, dichloromethane, tetrahydrofuran and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a suitable base, which may be selected from the group consisting of diethylamine and piperidine, preferably piperidine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 6 hours. Step 3: subjecting compound III-40 and compound III-41 to a condensation reaction to obtain compound III-42; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, 1,4-dioxane, dichloromethane, dimethylsulfoxide, N,N-dimethylformamide, and any combination thereof, and may be preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a condensing agent. The condensing agent may be selected from 1-hydroxybenzotriazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and may be preferably 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine, and 4-dimethylaminopyridine, and may be preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from -10 to 60°C. The condensation reaction is preferably carried out for a suitable time, for example, from 1 to 12 hours. Step 4: subjecting compound III-42 to a deprotection reaction to obtain compound III-43; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of 1,4-dioxane, dichloromethane, tetrahydrofuran and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable acid, which may be selected from the group consisting of hydrochloric acid, trifluoroacetic acid and zinc bromide, preferably zinc bromide. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours. Step 5: subjecting compound III-43 to a substitution reaction with compound III-44 to obtain compound III; The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of dichloromethane, tetrahydrofuran, 1,2-dichloroethane, diethyl ether, N,N-dimethylformamide and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a base. The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine and 4-dimethylaminopyridine, preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably -10 to 60°C. The reaction is preferably carried out for a suitable time, for example, 1 to 12 hours.

[0141] Definition of Terms Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. The techniques used herein are intended to mean techniques commonly understood in the art, including variations or equivalents that are obvious to those skilled in the art. In addition, the testing techniques used herein, such as genomics, nucleic acid chemistry and molecular biology, are routine procedures that are widely used in the corresponding fields. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to better explain the present invention.

[0142] As used herein, the term "antibody" refers to an immunoglobulin molecule that typically consists of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) light chains and λ (lambda) light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and the antibody isotypes can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domain is not directly involved in binding of the antibody to the antigen, but exhibits various effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including binding to the first component (C1q) of the classical complement system of various cells of the immune system (e.g., effector cells). The VH and VL regions are also subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form an antigen binding site, respectively. The assignment of amino acids to regions or domains can follow various numbering systems known in the art.

[0143] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The heavy and light chain variable regions each contain three CDRs, designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be determined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, one of skill in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev.Comparat.Immunol.27:55-77, 2003).

[0144] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, for example, the Kabat, Chothia, IMGT or AbM numbering systems. In certain embodiments, the antibody or antigen-binding fragment thereof comprises CDRs defined by the Chothia numbering system.

[0145] The term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0146] The term "antigen-binding fragment" of an antibody refers to a polypeptide that is a fragment of an antibody, such as a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that binds and / or competes with the full-length antibody for specific binding to the antigen, the term also being referred to as an "antigen-binding site." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab)', and the like. 2 , F(ab)' 3 , Fd, Fv, scFv, di-scFv, (scFv )2 , disulfide stabilized Fvs ("dsFvs"), single domain antibodies (sdAbs, nanobodies), and such polypeptides comprising at least a sufficient portion of an antibody to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0147] The term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "dAb" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab') 2 " refers to an antibody fragment comprising two Fab's connected by a disulfide bridge in the hinge region; the term "Fab'" refers to an antibody fragment comprising two Fab's connected by a disulfide bridge in the hinge region; 2It refers to a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in and consisting of an intact light chain and the Fd of the heavy chain (consisting of the VH and CH1 domains).

[0148] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. In general, the six CDRs are considered to confer antigen-binding specificity to the antibody. However, even the variable region (e.g., an Fd comprising only three antigen-specific CDRs) can recognize and bind antigen, although its affinity may be lower than that of the intact binding site.

[0149] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first and second heavy chains of an antibody. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0150] The term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, where the VL and VH are connected via a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the following general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH. A suitable linker in the prior art consists of a repeated GGGGS amino acid sequence or a variant thereof. For example, the amino acid sequence (GGGGS) 4can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by 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. In some cases, a disulfide bond can also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains can be positioned relative to each other in any suitable orientation. For example, an scFv can be configured with an NH 2 -VH-VH-COOH,NH 2 -VL-VL-COOH.

[0151] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and binds to and / or competes with the full-length antibody for specific binding to the antigen.

[0152] As used herein, when the term "antibody" is referred to, unless the context clearly indicates otherwise, it includes intact antibodies as well as antigen-binding fragments of antibodies.

[0153] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of an antibody are screened for specificity in the same manner as the intact antibody.

[0154] The term "mouse antibody" refers to an antibody obtained by the following method: fusing B cells of an immunized mouse with myeloma cells, selecting mouse hybrid fusion cells that can grow indefinitely and secrete antibodies, followed by screening, antibody preparation and antibody purification, or refers to an antibody secreted by plasma cells after B cells differentiate and grow after an antigen enters the mouse's body.

[0155] The term "humanized antibody" refers to a non-human antibody that has been genetically modified and has its amino acid sequence altered to increase sequence homology with human antibodies. Generally speaking, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (receptor antibody). A humanized antibody usually retains the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc. The donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody with the desired properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance immune response).

[0156] The twenty common amino acids included herein are prepared according to common usage. For example, see Immunology-A Synthesis (2nd ed., ES Golub and DR Gren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0157] The terms "comprise," "include," "having," "containing," or "involving" and other variations herein are inclusive or open-ended and do not exclude other unrecited elements or method steps.

[0158] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon obtained by removing one hydrogen atom, e.g., "C 1~20 Alkyl, C 1~10 Alkyl, C 1~6 Alkyl, C 1~4 Alkyl, C 1~3 Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, and the like.

[0159] The term "alkylene" refers to a straight or branched chain saturated hydrocarbon obtained by removing two hydrogen atoms, e.g., "C 1~20 Alkylene", "C 1~10 Alkylene", "C 3~10 Alkylene", "C 5~8 Alkylene", "C 1~6 Alkylene", "C 1~4 Alkylene", "C 1~3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene, and the like.

[0160] The term "cycloalkyl" refers to saturated cyclic hydrocarbons, including, but not limited to, monocycloalkyls and bicycloalkyls (e.g., spirocycloalkyls, fused cycloalkyls and bridged cycloalkyls). 3~10"Cycloalkyl" refers to a cycloalkyl group having 3 to 10 ring-forming carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0161] The term "heterocyclyl" refers to a saturated or partially saturated ring structure containing at least one ring member selected from the group consisting of N, O, and S. Particular examples include, but are not limited to, 3- to 12-membered heterocyclyl, 3- to 8-membered heterocyclyl, 5- to 6-membered heterocyclyl, and the like, for example, tetrahydrofuryl, pyrrolidinyl, piperidinyl, tetrahydropyranyl, and the like.

[0162] The term "heteroaryl" refers to an aromatic ring structure containing at least one ring member selected from the group consisting of N, O, and S. Particular examples include, but are not limited to, 5-10 membered heteroaryl, 5-6 membered heteroaryl, and the like, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, and the like.

[0163] The term "aryl" refers to, for example, C 6~10 Aryl refers to a group obtained by removing a hydrogen atom from a carbon atom of the aromatic nucleus of an aromatic hydrocarbon molecule. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, and the like.

[0164] As used herein, the term "fragment" or "structural fragment" refers to the remaining portion of a compound molecule after losing one or more atoms or radicals. For example, a "cytotoxic drug fragment" refers to the remaining portion of a cytotoxic drug obtained after losing a hydrogen atom or hydroxyl from a cytotoxic drug described herein and connecting it to a linker in an antibody-drug conjugate. In some detailed embodiments, a cytotoxic drug fragment is D 1 Similarly, a "TLR agonist fragment" refers to the remaining portion of a TLR agonist described herein that is obtained by removing one or more atoms or groups of atoms from the TLR and connecting them to a linker in an antibody-drug conjugate. In some specific embodiments, the TLR agonist fragment is represented by D 2 It is expressed by:

[0165] When a substituent or value is described as "independently selected from" a group or a group of values, each substituent or value is selected independently from the other groups. Thus, each substituent or value can be the same as or different from another (other) substituent or value.

[0166] The present invention also includes all pharma- ceutically acceptable isotopically labeled compounds that are identical to the compounds of the present invention except that one or more atoms are replaced with an atom having the same atomic number but an atomic mass or mass number different from the atomic mass predominant in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium D, tritium T); isotopes of carbon (e.g., 11 C. 13 C, and 14 C); isotopes of chlorine (e.g., 37 Cl); isotopes of fluorine (e.g., 18 F); isotopes of iodine (e.g., 123 I and 125 I); isotopes of nitrogen (e.g., 13 N and 15 N); isotopes of oxygen (e.g., 15 O.17 O and 18 O); isotopes of phosphorus (e.g., 32 P); and sulfur isotopes (e.g., 35 Certain isotopically labeled compounds of the present invention (e.g., those incorporating a radioactive isotope) can be used in drug and / or substrate tissue distribution studies (e.g., assays). The radioactive isotope tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) are particularly useful for this purpose because they are easy to incorporate and detect. Positron-emitting isotopes, such as 11 C. 18 F, 15 O, and 13 Substitution with N, etc., can be used to examine substrate receptor occupancy in positron emission tomography (PET) studies. Isotopically labeled compounds of the present invention can be prepared by methods analogous to those described in the accompanying schemes and / or examples, and by substituting the appropriate isotopically labeled reagent for the previously used non-labeled reagent. Pharmaceutically acceptable solvates of the present invention include those that are compatible with solvents of crystallization, e.g., D 2 O, acetone-d 6 or DMSO-d 6 can be substituted with an isotope.

[0167] The term "stereoisomer" refers to an isomer formed by at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers may occur. Some individual molecules may also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention may exist as a mixture of two or more structurally distinct forms (often called tautomers) that are in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, and imine-enamine tautomers, and the like. It goes without saying that the scope of the present application encompasses all such isomers or mixtures in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0168] The present invention includes all possible crystalline forms, or polymorphs, of the compounds of the present invention, which may be a single polymorph or a mixture of two or more polymorphs in any ratio.

[0169] It is also understood that some compounds of the present invention may exist in free form for use in therapy, or, where appropriate, as their pharma- ceutically acceptable derivatives. In the present invention, pharma-ceutically acceptable derivatives include, but are not limited to, pharma-ceutically acceptable salts, solvates, metabolites, or prodrugs, which can provide the compound of the present invention or its metabolites or residues directly or indirectly after administration to a patient in need thereof. Thus, when referring to the "compound of the present invention" herein, it is also intended to include various derivative forms of the compounds described above.

[0170] Pharmaceutically acceptable salts of the compounds of the present invention include acid and base addition salts thereof. Suitable acid addition salts are formed from acids that form pharma-ceutically acceptable salts. Suitable base addition salts are formed from bases that form pharma-ceutically acceptable salts. For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharma-ceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0171] The compounds of the present invention may exist in the form of solvates, preferably hydrates, which contain polar solvents as structural elements of the crystal lattice of the compounds. The amount of polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0172] Those skilled in the art will understand that not all nitrogen-containing heterocycles are capable of forming N-oxides, since nitrogen requires a pair of available electrons to oxidize and form an oxide; those skilled in the art will understand that those nitrogen-containing heterocycles capable of forming N-oxides can be recognized. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for the preparation of N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, and include using peroxyacids, such as peracetic acid and m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides, such as tert-butyl hydroperoxide, sodium perborate, and dioxiranes, such as dimethyldioxirane, to oxidize heterocycles and tertiary amines. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see, for example, T. L. Gilchrist, Comprehensive Organic Synthesis, Vol. 7, pp. 748-750; A. R. Katritzky and A. J. Boulton, eds., Academic Press; and G. W. H. Cheeseman and E. S. G. Werstiuk, Advances in Heterocyclic Chemistry, Vol. 22, pp. 390-392, A. R. Katritzky and A. J. Boulton, eds., Academic Press.

[0173] Also included within the scope of the invention are metabolic products of the compounds of the invention, i.e., substances formed in the body following administration of a compound of the invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, and the like, of the administered compound. Thus, the invention includes metabolites of compounds of the invention, including compounds prepared by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolite of a compound of the invention.

[0174] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are some derivatives of the compounds of the present invention, which may have little or no pharmacological activity themselves, and which can be converted to the compounds of the present invention having the desired activity after administration to the body or after administration to the body, for example, by hydrolytic cleavage. Typically, such prodrugs are functional derivatives of compounds that can be easily converted to compounds having the desired therapeutic activity in the body. Additional information regarding the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella), and "Bioreversible Carriers in Drug Design", Pergamon Press, 1987 (Ed. EB Roche, American Pharmaceutical Association). Prodrugs of the present invention can be prepared, for example, by replacing the appropriate functional groups present in the compounds of the present invention with some moieties known to those skilled in the art as "pro-moieties" (e.g., those described in "Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).

[0175] As used herein, in the antibody-drug conjugate of the present invention [ka] shows a specific method of attachment between a sulfhydryl in an antibody or antigen-binding fragment and a linker when the targeting moiety is an antibody or antigen-binding fragment.

[0176] As used herein, in the antibody-drug conjugate of the present invention [ka] shows a specific method of attachment between an amino group in an antibody or antigen-binding fragment and a linker when the targeting moiety is an antibody or antigen-binding fragment.

[0177] Antibody-drug conjugates are also characterized by the average loading of drug moieties (e.g., cytotoxic drugs and TLR agonists) relative to the antibody binding moiety, which is often referred to as the drug-to-antibody ratio (DAR) of the conjugate sample. For example, the average DAR of the ADC can be calculated from the LC-MS data of reduced and deglycosylated samples and based on the average loading of LC and HC chains. The DAR of a given antibody-drug conjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains.

[0178] Although the drug-to-antibody ratio will have a precise value (e.g., m in formula (I)) for a particular antibody-drug conjugate molecule, when used to describe a sample containing many molecules, this value will often be an average value, due, of course, to some degree of heterogeneity normally associated with the coupling step. The average loading of an antibody-drug conjugate sample is referred to herein as the drug-to-antibody ratio or "DAR."

[0179] In some embodiments, the DAR value of the antibody-drug conjugate (drug / antibody ratio of the conjugate sample) is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4- 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 to 8, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0 , 3.5~5.5, 3.5~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5. 0 to 5.5, 5.0 to 6.0, 5.0 to 6.5, 5.0 to 7.0, 5.0 to 7.5, 5.0 to 8.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.0, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.0, 7.0 to 7.5, 7.0 to 8.0, or 7.5 to 8.0.

[0180] In the present invention, "treatment" generally refers to the partial or complete stabilization or cure of a disease and / or side effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including (a) suppressing the symptoms of the disease, i.e., preventing its progression; or (b) alleviating the symptoms of the disease, i.e., causing regression of the disease or symptoms.

[0181] In the present invention, "prevention" refers to inhibiting and delaying the onset of a disease, including not only prevention before the onset of a disease, but also prevention of the recurrence of a disease after treatment.

[0182] In the present invention, a "subject" refers to a vertebrate. In some embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, farm animals (e.g., cows), pets (e.g., cats, dogs, and horses), primates, mice, and rats. In some embodiments, a mammal is a human.

[0183] In the present invention, "effective amount" refers to an amount effective at the dosage and time required to achieve the desired therapeutic effect. "Therapeutically effective amount" may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the active ingredient to elicit a desired response in the individual. A therapeutically effective amount also encompasses an amount of active ingredient in which the therapeutically beneficial effects outweigh any toxic or harmful consequences. In cancer treatment, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of a tumor; inhibit (i.e., slow down to a certain extent, preferably stop) the invasion of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent. EXAMPLES

[0184] Detailed Model for Carrying Out the Invention In order to make the objectives and technical solutions of the present invention clearer, the embodiments of the present invention will be described in detail below with reference to these examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no detailed conditions are shown in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturers. If no manufacturers of the reagents or equipment used are shown, they were all commercially available conventional products.

[0185] The structure of this compound was confirmed by nuclear magnetic resonance ( 1 The molecular weight was determined by 1 H NMR or mass spectrometry (MS). 1The H NMR measurement equipment was a JEOL Eclipse 400 nuclear magnetic device. The measurement solvent was deuterated methanol (CD 3 OD), deuterated chloroform (CDCl 3 ) or hexadeuterated dimethyl sulfoxide (DMSO-d 6 The internal standard was tetramethylsilane (TMS) and chemical shifts (δ) are given in parts per million (ppm).

[0186] The measuring instrument for MS was an Agilent (ESI) mass spectrometer. Manufacturer: Agilent, Model: Agilent 6120B.

[0187] Preparation method for HPLC: Instrument model: Agilent 1260; Chromatography column: Waters SunFire Prep C18 OBD (19 mm × 150 mm × 5.0 μm); Column temperature: 25 °C; Flow rate: 20.0 mL / min; Detection wavelength: 214 nm; Elution gradient: (0 min: A 10%, B 90%; 16.0 min: A 90%, B 10%); Mobile phase A: acetonitrile; Mobile phase B: 0.05% formic acid in water.

[0188] The silica gel plate for thin layer chromatography (TLC) used was an aluminum plate (20×20 cm) manufactured by Merck, and the specification of the silica gel plate for TLC separation and purification was GF254 (1 mm) manufactured by Yantai.

[0189] The reaction was monitored by thin layer chromatography (TLC) or LC-MS; the solvent systems used for development included dichloromethane / methanol, n-hexane / ethyl acetate, and petroleum ether / ethyl acetate, and the volume ratio of the solvents was adjusted according to the polarity of the compounds, or by adding triethylamine, etc.

[0190] Preparation method for reverse phase column chromatography: Preparation method A: Instrument model: Biotage rapid medium-pressure preparative chromatography; Chromatography column: Agela C18 reversed-phase column (spherical; 20-35 μm; 100A); Chromatography column temperature: 25°C; Flow rate: 28.0 mL / min; Detection wavelength: 220 nm; Mobile phase A: acetonitrile; Mobile phase B: water; Preparation method B: Instrument model: Biotage rapid medium pressure preparative chromatography; Chromatography column: Agela C18 reversed phase column (spherical; 20-35 μm; 100A); Chromatography column temperature: 25°C; Flow rate: 28.0 mL / min; Detection wavelength: 220 nm; Mobile phase A: acetonitrile; Mobile phase B: 0.05% formic acid in water; Preparation method C: Instrument model: Biotage rapid medium pressure preparative chromatography; Chromatography column: Agela C18 reversed phase column (spherical; 20-35 μm; 100A); Chromatography column temperature: 25°C; Flow rate: 28.0 mL / min; Detection wavelength: 220 nm; Mobile phase A: acetonitrile; Mobile phase B: 0.05% ammonium bicarbonate aqueous solution;

[0191] Microwave reactions were carried out by using a Biotage Initiator + (400W, RT approximately 300° C.) microwave reactor.

[0192] Silica gel of 200-300 mesh was usually used as the carrier for column chromatography. The eluent systems included dichloromethane / methanol and petroleum ether / ethyl acetate. The volume ratio of the solvents was adjusted according to the polarity of the compounds or by adding a small amount of triethylamine.

[0193] Unless otherwise specified in the examples, the reaction temperature was room temperature (20° C. to 35° C.).

[0194] Reagents used in this invention were purchased from Acros Organics, Aldrich Chemical Company, Tebo Chemical and other companies.

[0195] In the general preparative procedures, examples, and intermediate preparations, the meanings of abbreviations are as follows:

[0196] [Table 1]

[0197] 1. Examples of compounds Intermediate Preparation Example 1: Preparation of 2-((1-((4-((4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamoyl)cyclohexyl)methyl)-2,5-dioxopyrrolidin-3-yl)thio)acetic acid (compound Int1) [ka]

[0198] Step 1: Preparation of tert-butyl (4-((6-nitrothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate 7-Chloro-6-nitrothieno[3,2-b]pyridine (10.0 g, 46.59 mmol) and N-tert-butoxycarbonyl-1,4-butanediamine (10.53 g, 55.91 mmol) were dissolved in N,N-dimethylformamide (150 mL), cooled in an ice-water bath, N,N-diisopropylethylamine (12.04 g, 93.18 mmol) was added at 0° C., slowly warmed to room temperature, and stirred for 6 hours. The reaction solution was poured into water (1500 mL) and extracted three times with ethyl acetate (450 mL). The organic phases were combined, washed three times with saturated brine (150 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (16.61 g, yield: 97.3%). MS m / z(ESI): 367.1 [M+H] + .

[0199] Step 2: Preparation of tert-butyl (4-((6-aminothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate tert-Butyl (4-((6-nitrothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate (16.61 g, 49.37 mmol) was dissolved in a mixture of tetrahydrofuran (100 mL) and methanol (100 mL), 10% palladium on carbon (2.0 g, 11.97 mmol) was added, and the mixture was purged with hydrogen three times. The reaction was stirred at room temperature for 12 hours, and then suction filtered. The filter cake was washed with methanol (30 mL), and the filtrate was concentrated to give the title compound of this step (14.47 g, yield: 87.1%). MS m / z (ESI): 337.1 [M+H] + .

[0200] Step 3: Preparation of tert-butyl (4-((6-pentanamidothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate tert-Butyl (4-((6-aminothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate (14.47 g, 43.01 mmol) and N,N-diisopropylethylamine (11.12 g, 86.02 mmol) were dissolved in tetrahydrofuran (250 mL), cooled in an ice-water bath, n-pentanoyl chloride (7.78 g, 64.52 mmol) was slowly added dropwise at 0° C., slowly warmed to room temperature, and stirred for 4 hours. Water (5 mL) was added to the reaction system to quench the reaction. The reaction system was concentrated to remove as much tetrahydrofuran as possible. Water (300 mL) was added to the residue, and the mixture was extracted three times with ethyl acetate (300 mL). The organic phases were combined, washed three times with saturated brine (150 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (16.72 g, yield: 92.4%). MS m / z (ESI): 421.0 [M+H] + .

[0201] Step 4: Preparation of tert-butyl (4-(2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate tert-Butyl (4-((6-pentanamidothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate (16.72 g, 39.76 mmol) and o-chlorobenzoic acid (3.11 g, 19.88 mmol) were added to toluene (300 mL), heated to reflux, and reacted with stirring for 4 hours to separate water. After the reaction system was cooled to room temperature, the solvent was removed by concentration. The residue was purified by silica gel column chromatography (eluent: 100% ethyl acetate) to obtain the title compound of this step (14.76 g, yield: 92.2%). MS m / z (ESI): 403.2 [M+H] + .

[0202] Step 5: Preparation of tert-butyl (4-(7-bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate tert-Butyl (4-(2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate (5.41 g, 13.44 mmol) was completely dissolved in a mixture of N,N-dimethylformamide (75 mL) and glacial acetic acid (25 mL), cooled in an ice-water bath, N-bromosuccinimide (4.78 g, 26.88 mmol) was slowly added in batches to the reaction solution at 0° C., slowly warmed to room temperature, and stirred for 24 hours. The reaction solution was poured into water (300 mL) and extracted three times with ethyl acetate (300 mL). The organic phases were combined, washed three times with saturated aqueous sodium bicarbonate (150 mL), washed three times with saturated brine (150 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=1 / 1 (v / v)) to give the title compound of this step (4.95 g, yield: 76.5%). MS m / z (ESI): 481.0 [M+H] + .

[0203] Step 6: Preparation of tert-butyl (4-(2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate tert-Butyl (4-(7-bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate (1.0 g, 2.08 mmol), trimethylboroxine (50 wt% solution in tetrahydrofuran, 2.62 g, 10.4 mmol), potassium carbonate (575.0 mg, 4.16 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (307.3 mg, 0.42 mmol) were added sequentially to a mixture of 1,4-dioxane (10 mL) and water (1 mL). After bubbling nitrogen into the reaction system for 5 minutes, it was placed in a microwave oven, heated to 100°C, and stirred for 2 hours. After cooling the reaction system to room temperature, the solvent was removed by concentration. The residue was purified by silica gel column chromatography (eluent: 100% ethyl acetate) to give the title compound of this step (0.65 g, yield: 75.0%). MS m / z (ESI): 417.1 [M+H] + .

[0204] Step 7: Preparation of 1-(4-((tert-butoxycarbonyl)amino)butyl)-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide tert-Butyl (4-(2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate (0.65 g, 1.56 mmol) was dissolved in dichloromethane (10 mL) and cooled in an ice-water bath, m-chloroperoxybenzoic acid (538.5 mg, 3.12 mmol) was added slowly in batches to the reaction solution at 0° C., slowly warmed to room temperature, and stirred for 4 hours. The reaction solution was poured into water (100 mL) and extracted three times with dichloromethane (60 mL). The organic phases were combined, washed twice with saturated aqueous sodium bicarbonate (30 mL), twice with saturated aqueous sodium sulfite (30 mL), three times with saturated brine (45 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (640 mg, yield: 94.8%). MS m / z (ESI): 433.2 [M+H] + .

[0205] Step 8: Preparation of tert-butyl (4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate 1-(4-((Tert-butoxycarbonyl)amino)butyl)-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide (640 mg, 1.48 mmol) was dissolved in a mixture of dichloromethane (10 mL) and ammonia (5 mL), cooled in an ice-water bath, p-toluenesulfonyl chloride (564.1 mg, 2.96 mmol) was added slowly in batches to the reaction solution at 0° C., slowly warmed to room temperature, and stirred for 12 hours. Water (100 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (60 mL). The organic phases were combined, washed three times with saturated brine (45 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / methanol=15 / 1 (v / v)) to give the title compound of this step (266 mg, yield: 41.6%). MS m / z (ESI): 432.2 [M+H] + .

[0206] Step 9: Preparation of 1-(4-aminobutyl)-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (compound 1) tert-Butyl (4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamate (266 mg, 0.62 mmol) was dissolved in a mixture of methanol (10 mL) and hydrogen chloride in 1,4-dioxane (4N, 5 mL) and stirred at room temperature for 12 hours. The solvent was removed by concentration. A mixture of dichloromethane and methanol (volume ratio: 4:1, 10 mL) and saturated aqueous sodium bicarbonate solution (2 mL) were added to the residue in turn. After stirring at room temperature for 5 minutes, the solvent was removed by concentration. The obtained residue was purified by high performance liquid chromatography to obtain the title compound (130 mg, yield: 63.3%). MS m / z (ESI): 332.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6 ): δ 8.34(s, 2H), 6.98 (d, J = 1.2 Hz, 1H), 6.06 (s, 2H), 4.24 (t, J = 8.0 Hz, 2H), 2.86(t, J = 8.0 Hz, 2H), 2.78 (t, J = 8.0 Hz, 2H), 2.56 (d, J = 1.2 Hz, 1H),1.83-1.75 (m, 4H), 1.62-1.58 (m, 2H), 1.47-1.40 (m, 2H), 0.96 (t, J = 8.0 Hz,3H).

[0207] Step 10: Preparation of N-(4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 1-(4-aminobutyl)-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (200 mg, 0.60 mmol) and N,N-diisopropylethylamine (155 mg, 1.2 mmol) were added to N,N-dimethylformamide (2 mL), and 2,5-dioxopyrrolidin-1-yl-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (240 mg, 0.72 mmol) in dichloromethane (1 mL) was added dropwise, and the resulting reaction mixture was stirred at 25° C. for 12 hours. Water was added to the reaction, and it was extracted three times with dichloromethane. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give the crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol=10 / 1 (v / v)) to give the title compound of this step (282 mg, yield: 85.4%). MS m / z(ESI): 551.3 [M+H] + .

[0208] Step 11: Preparation of 2-((1-((4-((4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamoyl)cyclohexyl)methyl)-2,5-dioxopyrrolidin-3-yl)thio)acetic acid (Int1) N-(4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide (282 mg, 0.51 mmol) and thioglycolic acid (94 mg, 1.02 mmol) were added to dichloromethane (50 mL), and the resulting reaction mixture was stirred at 25° C. for 30 minutes. The reaction solution was concentrated to give a yellow solid, which was slurried with methyl tert-butyl ether to give the title compound of this step (237 mg, yield: 72.3%). MS m / z (ESI): 643.3 [M+H] + .

[0209] Intermediate Preparation Example 2: Preparation of N-(tert-butyl)-2-butyl-7-(piperidin-4-yl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (compound Int2) [ka]

[0210] Step 1: Preparation of 6-nitrothieno[3,2-b]pyridin-7-amine 7-Chloro-6-nitrothieno[3,2-b]pyridine (2.0 g, 9.23 mmol) was dissolved in methanol (20 mL), and ammonia in methanol solution (7N, 26.4 mL, 184.8 mmol) was added, heated to 50° C., and stirred for 4 hours. After the reaction was cooled to room temperature, the solvent was removed by concentration to give the title compound of this step (1.73 g, yield: 96.1%). MS m / z (ESI): 196.1 [M+H] + .

[0211] Step 2: Preparation of thieno[3,2-b]pyridine-6,7-diamine The synthetic route of Intermediate Preparation Example 1 was adopted. The raw material of step 2, tert-butyl (4-((6-nitrothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate, was replaced with 6-nitrothieno[3,2-b]pyridin-7-amine to obtain the title compound of this step (1.4 g, yield: 98.4%). MS m / z (ESI): 166.1 [M+H] + .

[0212] Step 3: Preparation of N-(7-aminothieno[3,2-b]pyridin-6-yl)pentanamide The synthetic route of Intermediate Preparation Example 1 was adopted. The raw material of step 3, tert-butyl (4-((6-aminothieno[3,2-b]pyridin-7-yl)amino)butyl)carbamate, was replaced with thieno[3,2-b]pyridine-6,7-diamine to obtain the title compound of this step (2.5 g, yield: 97.6%). MS m / z (ESI): 250.1 [M+H] + .

[0213] Step 4: Preparation of 2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine N-(7-aminothieno[3,2-b]pyridin-6-yl)pentanamide (2.5 g, 8.48 mmol) was added to ethanol (30 mL), followed by sodium hydroxide (1.7 g, 42.4 mmol) at room temperature, and the resulting mixture was heated to 50° C. and stirred for 6 h. The reaction was quenched with water (150 mL), adjusted to pH 6 with dilute hydrochloric acid, and extracted three times with ethyl acetate (150 mL). The organic phases were combined, washed twice with saturated aqueous sodium bicarbonate (30 mL), washed twice with saturated brine (60 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (1.5 g, yield: 76.5%). MS m / z (ESI): 232.0 [M+H] + .

[0214] Step 5: Preparation of 7-bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine The synthetic route of Intermediate Preparation Example 1 was adopted. The reaction material tert-butyl (4-(2-butyl-1H-imidazo[4,5-d]thiophene[3,2-b]pyridin-1-yl)butyl)carbamate in step 5 was replaced with 2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine to obtain the title compound of this step (1.4 g, yield: 65.7%). MS m / z (ESI): 310.0 [M+H] + .

[0215] Step 6: Preparation of a mixture of tert-butyl 7-bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-1-carboxylate and tert-butyl 7-bromo-2-butyl-3H-imidazo[4,5-d]thieno[3,2-b]pyridine-3-carboxylate 7-Bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine (1 g, 3.20 mmol) and 4-dimethylaminopyridine (40 mg, 0.32 mmol) were dissolved in tetrahydrofuran (10 mL), triethylamine (0.65 g, 6.40 mmol) and di-tert-butyl dicarbonate (1.1 g, 4.8 mmol) were added successively at room temperature, heated to 40° C., and stirred for 12 hours. The reaction solution was poured into water (100 mL) and extracted three times with dichloromethane (60 mL). The organic phases were combined, washed three times with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=1 / 1 (v / v)) to give the mixture of title compounds of this step (1.0 g, yield: 76.7%). MS m / z (ESI): 410.0 [M+H] + .

[0216] Step 7: Preparation of a mixture of 7-bromo-1-(tert-butoxycarbonyl)-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide and 7-bromo-3-(tert-butoxycarbonyl)-2-butyl-3H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide A mixture of tert-butyl 7-bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-1-carboxylate and tert-butyl 7-bromo-2-butyl-3H-imidazo[4,5-d]thieno[3,2-b]pyridine-3-carboxylate (1.0 g, 2.43 mmol) was dissolved in dichloromethane (10 mL), cooled in an ice-water bath, m-chloroperbenzoic acid (0.98 g, 4.86 mmol) was added slowly in batches to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 4 hours. The reaction solution was poured into water (100 mL) and extracted three times with dichloromethane (60 mL). The organic phases were combined, washed twice with saturated aqueous sodium bicarbonate (30 mL), twice with saturated aqueous sodium sulfite (30 mL), three times with saturated brine (45 mL), dried over anhydrous sodium sulfate, and concentrated to give a mixture of the title compounds of this step (1.0 g, yield: 90.9%). MS m / z (ESI): 426.0 [M+H] + .

[0217] Step 8: Preparation of a mixture of tert-butyl 7-bromo-2-butyl-4-(tert-butylamino)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-1-carboxylate and tert-butyl 7-bromo-2-butyl-4-(tert-butylamino)-3H-imidazo[4,5-d]thieno[3,2-b]pyridine-3-carboxylate A mixture of 7-bromo-1-(tert-butoxycarbonyl)-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide and 7-bromo-3-(tert-butoxycarbonyl)-2-butyl-3H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide (1.0 g, 2.34 mmol) and tert-butylamine (0.85 g, 11.7 mmol) was dissolved in dichloromethane (20 mL), cooled in an ice-water bath, p-toluenesulfonyl chloride (0.89 g, 4.68 mmol) was added slowly in batches to the reaction solution at 0° C., heated to room temperature, and stirred for 12 hours. Water (100 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (60 mL). The organic phases were combined, washed with saturated brine (45 mL) three times, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=5 / 1 (v / v)) to obtain a mixture of the title compounds of this step (0.6 g, yield: 53.1%). MS m / z (ESI): 481.2 [M+H] + .

[0218] Step 9: Preparation of tert-butyl 4-(2-butyl-4-(tert-butylamino)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate A mixture of tert-butyl 7-bromo-2-butyl-4-(tert-butylamino)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-1-carboxylate and tert-butyl 7-bromo-2-butyl-4-(tert-butylamino)-3H-imidazo[4,5-d]thieno[3,2-b]pyridine-3-carboxylate (1 g, 2.1 mmol) was added to a mixture of 1,4-dioxane (20 mL) and water (2 mL), followed by In the reaction mixture, tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (0.85 g, 2.73 mmol) and potassium carbonate (0.6 g, 4.2 mmol) were added, the mixture was gassed with nitrogen three times, 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (76 mg, 0.1 mmol) was added, and the mixture was heated to 90° C. under microwave for 2 hours. After the reaction was completed, the reaction system was cooled to room temperature, diluted with water, and then extracted with dichloromethane. The organic phases were combined and washed with brine. The crude product obtained by concentrating the combined organic phases was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate=3 / 1 (v / v)) to obtain the title compound of this step (0.8 g, yield: 80.0%). MS m / z (ESI): 484.3 [M+H] + .

[0219] Step 10: Preparation of tert-butyl 4-(2-butyl-4-(tert-butylamino)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)piperidine-1-carboxylate Under nitrogen atmosphere, 10% palladium on carbon (50 mg) was added to a solution of tert-butyl 4-(2-butyl-4-(tert-butylamino)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate (0.8 g, 1.66 mmol) in methanol (20 mL), and the mixture was gassed with hydrogen three times and stirred at room temperature for 24 hours. After the reaction was completed, the palladium on carbon was removed by filtration. The filtrate was concentrated. The obtained solid was dried under vacuum to give the title compound of this step (0.78 g, yield: 97.5%). MS m / z (ESI): 486.3 [M+H] + .

[0220] Step 11: Preparation of N-(tert-butyl)-2-butyl-7-(piperidin-4-yl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (Int2) To a solution of tert-butyl 4-(2-butyl-4-(tert-butylamino)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)piperidine-1-carboxylate (0.78 g, 1.61 mmol) in methanol (10 mL), hydrogen chloride in 1,4-dioxane (2 mL, 4 M, 8 mmol) was added and reacted at room temperature for 12 hours. After the reaction was completed, the reaction solution was concentrated. The obtained solid was dried under vacuum to obtain the title compound of this step (0.57 g, yield: 91.9%). MS m / z (ESI): 386.2 [M+H] + .

[0221] Intermediate Preparation Example 3: Preparation of Compound Int3 [ka]

[0222] Step 1: Preparation of compound Int3-2 To a solution of compound Int3-1 (2 g, 3.41 mmol) in dichloromethane (30 mL), triethylamine (0.69 g, 6.82 mmol) was added, followed by the addition of methylsulfonyl chloride (0.58 g, 5.12 mmol) in an ice-water bath, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was diluted with dichloromethane, then washed with sodium carbonate solution and brine in sequence, and the organic phase was concentrated to obtain the title compound of this step (2.2 g, yield: 95.6%). MS m / z (ESI): 682.4 [M+NH 4 ] + .

[0223] Step 2: Preparation of compound Int3-3 Compound Int3-2 (2.2 g, 3.37 mmol) and N,N-diisopropylethylamine (0.88 g, 6.74 mmol) were added to a solution of compound Int2 (1 g, 2.59 mmol) in N,N-dimethylformamide (15 mL), and the reaction mixture was reacted at 90° C. for 24 hours. After the reaction was completed, the reaction mixture was diluted with water and extracted with dichloromethane. The organic phases were combined and washed with brine. The crude product obtained by concentrating the combined organic phases was purified by silica gel column chromatography (eluent: dichloromethane / methanol=10 / 1 (v / v)) to obtain the title compound of this step (1.2 g, yield: 37.5%). MS m / z (ESI): 954.4 [M+H] + .

[0224] Step 3: Preparation of compound Int3 To a solution of compound Int3-3 (1.2 g, 1.26 mmol) in 1,4-dioxane (10 mL), a solution of hydrogen chloride in 1,4-dioxane (3.2 mL, 4 M, 12.6 mmol) was added. The reaction mixture was reacted at 90° C. for 12 hours. After the reaction was completed, the reaction solution was concentrated to obtain the crude title compound of this step (1 g, yield: 90.9%). MS m / z (ESI): 842.4 [M+H] + .

[0225] Intermediate Preparation Example 4: Preparation of 6-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)-N-(prop-2-yn-1-yl)hexanamide [ka]

[0226] N-Succinimidyl 6-(maleimido)hexanoate (631.58 mg, 2.05 mmol) was dissolved in N,N-dimethylformamide (4 mL), propargylamine (124.12 mg, 2.25 mmol) and triethylamine (414.87 mg, 4.1 mmol) were added, and the mixture was stirred at 25° C. for 12 hours and concentrated to obtain a crude product, which was then purified by silica gel column chromatography (eluent: dichloromethane / methanol=20 / 1 (v / v)) to obtain the title compound of this step (256 mg, yield: 50.2%). MS m / z (ESI): 249.1 [M+H] + .

[0227] Intermediate Preparation Example 5: Preparation of Compound Int5 [ka]

[0228] Step 1: Preparation of compound Int5-2 N-tert-Butoxycarbonyl-S-trityl-L-cysteine ​​(384 mg, 0.83 mmol) and 26-azido-3,6,9,12,15,18,21,24-octoxohexacosan-1-amine (435 mg, 0.99 mmol) were dissolved in N,N-dimethylformamide (4 mL), and 1-hydroxybenzotriazole (167.58 mg, 1.24 mmol), N, N-Diisopropylethylamine (320.43 mg, 2.48 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (238 mg, 1.24 mmol) were added under ice bath, and the mixture was stirred at 20° C. for 3 hours. The reaction solution was added dropwise to water, extracted with dichloromethane, and the organic phase was dried and concentrated to obtain the title compound of this step (0.69 g, yield: 78.4%). MS m / z (ESI): 884.4 [M+H] + .

[0229] Step 2: Preparation of compound Int5-3 Compound Int5-2 (0.69 g, 0.78 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added dropwise, stirred at room temperature for 2 hours, concentrated, and diluted with dichloromethane (20 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to give the title compound of this step (0.6 g, yield: 98%). MS m / z (ESI): 784.4 [M+H] + .

[0230] Step 3: Preparation of compound Int5-4 Compound Int1 (136 mg, 211 mmol) and compound Int5-3 (182 mg, 233 mmol) were dissolved in N,N-dimethylformamide (4 mL), 1-hydroxybenzotriazole (43.2 mg, 0.32 mmol), N,N-diisopropylethylamine (81.6 mg, 0.63 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (61.3 mg, 0.32 mmol) were added under ice bath, and the mixture was stirred at 20 ° C. for 3 hours. The reaction solution was added dropwise to water, extracted with dichloromethane, and the organic phase was dried and concentrated to obtain the title compound of this step (0.23 g, yield: 95.8%). MS m / z (ESI): 1408.6 [M+H] + .

[0231] Step 4: Preparation of compound Int5-5 Compound Int5-4 (0.23 g, 0.16 mmol) was dissolved in dichloromethane (2 mL) and triethylsilane (0.52 mL), trifluoroacetic acid (1 mL) was added dropwise, stirred at room temperature for 1 h, concentrated, and diluted with dichloromethane (20 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to give the title compound of this step (0.15 g, yield: 78.9%). MS m / z (ESI): 1166.5 [M+H] + .

[0232] Step 5: Preparation of compound Int5 Compound Int5-5 (0.15 g, 0.13 mmol) was dissolved in N,N-dimethylformamide (5 mL), MC-VC-PABC-MMAE (0.17 g, 0.13 mmol) and N,N-diisopropylethylamine (50.3 mg, 0.39 mmol) were added, and the mixture was stirred at room temperature for 1 hour, and then aqueous ammonium chloride solution (10 mL) was added, followed by extraction with dichloromethane (10 mL) three times. The organic phase was dried and concentrated to obtain the crude product, which was then purified by productive thin-layer silica gel plate (eluent: dichloromethane / methanol = 10 / 1 (v / v)) to obtain the title compound of this step (150 mg, yield: 47.0%). MS m / z (ESI): 1241.7 [M / 2+H] + .

[0233] Intermediate Preparation Example 6: Preparation of Compound Int6 [ka]

[0234] Step 1: Preparation of compound Int6-1 Compound Int5-3 (192 mg, 0.244 mmol) and N-tert-butoxycarbonyl-heptaethylene glycol-carboxylic acid (110.53 mg, 0.222 mmol) were dissolved in N,N-dimethylformamide (3 mL), 1-hydroxybenzotriazole (45 mg, 0.333 mmol), N,N-diisopropylethylamine (85.9 mg, 0.666 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (63.8 mg, 0.333 mmol) were added under ice bath, and the mixture was stirred for 6 hours at 20° C. The reaction solution was added dropwise to water (10 mL) and extracted three times with dichloromethane (10 mL). The organic phase was dried and concentrated to give the crude product, which was then purified by silica gel column chromatography (eluent: dichloromethane / methanol=10 / 1 (v / v)) to give the title compound of this step (145 mg, yield: 51.6%). MS m / z (ESI): 1263.6 [M+H] + .

[0235] Step 2: Preparation of compound Int6-2 Compound Int6-1 (145 mg, 0.11 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was added dropwise, stirred at room temperature for 2 hours, concentrated, and diluted with dichloromethane (10 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to give the title compound of this step (130 mg, yield: 97%). MS m / z (ESI): 1163.6 [M+H] + .

[0236] Step 3: Preparation of compound Int6-3 Compound Int1 (65 mg, 0.10 mmol) and compound Int6-2 (130 mg, 0.11 mmol) were dissolved in N,N-dimethylformamide (5 mL), 1-hydroxybenzotrizoate (20.3 mg, 0.15 mmol), N,N-diisopropylethylamine (38.7 mg, 0.3 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (28.8 mg, 0.15 mmol) were added under ice bath, and the mixture was stirred at 20° C. for 3 hours. The reaction solution was added dropwise to water (10 mL) and extracted three times with dichloromethane (10 mL). The organic phase was dried and concentrated to obtain the crude product, which was then purified by silica gel column chromatography (eluent: dichloromethane / methanol=10 / 1 (v / v)) to obtain the title compound of this step (110 mg, yield: 60.8%). MS m / z (ESI): 1787.8 [M+H] + .

[0237] Step 4: Preparation of compound Int6-4 Compound Int6-3 (110 mg, 0.06 mmol) was dissolved in dichloromethane (2 mL) and triethylsilane (0.4 mL), trifluoroacetic acid (1 mL) was added dropwise, stirred at room temperature for 1 h, concentrated, and diluted with dichloromethane (20 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to give the title compound of this step (95 mg, yield: 100%). MS m / z (ESI): 1545.7 [M+H] + .

[0238] Step 5: Preparation of compound Int6 Compound Int6-4 (95 mg, 0.06 mmol) was dissolved in N,N-dimethylformamide (25 mL), MC-VC-PABC-MMAE (79 mg, 0.06 mmol) and N,N-diisopropylethylamine (23.2 mg, 0.18 mmol) were added, and the mixture was stirred at room temperature for 1 hour, and then aqueous ammonium chloride solution (10 mL) was added, followed by extraction with dichloromethane (10 mL) three times. The organic phase was dried and concentrated to obtain the crude product, which was then purified by preparative thin-layer silica gel plate (eluent: dichloromethane / methanol = 8 / 1 (v / v)) to obtain the title compound of this step (73 mg, yield: 41.4%). MS m / z (ESI): 1431.3 [M / 2+H] + .

[0239] Intermediate Preparation Example 7: Preparation of Compound Int7 [ka]

[0240] Step 1: Preparation of compound Int7-2 2,5,8,11,14,17,20,23,26-Nonaoxaoctacosan-28-ol (2 g, 4.43 mmol) and p-toluenesulfonyl chloride (1.07 g, 5.32 mmol) were added to dichloromethane (10 mL), cooled to 0-10 ° C., potassium hydroxide (1.26 g, 22.17 mmol) was added, and the reaction was carried out at 25 ° C. for 12 hours. The reaction solution was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 (v / v)) to obtain the title compound of this step (2.5 g, yield: 91.9%). MS m / z (ESI): 583.5 [M+H] + .

[0241] Step 2: Preparation of compound Int7 Compound Int7-2 (1.30 g, 2.12 mmol), 2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-amine (1 g, 2.22 mmol), N,N-diisopropylethylamine (828.80 mg, 6.35 mmol, 1.06 mL) and potassium iodide (35.48 mg, 211.63 μmol) were added to N,N-dimethylformamide (15 mL) in sequence and reacted at 70° C. for 24 hours. The reaction solution was concentrated, and the resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=19 / 1 (v / v)) to obtain the title compound of this step (1.5 g, yield: 71.9%). MS m / z (ESI): 838.3 [M+H] + .

[0242] Intermediate Preparation Example 8: Preparation of Compound Int8 [ka]

[0243] Step 1: Preparation of compound Int8-2 Compound Int8-1 (155 mg, 131.07 μmol) and N-tert-butoxycarbonyl-hepta(ethylene glycol)-carboxylic acid (68.65 mg, 131.07 μmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL), N,N-diisopropylethylamine (42.78 mg, 327.68 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (75.53 mg, 196.61 μmol) were added, and the mixture was reacted at 25 ° C. for 5 hours. The reaction solution was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 (v / v)) to obtain the title compound of this step (193 mg, yield: 87.3%). MS m / z (ESI): 1603.0 [M+H] + .

[0244] Step 2: Preparation of compound Int8 Under nitrogen protection, compound Int8-2 (165 mg, 97.79 μmol) and zinc bromide (139.08 mg, 586.72 μmol) were added in sequence to anhydrous dichloromethane (2 mL) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the crude product of the title compound in this step, which was used directly in the next step. MS m / z (ESI): 1502.9 [M+H] + .

[0245] Intermediate Preparation Example 9: Preparation of Compound Int9 [ka]

[0246] Step 1: Preparation of compound Int9-2 Dess-Martin oxidant (1.23 g, 29.3 mmol) was added to a solution of compound Int9-1 (1 g, 19.5 mmol) in dichloromethane (20 mL) and stirred at room temperature overnight. The reaction solution was diluted with dichloromethane, and saturated sodium thiosulfate solution (10 mL) and sodium bicarbonate solution (10 mL) were added successively, stirred for 20 minutes, liquid separated, and extracted twice with dichloromethane. The organic phases were combined and washed with brine. The crude product obtained by concentrating the combined organic phases was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 12 / 1 (v / v)) to obtain the title compound of this step (0.9 g, yield: 90.9%). MS m / z (ESI): 512.3 [M+H] + .

[0247] Step 2: Preparation of compound Int9-3 Compound Int9-2 (0.86 g, 1.68 mmol), glacial acetic acid (0.16 g, 2.60 mmol) and sodium cyanoborohydride (0.42 g, 6.45 mmol) were added in sequence to a solution of compound Int2 (0.5 g, 1.29 mmol) in dichloroethane (10 mL), and the mixture was allowed to react at room temperature for 12 hours. The reaction solution was poured into water and extracted with dichloromethane. The organic phases were combined and washed with brine. The crude product obtained by concentrating the combined organic phases was purified by silica gel column chromatography (eluent: dichloromethane / methanol=10 / 1 (v / v)) to obtain the title compound of this step (0.6 g, yield: 52.6%). MS m / z (ESI): 825.5 [M+H] + .

[0248] Step 3: Preparation of compound Int9 A solution of hydrogen chloride in 1,4-dioxane (1.7 mL, 4 M, 6.8 mmol) was added to a solution of compound Int9-3 (0.6 g, 0.68 mmol) in 1,4-dioxane (4 mL), and the mixture was reacted at 70° C. for 12 hours. The crude product obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation Method A, elution gradient: A%=15%-25%) to obtain the title compound of this step (0.3 g, yield: 61.2%). MS m / z (ESI): 725.4 [M+H] + .

[0249] Intermediate Preparation Example 10: Preparation of Compound Int10 [ka]

[0250] Step 1: Preparation of compound Int10-1 Compound Int9 (186 mg, 243.74 μmol), ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (131 mg, 292.49 μmol), N,N-diisopropylethylamine (66.32 mg, 487.48 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (136.58 mg, 341.24 μmol) were added in sequence to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (270 mg, yield: 92.9%). MS m / z (ESI): 1132.6 [M+H] + .

[0251] Step 2: Preparation of compound Int10-2 Compound Int10-1 (340 mg, 285.23 μmol) and trifluoroacetic acid (2 mL) were added to dichloromethane (6 mL) and reacted at 25° C. for 2 hours. The reaction solution was concentrated. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=4 / 1 (v / v)) to obtain the title compound of this step (250 mg, yield: 77.4%). MS m / z (ESI): 1076.6 [M+H] + .

[0252] Step 3: Preparation of compound Int10-3 Compound Int10-2 (150 mg, 132.40 μmol), VC-PABC-MMAE (156.57 mg, 132.40 μmol), N,N-diisopropylethylamine (25.93 mg, 198.60 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (61.02 mg, 158.88 μmol) were added sequentially to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (250 mg, yield: 82.2%). MS m / z (ESI): 1091.1 [M / 2+H] + .

[0253] Step 4: Preparation of compound Int10 Compound Int10-3 (250 mg, 8.86 μmol) and piperidine (37.45 mg, 435.44 μmol) were added to N,N-dimethylformamide (4 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A%=20%-30%) to obtain the title compound of this step (185 mg, yield: 82.4%). MS m / z (ESI): 980.2 [M / 2+H] + .

[0254] Intermediate Preparation Example 11: Preparation of Compound Int11 [ka]

[0255] Step 1: Preparation of compound Int11-1 Compound Int8 (114 mg, 72.06 μmol) and N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl) - N 2-(tert-Butoxycarbonyl)-L-lysine (37.43 mg, 72.06 μmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), to which N,N-diisopropylethylamine (23.52 mg, 180.16 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (41.53 mg, 108.09 μmol) were added, and the mixture was reacted at 25° C. for 5 hours. The reaction solution was concentrated. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=50 / 1 (v / v)) to obtain the title compound of this step (110 mg, yield: 70.4%). MS m / z (ESI): 1953.1 [M+H] + .

[0256] Step 2: Preparation of compound Int11-2 Compound Int11-1 (100 mg, 46.07 μmol) was dissolved in dichloromethane (3 mL), diethylamine (67.40 mg, 921.47 μmol) was added, and the mixture was reacted at 20° C. for 10 hours. The reaction solution was concentrated. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=20 / 1 (v / v)) to obtain the title compound of this step (40 mg, yield: 42.8%). MS m / z (ESI): 1731.1 [M+H] + .

[0257] Step 3: Preparation of compound Int11-3 Compound Int11-2 (100 mg, 54.88 μmol) and compound Int3 (46.64 mg, 54.88 μmol) were added to anhydrous N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (10.75 mg, 82.31 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (25.29 mg, 65.85 μmol) were added, and the reaction was carried out at 25° C. for 5 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (60 mg, yield: 40.7%). MS m / z (ESI): 1277.8 [M / 2+H] + .

[0258] Step 4: Preparation of compound Int11 Under nitrogen protection, compound Int11-3 (45 mg, 7.04 μmol) and zinc bromide (6.68 mg, 28.18 μmol) were added sequentially to anhydrous dichloromethane (2 mL) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the crude product of the title compound in this step, which was used directly in the next step. MS m / z (ESI): 1227.8 [M / 2+H] + .

[0259] Intermediate Preparation Example 12: Preparation of Compound Int12 [ka]

[0260] Step 1: Preparation of compound Int12-2 Compound Int3 (150 mg, 169.23 μmol) and N-tert-butoxycarbonyl lysine methyl ester hydrochloride (46.37 mg, 169.23 μmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL), and then N,N-diisopropylethylamine (44.18 mg, 338.46 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (78.01 mg, 203.08 μmol) were added in sequence and reacted at 25° C. for 2 hours. The reaction solution was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=6 / 1 (v / v)) to obtain the title compound of this step (160 mg, yield: 95.8%). MS m / z (ESI): 1084.6 [M+H] + .

[0261] Step 2: Preparation of compound Int12 Compound Int12-2 (185 mg, 162.08 μmol) was dissolved in a mixed solvent of tetrahydrofuran (1 mL), methanol (1 mL) and water (1 mL), lithium hydroxide monohydrate (28.63 mg, 648.31 μmol) was added, and the mixture was reacted at 25° C. for 3 hours. The reaction solution was concentrated, and N,N-dimethylformamide (1 mL) was added. The resulting solution was purified by reverse phase column chromatography (preparation method A, elution gradient: A%=25%-35%) to obtain the title compound of this step (160 mg, yield: 87.6%). MS m / z (ESI): 1070.6 [M+H] + .

[0262] Intermediate Preparation Example 13: Preparation of Compound Int13 [ka]

[0263] Step 1: Preparation of benzyl 4-(((6-nitrothieno[3,2-b]pyridin-7-yl)amino)methyl)piperidine-1-carboxylate 7-Chloro-6-nitrothieno[3,2-b]pyridine (1.0 g, 4.67 mmol) and benzyl 4-(aminomethyl)tetrahydro-1(2H)-pyridine-carboxylate (1.38 g, 5.60 mmol) were dissolved in N,N-dimethylformamide (10 mL), cooled in an ice-water bath, N,N-diisopropylethylamine (1.08 g, 8.4 mmol) was added to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 6 hours. The reaction solution was poured into water (100 mL) and extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (1.91 g, yield: 96.3%). MS m / z (ESI): 427.0 [M+H] + .

[0264] Step 2: Preparation of benzyl 4-(((6-aminothieno[3,2-b]pyridin-7-yl)amino)methyl)piperidine-1-carboxylate Benzyl 4-(((6-nitrothieno[3,2-b]pyridin-7-yl)amino)methyl)piperidine-1-carboxylate (1.91 g, 4.47 mmol) was completely dissolved in absolute ethanol (30 mL), then water (5 mL) was added, cooled in an ice-water bath, sodium dithionite (0.8 g, 45 mmol) was added to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 4 hours. The reaction solution was poured into water (100 mL), stirred to precipitate a solid, and filtered. The solid was washed with water and dried to obtain the title compound of this step (1.5 g, yield: 84.2%). MS m / z (ESI): 397.1 [M+H] + .

[0265] Step 3: Preparation of benzyl 4-(((6-pentanamidothieno[3,2-b]pyridin-7-yl)amino)methyl)piperidine-1-carboxylate Benzyl 4-(((6-aminothieno[3,2-b]pyridin-7-yl)amino)methyl)piperidine-1-carboxylate (1.5 g, 3.78 mmol) and N,N-diisopropylethylamine (0.97 g, 7.57 mmol) were dissolved in tetrahydrofuran (20 mL), cooled in an ice-water bath, n-pentanoyl chloride (0.68 g, 5.67 mmol) was slowly added dropwise to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 4 hours. Water (10 mL) was added to the reaction system to quench the reaction. The reaction system was concentrated to remove as much tetrahydrofuran as possible. Water (50 mL) was added to the obtained residue, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phases were combined, washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (1.63 g, yield: 90%). MS m / z (ESI): 481.1 [M+H] + .

[0266] Step 4: Preparation of benzyl 4-((2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)methyl)piperidine-1-carboxylate Benzyl 4-(((6-pentanamidothieno[3,2-b]pyridin-7-yl)amino)methyl)piperidine-1-carboxylate (1.63 g, 3.38 mmol) and o-chlorobenzoic acid (260 mg, 1.7 mmol) were added to toluene (50 mL), heated to reflux, and reacted for 4 hours while stirring to separate water. After cooling the reaction system to room temperature, the solvent was removed by concentration. The obtained residue was purified by silica gel column chromatography (eluent: 100% ethyl acetate) to obtain the title compound of this step (1.39 g, yield: 89.3%). MS m / z (ESI): 463.1 [M+H] + .

[0267] Step 5: Preparation of benzyl 4-((7-bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)methyl)piperidine-1-carboxylate Benzyl 4-((2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)methyl)piperidinecarboxylate (1.1 g, 2.37 mmol) was completely dissolved in a mixture of N,N-dimethylformamide (9 mL) and glacial acetic acid (3 mL), cooled in an ice-water bath, N-bromosuccinimide (0.63 g, 3.55 mmol) was added slowly in batches to the reaction solution at 0° C., slowly warmed to room temperature, and stirred for 24 hours. The reaction solution was poured into water (100 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined, washed three times with saturated aqueous sodium bicarbonate (50 mL), washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=1 / 1 (v / v)) to give the title compound of this step (0.87 g, yield: 68.5%). MS m / z (ESI): 541.1 [M+H] + .

[0268] Step 6: Preparation of benzyl 4-((2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)methyl)piperidine-1-carboxylate Benzyl 4-((7-bromo-2-butyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)methyl)piperidine-1-carboxylate (0.87 g, 1.61 mmol), trimethylboroxine (50 wt% solution in tetrahydrofuran, 2.0 g, 8.05 mmol), potassium carbonate (0.67 g, 4.83 mmol), and 1,1'-bis(diphenylphosphine)ferrocenepalladium dichloride (0.23 g, 0.32 mmol) were added sequentially to a mixture of 1,4-dioxane (10 mL) and water (1 mL), and the reaction was sparged with nitrogen for 5 minutes, heated to 100° C. under microwave, and stirred for 2 hours. The reaction was cooled to room temperature, and the solvent was removed by concentration. The residue was purified by silica gel column chromatography (eluent: 100% ethyl acetate) to give the title compound of this step (0.54 g, yield: 71.0%). MS m / z (ESI): 477.1 [M+H] + .

[0269] Step 7: Preparation of 1-((1-((benzyloxy)carbonyl)piperidin)-4-yl)methyl)-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide Benzyl 4-((2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)piperidine-1-carboxylate (0.5 g, 1.04 mmol) was dissolved in dichloromethane (10 mL) and cooled in an ice-water bath, m-chloroperoxybenzoic acid (0.42 g, 2.05 mmol) was slowly added to the reaction solution at 0° C., slowly warmed to room temperature, and stirred for 4 hours. The reaction solution was poured into water (100 mL) and extracted three times with dichloromethane (20 mL). The organic phases were combined, washed twice with saturated aqueous sodium bicarbonate (30 mL), twice with saturated aqueous sodium sulfite (30 mL), three times with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (0.52 g, yield: 100%). MS m / z (ESI): 493.2 [M+H] + .

[0270] Step 8: Preparation of benzyl 4-((4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)methyl)piperidine-1-carboxylate 1-((1-(((benzyloxy)carbonyl)piperidine)-4-yl)methyl)-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide (0.52 g, 1.04 mmol) was dissolved in a mixture of dichloromethane (10 mL) and aqueous ammonia (5 mL), cooled in an ice-water bath, p-toluenesulfonyl chloride (0.4 g, 2.09 mmol) was added slowly in batches to the reaction solution at 0° C., slowly warmed to room temperature, and stirred for 12 hours. Water (100 mL) was added to the reaction system, and it was extracted three times with dichloromethane (20 mL). The organic phases were combined, washed three times with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / methanol=20 / 1 (v / v)) to give the title compound of this step (230 mg, yield: 45%). MS m / z (ESI): 492.2 [M+H] + .

[0271] Step 9: Preparation of 2-butyl-7-methyl-1-(piperidin-4-ylmethyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (Int13) Benzyl 4-((4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)methyl)piperidine-1-carboxylate (150 mg, 0.31 mmol) was added to trifluoroacetic acid (6 mL), heated to 50° C., and stirred for 3 hours. The solvent was removed by concentration. A mixed solvent of dichloromethane and methanol (volume ratio: 4:1, 10 mL) and a saturated aqueous sodium bicarbonate solution (10 mL) were added to the residue, which was stirred at room temperature for 5 minutes, and then concentrated to remove the solvent. The obtained residue was purified by preparative high performance liquid chromatography to obtain the title compound of this step (22 mg, yield: 19.9%). MS m / z (ESI): 358.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6): δ 8.24(s, 1H), 6.99-6.98 (m, 1H), 6.11 (br, 2H), 4.14 (d, J = 8.0 Hz, 2H), 3.26-3.23(m, 2H), 2.88-2.76 (m, 4H), 2.57-2.55 (m, 3H), 2.19-2.12(m, 1H), 1.87-1.76 (m,2H), 1.68-1.62(m, 2H), 1.56-1.39 (m, 4H), 0.96 (t, J = 8.0 Hz, 3H).

[0272] Intermediate Preparation Example 14: Preparation of Compound Int14 [ka]

[0273] Step 1: Preparation of compound Int14-1 Compound Int3-1 (1 g, 1.7 mmol) and Dess-Martin oxidant (1.08 g, 2.56 mmol) were added to dichloromethane (15 mL) and reacted at 25° C. for 10 hours. Saturated sodium thiosulfate solution (5 mL) and saturated aqueous sodium bicarbonate solution (10 mL) were added to the reaction solution, stirred until clear, then extracted with dichloromethane three times, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=10 / 1 (v / v)) to obtain the title compound of this step (900 mg, yield: 91.3%). MS m / z(ESI): 585.5 [M+H] + .

[0274] Step 2: Preparation of compound Int14-2 Compound Int14-1 (472.48 mg, 799.99 μmol), compound Int13 (191 mg, 533.33 μmol), sodium cyanoborohydride (99.08 mg, 1576.7 μmol) and glacial acetic acid (51.72 mg, 853.33 μmol) were added in sequence to a mixed solvent of 1,2-dichloroethane (6 mL) and methanol (3 mL), and reacted at 25° C. for 4 hours. The crude product obtained by concentrating the reaction solution was dissolved in methanol (3 mL) and purified by reverse phase column chromatography (preparation method B, elution gradient: A%=15%-25%) to obtain the title compound of this step (250 mg, yield: 50.6%). MS m / z (ESI): 926.5 [M+H] + .

[0275] Step 3: Preparation of compound Int14 Compound Int14-2 (250 mg, 269.83 μmol) was dissolved in dioxane hydrochloride solution (6 mL, 4 M) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (203 mg, yield: 86.5%). MS m / z (ESI): 870.4 [M+H] + .

[0276] Intermediate Preparation Example 15: Preparation of Compound Int15 [ka]

[0277] Step 1: Preparation of compound Int15-1 Compound Int9-2 (390.27 mg, 794.96 μmol), compound Int13 (200 mg, 529.98 μmol), sodium cyanoborohydride (159.79 mg, 2.52 mmol) and glacial acetic acid (7.88 mg, 1.51 mmol) were added in sequence to a mixed solvent of 1,2-dichloroethane (6 mL) and methanol (3 mL), and reacted at 25° C. for 4 hours. The crude product obtained by concentrating the reaction solution was dissolved in methanol (3 mL) and purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (330 mg, yield: 76.8%). MS m / z (ESI): 853.5 [M+H] + .

[0278] Step 2: Preparation of compound Int15 Compound Int15-1 (330 mg, 386.82 μmol) was dissolved in dioxane hydrochloride solution (6 mL, 4 M) and reacted for 5 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (250 mg, yield: 86.2%). MS m / z (ESI): 753.4 [M+H] + .

[0279] Intermediate Preparation Example 16: Preparation of Compound Int16 [ka]

[0280] Step 1: Preparation of compound Int16-2 Compound Int16-1 (270 mg, 428.06 μmol), p-aminobenzyl alcohol (106.50 mg, 856.13 μmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (213.60 mg, 856.13 μmol) were added to anhydrous methanol (1 mL) and anhydrous dichloromethane (3 mL) and stirred at room temperature overnight. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 45% to 55%) to obtain the title compound of this step (130 mg, yield: 40.62%). MS m / z (ESI): 673.3 [M+H] + .

[0281] Step 2: Preparation of compound Int16-3 Compound Int16-2 (125 mg, 167.21 μmol), bis(p-nitrophenyl)carbonate (77.07 mg, 250.81 μmol) and N,N-diisopropylethylamine (54.47 mg, 418.02 μmol) were added sequentially to N,N-dimethylformamide (2 mL) and stirred at room temperature for 5 hours. The reaction solution was directly purified by reversed-phase column chromatography (preparation method B, elution gradient: A% = 65% to 75%) to obtain the title compound of this step (95 mg, yield: 64.42%). 1 H-NMR (400MHz, DMSO-d 6): δ 10.14(br, 1H), 8.31 (d, J = 8Hz, 2H), 8.12 (d, J = 8Hz, 1H), 7.90 (d, J = 4Hz, 2H),7.76-7.72 (m, 2H), 7.65 (d, J = 8Hz, 2H), 7.58 (d, J = 8Hz, 2H), 7.48-7.39 (m,5H), 7.32 (t, J = 8Hz, 2H), 6.76 (br, 1H), 5.24 (s, 2H), 4.38-4.22 (m, 4H),3.92 (t, J = 8Hz, 1H), 2.88 (d, J = 4Hz, 2H), 2.01- 1.96 (m, 1H), 1.68-1.59 (m,2H), 1.40-1.24 (m, 15H), 0.89-0.85 (m, 6H) MS m / z (ESI): 838.3 [M+H] + .

[0282] Step 3: Preparation of compound Int16-4 Monomethyl auristatin E (77 mg, 101.88 μmol), compound Int16-3 (94.85 mg, 101.88 μmol), 1-hydroxybenzotriazole (2.09 mg, 15.28 μmol) and N,N-diisopropylethylamine (26.55 mg, 203.77 μmol) were added to N,N-dimethylformamide (1 mL) and stirred at 35° C. for 5 hours. The reaction solution was directly purified by reversed-phase column chromatography (preparation method B, elution gradient: A%=50%-60%) to obtain the title compound of this step (65 mg, yield: 42.78%). MS m / z (ESI): 1416.8 [M+H] + .

[0283] Step 4: Preparation of compound Int16-5 Compound Int16-4 (30 mg, 19.06 μmol) and anhydrous zinc bromide (21.68 mg, 95.29 μmol) were added to anhydrous dichloromethane (2 mL) and stirred for 2 hours at 35° C. The reaction solution was diluted with ethyl acetate (10 mL), washed with saturated brine (3 mL×2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound of this step (31 mg, yield: 98.84%). MS m / z (ESI): 1316.8 [M+H] + .

[0284] Step 5: Preparation of compound Int16-6 Compound Int3 (20 mg, 21.38 μmol), compound Int16-5 (31.0 mg, 21.38 μmol), N,N-diisopropylethylamine (5.57 mg, 42.75 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.85 mg, 25.65 μmol) were added to N,N-dimethylformamide (1 mL) and stirred at room temperature for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 40% to 50%) to obtain the title compound of this step (29 mg, yield: 57.04%). MS m / z (ESI): 1070.6 [M / 2+H] + .

[0285] Step 6: Preparation of compound Int16 Compound Int16-6 (29 mg, 12.19 μmol) and piperidine (2.10 mg, 24.38 μmol) were added to N,N-dimethylformamide (1 mL) and stirred at room temperature for 2 hours. After concentrating the reaction solution, methyl tert-butyl ether (5 mL) was added, sonicated for 5 minutes, then allowed to stand, filtered, and the solid was dried to obtain the title compound of this step (22 mg, yield: 94.06%). MS m / z (ESI): 959.6 [M / 2+H] + .

[0286] Intermediate Preparation Example 17: Preparation of Compound Int17 [ka]

[0287] Step 1: Preparation of compound Int17-1 (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (85.29 mg, 200.46 μmol), N,N-diisopropylethylamine (45.2 mg, 346.26 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (103.94 mg, 270.22 μmol) and compound Int15 (150 mg, 182.24 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (170 mg, yield: 79.8%). MS m / z (ESI): 1160.6 [M+H] + .

[0288] Step 2: Preparation of compound Int17 Compound Int17-1 (170 mg, 138.15 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (3 mL) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (133 mg, yield: 86.3%). MS m / z (ESI): 1104.6 [M+H] + .

[0289] Intermediate Preparation Example 18: Preparation of Compound Int18 [ka]

[0290] Step 1: Preparation of compound Int18-1 Compound Int15 (60 mg, 79.78 μmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutyric acid (32.78 mg, 79.78 μmol), N,N-diisopropylethylamine (25.73 mg, 199.46 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (36.38 mg, 95.73 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and stirred at room temperature for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 40%) to obtain the title compound of this step (52 mg, yield: 56.94%). MS m / z (ESI): 1146.7 [M+H] + .

[0291] Step 2: Preparation of compound Int18-2 Compound Int18-1 (50 mg, 43.60 μmol) and 48% aqueous hydrobromic acid (0.5 mL) were added to acetonitrile (1 mL) in sequence and stirred at room temperature for 3 hours. The reaction solution was concentrated to obtain the title compound of this step (35 mg, yield: 73.42%). MS m / z (ESI): 1090.6 [M+H] + .

[0292] Step 3: Preparation of compound Int18-3 Compound Int18-2 (35 mg, 32.09 mmol), VC-PABC-MMAE (36.04 mg, 32.09 μmol), N,N-diisopropylethylamine (10.34 mg, 80.22 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (14.63 mg, 38.50 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and stirred at room temperature for 2 hours. The reaction solution was directly purified by reversed-phase column chromatography (preparation method B, elution gradient: A% = 40% to 45%) to obtain the title compound of this step (63 mg, yield: 89.31%). MS m / z (ESI): 1098.1 [M / 2+H] + .

[0293] Step 4: Preparation of compound Int18 Compound Int18-3 (63 mg, 28.7 μmol) and piperidine (4.82 mg, 57.40 μmol) were added to N,N-dimethylformamide (1 mL) and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (28 mg, yield: 49.45%). MS m / z (ESI): 1973.2 [M+H] + .

[0294] Intermediate Preparation Example 19: Preparation of Compound Int19 [ka]

[0295] Step 1: Preparation of compound Int19-1 (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutyric acid (35.52 mg, 86.33 μmol), N,N-diisopropylethylamine (14.16 mg, 109.56 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (38.88 mg, 102.25 μmol) and compound Int15 (65 mg, 86.33 μmol) were added sequentially to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (50 mg, yield: 53.2%). MS m / z (ESI): 1146.6 [M+H] + .

[0296] Step 2: Preparation of compound Int19-2 Compound Int19-1 (50 mg, 43.18 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (3 mL) and trifluoroacetic acid (1.5 mL) and reacted for 4 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (45 mg, yield: 89.2%). MS m / z (ESI): 1090.5 [M+H] + .

[0297] Step 3: Preparation of compound Int19-3 Compound Int19-2 (41.53 mg, 38.09 μmol), N,N-diisopropylethylamine (6.82 mg, 52.37 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (18.07 mg, 47.05 μmol) and VC-PBAC-MMAE (42.8 mg, 38.09 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 25%) to obtain the title compound of this step (52 mg, yield: 65.4%). MS m / z (ESI): 1098.1 [M / 2+H] + .

[0298] Step 4: Preparation of compound Int19 Compound Int19-3 (52 mg, 23.69 μmol) and piperidine (6.52 mg, 76.54 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (31 mg, yield: 82.9%). MS m / z (ESI): 658.4 [M / 3+H] + .

[0299] Intermediate Preparation Example 20: Preparation of Compound Int20 [ka]

[0300] VC-PABC-MMAE (150 mg, 133.52 μmol), N,N-diisopropylethylamine (22.43 mg, 173.58 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (66 mg, 173.58 μmol), and N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 -((tert-butoxy)carbonyl)-L-lysine (62.56 mg, 133.52 μmol) was added sequentially to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0° C. for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=60%-70%) to obtain the title compound of this step (170 mg, yield: 85.1%). MS m / z (ESI): 1573.9 [M+H] + .

[0301] Intermediate Preparation Example 21: Preparation of Compound Int21 [ka]

[0302] Step 1: Preparation of compound Int21-1 Compound 8-3 (86 mg, 72.3 μmol), N,N-diisopropylethylamine (12.15 mg, 94 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (35.74 mg, 94 μmol) and GGFG-Dxd (60.8 mg, 72.3 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (60 mg, yield: 43.4%). MS m / z (ESI): 1006.5 [M / 2+H] + .

[0303] Step 2: Preparation of compound Int21 Compound Int21-1 (60 mg, 29.82 μmol) and diethylamine (10.46 mg, 141.64 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=15%-25%) to obtain the title compound of this step (45 mg, yield: 88.8%). MS m / z (ESI): 1789.9 [M+H] + .

[0304] Intermediate Preparation Example 22: Preparation of Compound Int22 [ka]

[0305] Step 1: Preparation of compound Int22-1 Compound Int14 (280 mg, 321.84 μmol), N,N-diisopropylethylamine (68.42 mg, 524.08 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (114.8 mg, 350 μmol) and N-tert-butoxycarbonyl lysine methyl ester hydrochloride (92 mg, 350 μmol) were added sequentially to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (300 mg, yield: 83.8%). MS m / z (ESI): 1112.6 [M+H] + .

[0306] Step 2: Preparation of compound Int22-2 Compound Int22-1 (300 mg, 269.68 μmol) was dissolved in a mixed solvent of methanol (6 mL) and water (2 mL), and then lithium hydroxide (56.58 mg, 1348.4 μmol) was added and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (280 mg, yield: 94.5%). MS m / z (ESI): 1098.6 [M+H] + .

[0307] Step 3: Preparation of compound Int22-3 Compound Int22-2 (280 mg, 254.93 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (3 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (180 mg, yield: 74.5%). MS m / z (ESI): 998.6 [M+H] + .

[0308] Step 4: Preparation of compound Int22 Compound Int22-3 (180 mg, 178.51 μmol) and 9-fluorenylmethyl-N-succinimidyl carbonate (91.24 mg, 267.76 μmol) were dissolved in anhydrous dichloromethane (3 mL), and then N,N-diisopropylethylamine (46.14 mg, 357.02 μmol) was added and reacted at 25° C. for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (180 mg, yield: 82.9%). MS m / z (ESI): 1220.6 [M+H] + .

[0309] Intermediate Preparation Example 23: Preparation of Compound Int23 [ka]

[0310] Step 1: Preparation of 6-nitro-N-((tetrahydro-2H-pyran-4-yl)methyl)thieno[3,2-b]pyridin-7-amine 7-Chloro-6-nitrothieno[3,2-b]pyridine (2 g, 9.34 mmol) and 4-aminomethyltetrahydropyran (1.29 g, 11.2 mmol) were dissolved in N,N-dimethylformamide (20 mL), cooled in an ice-water bath, and N,N-diisopropylethylamine (1.81 g, 14 mmol) was added to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 6 hours. The reaction solution was poured into water (100 mL) and extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the title compound of this step (2.52 g, yield: 92.3%). MS m / z (ESI): 294.1 [M+H] + .

[0311] Step 2:N 7 Preparation of -((tetrahydro-2H-pyran-4-yl)methyl)thieno[3,2-b]pyridine-6,7-diamine 6-Nitro-N-((tetrahydro-2H-pyran-4-yl)methyl)thieno[3,2-b]pyridin-7-amine (2.52 g, 8.53 mmol) was dissolved in tetrahydrofuran (50 mL) and methanol (50 mL), 10% palladium on carbon (0.2 g) was added, and the mixture was purged with hydrogen three times. The reaction was stirred at room temperature for 12 hours, suction filtered, the filter cake was washed with methanol (30 mL), and the filtrate was concentrated to give the title compound of this step (2.1 g, yield: 93.7%). MS m / z (ESI): 264.1 [M+H] + .

[0312] Step 3: Preparation of N-(7-(((tetrahydro-2H-pyran-4-yl)methyl)amino)thieno[3,2-b]pyridin-6-yl)pentanamide N 7-((tetrahydro-2H-pyran-4-yl)methyl)thieno[3,2-b]pyridine-6,7-diamine (2.1 g, 7.98 mmol) and N,N-diisopropylethylamine (2.1 g, 15.9 mmol) were dissolved in tetrahydrofuran (40 mL), cooled in an ice-water bath, n-pentanoyl chloride (1.45 g, 11.97 mmol) was added dropwise to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 4 hours. Water (10 mL) was added to the reaction system to quench the reaction. The reaction system was concentrated to remove tetrahydrofuran as much as possible. Water (50 mL) was added to the residue, and extracted three times with ethyl acetate (30 mL). The organic phases were combined, washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (2.8 g, yield: 100%). MS m / z (ESI): 348.1 [M+H] + .

[0313] Step 4: Preparation of 2-butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine N-(7-(((tetrahydro-2H-pyran-4-yl)methyl)amino)thieno[3,2-b]pyridin-6-yl)pentanamide (2.8 g, 7.98 mmol) was added to ethanol (30 mL), sodium hydroxide (1.3 g, 31.9 mmol) was added at room temperature, heated to 50° C., and stirred for 6 hours. Water (150 mL) was added to the reaction system to quench the reaction. The reaction system was adjusted to pH=6 with hydrochloric acid and extracted three times with ethyl acetate (100 mL). The organic phases were combined, washed twice with saturated sodium bicarbonate solution (30 mL), washed twice with saturated brine (60 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound of this step (1.9 g, yield: 72.2%). MS m / z (ESI): 330.1 [M+H] + .

[0314] Step 5: Preparation of 7-bromo-2-butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine 2-Butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine (1.9 g, 5.75 mmol) was completely dissolved in a mixture of N,N-dimethylformamide (20 mL) and glacial acetic acid (6 mL), cooled in an ice-water bath, N-bromosuccinimide (1.54 g, 8.63 mmol) was slowly added in batches to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 24 hours. The reaction solution was poured into water (100 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined, washed three times with saturated aqueous sodium bicarbonate solution (50 mL), washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=1 / 1 (v / v)) to obtain the title compound of this step (1.64 g, yield: 70%). MS m / z (ESI): 408.0 [M+H] + .

[0315] Step 6: Preparation of 7-bromo-2-butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide 7-Bromo-2-butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine (1.64 g, 4.0 mmol) was dissolved in dichloromethane (30 mL), cooled in an ice-water bath, m-chloroperoxybenzoic acid (1.63 g, 8.0 mmol) was added slowly in batches to the reaction solution at 0° C., slowly heated to room temperature, and stirred for 4 hours. The reaction solution was poured into water (100 mL) and extracted three times with dichloromethane (30 mL). The organic phases were combined, washed twice with saturated aqueous sodium bicarbonate solution (30 mL), washed twice with saturated aqueous sodium sulfite solution (30 mL), washed three times with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound (1.7 g, yield: 100%). MS m / z (ESI): 424.0 [M+H] + .

[0316] Step 7: Preparation of 7-bromo-N-(tert-butyl)-2-butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine 7-Bromo-2-butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridine-5-oxide (1.7 g, 4.0 mmol) and tert-butylamine (0.73 g, 20 mmol) were dissolved in dichloromethane (20 mL), cooled in an ice-water bath, and p-toluenesulfonyl chloride (1.53 g, 8.0 mmol) was added slowly in batches to the reaction solution at 0° C., slowly warmed to room temperature, and stirred for 12 hours. Water (100 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (30 mL). The organic phases were combined, washed three times with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=5 / 1 (v / v)) to give the title compound of this step (1.1 g, yield: 57.3%). MS m / z (ESI): 479.0 [M+H] + .

[0317] Step 8: Preparation of tert-butyl 4-(2-butyl-4-(tert-butylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate 7-Bromo-N-(tert-butyl)-2-butyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (0.8 g, 1.71 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6,-dihydropyridine-1(2H)-carboxylate (0.8 g, 2.56 mmol), potassium carbonate (4.73 g, 3.4 mmol) and 1,1'-bis(diphenylphosphine)ferrocenepalladium dichloride (62 mg, 0.08 mmol) were added to a mixture of 1,4-dioxane (10 mL) and water (2 mL). The reaction was sparged with nitrogen for 5 min, then heated to 100° C. and stirred for 2 h. After the reaction was cooled to room temperature, the solvent was removed by concentration, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate=1 / 3 (v / v)) to give the title compound of this step (0.5 g, yield: 50.5%). MS m / z (ESI): 582.3 [M+H] + .

[0318] Step 9: Preparation of tert-butyl 4-(2-butyl-4-(tert-butylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)piperidine-1-carboxylate tert-Butyl 4-(2-butyl-4-(tert-butylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate (0.5 g, 0.86 mmol), ammonium formate (1.08 g, 17.2 mmol) and 10% palladium on carbon (20 mg, 0.019 mmol) were added successively in absolute ethanol (10 mL), slowly heated to 80° C. and stirred for 12 h. The reaction was cooled to room temperature, then suction filtered, the filter cake was washed with ethanol (15 mL), the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=10:1 (v / v)) to obtain the title compound of this step (0.40 g, yield: 79.7%). MS m / z (ESI): 584.3 [M+H] + .

[0319] Step 10: Preparation of 2-butyl-7-(piperidin-4-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (Int23) tert-Butyl 4-(2-butyl-4-(tert-butylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-7-yl)piperidine-1-carboxylate (0.3 g, 0.51 mmol) was dissolved in methanol (3 mL), concentrated hydrochloric acid (0.5 mL) was added at room temperature, heated to 50 ° C., and stirred for 4 hours. The reaction was cooled to room temperature, then concentrated to remove the solvent, and the residue was purified by preparative high performance liquid chromatography to obtain the title compound of this step (0.19 g, yield: 87.1%). MS m / z (ESI): 428.3 [M+H] + . 1H NMR (400 MHz, CD3OD) δ: 7.11 (s, 1H), 4.23(d, J = 7.6 Hz, 2H), 3.95-3.92 (m, 2H), 3.47-3.44 (m, 2H), 3.15-3.08 (m, 2H),2.98-2.94 (m, 2H), 2.31-2.15 (m, 3H), 2.02-1.86 (m, 4H), 1.57-1.49 (m, 5H),1.37-1.26 (m, 4H), 1.03 (t, J = 7.6 Hz, 3H).

[0320] Intermediate Preparation 24: Preparation of Compound Int24 [ka]

[0321] Step 1: Preparation of compound Int24-1 Compound Int14-1 (472.48 mg, 799.99 μmol), compound Int23 (250 mg, 533.33 μmol), sodium cyanoborohydride (99.08 mg, 1576.7 μmol) and glacial acetic acid (51.72 mg, 853.33 μmol) were added in sequence to a mixed solvent of 1,2-dichloroethane (6 mL) and methanol (3 mL), and reacted at 25 ° C. for 4 hours. The crude product obtained by concentrating the reaction solution was dissolved in methanol (3 mL) and purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 30%) to obtain the title compound of this step (475 mg, yield: 89.4%). MS m / z(ESI): 996.6 [M+H] + .

[0322] Step 2: Preparation of compound Int24-2 Compound Int24-1 (475 mg, 476.76 μmol) was dissolved in dioxane hydrochloride solution (6 mL, 4 M) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (400 mg, yield: 99.3%). MS m / z (ESI): 940.6 [M+H] +.

[0323] Step 3: Preparation of compound Int24-3 Compound Int24-2 (280 mg, 297.81 μmol), N,N-diisopropylethylamine (116.63 mg, 893.44 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (160.13 mg, 416.94 μmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine tert-butyl ester (133.08 mg, 313.48 μmol) were added sequentially to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (248 mg, yield: 61.8%). MS m / z (ESI): 1346.7 [M+H] + .

[0324] Step 4: Preparation of compound Int24 Compound Int24-3 (94.74 mg, 70.34 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (4 mL) and trifluoroacetic acid (2 mL) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (70 mg, yield: 81.2%). MS m / z (ESI): 1290.7 [M+H] + .

[0325] Intermediate Preparation 25: Preparation of Compound Int25 [ka]

[0326] Step 1: Preparation of compound Int25-1 (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (37.29 mg, 87.65 μmol), N,N-diisopropylethylamine (14.16 mg, 109.56 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (38.88 mg, 102.25 μmol) and compound Int15 (55 mg, 73.04 μmol) were added sequentially to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (80 mg, yield: 99.4%). MS m / z (ESI): 1160.6 [M+H] + .

[0327] Step 2: Preparation of compound Int25-2 Compound Int25-1 (50 mg, 43.09 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (3 mL) and trifluoroacetic acid (1.5 mL) and reacted for 4 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (45 mg, yield: 62.2%). MS m / z (ESI): 1104.6 [M+H] + .

[0328] Step 3: Preparation of compound Int25-3 Compound Int25-2 (45 mg, 40.74 μmol), N,N-diisopropylethylamine (7.58 mg, 58.07 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (20.81 mg, 54.19 μmol) and VC-PABC-MMAE (45.78 mg, 40.74 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 30%) to obtain the title compound of this step (30 mg, yield: 35.1%). MS m / z (ESI): 1105.2 [M / 2+H] + .

[0329] Step 4: Preparation of compound Int25 Compound Int25-3 (30 mg, 13.44 μmol) and diethylamine (4.58 mg, 53.76 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (25 mg, yield: 93.6%). MS m / z (ESI): 994.1 [M / 2+H] + .

[0330] Intermediate Preparation 26: Preparation of Compound Int26 [ka]

[0331] Step 1: Preparation of compound Int26-1 N-tert-butoxycarbonyl-hepta(ethylene glycol)-carboxylic acid (450 mg, 895.34 μmol), N,N-diisopropylethylamine (466.66 mg, 3.58 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (412.4 mg, 1.07 mmol) and compound Int23 (419.69 mg, 895.34 μmol) were added sequentially to anhydrous N,N-dimethylformamide (6 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 55% to 65%) to obtain the title compound of this step (800 mg, yield: 98.5%). MS m / z (ESI): 907.5 [M+H] + .

[0332] Step 2: Preparation of compound Int26-2 Compound Int26-1 (800 mg, 881.87 μmol) was dissolved in dioxane hydrochloride solution (6 mL, 4 M) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (610 mg, yield: 90.2%). MS m / z (ESI): 807.5 [M+H] + .

[0333] Step 3: Preparation of compound Int26-3 (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (276.97 mg, 644.45 μmol), N,N-diisopropylethylamine (209.93 mg, 1610 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (296.84 mg, 773.34 μmol) and compound Int26-2 (604 mg, 644.45 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0° C. for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (747.9 mg, yield: 95.6%). MS m / z (ESI): 1214.6 [M+H] + .

[0334] Step 4: Preparation of compound Int26-4 Compound Int26-3 (460 mg, 378.75 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (3 mL) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (400 mg, yield: 91.2%). MS m / z (ESI): 1158.6 [M+H] + .

[0335] Step 5: Preparation of compound Int26-5 Compound Int26-4 (113.74 mg, 91.19 μmol), N,N-diisopropylethylamine (23.24 mg, 178.03 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (51.28 mg, 133.52 μmol) and VC-PABC-MMAE (95 mg, 84.56 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 55% to 65%) to obtain the title compound of this step (170 mg, yield: 93.5%). MS m / z (ESI): 1132.2 [M / 2+H] + .

[0336] Step 6: Preparation of compound Int26 Compound Int26-5 (170 mg, 75.09 μmol) and diethylamine (27.74 mg, 375.47 μmol) were added to N,N-dimethylformamide (4 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=55%-65%) to obtain the title compound of this step (110 mg, yield: 71.9%). MS m / z (ESI): 1021.2 [M / 2+H] + .

[0337] Intermediate Preparation 27: Preparation of Compound Int27 [ka]

[0338] Step 1: Preparation of compound Int27-1 Compound Int26-4 (440 mg, 379.83 μmol), N,N-diisopropylethylamine (98.18 mg, 759.66 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (173.31 mg, 455.8 μmol) and GGFG-Dxd (319.38 mg, 379.83 μmol) were added to anhydrous N,N-dimethylformamide (6 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (250 mg, yield: 35.0%). MS m / z (ESI): 991.2 [M / 2+H] + .

[0339] Step 2: Preparation of compound Int27 Compound Int27-1 (205 mg, 103.47 μmol) and diethylamine (36.31 mg, 491.49 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (160 mg, yield: 93.1%). MS m / z (ESI): 586.9 [M / 3+H] + .

[0340] Intermediate Preparation 28: Preparation of Compound Int28 [ka]

[0341] Step 1: Preparation of compound Int28-2 Hepta(ethylene glycol) monomethyl ether (1 g, 2.91 mmol) and Dess-Martin oxidant (1.87 g, 4.36 mmol) were added to dichloromethane (15 mL) and reacted at 25° C. for 10 hours. Saturated sodium thiosulfate solution (5 mL) and saturated aqueous sodium bicarbonate solution (10 mL) were added to the reaction solution, stirred until clear, then extracted with dichloromethane three times, washed with saturated brine, dehydrated with anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The obtained crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol=15 / 1 (v / v)) to obtain the title compound of this step (890 mg, yield: 90.4%). MS m / z (ESI): 339.2 [M+H] + .

[0342] Step 2: Preparation of compound Int28 Compound Int28-2 (600 mg, 1.78 mmol), sodium cyanoborohydride (143.5 mg, 2.26 mmol) and glacial acetic acid (137.13 mg, 2.26 mmol) were added to a mixed solvent of 1,2-dichloroethane (6 mL) and methanol (4 mL) and stirred uniformly at 25° C., and then 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (592.33 mg, 1.74 mmol) was dissolved in 1,2-dichloroethane (6 mL) and added dropwise to the above system within 3 hours by using a constant pressure dropping funnel. The filtrate was concentrated. The obtained crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol=17 / 3 (v / v)) to obtain the title compound of this step (650 mg, yield: 65.2%). MS m / z (ESI): 662.3 [M+H] + .

[0343] Intermediate Preparation 29: Preparation of Compound Int29 [ka]

[0344] Step 1: Preparation of compound Int29-1 (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (496.25 mg, 1.17 mmol), N,N-diisopropylethylamine (188.43 mg, 1.39 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (554.36 mg, 1.39 mmol) and compound Int28 (650 mg, 982.14 μmol) were added to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (650 mg, yield: 65.8%). MS m / z (ESI): 1069.6 [M+H] + .

[0345] Step 2: Preparation of compound Int29-2 Compound Int29-1 (235.79 mg, 220.51 μmol) was dissolved in dioxane hydrochloride solution (5 mL, 4 M) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (180 mg, yield: 84.8%). MS m / z (ESI): 1013.5 [M+H] + .

[0346] Step 3: Preparation of compound Int29-3 Compound Int29-2 (151.82 mg, 149.85 μmol), N,N-diisopropylethylamine (25.18 mg, 194.8 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (62.67 mg, 164.83 μmol) and GGFG-Dxd (126 mg, 149.85 μmol) were added to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (160 mg, yield: 61.2%). MS m / z (ESI): 1835.8 [M+H] + .

[0347] Step 4: Preparation of compound Int29 Compound Int29-3 (160 mg, 87.16 μmol) and diethylamine (63.74 mg, 871.6 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (145 mg, yield: 596.7%). MS m / z (ESI): 1613.8 [M+H] + .

[0348] Intermediate Preparation 30: Preparation of Compound Int30 [ka]

[0349] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (400 mg, 930.73 μmol), N,N-diisopropylethylamine (243 mg, 1.86 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (500.45 mg, 1.3 mmol) and propargylamine (77.67 mg, 1.4 mmol) were added to anhydrous N,N-dimethylformamide (4 mL) and reacted at 25 ° C. for 3 hours. The crude product obtained by concentrating the reaction solution was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 2 (v / v)) to obtain the title compound of this step (420 mg, yield: 97.7%). MS m / z (ESI): 463.2 [M+H] + .

[0350] Intermediate Preparation 31: Preparation of Compound Int31 [ka]

[0351] Step 1: Preparation of compound Int31-1 4-Hydroxymethyl-1-cyclohexanecarboxylic acid (125.94 mg, 788.14 μmol), N,N-diisopropylethylamine (292.42 mg, 2.26 mmol, 373.94 μL), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (315.45 mg, 788.14 μmol) and compound Int23 (350 mg, 754.2 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and reacted at 25 ° C for 3 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (380 mg, yield: 93.4%). MS m / z (ESI): 568.3 [M+H] + .

[0352] Step 2: Preparation of compound Int31-2 Compound Int31-1 (325 mg, 503.19 μmol) was dissolved in anhydrous pyridine (5 mL), and methylsulfonyl chloride (65.43 mg, 1006.39 μmol) was added in batches within 1 h. After addition, the reaction was carried out at 25° C. for 3 h. The reaction solution was concentrated, then diluted with N,N-dimethylformamide (4 mL), and directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=60%-70%) to obtain the title compound of this step (325 mg, yield: 82.5%). MS m / z (ESI): 646.3 [M+H] + .

[0353] Step 3: Preparation of compound Int31-3 Compound Int31-2 (325 mg, 503.19 μmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL), and then sodium azide (65.43 mg, 1006.39 μmol) was added and reacted at 80° C. for 4 hours. Water (20 mL) was added to the reaction solution, extracted with ethyl acetate (20 mL×3), washed with saturated brine, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the title compound of this step (260 mg, yield: 91.9%). MS m / z (ESI): 593.3 [M+H] + .

[0354] Step 4: Preparation of compound Int31-4 Compound Int31-3 (230.69 mg, 389.16 μmol), compound Int30 (180 mg, 389.16 μmol), sodium ascorbate (154.19 mg, 778.32 μmol) and anhydrous copper sulfate (291.5 mg, 1170 μmol) were added to a mixed solvent of water (0.25 mL), dimethyl sulfoxide (2 mL) and tetrahydrofuran (1 mL) and reacted at 0 ° C. for 3 hours. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (405 mg, yield: 98.8%). MS m / z (ESI): 1055.5 [M+H] + .

[0355] Step 5: Preparation of compound Int31-5 Compound Int31-4 (190 mg, 180.04 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (2 mL) and reacted for 2 hours at 25° C. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (preparation method B, elution gradient: A%=20%-30%) to obtain the title compound of this step (150 mg, yield: 84.7%). MS m / z (ESI): 999.6 [M+H] + .

[0356] Step 6: Preparation of compound Int31-6 Compound Int31-5 (24.77 mg, 24.79 μmol), N,N-diisopropylethylamine (4.16 mg, 32.22 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.31 mg, 29.75 μmol) and compound Int29 (40 mg, 24.79 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (50 mg, yield: 81.8%). MS m / z (ESI): 1297.6 [M / 2+H] + .

[0357] Step 7: Preparation of compound Int31 Compound Int31-6 (50 mg, 19.26 μmol) and diethylamine (6.76 mg, 91.52 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (25 mg, yield: 57.6%). MS m / z (ESI): 1186.6 [M / 2+H] + .

[0358] Intermediate Preparation 32: Preparation of Compound Int32 [ka]

[0359] Step 1: Preparation of compound Int32-2 Compound Int32-1 (75 mg, 138.75 μmol), N,N-diisopropylethylamine (26.9 mg, 208.12 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (65.58 mg, 180.36 μmol) and exatecan mesylate (73.75 mg, 138.75 μmol) were added to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (115 mg, yield: 91.1%). MS m / z (ESI): 958.3 [M+H] + .

[0360] Step 2: Preparation of compound Int32 Compound Int32-2 (115 mg, 120.04 μmol) and diethylamine (42.13 mg, 570.2 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=20%-30%) to obtain the title compound of this step (75 mg, yield: 89.4%). MS m / z (ESI): 736.3 [M+H] + .

[0361] Intermediate Preparation 33: Preparation of Compound Int33 [ka]

[0362] Step 1: Preparation of compound Int33-1 Compound Int26-4 (75.54 mg, 61.78 μmol), N,N-diisopropylethylamine (15.97 mg, 123.56 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (30.54 mg, 80.32 μmol) and compound Int32 (50 mg, 67.96 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (85 mg, yield: 77.2%). MS m / z (ESI): 1875.9 [M+H] + .

[0363] Step 2: Preparation of compound Int33 Compound Int33-1 (85 mg, 45.3 μmol) and diethylamine (15.9 mg, 215.2 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=45%-55%) to obtain the title compound of this step (70 mg, yield: 98.3%). MS m / z (ESI): 1653.8 [M+H] + .

[0364] Intermediate Preparation 34: Preparation of Compound Int34 [ka]

[0365] Step 1: Preparation of compound Int34-2 Compound Int34-1 (800 mg, 1.67 mmol) and piperidine (287.11 mg, 3.34 mmol) were added to N,N-dimethylformamide (15 mL) and reacted at room temperature for 2 hours. After concentrating the reaction solution, methyl tert-butyl ether (20 mL) was added, sonicated for 5 minutes, allowed to stand, filtered, and the solid was dried to obtain the title compound of this step (315 mg, yield: 73.47%). MS m / z (ESI): 253.1 [M+H] + .

[0366] Step 2: Preparation of compound Int34-3 Compound Int34-2 (90mg, 321.09μmol), N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6-(tert-Butoxycarbonyl)-L-lysine (202.53 mg, 321.09 μmol), N,N-diisopropylethylamine (104.60 mg, 802.72 μmol), 1-hydroxybenzotriazole (49.23 mg, 353.20 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (77.43 mg, 401.36 μmol) were added to N,N-dimethylformamide (3 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50% to 55%) to obtain the title compound of this step (142 mg, yield: 47.43%). MS m / z (ESI): 802.4 [M+H] + .

[0367] Step 3: Preparation of compound Int34-4 Compound Int34-3 (142 mg, 159.37 μmol), ammonium formate (50.75 mg, 796.84 μmol) and 10% palladium on carbon (15 mg) were added to anhydrous methanol (4 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reversed-phase column chromatography (preparation method B, elution gradient: A%=30%-35%) to obtain the title compound of this step (101 mg, yield: 80.13%). MS m / z (ESI): 712.4 [M+H] + .

[0368] Step 4: Preparation of compound Int34-5 Compound Int34-4 (95 mg, 133.46 μmol), exatecan mesylate (70.94 mg, 133.46 μmol), N,N-diisopropylethylamine (43.04 mg, 333.66 μmol) and bis(2-oxo-3-oxazolidinyl)phosphonic acid chloride (50.85 mg, 200.20 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 45% to 55%) to obtain the title compound of this step (105 mg, yield: 69.67%). MS m / z (ESI): 1129.5 [M+H] + .

[0369] Step 5: Preparation of compound Int34 Compound Int34-5 (105 mg, 92.98 μmol) and piperidine (15.83 mg, 185.97 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A% = 27% to 33%) to obtain the title compound of this step (46 mg, yield: 54.54%). MS m / z (ESI): 907.4 [M+H] + .

[0370] Intermediate Preparation 35: Preparation of Compound Int35 [ka]

[0371] Step 1: Preparation of compound Int35-2 (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-D-alanine (537.38 mg, 1.33 mmol), N,N-diisopropylethylamine (345.07 mg, 3.67 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (641.18 mg, 16.84 mmol) and compound Int35-1 (300 mg, 1.33 mmol) were added to anhydrous N,N-dimethylformamide (6 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 45% to 55%) to obtain the title compound of this step (400 mg, yield: 52.9%). MS m / z (ESI): 567.3 [M+H] + .

[0372] Step 2: Preparation of compound Int35-3 Compound Int35-2 (400 mg, 635.32 μmol) was dissolved in dioxane hydrochloride solution (4 mL, 4 M) and reacted for 3 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (310 mg, yield: 95.6%). MS m / z (ESI): 511.2 [M+H] + .

[0373] Step 3: Preparation of compound Int35-4 Under nitrogen protection, compound Int35-3 (350 mg, 651.27 μmol), lead tetraacetate (437.51 mg, 976.91 μmol), copper acetate (13.13 mg, 64.13 μmol) and glacial acetic acid (59.26 mg, 976.91 μmol) were added sequentially to anhydrous N,N-dimethylformamide (6 mL) and reacted at 60 ° C for 0.5 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 45% to 55%) to obtain the title compound of this step (285 mg, yield: 83.4%). MS m / z (ESI): 547.2 [M+Na] + .

[0374] Step 4: Preparation of compound Int35-5 Compound Int35-4 (280 mg, 533.76 μmol) and benzyl glycolate (373.47 mg, 2.14 mmol) were added to a mixed solvent of dichloromethane (6 mL) and trifluoroacetic acid (2 mL) and reacted for 0.5 hours at 25° C. The crude product obtained by concentrating the reaction solution was purified by silica gel column chromatography (eluent: dichloromethane / methanol=7 / 3 (v / v)) to obtain the title compound of this step (30 mg, yield: 98.1%). MS m / z (ESI): 653.3 [M+Na] + .

[0375] Step 5: Preparation of compound Int35-6 Compound Int35-5 (160 mg, 237.84 μmol), 10% palladium on carbon (28.89 mg) and ammonium acetate (89.99 mg, 1430 μmol) were added to methanol (6 mL) and reacted for 3 hours at 25° C. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to give the title compound of this step (30 mg, yield: 23.3%). MS m / z (ESI): 541.2 [M+H] + .

[0376] Step 6: Preparation of compound Int35-7 Compound Int35-6 (30 mg, 55.49 μmol), N,N-diisopropylethylamine (11.09 mg, 85.84 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (22.85 mg, 60.08 μmol) and exatecan mesylate (32.45 mg, 138.75 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 45% to 55%) to obtain the title compound of this step (45 mg, yield: 89.1%). MS m / z (ESI): 958.4 [M+H] + .

[0377] Step 7: Preparation of compound Int35 Compound Int35-7 (39.8 mg, 41.55 μmol) and diethylamine (15.35 mg, 207.74 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=55%-65%) to obtain the title compound of this step (30 mg, yield: 98.1%). MS m / z (ESI): 736.2 [M+H] + .

[0378] Example 1-1: Preparation of compound C-1 [ka]

[0379] Compound Int5 (50 mg, 0.02 mmol) was dissolved in DMSO (6 mL) and water (0.6 mL), and 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(prop-2-yn-1-yl)hexanamide (5.99 mg, 0.024 mmol), sodium ascorbate (5.94 mg, 0.03 mmol), and anhydrous copper sulfate (6.4 mg, 0.04 mmol) were added under nitrogen protection, and the mixture was stirred at room temperature for 1 hour, and extracted with water and ethyl acetate. The organic phase was dried and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography to obtain the title compound of this step (15 mg, yield: 27.2%). MS m / z (ESI): 1365.7 [M / 2+H] + .

[0380] Example-2: Preparation of compound C-2 [ka]

[0381] The synthetic route of Example 1-1 was adopted. The reaction raw material 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(prop-2-yn-1-yl)hexanamide was replaced with 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide to obtain the title compound of this step (3 mg, yield: 5.6%). MS m / z (ESI): 1394.2 [M / 2+H] + .

[0382] Example 1-3: Preparation of compound C-3 [ka]

[0383] The synthetic route of Example 1-1 was adopted. The raw material compound Int5 was replaced with compound Int6, and 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(prop-2-yn-1-yl)hexanamide was replaced with 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hexa-5-ynamide, to obtain the title compound of this step (5 mg, yield: 8%). MS m / z (ESI): 1583.8 [M / 2+H] + .

[0384] Example 1-4: Preparation of compound C-4 [ka]

[0385] The synthetic route of Example 1-1 was adopted, and the raw material compound Int5 was replaced with compound Int6 to obtain the title compound of this step (10 mg, yield: 6%). MS m / z (ESI): 1555.3 [M / 2+H] + .

[0386] Examples 1-5: Preparation of Compound C-5 [ka]

[0387] Step 1: Preparation of compound 5-1 The synthetic route of compound Int5 was adopted, and the reactant MC-VC-PABC-MMAE was replaced with MC-GGFG-Dxd to obtain the title compound of this step (30 mg, yield: 21.3%). MS m / z (ESI): 1100.5 [M / 2+H] + .

[0388] Step 2: Preparation of compound C-5 Compound 5-1 (30 mg, 0.014 mmol) was dissolved in DMSO (6 mL) and water (0.3 mL), and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hexa-5-ynamide (4.99 mg, 0.016 mmol), sodium ascorbate (4.16 mg, 0.021 mmol), and anhydrous copper sulfate (4.5 mg, 0.028 mmol) were added under nitrogen protection, and the mixture was stirred at room temperature for 1 hour. Water and ethyl acetate were added for extraction, and the organic phase was dried and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography to obtain the title compound of this step (3 mg, yield: 8.8%). MS m / z (ESI): 1252.0 [M / 2+H] + .

[0389] Examples 1-6: Preparation of Compound C-6 [ka]

[0390] Compound Int11 (20 mg, 7.74 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (2.17 mg, 7.74 μmol) and N,N-diisopropylethylamine (1.52 mg, 11.61 μmol) were added to anhydrous N,N-dimethylformamide (1.5 mL) in that order and reacted for 3 hours at 25° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (0.64 mg, yield: 3%). MS m / z (ESI): 1303.3 [M / 2+H] + .

[0391] Examples 1-7: Preparation of Compound C-7 [ka] TIFF2025513163000172.tif224149 TIFF2025513163000173.tif90149

[0392] Step 1: Preparation of compound 7-1 Compound Int12 (115 mg, 102.07 μmol) and compound Int7 (135.06 mg, 153.11 μmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL), N,N-diisopropylethylamine (27.77 mg, 204.14 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (61.29 mg, 153.11 μmol) were added in sequence, and the mixture was reacted at 0 ° C for 1 hour. The crude product obtained by concentrating the reaction solution was purified by reverse phase column chromatography (preparation method A, elution gradient: A% = 40% to 50%) to obtain the title compound of this step (125 mg, yield: 58.3%). MS m / z (ESI): 945.6 [M / 2+H] + .

[0393] Step 2: Preparation of compound 7-2 Compound 7-1 (125 mg, 59.51 μmol) was dissolved in a solution of hydrogen chloride in 1,4-dioxane (2 mL, 4 M) under nitrogen protection and reacted for 2 hours at 25° C. The residue obtained by concentrating the reaction solution was dissolved in dichloromethane (15 mL) and concentrated again under reduced pressure to remove the solvent, which was repeated three times to obtain the title compound of this step (140 mg, yield: 90.4%). MS m / z (ESI): 895.6 [M / 2+H] + .

[0394] Step 3: Preparation of compound 7-3 Compound 7-2 (95 mg, 50.41 μmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (135.06 mg, 153.11 μmol), N,N-diisopropylethylamine (13.72 mg, 100.82 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (30.27 mg, 75.62 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A% = 40% to 50%) to obtain the title compound of this step (120 mg, yield: 97.5%). MS m / z (ESI): 1099.0 [M / 2+H] + .

[0395] Step 4: Preparation of compound 7-4 Compound 7-3 (110 mg, 45.05 μmol) and trifluoroacetic acid (2 mL) were dissolved in dichloromethane (6 mL) and reacted for 2 hours at 25° C. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (76 mg, yield: 74.8%). MS m / z (ESI): 1071.1 [M / 2+H] + .

[0396] Step 5: Preparation of compound 7-5 Compound 7-4 (60 mg, 26.62 μmol), VC-PABC-MMAE (43.19 mg, 34.60 μmol), N,N-diisopropylethylamine (5.21 mg, 39.92 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (14.31 mg, 37.26 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted at 0 ° C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (65 mg, yield: 71.5%). MS m / z (ESI): 1623.5 [M / 2+H] + .

[0397] Step 6: Preparation of compound 7-6 Compound 7-5 (65 mg, 19.02 μmol) and piperidine (3.27 mg, 129.16 μmol) were added sequentially to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A%=25%-35%) to obtain the title compound of this step (55.6 mg, yield: 91.8%). MS m / z (ESI): 1008.6 [M / 3+H] + .

[0398] Step 7: Preparation of compound C-7 Compound 7-6 (58 mg, 18.22 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (5.10 mg, 18.22 μmol) and N,N-diisopropylethylamine (3.57 mg, 27.32 μmol) were dissolved in anhydrous N,N-dimethylformamide (1.5 mL) and reacted for 3 hours at 25° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (28.33 mg, yield: 46.5%). MS m / z (ESI): 1587.9 [M / 2+H] + .

[0399] Examples 1-8: Preparation of Compound C-8 [ka] TIFF2025513163000175.tif226149

[0400] Step 1: Preparation of compound 8-2 Compound Int7 (300 mg, 340.08 μmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (228.47 mg, 425.47 μmol), N,N-diisopropylethylamine (92.53 mg, 680.17 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (204.22 mg, 510.13 μmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL) in order and reacted at 25° C. for 2 hours. The reaction solution was concentrated. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=9 / 1 (v / v)) to give the title compound of this step (371 mg, yield: 78.8%). MS m / z (ESI): 1245.7 [M+H] + .

[0401] Step 2: Preparation of compound 8-3 Compound 8-2 (350 mg, 252.91 μmol) and trifluoroacetic acid (2 mL) were added to dichloromethane (6 mL) and reacted for 2 hours at 25° C. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (preparation method A, elution gradient: A%=35%-45%) to obtain the title compound of this step (250 mg, yield: 74.8%). MS m / z (ESI): 1189.6 [M+H] + .

[0402] Step 3: Preparation of compound 8-4 Compound 8-3 (105 mg, 83.87 μmol), VC-PABC-MMAE (110.85 mg, 83.87 μmol), N,N-diisopropylethylamine (19.71 mg, 150.96 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (45.11 mg, 117.44 μmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A% = 45% to 55%) to obtain the title compound of this step (170 mg, yield: 79.5%). MS m / z (ESI): 1147.7 [M / 2+H] + .

[0403] Step 4: Preparation of compound 8-5 Compound 8-4 (156 mg, 64.58 μmol) and piperidine (11.11 mg, 129.16 μmol) were added to N,N-dimethylformamide (3 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A%=35%-45%) to obtain the title compound of this step (125 mg, yield: 88.7%). MS m / z (ESI): 1036.7 [M / 2+H] + .

[0404] Step 5: Preparation of compound 8-6 Compound 8-5 (120.05 mg, 55.03 μmol), compound Int12 (62 mg, 55.03 μmol), N,N-diisopropylethylamine (10.78 mg, 82.54 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (25.36 mg, 66.02 μmol) were added in sequence, dissolved in anhydrous N,N-dimethylformamide (4 mL), and reacted at 0 ° C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (170 mg, yield: 89.0%). MS m / z (ESI): 1562.6 [M / 2+H] + .

[0405] Step 6: Preparation of compound 8-7 Compound 8-6 (160 mg, 48.64 μmol) and zinc bromide (46.12 mg, 194.67 μmol) were added to anhydrous dichloromethane (2 mL) under nitrogen protection and reacted for 2 hours at 25° C. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (preparation method B, elution gradient: A%=30%-40%) to obtain the title compound of this step (140 mg, yield: 90.4%). MS m / z (ESI): 1512.4 [M / 2+H] + .

[0406] Step 7: Preparation of compound C-8 Compound 8-7 (80 mg, 125.13 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (7.04 mg, 25.13 μmol) and N,N-diisopropylethylamine (4.92 mg, 37.69 μmol, 6.29 μL) were added to anhydrous N,N-dimethylformamide (1.5 mL) in sequence and reacted for 3 hours at 25° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (19.4 mg, yield: 23.1%). MS m / z (ESI): 1587.9 [M / 2+H] + .

[0407] Example 1-9: Preparation of Compound C-9 [ka]

[0408] Compound Int10 (50 mg, 24.24 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (20.38 mg, 72.72 μmol) and N,N-diisopropylethylamine (4.75 mg, 36.36 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) in sequence and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (11 mg, yield: 20.2%). MS m / z (ESI): 1055.5 [M / 2+H] + .

[0409] Examples 1-10: Preparation of Compound C-10 [ka]

[0410] Compound Int10 (60 mg, 29.09 μmol), 6-(maleimido)hexanoic acid succinimide ester (9.44 mg, 09.09 μmol) and N,N-diisopropylethylamine (45.70 mg, 43.63 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (11 mg, yield: 16.3%). MS m / z (ESI): 1076.6 [M / 2+H] + .

[0411] Examples 1-11: Preparation of Compound C-11 [ka]

[0412] Compound Int10 (60 mg, 29.09 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynolate (22.38 mg, 58.18 μmol), N,N-diisopropylethylamine (5.7 mg, 43.63 μmol), and 1-hydroxybenzotrizoate (5.09 mg, 14.55 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 25° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (4 mg, yield: 5.8%). MS m / z (ESI): 1105.1 [M / 2+H] + .

[0413] Examples 1-12: Preparation of Compound C-14 [ka]

[0414] Step 1: Preparation of compound C-14 Compound Int16 (22 mg, 10.32 μmol), 6-(maleimido)hexanoic acid succinimide ester (4.82 mg, 15.48 μmol) and N,N-diisopropylethylamine (3.36 mg, 25.80 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 30%) to obtain the title compound of this step (12.73 mg, yield: 54.31%). MS m / z (ESI): 1056.1 [M / 2+H] + .

[0415] Examples 1-13: Preparation of Compound C-15 [ka]

[0416] Step 1: Preparation of compound 15-1 Compound Int12 (50 mg, 42.04 μmol), VC-PABC-MMAE (52.48 mg, 42.04 μmol), N,N-diisopropylethylamine (13.70 mg, 105.11 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (19.37 mg, 50.45 μmol) were added sequentially to N,N-dimethylformamide (1.5 mL) and reacted at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 45% to 55%) to obtain the title compound of this step (41 mg, yield: 40.34%). MS m / z (ESI): 1088.2 [M / 2+H] + .

[0417] Step 2: Preparation of compound 15-2 Compound 15-1 (38 mg, 15.72 μmol) and anhydrous zinc bromide (17.88 mg, 78.59 μmol) were added to anhydrous dichloromethane (3 mL) and reacted at room temperature for 5 hours. The crude product obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 20% to 30%) to obtain the title compound of this step (30 mg, yield: 82.76%). MS m / z (ESI): 1038.1 [M / 2+H] + .

[0418] Step 3: Preparation of compound C-15 Compound 15-2 (30 mg, 13.01 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (5.25 mg, 19.51 μmol) and N,N-diisopropylethylamine (2.54 mg, 19.51 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and reacted at room temperature for 1.5 hours. The reaction solution was directly purified by reversed-phase column chromatography (preparation method B, elution gradient: A% = 25% to 40%) to obtain the title compound of this step (9.81 mg, yield: 31.52%). MS m / z (ESI): 1113.7 [M / 2+H]+ .

[0419] Examples 1-14: Preparation of Compound C-16 [ka]

[0420] Compound Int25 (25 mg, 12.58 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (10.05 mg, 37.74 μmol) and N,N-diisopropylethylamine (2.34 mg, 17.92 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (8 mg, yield: 31.3%). MS m / z (ESI): 1069.6 [M / 2+H] + .

[0421] Examples 1-15: Preparation of Compound C-17 [ka]

[0422] Step 1: Preparation of compound 17-2 VC-PABC-MMAE (60mg, 53.41μmol), N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-N 6-(tert-Butoxycarbonyl)-L-lysine (25 mg, 53.41 μmol), N,N-diisopropylethylamine (17.2 mg, 133.52 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (24.35 mg, 64.09 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and reacted at room temperature for 1 hour. The reaction was poured into ice water (10 mL) and extracted with ethyl acetate (5 mL x 3). The organic phases were combined, backwashed with saturated brine (3 mL x 2), dried over anhydrous sodium sulfate and concentrated to give the title compound of this step (82 mg, crude product). MS m / z (ESI): 1574.0 [M+H] + .

[0423] Step 2: Preparation of compound 17-3 Compound 17-2 (82.00 mg, crude product) and piperidine (8.07 mg, 93.78 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and reacted at room temperature for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (41 mg, total yield for two steps: 56.75%). MS m / z (ESI): 1351.9 [M+H] + .

[0424] Step 3: Preparation of compound 17-4 Compound 17-3 (41.00 mg, 30.32 μmol), compound Int3 (25.52 mg, 30.32 μmol), N,N-diisopropylethylamine (9.78 mg, 75.81 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (17.28 mg, 45.48 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 45% to 50%) to obtain the title compound of this step (35 mg, yield: 53.06%). MS m / z (ESI): 1088.2 [M / 2+H] + .

[0425] Step 4: Preparation of compound 17-5 Compound 17-4 (35 mg, 16.09 μmol) and anhydrous zinc bromide (14.28 mg, 64.36 μmol) were added to dichloromethane (2 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25% to 30%) to obtain the title compound of this step (25 mg, yield: 74.87%). MS m / z (ESI): 1038.2 [M / 2+H] + .

[0426] Step 5: Preparation of compound C-17 Compound 17-5 (25 mg, 12.05 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (4.37 mg, 16.26 μmol) and N,N-diisopropylethylamine (3.12 mg, 24.10 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reversed-phase column chromatography (preparation method B, elution gradient: A% = 30% to 45%) to obtain the title compound of this step (11.1 mg, yield: 41.38%). MS m / z (ESI): 1113.7 [M / 2+H] + .

[0427] Examples 1-16: Preparation of Compound C-18 [ka]

[0428] Compound Int16 (16 mg, 8.34 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (4.57 mg, 12.51 μmol) and N,N-diisopropylethylamine (2.15 mg, 16.68 μmol) were added sequentially to N,N-dimethylformamide (0.5 mL) and reacted at room temperature for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 40%) to obtain the title compound of this step (2.87 mg, yield: 15.87%). MS m / z (ESI): 1084.6 [M / 2+H] + .

[0429] Examples 1-17: Preparation of Compound C-19 [ka]

[0430] Step 1: Preparation of compound 19-1 Compound Int17 (25 mg, 22.64 μmol), N,N-diisopropylethylamine (4.21 mg, 32.26 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.91 mg, 25.81 μmol) and GGFG-Dxd (20.09 mg, 22.64 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method C, elution gradient: A% = 15% to 25%) to obtain the title compound of this step (38 mg, yield: 91.7%). MS m / z (ESI): 964.0 [M / 2+H] + .

[0431] Step 2: Preparation of compound 19-2 Compound 19-1 (31 mg, 16.08 μmol) and diethylamine (6.85 mg, 80.4 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=50%-60%) to obtain the title compound of this step (20 mg, yield: 72.9%). MS m / z (ESI): 852.8 [M / 2+H] + .

[0432] Step 3: Preparation of compound C-19 Compound 19-2 (20 mg, 11.73 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (6.25 mg, 23.46 μmol) and N,N-diisopropylethylamine (2.18 mg, 16.72 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (15 mg, yield: 72.5%). MS m / z (ESI): 928.4 [M / 2+H] + .

[0433] Example 1-18: Preparation of compound C-20 [ka]

[0434] Compound Int18 (28 mg, 14.19 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (5.66 mg, 21.28 μmol) and N,N-diisopropylethylamine (2.75 mg, 21.28 μmol) were added to N,N-dimethylformamide (0.5 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 35%) to obtain the title compound of this step (7.64 mg, yield: 25.34%). MS m / z (ESI): 1062.6 [M / 2+H]+ .

[0435] Example 1-19: Preparation of Compound C-21 [ka]

[0436] Compound Int19 (23.15 mg, 11.73 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (6.25 mg, 23.46 μmol) and N,N-diisopropylethylamine (2.18 mg, 16.72 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (12 mg, yield: 50.7%). MS m / z (ESI): 1062.7 [M / 2+H] + .

[0437] Examples 1-20: Preparation of Compound C-22 [ka]

[0438] Step 1: Preparation of compound 22-2 Nona(ethylene glycol) (1.2 g, 2.9 mmol) and p-toluenesulfonyl chloride (250.52 mg, 2.9 mmol) were dissolved in anhydrous dichloromethane (20 mL), and then potassium hydroxide (324.89 mg, 5.79 mmol) was added and reacted for 10 hours at 25° C. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=30 / 1 (v / v)) to obtain the title compound of this step (1.1 g, yield: 66.7%). MS m / z (ESI): 586.4 [M+NH 4 ] + .

[0439] Step 2: Preparation of compound 22-3 Compound 22-2 (460 mg, 808.9 μmol) and N-tert-butoxycarbonyl-L-cysteine ​​ethyl ester (242 mg, 970.67 μmol) were dissolved in N,N-dimethylformamide (6 mL), and then potassium iodide (13.43 mg, 80.89 μmol) and N,N-diisopropylethylamine (150 mg, 1152.68 μmol) were added and reacted for 10 hours at 25° C. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=5 / 1 (v / v)) to obtain the title compound of this step (320 mg, yield: 64.5%). MS m / z (ESI): 646.1 [M+H] + .

[0440] Step 3: Preparation of compound 22-4 Compound 22-3 (220 mg, 337.26 μmol) and Dess-Martin oxidant (216.66 mg, 505.89 μmol) were added to dichloromethane (15 mL) and reacted at 25° C. for 10 hours. Saturated sodium thiosulfate solution (5 mL) and saturated aqueous sodium bicarbonate solution (10 mL) were added to the reaction solution, stirred until clear, extracted with dichloromethane three times, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol=20 / 1 (v / v)) to obtain the title compound of this step (185 mg, yield: 85.2%). MS m / z (ESI): 644.3 [M+H] + .

[0441] Step 4: Preparation of compound 22-5 Compound 22-4 (146.31 mg, 227.26 μmol), compound Int13 (65 mg, 181.81 μmol), sodium cyanoborohydride (31.56 mg, 502.2 mmol) and glacial acetic acid (31.4 mg, 518.16 mmol) were added in sequence to a mixed solvent of 1,2-dichloroethane (4 mL) and methanol (2 mL), and reacted at 25° C. for 4 hours. The crude product obtained by concentrating the reaction solution was dissolved in methanol (3 mL) and purified by reverse phase column chromatography (preparation method B, elution gradient: A%=20%-30%) to obtain the title compound of this step (109 mg, yield: 63.8%). MS m / z (ESI): 985.5 [M+H] + .

[0442] Step 5: Preparation of compound 22-6 Compound 22-5 (100 mg, 101.49 μmol) was dissolved in methanol (4 mL), and lithium hydroxide aqueous solution (21.31 mg, 507.46 μmol, 1 mL) was added and reacted for 4 hours at 25 ° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (85 mg, yield: 87.5%). MS m / z (ESI): 957.5 [M+H] + .

[0443] Step 6: Preparation of compound 22-7 Compound 22-6 (65 mg, 67.91 μmol), N,N-diisopropylethylamine (12.63 mg, 96.76 μmol, 16.15 μL), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (34.69 mg, 90.31 μmol) and VC-PABC-MMAE (76.28 mg, 67.91 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 30% to 45%) to obtain the title compound of this step (85 mg, yield: 63.9%). MS m / z (ESI): 1031.6 [M / 2+H] + .

[0444] Step 7: Preparation of compound 22-8 Compound 22-7 (38 mg, 18.42 μmol) and zinc bromide (31.85 mg, 140.01 μmol) were added to anhydrous dichloromethane (2 mL) and reacted for 4 hours at 25° C. The crude product obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=20%-30%) to obtain the title compound of this step (30 mg, yield: 87.0%). MS m / z (ESI): 981.5 [M / 2+H] + .

[0445] Step 8: Preparation of compound C-22 Compound 22-8 (30 mg, 15.28 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (8.94 mg, 24.46 μmol) and N,N-diisopropylethylamine (2.84 mg, 21.75 μmol, 3.64 μL) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (20 mg, yield: 62.2%). MS m / z (ESI): 1106.6 [M / 2+H] + .

[0446] Examples 1-21: Preparation of Compound C-23 [ka]

[0447] Step 1: Preparation of compound 23-1 Compound Int17 (51 mg, 46.19 μmol), VC-PABC-MMAE (51.88 mg, 46.19 μmol), N,N-diisopropylethylamine (24.37 mg, 187.03 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (19.14 mg, 49.88 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and reacted at room temperature for 1.5 hours. The reaction was poured into ice water (10 mL) and extracted with ethyl acetate (5 mL x 3). The organic phases were combined, backwashed with saturated brine (3 mL x 2), dried over anhydrous sodium sulfate and concentrated to give the title compound of this step (86 mg, yield: 84.27%). MS m / z (ESI): 1105.2 [M / 2+H] + .

[0448] Step 2: Preparation of compound 23-2 Compound 23-1 (86 mg, 38.93 μmol) and piperidine (5.36 mg, 62.27 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=20%-25%) to obtain the title compound of this step (47 mg, yield: 60.79%). MS m / z (ESI): 1987.2 [M+H] + .

[0449] Step 3: Preparation of compound C-23 Compound 23-2 (47 mg, 23.66 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (12.99 mg, 35.49 μmol) and N,N-diisopropylethylamine (5.55 mg, 42.57 μmol) were added to N,N-dimethylformamide (0.5 mL) in sequence and reacted at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 35%) to obtain the title compound of this step (37.38 mg, yield: 70.60%). MS m / z (ESI): 1119.1 [M / 2+H] + .

[0450] Examples 1-22: Preparation of Compound C-24 [ka]

[0451] Compound Int25 (28.80 mg, 14.49 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.88 mg, 14.49 μmol) and N,N-diisopropylethylamine (3.78 mg, 28.98 μmol) were added sequentially to N,N-dimethylformamide (0.5 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 35%) to obtain the title compound of this step (13.57 mg, yield: 41.83%). MS m / z (ESI): 1119.1 [M / 2+H] + .

[0452] Examples 1-23: Preparation of Compound C-25 [ka]

[0453] Compound Int19 (24.7 mg, 12.52 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.08 mg, 12.52 μmol) and N,N-diisopropylethylamine (3.26 mg, 25.03 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 35%) to obtain the title compound of this step (17.16 mg, yield: 61.66%). MS m / z (ESI): 1112.1 [M / 2+H] + .

[0454] Examples 1-24: Preparation of Compound C-26 [ka]

[0455] Step 1: Preparation of compound 26-1 Compound Int20 (87 mg, 55.27 μmol) and zinc bromide (95.56 mg, 420.09 μmol) were added to anhydrous dichloromethane (3 mL) and reacted for 3 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (61 mg, yield: 78.8%). MS m / z (ESI): 1473.8 [M+H] + .

[0456] Step 2: Preparation of compound 26-2 Compound Int14 (21.25 mg, 24.42 μmol), N,N-diisopropylethylamine (4.54 mg, 34.81 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (12.48 mg, 32.49 μmol) and compound 26-1 (36 mg, 24.42 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 35%) to obtain the title compound of this step (45 mg, yield: 83.4%). MS m / z (ESI): 1163.2 [M / 2+H] + .

[0457] Step 3: Preparation of compound 26-3 Compound 26-2 (40 mg, 17.03 μmol) and diethylamine (6.29 mg, 85.13 μmol) were added to N,N-dimethylformamide (4 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reversed-phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (30 mg, yield: 83.8%). MS m / z (ESI): 1052.3 [M / 2+H] + .

[0458] Step 4: Preparation of compound C-26 Compound 26-3 (20 mg, 9.50 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.56 mg, 15.21 μmol) and N,N-diisopropylethylamine (1.77 mg, 13.67 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (8 mg, yield: 37.6%). MS m / z (ESI): 1177.2 [M / 2+H] + .

[0459] Examples 1-25: Preparation of Compound C-27 [ka]

[0460] Step 1: Preparation of compound 27-1 Compound Int20 (85 mg, 53.46 μmol) and diethylamine (19.75 mg, 267.32 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (73 mg, yield: 98.6%). MS m / z (ESI): 1351.8 [M+H] + .

[0461] Step 2: Preparation of compound 27-2 Compound Int14 (55.52 mg, 63.81 μmol), N,N-diisopropylethylamine (10.32 mg, 79.07 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (28.34 mg, 73.8 μmol) and compound 27-1 (75 mg, 55.48 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 40%) to obtain the title compound of this step (38 mg, yield: 32.7%). MS m / z (ESI): 1102.2 [M / 2+H] + .

[0462] Step 3: Preparation of compound 27-3 Compound 27-2 (38 mg, 17.24 μmol) and zinc bromide (29.81 mg, 131.05 μmol) were added to anhydrous dichloromethane (3 mL) and reacted for 3 hours at 25° C. The crude product obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=20%-30%) to obtain the title compound of this step (61 mg, yield: 78.8%). MS m / z (ESI): 701.8 [M / 3+H] + .

[0463] Step 4: Preparation of compound C-27 Compound 27-3 (20 mg, 9.50 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.56 mg, 15.21 μmol) and N,N-diisopropylethylamine (1.77 mg, 13.67 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (6 mg, yield: 28.2%). MS m / z (ESI): 1177.2 [M / 2+H] + .

[0464] Examples 1-26: Preparation of Compound C-28 [ka] TIFF2025513163000194.tif186149

[0465] Step 1: Preparation of compound 28-1 Compound Int22 (10.23 mg, 8.38 μmol), N,N-diisopropylethylamine (1.62 mg, 12.57 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.82 mg, 10.04 μmol) and compound Int21 (15 mg, 8.38 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (16 mg, yield: 67.2%). MS m / z (ESI): 1496.3 [M / 2+H] + .

[0466] Step 2: Preparation of compound 28-2 Compound 28-1 (16 mg, 5.35 μmol) and diethylamine (1.88 mg, 25.40 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=20%-35%) to obtain the title compound of this step (14 mg, yield: 99.5%). MS m / z (ESI): 1385.2 [M / 2+H] + .

[0467] Step 3: Preparation of compound C-28 Compound 28-2 (14 mg, 5.05 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (2.95 mg, 8.08 μmol) and N,N-diisopropylethylamine (0.94 mg, 7.22 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (1.61 mg, yield: 11.4%). MS m / z (ESI): 1510.3 [M / 2+H] + .

[0468] Example 1-27: Preparation of compound C-29 [ka] TIFF2025513163000196.tif166149

[0469] Step 1: Preparation of compound 29-1 Compound Int22 (16.25 mg, 13.31 μmol), N,N-diisopropylethylamine (2.06 mg, 15.81 μmol, 2.64 μL), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.26 mg, 713.71 μmol) and compound 8-5 (23 mg, 11.1 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (16 mg, yield: 46.3%). MS m / z (ESI): 1092.0 [M / 3+H] + .

[0470] Step 2: Preparation of compound 29-2 Compound 29-1 (16 mg, 4.88 μmol) and diethylamine (1.71 mg, 23.21 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (9 mg, yield: 63.5%). MS m / z (ESI): 1017.9 [M / 3+H] + .

[0471] Step 3: Preparation of compound C-29 Compound 29-2 (9 mg, 2.95 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (1.72 mg, 4.71 μmol) and N,N-diisopropylethylamine (0.55 mg, 4.2 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) in sequence and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (4.59 mg, yield: 48.9%). MS m / z (ESI): 1101.3 [M / 3+H] + .

[0472] Examples 1-28: Preparation of Compound C-30 [ka]

[0473] Compound Int18 (27.90 mg, 14.14 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.22 mg, 14.14 μmol) and N,N-diisopropylethylamine (3.68 mg, 28.27 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 35%) to obtain the title compound of this step (7.88 mg, yield: 25.07%). MS m / z (ESI): 1112.1 [M / 2+H] + .

[0474] Examples 1-29: Preparation of Compound C-31 [ka]

[0475] Step 1: Preparation of compound 31-1 Compound Int9-2 (727.61 mg, 1347.36 μmol), compound Int23 (400 mg, 853.33 μmol), sodium cyanoborohydride (99.08 mg, 1576.7 μmol) and glacial acetic acid (51.72 mg, 853.33 μmol) were added in sequence to a mixed solvent of 1,2-dichloroethane (6 mL) and methanol (3 mL), and reacted at 25° C. for 4 hours. The crude product obtained by concentrating the reaction solution was dissolved in methanol (3 mL) and purified by reverse phase column chromatography (preparation method B, elution gradient: A%=20%-35%) to obtain the title compound of this step (600 mg, yield: 76.2%). MS m / z (ESI): 923.5 [M+H] + .

[0476] Step 2: Preparation of compound 31-2 Compound 31-1 (180 mg, 194.97 μmol) was added to a dioxane hydrochloride solution (6 mL, 4 M) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (150 mg, yield: 93.3%). MS m / z (ESI): 823.6 [M+H] + .

[0477] Step 3: Preparation of compound 31-3 (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (85.29 mg, 200.46 μmol), N,N-diisopropylethylamine (45.2 mg, 346.26 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (103.94 mg, 270.22 μmol) and compound 31-2 (150 mg, 182.24 μmol) were added in sequence to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 25% to 35%) to obtain the title compound of this step (170 mg, yield: 79.8%). MS m / z (ESI): 1230.6 [M+H] + .

[0478] Step 4: Preparation of compound 31-4 Compound 31-3 (170 mg, 138.15 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (3 mL) and reacted for 2 hours at 25° C. The reaction solution was concentrated to obtain the title compound of this step (133 mg, yield: 86.3%). MS m / z (ESI): 1174.6 [M+H] + .

[0479] Step 5: Preparation of compound 31-5 Compound 31-4 (132.05 mg, 112.44 μmol), N,N-diisopropylethylamine (23.24 mg, 178.03 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (51.28 mg, 133.52 μmol) and VC-PABC-MMAE (105.26 mg, 93.69 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 55% to 65%) to obtain the title compound of this step (110 mg, yield: 54.2%). MS m / z (ESI): 1140.2 [M / 2+H] + .

[0480] Step 6: Preparation of compound 31-6 Compound 31-5 (80 mg, 35.09 μmol) and diethylamine (12.19 mg, 166.68 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=55%-65%) to obtain the title compound of this step (50 mg, yield: 72.9%). MS m / z (ESI): 686.4 [M / 3+H] + .

[0481] Step 7: Preparation of compound C-31 Compound 31-6 (50 mg, 24.31 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (7.76 mg, 29.16 μmol) and N,N-diisopropylethylamine (4.52 mg, 34.63 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) in sequence and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (21.5 mg, yield: 41.3%). MS m / z (ESI): 1104.6 [M / 2+H] + .

[0482] Examples 1-30: Preparation of Compound C-32 [ka] TIFF2025513163000200.tif170149

[0483] Step 1: Preparation of compound 32-1 Compound Int24 (31.38 mg, 22.31 μmol), N,N-diisopropylethylamine (3.77 mg, 28.88 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.61 mg, 25.03 μmol) and compound 8-5 (42 mg, 20.26 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (38 mg, yield: 59.0%). MS m / z (ESI): 1672.5 [M / 2+H] + .

[0484] Step 2: Preparation of compound 32-2 Compound 32-1 (38 mg, 11.36 μmol) and diethylamine (3.95 mg, 53.96 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (12 mg, yield: 61.2%). MS m / z (ESI): 1041.3 [M / 3+H] + .

[0485] Step 3: Preparation of compound C-32 Compound 32-2 (10 mg, 3.21 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (1.02 mg, 3.84 μmol) and N,N-diisopropylethylamine (0.6 mg, 4.56 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) in sequence and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (6.18 mg, yield: 35.6%). MS m / z (ESI): 1637.0 [M / 2+H] + .

[0486] Examples 1-31: Preparation of Compound C-33 [ka] TIFF2025513163000202.tif167149

[0487] Step 1: Preparation of compound 33-1 Compound Int24 (39.66 mg, 27.94 μmol), N,N-diisopropylethylamine (6.93 mg, 53.07 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (15.93 mg, 33.48 μmol) and compound Int21 (50 mg, 27.94 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 30%) to obtain the title compound of this step (66 mg, yield: 81.2%). MS m / z (ESI): 1531.3 [M / 2+H] + .

[0488] Step 2: Preparation of compound 33-2 Compound 33-1 (66 mg, 21.55 μmol) and diethylamine (7.56 mg, 102.37 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (22 mg, yield: 37.8%). MS m / z (ESI): 1420.3 [M / 2+H] + .

[0489] Step 3: Preparation of compound C-33 Compound 33-2 (14 mg, 4.93 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (2.88 mg, 7.88 μmol) and N,N-diisopropylethylamine (0.92 mg, 7.02 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (6.28 mg, yield: 43.4%). MS m / z (ESI): 1545.3 [M / 2+H] + .

[0490] Examples 1-32: Preparation of Compound C-34 [ka]

[0491] Step 1: Preparation of compound 34-1 Compound Int24 (85 mg, 65.86 μmol), N,N-diisopropylethylamine (14.85 mg, 113.76 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (29.6 mg, 77.83 μmol) and GGFG-Dxd (50.34 mg, 59.87 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 15% to 25%) to obtain the title compound of this step (66 mg, yield: 54.9%). MS m / z (ESI): 1057.0 [M / 2+H] + .

[0492] Step 2: Preparation of compound 34-2 Compound 34-1 (65 mg, 30.76 μmol) and diethylamine (4.5 mg, 61.51 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=35%-45%) to obtain the title compound of this step (36 mg, yield: 65.1%). MS m / z (ESI): 946.0 [M / 2+H] + .

[0493] Step 3: Preparation of compound C-34 Compound 34-2 (36 mg, 19.03 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (10.43 mg, 28.56 μmol) and N,N-diisopropylethylamine (3.54 mg, 27.13 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) in sequence and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (8.47 mg, yield: 21.9%). MS m / z (ESI): 1071.0 [M / 2+H] + .

[0494] Examples 1-33: Preparation of Compound C-35 [ka]

[0495] Compound Int26 (113.67 mg, 55.68 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (22.27 mg, 60.95 μmol) and N,N-diisopropylethylamine (11.63 mg, 89.08 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (55 mg, yield: 53.9%). MS m / z (ESI): 1146.1 [M / 2+H] + .

[0496] Examples 1-34: Preparation of Compound C-36 [ka]

[0497] Compound Int27 (90 mg, 51.17 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (23.02 mg, 63.01 μmol) and N,N-diisopropylethylamine (12.02 mg, 92.1 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (27 mg, yield: 29.2%). MS m / z (ESI): 1005.0 [M / 2+H] + .

[0498] Examples 1-35: Preparation of Compound C-37 [ka]

[0499] Compound Int31 (12 mg, 5.05 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (2.4 mg, 6.58 μmol) and N,N-diisopropylethylamine (1.25 mg, 9.61 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (1.67 mg, yield: 13.3%). MS m / z (ESI): 1311.6 [M / 2+H] + .

[0500] Examples 1-36: Preparation of Compound C-38 [ka]

[0501] Compound Int31 (12 mg, 5.05 μmol), 3-maleimidopropionic acid hydroxysuccinimide ester (1.75 mg, 6.58 μmol) and N,N-diisopropylethylamine (1.25 mg, 9.61 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) and reacted for 2 hours at 0° C. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (1.92 mg, yield: 15.8%). MS m / z (ESI): 1262.1 [M / 2+H] + .

[0502] Examples 1-37: Preparation of Compound C-39 [ka]

[0503] Compound Int33 (70 mg, 42.33 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (20.1 mg, 55.02 μmol) and N,N-diisopropylethylamine (7.87 mg, 60.31 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (2.53 mg, yield: 7.1%). MS m / z (ESI): 1903.8 [M+H] + .

[0504] Example 1-38: Preparation of compound C-40 [ka]

[0505] Step 1: Preparation of compound 40-1 Compound Int26-3 (250 mg, 205.84 μmol) and diethylamine (73.14 mg, 1 mmol) were added to N,N-dimethylformamide (4 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (175 mg, yield: 85.7%). MS m / z (ESI): 992.6 [M+H] + .

[0506] Step 2: Preparation of compound 40-2 Monomethyl succinate (26.47 mg, 200.35 μmol), N,N-diisopropylethylamine (52.71 mg, 407.85 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (62.03 mg, 163.14 μmol) and compound 40-1 (142 mg, 143.11 μmol) were added sequentially to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (120 mg, yield: 79.8%). MS m / z (ESI): 1106.6 [M+H] + .

[0507] Step 3: Preparation of compound 40-3 Compound 40-2 (145 mg, 131.06 μmol) was dissolved in methanol (5 mL), and lithium hydroxide aqueous solution (15.06 mg, 622.53 μmol, 2 mL) was added and reacted for 4 hours at 25 ° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (100 mg, yield: 73.5%). MS m / z (ESI): 1092.6 [M+H] + .

[0508] Step 4: Preparation of compound 40-4 Compound 40-3 (50 mg, 47.16 μmol), N,N-diisopropylethylamine (12.63 mg, 96.76 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (119.84 mg, 52.18 μmol) and 4-piperidine carboxylic acid methyl ester (6.55 mg, 47.16 μmol) were added in sequence to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 30%) to obtain the title compound of this step (50 mg, yield: 94.4%). MS m / z (ESI): 1217.7 [M+H] + .

[0509] Step 5: Preparation of compound 40-5 Compound 40-4 (50 mg, 41.06 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (3 mL) and trifluoroacetic acid (1 mL) and reacted for 4 hours at 25° C. The crude product obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=40%-55%) to obtain the title compound of this step (40 mg, yield: 88.3%). MS m / z (ESI): 1161.6 [M+H] + .

[0510] Step 6: Preparation of compound 40-6 Compound 40-5 (42 mg, 36.16 μmol), N,N-diisopropylethylamine (12.63 mg, 96.76 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (15.68 mg, 41.23 μmol) and VC-PABC-MMAE (40.63 mg, 36.16 μmol) were added in sequence to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The crude product obtained by concentrating the reaction solution was purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 20% to 30%) to obtain the title compound of this step (60 mg, yield: 77.1%). MS m / z (ESI): 1133.7 [M / 2+H] + .

[0511] Step 7: Preparation of compound 40-7 Compound 40-6 (60 mg, 26.47 μmol) was dissolved in methanol (3 mL), and lithium hydroxide aqueous solution (3.04 mg, 125.73 μmol, 1 mL) was added and reacted for 4 hours at 25 ° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 35% to 45%) to obtain the title compound of this step (40 mg, yield: 70.6%). MS m / z (ESI): 1126.7 [M / 2+H] + .

[0512] Step 8: Preparation of compound C-40 Compound 40-7 (10 mg, 4.44 μmol), pentafluorophenol (1.62 mg, 8.8 μmol) and N,N'-dicyclohexylcarbodiimide (1.83 mg, 8.88 μmol) were added to anhydrous dichloromethane (2 mL) in that order and reacted for 3 hours at 25° C. The crude product obtained by concentrating the reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (3 mg, yield: 29.4%). MS m / z (ESI): 1209.7 [M / 2+H] + .

[0513] Examples 1-39: Preparation of Compound C-41 [ka]

[0514] Step 1: Preparation of compound 41-1 Compound Int26-4 (50 mg, 41.85 μmol), compound Int34 (37.95 mg, 41.85 μmol), N,N-diisopropylethylamine (13.63 mg, 104.61 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (19.27 mg, 50.21 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A% = 40% to 48%) to obtain the title compound of this step (43 mg, yield: 50.19%). MS m / z (ESI): 1024.0 [M / 2+H] + .

[0515] Step 2: Preparation of compound 41-2 Compound 41-1 (40 mg, 19.54 μmol) and piperidine (3.33 mg, 39.07 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. After concentrating the reaction solution, methyl tert-butyl ether (5 mL) was added, sonicated for 5 minutes, allowed to stand, filtered, and the solid was dried to obtain the title compound of this step (28 mg, yield: 70.67%). MS m / z (ESI): 1824.9 [M+H] + .

[0516] Step 3: Preparation of compound 41-3 Compound 41-2 (28 mg, 15.34 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (8.41 mg, 23.01 μmol) and N,N-diisopropylethylamine (4.00 mg, 30.68 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A% = 35% to 40%) to obtain the title compound of this step (30 mg, yield: 94.22%). MS m / z (ESI): 1038.0 [M / 2+H] + .

[0517] Step 4: Preparation of compound C-41 Compound 41-3 (30 mg, 13.01 μmol) and anhydrous zinc bromide (11.72 mg, 52.04 μmol) were added to anhydrous dichloromethane (2 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method A, elution gradient: A%=25%-35%) to obtain the title compound of this step (4.65 mg, yield: 16.80%). MS m / z (ESI): 1974.9 [M+H] + .

[0518] Examples 1-40: Preparation of Compound C-42 [ka]

[0519] Step 1: Preparation of compound 42-1 Compound Int26-4 (51.95 mg, 44.85 μmol), N,N-diisopropylethylamine (11.09 mg, 85.84 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (22.85 mg, 60.08 μmol) and compound Int35 (30 mg, 40.77 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0 ° C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A% = 45% to 50%) to obtain the title compound of this step (56 mg, yield: 73.2%). MS m / z (ESI): 1875.8 [M+H] + .

[0520] Step 2: Preparation of compound 42-2 Compound 42-1 (57 mg, 30.38 μmol) and diethylamine (11.22 mg, 151.91 μmol) were added to N,N-dimethylformamide (2 mL) and reacted for 2 hours at 25° C. The reaction solution was directly purified by reverse phase column chromatography (preparation method B, elution gradient: A%=45%-55%) to obtain the title compound of this step (30 mg, yield: 59.7%). MS m / z (ESI): 827.4 [M / 2+H] + .

[0521] Step 3: Preparation of compound C-42 Compound 42-2 (30 mg, 18.14 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (8.62 mg, 23.58 μmol) and N,N-diisopropylethylamine (3.55 mg, 27.21 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0° C. for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound of this step (11 mg, yield: 31.8%). MS m / z(ESI): 952.4 [M / 2+H] + .

[0522] 2. Preparation of Immunomodulatory Antibody-Drug Conjugates (iADCs) Example 2-1: Trastuzumab-C-7 [ka]

[0523] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.59 using 1M phosphoric acid solution to obtain trastuzumab monoclonal antibody buffer solution 9.5mg / mL. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer solution, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at 21℃ for 4 hours, then compound C-7 DMSO solution (10mM) was added thereto in a molar equivalent amount of 5 times that of trastuzumab monoclonal antibody, shaken thoroughly, reacted at room temperature for 2 hours, and allowed to stand overnight at 4℃, purified with NAP-5 gel column, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-7 in histidine hydrochloride buffer (2.4mg / mL, 1.5mL), which was stored at -40℃.

[0524] Example 2-2: Trastuzumab-C-8 [ka]

[0525] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.55 using 1M phosphoric acid solution to obtain trastuzumab monoclonal antibody buffer solution 9.5mg / mL. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer solution, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at 21℃ for 4 hours, then compound C-8 DMSO solution (10mM) was added thereto in a molar equivalent amount of 5 times that of trastuzumab monoclonal antibody, shaken thoroughly, reacted at room temperature for 2 hours, and allowed to stand overnight at 4℃, purified with NAP-5 gel column, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-8 in histidine hydrochloride buffer (2.3mg / mL, 1.5mL), which was stored at -40℃.

[0526] Example 2-3: Trastuzumab-C-9 [ka]

[0527] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.55 using 1M phosphoric acid solution to obtain trastuzumab monoclonal antibody buffer solution 9.5mg / mL. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer solution, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at 21℃ for 4 hours, then compound C-9 DMSO solution (10mM) was added thereto in a molar equivalent amount of 5 times that of trastuzumab monoclonal antibody, shaken thoroughly, reacted at room temperature for 2 hours, and allowed to stand overnight at 4℃, purified with NAP-5 gel column, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-9 in histidine hydrochloride buffer (3.8mg / mL, 2.5mL), which was stored at -40℃.

[0528] Example 2-4: Trastuzumab-C-10 [ka]

[0529] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.53 using 1M phosphoric acid solution to obtain trastuzumab monoclonal antibody buffer solution 9.5mg / mL. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer solution, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at 21℃ for 4 hours, then compound C-10 DMSO solution (10mM) was added thereto in a molar equivalent amount of 5 times that of trastuzumab monoclonal antibody, shaken thoroughly, reacted at room temperature for 2 hours, and allowed to stand overnight at 4℃, purified with NAP-5 gel column, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-10 in histidine hydrochloride buffer (3.4mg / mL, 2.5mL), which was stored at -40℃.

[0530] Example 2-5: Trastuzumab-C-11 [ka]

[0531] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.59 using 1M phosphoric acid solution to obtain trastuzumab monoclonal antibody buffer solution 9.5mg / mL. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer solution, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at 21℃ for 4 hours, then compound C-11 DMSO solution (10mM) was added thereto in a 5-fold molar equivalent amount of trastuzumab monoclonal antibody, shaken thoroughly, reacted at room temperature for 2 hours, left to stand overnight at 4℃, purified with NAP-5 gel column, washed with histidine hydrochloride buffer solution (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-10 (4.0mg / mL, 2.5mL) in histidine hydrochloride buffer solution, which was stored at -40℃.

[0532] Example 2-6: Trastuzumab-C-15 [ka]

[0533] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.56 using 1M disodium hydrogen phosphate solution, and then the volume was adjusted to 10 mg / mL using phosphate buffered saline (PBS, pH=6.5-6.6) to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at room temperature for 2 hours. Then, a DMSO solution (10mM) of compound C-15 was added thereto in an amount 5.5 times the molar equivalent of trastuzumab monoclonal antibody, shaken thoroughly, and reacted at room temperature for 2 hours. The mixture was purified with NAP-10 and NAP-5 gel columns, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-15 in histidine hydrochloride buffer (3.4mg / mL, 2.5mL), which was then stored at 4℃.

[0534] Example 2-7: Trastuzumab-C-16 [ka]

[0535] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.54 using 1M disodium hydrogen phosphate solution, and then the volume was adjusted to 10 mg / mL using phosphate buffered saline (PBS, pH=6.5-6.6) to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at room temperature for 2 hours. Then, a DMSO solution (10mM) of compound C-16 was added thereto in an amount 5 times the molar equivalent of trastuzumab monoclonal antibody, shaken thoroughly, and reacted at room temperature for 2 hours. The mixture was purified with NAP-10 and NAP-5 gel columns, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-16 in histidine hydrochloride buffer (3.1mg / mL, 2.5mL), which was then stored at 4°C.

[0536] Example 2-8: Trastuzumab-C-17 [ka]

[0537] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.57 using 1M disodium hydrogen phosphate solution, and then the volume was adjusted to 10 mg / mL using phosphate buffered saline (PBS, pH=6.5-6.6) to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at room temperature for 2 hours. Then, a DMSO solution (10mM) of compound C-17 was added thereto in an amount 5 times the molar equivalent of trastuzumab monoclonal antibody, shaken thoroughly, and reacted at room temperature for 2 hours. The mixture was purified with NAP-10 and NAP-5 gel columns, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-17 in histidine hydrochloride buffer (3.5mg / mL, 2.5mL), which was then stored at 4℃.

[0538] Example 2-9: Trastuzumab-C-20 [ka]

[0539] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.55 using 1M disodium hydrogen phosphate solution, and then the volume was adjusted to 10 mg / mL using phosphate buffered saline (PBS, pH=6.5-6.6) to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at room temperature for 2 hours. Then, a DMSO solution (10mM) of compound C-20 was added thereto in an amount 5.5 times the molar equivalent of trastuzumab monoclonal antibody, shaken thoroughly, and reacted at room temperature for 2 hours. The mixture was purified with NAP-10 and NAP-5 gel columns, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-20 in histidine hydrochloride buffer (3.0mg / mL, 2.5mL), which was then stored at 4°C.

[0540] Example 2-10: Trastuzumab-C-21 [ka]

[0541] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.55 using 1M disodium hydrogen phosphate solution, and then the volume was adjusted to 10 mg / mL using phosphate buffered saline (PBS, pH=6.5-6.6) to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at room temperature for 2 hours. Then, a DMSO solution (10mM) of compound C-21 was added thereto in an amount 5.5 times the molar equivalent of trastuzumab monoclonal antibody, shaken thoroughly, and reacted at room temperature for 2 hours. The mixture was purified with NAP-10 and NAP-5 gel columns, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-21 in histidine hydrochloride buffer (3.2mg / mL, 2.5mL), which was then stored at 4°C.

[0542] Example 2-11: Trastuzumab-C-22 [ka]

[0543] The trastuzumab monoclonal antibody stock solution was adjusted to pH=6.55 using 1M disodium hydrogen phosphate solution, and then the volume was adjusted to 10 mg / mL using phosphate buffered saline (PBS, pH=6.5-6.6) to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained trastuzumab monoclonal antibody buffer, and TCEP (2.5eq, 10mM) was added at the same time, shaken thoroughly, and reacted at room temperature for 2 hours. Then, a DMSO solution (10mM) of compound C-22 was added thereto in an amount 5.5 times the molar equivalent of trastuzumab monoclonal antibody, shaken thoroughly, and reacted at room temperature for 2 hours. The mixture was purified with NAP-10 and NAP-5 gel columns, washed with histidine hydrochloride buffer (pH=6.0-6.1), and the filtrate was collected to obtain trastuzumab-C-22 in histidine hydrochloride buffer (3.8mg / mL, 2.5mL), which was then stored at 4°C.

[0544] 3. Determining Drug / Antibody Ratio:DAR Values ​​for Immunomodulatory Antibody-Drug Conjugates (iADCs) The molecular weight of the iADC was determined using LC-MS, and the drug / antibody ratio DAR value was calculated. Chromatography conditions: Liquid chromatography column: Thermo MAbPac RP 3.0 × 100 mm; Mobile phase A: 0.1% FA / H 2 O; Mobile phase B: 0.1% FA / ACN; Flow rate: 0.25 ml / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μl;

[0545] [Table 2]

[0546] Mass spectrometry conditions: Mass spectrometer model: AB Sciex Triple TOF 5600+; GS1 35;GS2 35;CUR 30;TEM 350;ISVF 5500;DP 250;CE 10;Integration time 0.5s; m / z 600–4000; time bins – 40 in total.

[0547] CE-SDS calculations showed that antibody-drug conjugates trastuzumab-C-7 had a DAR value of 4.42; trastuzumab-C-8 had a DAR value of 3.56; trastuzumab-C-9 had a DAR value of 3.53; trastuzumab-C-10 had a DAR value of 3.3; trastuzumab-C-11 had a DAR value of 2.3; trastuzumab-C-15 had a DAR value of 3.58; trastuzumab-C-16 had a DAR value of 2.84; trastuzumab-C-17 had a DAR value of 3.37; trastuzumab-C-20 had a DAR value of 3.53; trastuzumab-C-21 had a DAR value of 2.89; and trastuzumab-C-22 had a DAR value of 3.38.

[0548] Biological assays Experimental Example 1: Inhibitory effect of immunomodulatory antibody-drug conjugates (iADCs) on the proliferation of HER2-positive and HER2-negative tumor cells Experimental steps: 1. HER2-positive tumor cells HCC1954 (Jiangyin Concortis Biotechnology Co, Ltd) and NCI-N87 (ATCC) in the logarithmic growth phase, and HER2-negative tumor cells NCI-H358 (Nanjing Co-Bioer) were harvested and counted. 2. Three types of tumor cells were seeded in a 96-well plate (Corning) at 2,000 cells (HCC1954 or NCI-H358) or 3,000 cells (NCI-N87) per well in a volume of 80 μL. The cells were incubated at 37°C and CO 2 The cells were allowed to adhere (18-24 hours) by overnight incubation in a 5% incubator. 3. 5x drug standard solutions with concentration gradient were prepared according to the experimental design. The 96-well cell plate was taken out and 20μL of standard solution was transferred from the compound plate to the cell plate. The cell plate was incubated at 37℃ and CO 2 The cells were then cultured for 72 minutes in a 5% incubator. 4. Test plate: The cell plate was taken out and the state of the cells was observed under a microscope. After observation, the detection solution was added to the cell plate according to the instructions of the Cell Counting-Lite® 2.0 kit (Nuoweizan), and raw data was obtained by PHERA Star FS. 5. The inhibition rate was calculated: (Inhibition rate, IR%)=1-(mean luminescence value of experimental wells / mean luminescence value of DMSO control wells)×100%. Graphpad Prism 8.0 software was used for data analysis and graphing. The logarithm of cell viability and drug concentration were used to fit a four-parameter curve to determine the IC 50 The value was calculated.

[0549] Test Results: The inhibitory effect of each iADC on the proliferation of three tumor cells was determined according to the above method, as shown in Table 1:

[0550] [Table 3]

[0551] These results show that each iADC exhibited a strong inhibitory effect on the proliferation of HCC1954 cells and NCI-N87 cells (HER2 positive), but the inhibitory effect was significantly weakened on NCI-H358 cells (HER2 negative), indicating that each iADC has a selective killing effect on HER2-positive cells.

[0552] Experimental Example 2: Determining the ability of immunomodulatory antibody-drug conjugates (iADCs) to stimulate peripheral blood mononuclear cells (PBMCs) to secrete TNF-α in the presence of tumor cells Experimental steps: 1. HER2-expressing HCC1954 tumor cells (Jiangyin Concortis Biotechnology Co., Ltd.) in logarithmic growth phase were harvested, washed twice with PBS, and resuspended in RPMI1640 + 10% heat-inactivated FBS complete medium to a cell density of 2 × 10 5HCC1954 tumor cells were cultured in a 96-well plate (Corning) at 1 × 10 cells / mL. 4 Single cells were inoculated. 2. Thaw PBMCs (Sail Bio) and resuspend the cells in RPMI 1640 + 10% heat-inactivated FBS complete medium to a density of 6 × 10 5 The tumor cells were inoculated into a 96-well plate, and 3 × 10 cells / mL were added per well. 4 PBMC cells were added. 3. 100 μL of different concentrations of test drugs were added to the well plate to achieve final concentrations of 500 nM, 166.7 nM, 55.6 nM, 18.52 nM, 6.17 nM, 2.06 nM, and 0.69 nM, and 100 μL of RPMI1640 + 10% heat-inactivated FBS complete medium was added to blank wells. 4. Incubate the Plate with CO 2 After incubation in a 5% CO2, 37°C incubator for 24 hours, 50 μL of the supernatant was taken from each well for detection, and the primary antibody and HRP-labeled secondary antibody were incubated according to the steps of the Human TNF-α ELISA Detection Kit (Invitrogen). After color development, the absorbance was read at a wavelength of 450 nm. The concentration of TNF-α was calculated based on the standard curve using Graphpad Prism software.

[0553] Test Results: The ability of each immunomodulatory antibody-drug conjugate (iADC) to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete TNF-α in the presence of HCC1954 tumor cells was determined according to the methods described above. The results are shown in Table 2:

[0554] [Table 4]

[0555] These results demonstrated that each iADC could effectively stimulate PBMCs to secrete TNF-α in an in vitro co-incubation system of HCC1954 tumor cells and PBMCs.

[0556] Various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description, and such modifications are also intended to fall within the scope of the appended claims.

Claims

1. Equation (I): 【Chemistry 1】 [In the formula, Ab represents the targeting portion; M represents the connecting part connected to the targeting part; X represents the linker connecting M and Aa; Aa represents an amino acid fragment, or a peptide fragment formed from two or more amino acids; L １ Aa and D １ It represents a covalent bond or linker that connects; L ２ Aa and D ２ Represents the linker that connects them; D １ It is a cytotoxic drug fragment; D ２ This is a TLR agonist fragment; m is an antibody-drug conjugate selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

2. The antibody-drug conjugate according to claim 1, wherein Ab is a targeting portion that targets a cell surface receptor or a tumor surface antigen.

3. The antibody-drug conjugate according to claim 1, wherein Ab is an antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment comprises a monoclonal antibody, a polyclonal antibody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a mouse antibody, a humanized antibody, a fully human antibody, or a fusion protein containing the antigen-binding region of an antibody.

4. Ab is an antibody or its antigen-binding fragment, and the antigen-binding fragment is Fab, Fab', F(ab'). ２ The antibody-drug conjugate according to claim 1, selected from the group consisting of Fv, Fv linked by a disulfide bond, and scFv.

5. The antibody-drug conjugate according to claim 1, wherein the antibody or its antigen-binding fragment is selected from the group consisting of anti-Her-2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-Trop-2 antibody.

6. M is either covalent or has the following structure: 【Chemistry 2】 Selected from the group consisting of; The antibody-drug conjugate according to claim 1, wherein each a is an integer independently selected from 1 to 5, b is an integer selected from 1 to 3, position 1 of M is connected to Ab, and position 2 of M is connected to X.

7. M is either covalent or has the following structure: 【Transformation 3】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the following.

8. Whether X is a covalent bond or C １～６ alkylene, -NH-(CH ２ ) ｃ -C(O)-, 【Chemistry 4】 Selected from the group consisting of; The antibody-drug conjugate according to claim 1, wherein each c is an integer independently selected from 1 to 10, position 3 of X is connected to M, and position 4 of X is connected to Aa.

9. Aa is selected from the group consisting of amino acid fragments and peptide fragments formed from two or more amino acids, and the amino acids are selected from the group consisting of Gly, Phe, Ala, Val, Ser, Thr, His, Trp, Cys, Asp, Glu, Lys, Tyr, and Arg, and each amino acid is optionally C １～６ Independently modified by alkyl or polyethylene glycol hydrophilic structural units; Preferably, the polyethylene glycol hydrophilic structural unit is 【Transformation 5】 The antibody-drug conjugate according to claim 1, wherein each c is an integer independently selected from the group consisting of 1 to 10.

10. Aa is selected from the group consisting of the following amino acid fragments: Gly, Phe, Ala, Val, Cys, Asp, Glu, Lys, and Arg, and peptide fragments formed from any combination of two or more of these amino acids, and each amino acid is C １～６ Alkyl or polyethylene glycol hydrophilic structural units are optionally and independently modified; Preferably, the polyethylene glycol hydrophilic structural unit is 【Transformation 6】 The antibody-drug conjugate according to claim 1, wherein each c is an integer independently selected from the group consisting of 1 to 10.

11. Aa has the following structure: 【Transformation 7】 Selected from the group consisting of; Each R ７ However, H, C １～６ Independently selected from the group consisting of alkyl and polyethylene glycol hydrophilic structural units; each R ８ However, it is independently selected from the group consisting of hydroxyl and polyethylene glycol hydrophilic structural units; position 5 is connected to X, and position 6 is connected to L １ and L ２ It is connected to one of the two, and position 7 is L １ and L ２ It is connected to the other side; preferably, position 5 is connected to X, and position 6 is connected to L １ It is connected to position 7, L ２ Connected; Preferably, the polyethylene glycol hydrophilic structural unit is 【Transformation 8】 The antibody-drug conjugate according to claim 1, wherein each c is an integer independently selected from the group consisting of 1 to 10.

12. L １ The antibody-drug conjugate according to claim 1, wherein the linker is covalent, non-cleavable, or cleavable, and the cleavable linker is capable of being cleaved by an enzyme present in the pathological environment, and the enzyme is selected from the group consisting of proteases, phosphatases, pyrophosphatases, β-glucuronidases, β-galactosidases, and sulfatases.

13. L １ However, covalent bond or -L ａ -L ｂ -L ｃ - and L ａ However, whether it is a covalent bond or C １～６ Alkylene, -NH-(CH ２ ) ｃ -C(O)-, 【Chemistry 9】 Selected from the group consisting of; Each c is an integer independently selected from 1 to 10; L ｂ The amino acids are either covalently bonded or selected from the group consisting of amino acid fragments and peptide fragments formed from two or more amino acids, and the amino acids are selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala, and Asn; Preferably, L ｂ However, it is either a covalent bond or has the following structure: 【Chemistry 10】 Selected from the group consisting of; Each R ９ However, it is selected from the group consisting of H, acetyl, fluorenyloxycarbonyl, trityl, and polyethylene glycol hydrophilic structural units; preferably, the polyethylene glycol hydrophilic structural unit is 【Chemistry 11】 And each c is an integer independently selected from 1 to 10; L ｃ is a covalent bond, -NH-CH ２ - or the following structure: 【Chemistry 12】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the following.

14. L １ However, it is either a covalent bond or has the following structure: 【Chemistry 13】 Selected from the group consisting of; Position 8 is connected to Aa, and position 9 is connected to D １ Connected; Preferably, L １ teeth, 【Chemistry 14】 The antibody-drug conjugate according to claim 1.

15. L ２ The antibody-drug conjugate according to claim 1, wherein the linker is either non-cleavable or cleavable, and the cleavable linker is capable of being cleaved by an enzyme present in the pathological environment, and the enzyme is selected from the group consisting of proteases, phosphatases, pyrophosphatases, β-glucuronidases, β-galactosidases, and sulfatases.

16. L ２ However, -L ｄ -L ｅ -L ｆ - and L ｄ However, covalent or the following: C １～６ Alkilen, 【Chemistry 15】 、-C(O)-(CH ２ ) ｄ -NHH-、 【Chemistry 16】 A divalent structure consisting of one or more elements, selected from the group consisting of; Each d is an integer independently selected from 1 to 12; L ｅ The amino acids are either covalently bonded or selected from the group consisting of amino acid fragments and peptide fragments formed from two or more amino acids, and the amino acids are selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala, and Asn; Preferably, L ｅ However, it is either a covalent bond or has the following structure: 【Chemistry 17】 Selected from the group consisting of; Each R １０ However, it is independently selected from the group consisting of H, acetyl, fluorenyloxycarbonyl, trityl, and polyethylene glycol hydrophilic structural units; preferably, the polyethylene glycol hydrophilic structural unit is [Chemistry 18] And each d is an integer independently selected from 1 to 10; L ｆ is a covalent bond, -NH-CH ２ - Or the following structure: 【Chemistry 19】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the following.

17. L ２ However, the following structure: 【Chemistry 20】 Selected from the group consisting of; Position 10 is connected to Aa, and position 11 is connected to D ２ Connected; Preferably, L ２ However, the following structure: 【Chemistry 21】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the following. 【Request Item 18】 【Chemistry 22】 However, the following structure: 【Chemistry 23】 【change】 【change】 Selected from the group consisting of; Position 1 is connected to Ab, position 6 is connected to L1, and position 7 is connected to L2. The antibody-drug conjugate according to claim 1. 【Request Item 19】 【Chemistry 24】 However, the following structure: 【Chemistry 25】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 Selected from the group consisting of; Position 1 is connected to Ab, position 9 is connected to D1, and position 11 is connected to D2. The antibody-drug conjugate according to claim 1.

20. D １ The antibody-drug conjugate according to claim 1, wherein the antibody is selected from the group consisting of cytotoxic drug fragments, the cytotoxic drug is selected from the group consisting of tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, the tubulin inhibitor comprises drastatin, auristatin, mytansin, tubulidine and cryptomycin, the DNA damaging agent comprises PBD, duocalmycin and calicheamycin, and the topoisomerase inhibitor comprises camptothecin and its derivatives.

21. The antibody-drug conjugate according to claim 20, wherein the tubulin inhibitor is selected from the group consisting of drastatin 10, MMAE, MMAF, meitansine, DM1, DM3, and DM4, and the topoisomerase inhibitor is selected from the group consisting of camptothecin, SN-38, exatecan, topotecan, berotecan, 10-hydroxycamptothecin, 9-aminocamptothecin, doxorubicin, epirubicin, and PNU-159682.

22. D １ However, the following structure: 【Chemistry 26】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the following.

23. D ２ The antibody-drug conjugate according to claim 1, wherein the TLR agonist is a TLR agonist fragment, and the TLR agonist is selected from the group consisting of TLR2 agonists, TLR4 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR7 / 8 agonists, and TLR9 agonists.

24. D ２ The antibody-drug conjugate according to claim 1, wherein the TLR agonist is a TLR agonist fragment, and the TLR agonist is selected from the group consisting of TLR7 agonists, TLR8 agonists, and TLR7 / 8 agonists.

25. D ２ However, it is a TLR agonist fragment, and the TLR agonist is given by equation (II): 【Chemistry 27】 [In the formula, X １ is N or C; X ２ is N or C; X １ and X ２ At least one of them is N; X ３ It is selected from the group consisting of O, S, and C; X ４ O, S, N and CR ４ Selected from the group consisting of; R １ C １～６ Alkyl and -C １～６ Alkylene-OC １～６ Selected from the group consisting of alkyl groups; R ２ is hydrogen or formula -L ３ -L ４ -L ５ -L ６ And; L ３ is a covalent bond or -C １～６ It is alkylene; L ４ Covalent bond, C ３～１０ Cycloalkyl, 3-12 membered heterocyclyl, C ６～１０ Selected from the group consisting of aryls and 5-10 membered heteroaryls, cycloalkyls, heterocyclyls, aryls, and heteroaryls are hydrogen, halogens, cyanos, and C １～６ Alkyl, C １～６ Alkoxy and -C １～６ Alkylene-NH ２ It is optionally replaced by one or more elements selected from the group consisting of; L ５ is selected from the group consisting of a covalent bond, -O-, -S-, -C(O)-, -O-C(O)-, -C(O)-O-, -O-C(O)-O-, -NR ５ -, -C(O)-NR ５ -, -NR ５ -C(O)-, -O-C(O)-NR ５ -, -NR ５ -C(O)-O-, -NR ５ -C(O)-NR ５ -, -S(O) ｒ -NR ５ -, -NR ５ -S(O) ｒ -, -NR ５ -S(O) ｒ -NR ５ -; L ６ is hydrogen, C １～６ Alkyl, -(O-CH ２ CH ２ ) ｎ -O-C １～６ Alkyl and -C １～６ Alkylene-(O-CH) ２ CH ２ ) ｎ -O-C １～６ Selected from the group consisting of alkyl groups, where alkyl is hydrogen, halogen, hydroxyl, -NH ２ , -NH-C(O)-OC １～６ Alkyl, C １～６ Alkoxy, carboxyl, and -C(O)-O-C １～６ Optionally substituted with one or more groups selected from the group consisting of alkyl groups; R ３ is hydrogen or formula -L ７ -L ８ -L ９ -L １０ And; L ７ Covalent bonds and C １～６ Selected from the group consisting of alkylenes; L ８ Covalent bond, C ３～１０ Cycloalkyl, 3-12 membered heterocyclyl, C ６～１０ Selected from the group consisting of aryls and 5- to 10-membered heteroaryls, cycloalkyls, heterocyclyls, aryls, and heteroaryls are hydrogen, halogens, cyanos, and C １～６ Alkyl, C １～６ Alkoxy and -C １～６ Alkylene-NH ２ It is optionally replaced by one or more elements selected from the group consisting of; L ９ is a covalent bond, -O-, -S-, -C(O)-, -O-C(O)-, -C(O)-O-, -O-C(O)-O-, -NR ６ -, -C(O)-NR ６ -, -NR ６ -C(O)-, -OC(O)-NR ６ -, -NR ６ -C(O)-O-, -NR ６ -C(O)-NR ６ -, -S(O) ｐ -NR ６ -, -NR ６ -S(O) ｐ - and -NR ６ -S(O) ｐ -NR ６ Selected from the group consisting of: L １０ is hydrogen, C １～６ Alkyl, -(O-CH ２ CH ２ ) ｑ -O-C １～６ Alkyl and -C １～６ Alkylene-(O-CH) ２ CH ２ ) ｑ -O-C １～６ Selected from the group consisting of alkyl groups, where alkyl is hydrogen, halogen, hydroxyl, -NH ２ , -NH-C(O)-OC １～６ Alkyl, C １～６ Alkoxy, carboxyl, and -C(O)-O-C １～６ Optionally substituted with one or more groups selected from the group consisting of alkyl groups; R ４ These are hydrogen, halogen, cyano, hydroxyl, and C １～６ Alkyl, C １～６ Haloalkyl, C １～６ Alkoxy and C ３～１０ Selected from the group consisting of cycloalkyl groups; Each R ５ These are, independently, hydrogen or C １～６ It is alkyl; Each R ６ These are, independently, hydrogen or C １～６ It is alkyl; Each r is independently either 1 or 2; Each n is an integer independently selected from 1 to 25; Each p is independently either 1 or 2; Each q is an integer independently selected from 1 to 25; D ２ In equation (II), R ２ or R ３ via L ２ The antibody-drug conjugate according to claim 1, which is a compound represented by [connected to].

26. D ２ However, the following structure: 【Chemistry 28】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the following.

27. Next structure: 【Chemistry 29】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 Selected from the group consisting of; Each m is independently selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, Ab is trastuzumab; Preferably, the antibody-drug conjugate has a DAR value of 2.0 to 5.0, for example, 2.0 to 2.5, 2.0 to 3.0, 2.0 to 3.5, 2.0 to 4.0, 2.0 to 4.5, 2.0 to 5.0, 2.5 to 3.0, 2.5 to 3.5, 2.5 to 4.0, 2.5 to 4.5, 2.5 to 5.0, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 4.0 to 4.5, 4.0 to 5.0, or 4.0 to 5.0, according to claim 1.

28. Formula (III): 【Transformation 30】 [In the formula, M １ This is a precursor of a connecting portion connected to a targeting portion, the targeting portion being as defined in claim 1; X is a linker connecting M and Aa; Aa is an amino acid fragment, or a peptide fragment formed from two or more amino acids; L １ is a covalent bond or Aa and D １ It is a linker that connects; L ２ Aa and D ２ It is a linker that connects; D １ It is a cytotoxic drug fragment; D ２ This is a TLR agonist fragment; Preferably, X, Aa, L １ , L ２ , D １ and D ２ A compound of [as defined in claim 1], or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof.

29. M １ However, the following structure: 【Chemistry 31】 Selected from the group consisting of; Each a is an integer independently selected from 1 to 5, and b is an integer selected from 1 to 3, M ａ However, it is a carboxyl-active group (e.g., pentafluorophenoxy); Preferably, M １ However, it is either a covalent bond or has the following structure: 【Chemistry 32】 A compound according to claim 28, selected from the group consisting of the following. 【Request Item 30】 【Chemistry 33】 However, the following structure: 【Transformation 34】 【change】 【change】 【change】 Selected from the group consisting of; Position 5 is connected to X, position 6 is connected to one of L1 and L2, and position 7 is connected to the other of L1 and L2; preferably, position 5 is connected to X, position 6 is connected to L1, and position 7 is connected to L2. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug according to claim 28. 【Request Item 31】 【Chemistry 35】 However, the following structure: 【Transformation 36】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 Selected from the group consisting of; Position 9 is connected to D1, and position 11 is connected to D2. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug according to claim 28.

32. Next structure: 【Chemistry 37】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, selected from the group consisting of the above. 【Request Item 33】 【Chemistry 38】 However, the following structure: 【Chemistry 39】 【change】 【change】 【change】 【change】 Selected from the group consisting of; Position 5 is connected to X, position 9 is connected to D1, and position 7 is connected to L2. An antibody-drug conjugate according to claim 1, or a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 28. 【Request Item 34】 【Chemistry 40】 However, the following structure: 【Chemistry 41】 【change】 【change】 【change】 【change】 Selected from the group consisting of; Position 5 is connected to X, position 6 is connected to L1, and position 11 is connected to D2. An antibody-drug conjugate according to claim 1, or a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 28. 【Request Item 35】 【Chemistry 42】 However, the following structure: 【Chemistry 43】 【change】 【change】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the above, or a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug according to claim 28. 【Request Item 36】 【Chemistry 44】 However, the following structure: 【Chemistry 45】 【change】 【change】 Selected from the group consisting of, An antibody-drug conjugate according to claim 1, or a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 28. 【Request Item 37】 【Chemistry 46】 However, the following structure: 【Chemistry 47】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 Selected from the group consisting of; Position 5 is connected to X, position 9 is connected to D1, and position 11 is connected to D2. An antibody-drug conjugate according to claim 1, or a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 28.

38. The following compound: 【Chemistry 48】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 An antibody-drug conjugate according to claim 1, selected from the group consisting of the above, or an intermediate for producing a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug according to claim 28.

39. Formula: 【Chemistry 49】 or [Transformation 50] A linker which is a compound of 【Chemistry 51】 but, 【Chemistry 52】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 Selected from the group consisting of, 【Chemistry 53】 but, 【Chemistry 54】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 A linker selected from the group consisting of the following.

40. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the compound comprises a linker as described in claim 39 linked to at least one of D1, D2 or M1-X-, where D1 is linked to position 9 of the linker, D2 is linked to position 11 of the linker, M1-X- is linked to position 5 of the linker, X, D1 and D2 are as defined in any one of claims 1 to 27, and M1 is as defined in any one of claims 28 to 32.

41. A pharmaceutical composition for treating and / or preventing cancer (e.g., HER2-positive cancer, e.g., HER2-positive gastric cancer, breast cancer, or non-small cell lung cancer), comprising the antibody-drug conjugate described in claim 1, and optionally a compound described in claim 28 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, and one or more pharmaceutical excipients.

42. Use in the manufacture of a pharmaceutical product for treating and / or preventing cancer (e.g., HER2-positive cancer, e.g., HER2-positive gastric cancer, breast cancer, or non-small cell lung cancer) the antibody-drug conjugate described in claim 1, the compound described in claim 28 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition containing the antibody-drug conjugate or compound.

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