Deuterated camptothecin compound, and preparation and use thereof

Abstract

A compound represented by formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, a preparation method therefor, a pharmaceutical composition containing the compound represented by formula I or a pharmaceutically acceptable salt of the composition or a stereoisomer of the composition, and a use thereof in treating tumor-related diseases.

Description

A deuterated camptothecin compound and its preparation and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefits and priority of patent application No. 202311545719.9 filed with the State Intellectual Property Office of China on November 16, 2023, and patent application No. 202411561506.X filed with the State Intellectual Property Office of China on November 1, 2024, the entire contents of which are hereby incorporated by reference into this document in their entirety. Technical Field

[0003] The present disclosure belongs to the field of pharmaceutical chemistry, and specifically relates to a class of deuterated camptothecin compounds and pharmaceutically acceptable salts thereof, methods for preparing the same, pharmaceutical compositions containing the deuterated camptothecin compounds or pharmaceutically acceptable salts thereof, and uses of the deuterated camptothecin compounds or pharmaceutically acceptable salts thereof or the pharmaceutical compositions in treating tumor-related diseases. Background Art

[0004] Camptothecin (CPT), a pyrroloquinoline cytotoxic alkaloid, is one of the most studied natural antitumor drugs besides paclitaxel. It is primarily found in the fruit or root bark of the Davidia involucrata plant, a species endemic to my country. In 1985, Hsiang et al. discovered that camptothecin and its derivatives exert their anticancer effects by inhibiting DNA synthesis through topoisomerase I (Topoisomerase I). This led to renewed interest in camptothecin, which subsequently garnered numerous derivatives and became a new hotspot in anticancer research. Since then, a new generation of camptothecins, including 10-hydroxycamptothecin (HCPT), irinotecan, topotecan, SN-38, and belotecan, have been approved for the treatment of various tumors, including colorectal cancer, small cell lung cancer, and ovarian cancer. Research into their indications and dosage forms has also yielded promising results.

[0005] With the development of drug delivery systems, a series of camptothecins that were previously unavailable and had significant side effects have been revived. However, the problem of in vivo toxicity of camptothecins has not been completely eliminated. How to further reduce the toxicity of these drugs remains a major concern.

[0006] Summary of the Invention

[0007] In one aspect, the present disclosure provides a compound of Formula I or a pharmaceutically acceptable salt or stereoisomer thereof:

[0008] Wherein, Ab represents an antibody or antigen-binding fragment; L represents a linker connecting Ab and the warhead drug molecule; n is selected from 1-12;

[0009] R1 is selected from H, halogen, OH, SH, NH2, C 1-4 Alkyl, C 1-4 Haloalkyl or C 1-4 alkoxy;

[0010] R2 is selected from H, halogen, C 1-4 Alkyl or C 1-4 alkoxy;

[0011] Or R1 and R2 are cyclized to -O-(CH2) m -O-, wherein m is selected from 1, 2, 3;

[0012] R3 and R4 are each independently selected from H, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Haloalkyl or C 1-4 haloalkoxy;

[0013] Or R3 and R4 cyclize to -(CH2) k -, wherein k is selected from 1, 2, 3, 4;

[0014] X is selected from H, OH, HO-CH(R5)-(CH2) p -CO-NH- or -N(R6)(R7), wherein p is selected from 0, 1, 2;

[0015] R5 is selected from H, C 1-4 Alkyl, C 1-4 Haloalkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl;

[0016] R6 is selected from H, C 1-4 Alkyl or C 1-4 alkyl halide;

[0017] R7 is selected from H or R8-S(O)2-;

[0018] R8 is selected from C 1-4 Alkyl; and

[0019] t is selected from 0, 1, 2, 3, 4, 5.

[0020] In the present disclosure, the expression "R1 and R2 are cyclized to form -O-(CH2) m -O-" means R1 and R2 are connected to form -O-(CH2) m -O-. Expression "R3 and R4 are cyclized to -(CH2) k-" means that R3 and R4 are connected to form -(CH2) k -.

[0021] In any embodiment of the compounds of formula I disclosed herein, R1 is C 1-4 alkyl, and R2 is halogen.

[0022] In any of the embodiments of the compounds of formula I disclosed herein, R1 is methyl and R2 is F.

[0023] In any of the embodiments of the compounds of formula I disclosed herein, R1 is H, and R2 is H.

[0024] In any of the disclosed embodiments of the compounds of formula I, R1 and R2 are cyclized to -O-CH2-O-.

[0025] In any of the embodiments of the compounds of formula I disclosed herein, R1 is NH2, and R2 is H or halogen.

[0026] In any of the embodiments of the compounds of formula I disclosed herein, R1 is NH2, and R2 is H or F.

[0027] In any of the embodiments of the compounds of formula I disclosed herein, R3 is H, and R4 is H.

[0028] In any embodiment of the compounds of formula I disclosed herein, R3 is H, and R4 is C 1-4 alkyl.

[0029] In any of the disclosed embodiments of the compounds of formula I, R3 is H, and R4 is methyl.

[0030] In any of the disclosed embodiments of the compounds of formula I, R3 and R4 are cyclized to -CH2-CH2-.

[0031] In any embodiment of the compounds of formula I disclosed herein, X is HO-CH(R5)-(CH2) p -CO-NH-, wherein p is selected from 0, 1, and 2.

[0032] In any embodiment of the compounds of formula I disclosed herein, X is selected from OH or NH 2。

[0033] In any of the disclosed embodiments of the compounds of formula I, X is H, and t is 0.

[0034] In any of the disclosed embodiments of the compounds of formula I, X is -N(R6)(R7).

[0035] In any embodiment of the compounds of formula I disclosed herein, n is selected from 4-9, or 6-8, or 7-8, or 7.4-8.0, or 7.5-8.5; or n is selected from 6, 7, 8. In any embodiment of the compounds of formula I disclosed herein, n is selected from 7-8, or 7.3-7.9, or 7.4-7.8, or 7.5-7.7, or 7.5-7.6, or 7.5-7.8, or 7.4-7.7.

[0036] In one embodiment of the compound of formula I disclosed herein, it is a compound of formula Ia or a pharmaceutically acceptable salt or stereoisomer thereof;

[0037] wherein R1, R2, R3, and R4 are as defined in any embodiment of the compound of formula I; X1 is selected from a chemical bond, -O-CH(R5)-(CH2) p -CO-, wherein -CO- is connected to -NH-; p is selected from 0, 1, 2; R5 is selected from H, C 1-4 alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl.

[0038] In any of the embodiments of the compounds of Formula Ia disclosed herein, R1 is selected from methyl or methoxy; or R1 is methyl.

[0039] In any of the embodiments of the compounds of formula Ia disclosed herein, R2 is selected from F or Cl; or R2 is F.

[0040] In any of the disclosed embodiments of the compounds of formula Ia, R1 is methyl and R2 is F.

[0041] In any embodiment of the compounds of formula Ia disclosed herein, R3 and R4 are cyclized to -(CH2) k -, where k is 2.

[0042] In any of the disclosed embodiments of the compounds of Formula Ia, R3 is H; and R4 is H.

[0043] In any embodiment of the compounds of formula Ia disclosed herein, X1 is -O-CH(R5)-(CH2) p -CO-, wherein the -CO- end is connected to -NH-; wherein p is selected from 0 or 1, and R5 is selected from H, C 1-4 Alkyl or 3-6 membered cycloalkyl.

[0044] In any embodiment of the compounds of formula Ia disclosed herein, X1 is -O-CH(R5)-(CH2) p -CO-, wherein the -CO- end is connected to -NH-; wherein p is selected from 0 or 1, and R5 is selected from H, methyl or cyclopropyl.

[0045] In any embodiment of the compounds of Formula Ia disclosed herein, X1 is -O-CH(R5)-CO-, wherein the -CO- terminus is linked to -NH-; and R5 is selected from H or a 3-6 membered cycloalkyl group.

[0046] In any of the disclosed embodiments of the compounds of Formula Ia, X1 is -O-CH(R5)-CO-, wherein the -CO- terminus is linked to -NH-; and R5 is selected from H or cyclopropyl.

[0047] In any embodiment of the compounds of formula Ia disclosed herein, X1 is -O-CH(R5)-CH2-CO-, wherein the -CO- terminus is connected to -NH-; and R5 is C 1-4 alkyl.

[0048] In any of the disclosed embodiments of the compounds of Formula Ia, X1 is -O-CH(R5)-CH2-CO-, wherein the -CO- terminus is linked to -NH-; and R5 is methyl.

[0049] In any of the embodiments of the compounds of formula Ia disclosed herein, X1 is selected from the following groups: The -CO- end is connected to the -NH.

[0050] In some embodiments of the compounds of Formula Ia disclosed herein, the compound of Formula Ia is selected from the following compounds of Formula Ia-1 or Formula Ia-2, or pharmaceutically acceptable salts thereof, or stereoisomers thereof;

[0051] wherein Ab, L, R1, R2, X1, and n are as defined in any embodiment of the compound of formula Ia.

[0052] In any of the embodiments of the compounds of Formula Ia disclosed herein, Selected from the following compound fragments:

[0053] in It indicates that the position is connected to the linker L through a chemical bond.

[0054] In one embodiment of the compounds of formula I disclosed herein, the compound of formula I is a compound of formula Ib or a pharmaceutically acceptable salt or stereoisomer thereof;

[0055] wherein Ab, L, and n are as defined in any embodiment of the compound of formula I;

[0056] R1 is selected from H, OH, halogen, NH2, C 1-4 Alkyl, C1-4 Haloalkyl or C 1-4 alkoxy;

[0057] R2 is selected from H, halogen, C 1-4 Alkyl or C 1-4 alkoxy;

[0058] Or R1 and R2 are cyclized to -O-(CH2) m -O-, wherein m is selected from 1, 2, 3;

[0059] R3 and R4 are each independently selected from H, C 1-4 Alkyl or C 1-4 alkoxy;

[0060] Or R3 and R4 cyclize to -(CH2) k -, wherein k is selected from 1, 2, 3;

[0061] X2 is selected from O or -N(R6)-;

[0062] R6 is selected from H or C 1-4 alkyl;

[0063] q is selected from 0, 1, 2, 3, 4.

[0064] In any of the disclosed embodiments of the compound of formula Ib, R1 is selected from methyl, and R2 is selected from H, Cl, F.

[0065] In any embodiment of the compounds of formula Ib disclosed herein, R1 and R2 are cyclized to -O-(CH2) m -O-, wherein m is selected from 1 or 2.

[0066] In any of the disclosed embodiments of the compound of formula Ib, R3 and R4 are both H.

[0067] In any of the disclosed embodiments of the compounds of Formula Ib, R3 is H, and R4 is methyl.

[0068] In any of the disclosed embodiments of the compounds of Formula Ib, X2 is selected from -O- or -NH-.

[0069] In any of the disclosed embodiments of the compounds of Formula Ib, X2 is selected from -O- or -NH-, and q is 0, 1, 2, 3, or 4.

[0070] In any embodiment of the compounds of formula Ib disclosed herein, X2 is selected from -O- or -NH-, q is 1, 2, 3 or 4; R3 and R4 are both H; R1 is selected from methyl and R2 is selected from H, Cl or F, or R1 and R2 are cyclized to -O-(CH2) m -O-, wherein m is selected from 1 or 2.

[0071] In some embodiments of the compound of Formula Ib disclosed herein, the compound of Formula Ib is a compound of Formula Ib-1.

[0072] In the compound of formula Ib-1, Ab, L, n, X2, and q are as defined in any of the embodiments of the compound of formula Ib.

[0073] In any embodiment of Formula Ib, Selected from the following compound fragments;

[0074] in It indicates that the position is connected to the linker L through a chemical bond.

[0075] In one embodiment of the compounds of formula I disclosed herein, the compound of formula I is a compound of formula Ic or a pharmaceutically acceptable salt thereof:

[0076] wherein X3 is selected from NH, O, S; and Ab, L, n, R2, R3, and R4 are as defined in any embodiment of the compound of formula I disclosed herein.

[0077] In any of the disclosed embodiments of the compounds of formula Ic, R2 is selected from H or halogen, or R2 is selected from H or F.

[0078] In any embodiment of the compounds of formula Ic disclosed herein, R3 is H, and R4 is C 1-4 alkyl; or, R3 is selected from H, and R4 is selected from methyl.

[0079] In any embodiment of the compounds of formula Ic disclosed herein, R3 and R4 are cyclized to -(CH2) k -, k is selected from 1, 2, 3.

[0080] In any of the disclosed embodiments of the compounds of Formula Ic, R3 and R4 are cyclized to -(CH2)2-.

[0081] In any of the embodiments of the compounds of formula Ic disclosed herein, X3 is selected from NH or O; or X3 is selected from NH, or X3 is selected from O;

[0082] In any of the embodiments of the compounds of formula Ic disclosed herein, Selected from the following compound fragments:

[0083] In one embodiment of the compounds of formula I disclosed herein, the compound of formula I is a compound of formula Id or a pharmaceutically acceptable salt thereof:

[0084] wherein R1, R2, R3, R4, R6, and R7 are as defined in any embodiment of the compound of formula I disclosed herein; Ab, L, and n are as defined in any embodiment of the compound of formula I disclosed herein; and r is selected from 0, 1, 2, and 3.

[0085] In any of the disclosed embodiments of the compound of formula Id, R1 and R2 are both H.

[0086] In any of the disclosed embodiments of the compound of formula Id, R3 and R4 are both H.

[0087] In any of the disclosed embodiments of the compounds of formula Id, r is 1.

[0088] In any embodiment of the compound of formula Id disclosed herein, R6 is selected from methyl, ethyl, isopropyl; or, R6 is isopropyl.

[0089] In any of the disclosed embodiments of the compounds of formula Id, R7 is selected from H.

[0090] In any embodiment of the compounds of formula Id disclosed herein, R7 is selected from R8-S(O)2-, wherein R8 is selected from methyl, ethyl; or, R8 is selected from methyl.

[0091] In any of the embodiments of the compounds of formula Id disclosed herein, Selected from the following compound fragments:

[0092] In one embodiment of the compounds of Formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id of the present disclosure, Ab is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody.

[0093] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic or Formula Id disclosed herein, the Ab is an anti-Her2 antibody having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0094] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic or Formula Id disclosed herein, the Ab is an anti-Her2 antibody having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0095] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic or Formula Id of the present disclosure, the Ab is an anti-Her2 antibody having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0096] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic or Formula Id disclosed herein, the Ab is an anti-Her2 antibody having a heavy chain variable region shown in SEQ ID NO: 4.

[0097] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic or Formula Id disclosed herein, the Ab is an anti-Her2 antibody having a light chain variable region shown in SEQ ID NO:9.

[0098] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic or Formula Id disclosed herein, the Ab is an anti-Her2 antibody having a heavy chain variable region set forth in SEQ ID NO: 4 and a light chain variable region set forth in SEQ ID NO: 9.

[0099] In any embodiment of the compounds of Formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, the Ab is an anti-Her2 antibody having a heavy chain as shown in SEQ ID NO:5.

[0100] In any embodiment of the compounds of Formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, the Ab is an anti-Her2 antibody having a light chain as shown in SEQ ID NO: 10.

[0101] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic or Formula Id disclosed herein, the Ab is an anti-Her2 antibody having a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10.

[0102] In any of the disclosed embodiments of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic, or Formula Id, Ab is Trastuzumab.

[0103] In one embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic, or Formula Id of the present disclosure, Ab is selected from Pertuzumab, Sacituzumab, zolbetuximab.

[0104] In one embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic, or Formula Id disclosed herein, L is L1-L2-L3-L4, wherein one end of L1 is connected to Ab, and one end of L4 is connected to the warhead drug molecule D;

[0105] L1 is selected from the following groups, and the end of L1 marked with an asterisk * is connected to Ab:

[0106] L2 is selected from a chemical bond, -N(R 10 )-CH2-CO-, One end of CO of L2 is connected to L3, and the other end is connected to L1; R 10 Selected from C 1-4 Alkyl, C 1-4 alkyl halide;

[0107] L3 is selected from a polypeptide residue consisting of 2-6 amino acids, and the C-terminus of the polypeptide residue is connected to L4;

[0108] L4 is selected from a chemical bond, -NH-CH-, One end of the CH is connected to the warhead drug molecule, and the other end of the -NH- is connected to L3.

[0109] In any of the disclosed embodiments of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic, or Formula Id, L is L1-L2-L3-L4, and L2 is a bond.

[0110] In any of the embodiments of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic, or Formula Id disclosed herein, L is L1-L2-L3-L4, and L2 is -N(CH3)-CH2-CO-.

[0111] In any of the disclosed compounds of Formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id, L is L1-L2-L3-L4, and L3 is selected from the following polypeptide residues: GFG, GGFG, GGGFG, GGVA, V-Cit, VA.

[0112] In any embodiment of the compounds of Formula I, Formula Ia, Formula Ia-1, Formula Ia-2, Formula Ib, Formula Ib-1, Formula Ic, or Formula Id disclosed herein, L is L1-L2-L3-L4, and L1 is selected from L2 is selected from a chemical bond or -N(CH3)-CH2-CO-, L3 is selected from GFG, GGFG, GGGFG, and L4 is -NH-CH-.

[0113] In any of the disclosed compounds of Formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id, L is selected from the following fragments:

[0114] Wherein, "*" indicates the position where the linker is connected to the warhead drug molecule, and the other end indicates the position where the linker is connected to the Ab. In any embodiment of the compound of formula I disclosed herein, Selected from the following structural fragments:

[0115] In one embodiment of the compound of formula I disclosed herein, the compound of formula I is a compound selected from the following:

[0116] wherein n has the same meaning as in Formula I; for example, n is 7.0-8.0, or n is 7.4-7.8.

[0117] In a second aspect, the present disclosure provides the following compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0118] In a third aspect, the present disclosure provides the following structural fragments:

[0119] The asterisk * end indicates the position connected to the warhead drug molecule, and the other end indicates the position where the linker is connected to the Ab.

[0120] On the other hand, the present disclosure provides a pharmaceutical composition comprising the compound of Formula I, Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ia-1, Formula Ia-2, Formula Ib-1, its stereoisomers or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient.

[0121] In another aspect, the present disclosure provides the use of the compounds of Formula I, Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ia-1, Formula Ia-2, Formula Ib-1, their stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical compositions in the preparation of drugs for treating tumors.

[0122] In another aspect, the present disclosure provides a method for treating tumors, comprising administering to a patient in need thereof an effective amount of the compound of Formula I, Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ia-1, Formula Ia-2, Formula Ib-1, its stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition.

[0123] In another aspect, the present disclosure provides the compound of Formula I, Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ia-1, Formula Ia-2, Formula Ib-1, its stereoisomer or pharmaceutically acceptable salt or the pharmaceutical composition for treating tumors.

[0124] In another aspect, the present disclosure provides use of the compounds of Formula I, Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ia-1, Formula Ia-2, Formula Ib-1, their stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical compositions in treating tumors.

[0125] The payload compound (i.e., warhead drug molecule) in the compounds of Formula I, Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ia-1, Formula Ia-2, and Formula Ib-1 disclosed herein has better stability in plasma, a shorter half-life in vivo, and lower in vivo exposure. Therefore, the payload of the compounds disclosed herein is more easily eliminated after shedding in vivo, resulting in lower toxicity. In addition, toxicity tests disclosed herein have demonstrated that the compounds disclosed herein have lower in vivo toxicity and hematotoxicity, exhibiting better safety.

[0126] Terminology and Description

[0127] Unless otherwise indicated, the terms used herein have their ordinary meanings in the art. A particular term or phrase should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0128] In the present disclosure, the antibody Trastuzumab has the sequence shown in Table S1 below.

[0129] Table S1 Sequence of antibody Trastuzumab

[0130] More specifically, the antibody Trastuzumab has a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10:

[0131] In the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are all encoded using the Kabat scheme; the Kabat encoding scheme refers to an encoding scheme based on sequence variability.

[0132] In any embodiment of the compounds of formula I disclosed herein, when R1 and R2 are cyclized to -O-(CH2) m -O-, the compound of formula I is the following compound of formula I-1. In the compound of formula I disclosed herein or any embodiment thereof, when R3 and R4 are cyclized to -(CH2) k -, it is in the form of the following formula I-2.

[0133] In the compounds of formula I disclosed herein, This means that the linker L can be chemically linked to any connectable site of the warhead drug molecule D in Formula I, as long as it is chemically feasible and a robust drug can be obtained.

[0134] It should be known to those skilled in the art that in Formula I, Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ia-1, Formula Ia-2, Formula Ib-1, or specific compounds thereof disclosed herein, the linker L or its corresponding specific structure is connected to Ab via a sulfur atom from Ab, and the sulfur atom S may be reflected in the structural formula or chemical formula of L or L1, or may be omitted, hidden, or not reflected. For example Can also be written as Both represent the same structure and meaning; It can also be written as Can be written as It can also be written as The same understanding can be applied to other L1 segments.

[0135] The term "warhead drug molecule," also known as "payload," "drug payload," or "payload," refers to a substance that has the potential to prevent or treat disease. The drug in an antibody-drug conjugate is typically a cytotoxic drug, a chemical molecule that has the potential to disrupt the normal growth of tumor cells.

[0136] The term "linker" refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being connected to the drug.

[0137] The term "drug-linker conjugate" is also referred to as payload-linker conjugate, linker-payload conjugate, and has the same meaning in the present disclosure.

[0138] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes both occurrence and non-occurrence of the event or circumstance. For example, the term "optionally" substituted means that the group may be unsubstituted or substituted with the specified substituent; whether or not substituted is within the scope of "optionally".

[0139] The term "independently selected" or "each independently selected" means that each substituent in the aforementioned substituent may select a different optional variable from a specified range of options, and is not affected by the selection of other substituents. When a substituent that can be "independently selected" exists in a general chemical formula and has multiple identical substituents, they may still select the same or different optional variables.

[0140] The term "halogen" or "halogen atom" refers to fluorine, chlorine, bromine, or iodine.

[0141] The term "halo" refers to a group in which one or more hydrogen atoms in a substituent are replaced by halogen atoms.

[0142] The term "alkyl" refers to a straight or branched hydrocarbon group in which carbon atoms are connected by single bonds. 1-4 or C 1-6 Alkyl. C 1-4 "Alkyl" means a straight or branched chain alkyl group having 1, 2, 3 or 4 carbon atoms. 1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl. 1-6 "Alkyl" means a straight or branched chain alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms. 1-6 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl.

[0143] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen atom. 1-4 Examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl.

[0144] The term "alkoxy" refers to an alkyl-O- group, wherein alkyl is as defined above and includes C 1-4 Alkoxy or C 1-6 Alkoxy. C1-4 Alkoxy can be understood as "C 1-4 Alkyl-O-", examples of which include but are not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy. 1-6 Alkoxy can be understood as "C 1-6 Alkyl-O-", examples of which include the aforementioned C 1-4 Specific examples of alkoxy include, but are not limited to, n-pentyloxy, neopentyloxy, n-hexyloxy, and the like.

[0145] The term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by a halogen atom. 1-4 Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, 2,2,2-trichloroethoxy.

[0146] The term "cycloalkyl" refers to a cyclic, saturated, monocyclic or polycyclic hydrocarbon group whose carbon atoms are connected by single bonds. The H atoms of the ring carbon atoms are optionally oxo-substituted, i.e., the "-CH2-" radical in the ring is optionally oxo-substituted to form "-C(O)-". A 3- to 6-membered cycloalkyl group refers to a 3-, 4-, 5-, or 6-membered cycloalkyl group, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0147] The term "heterocycloalkyl" refers to a cycloalkyl group in which one or more (e.g., 2, 3, or 4) ring carbon atoms are substituted by heteroatoms or heteroatom groups (i.e., heteroatom groups containing heteroatoms). The ring carbon atoms refer to carbon atoms that constitute the cyclic skeleton structure; the heteroatoms refer to atoms other than C and H in an organic compound, such as nitrogen atoms (N), oxygen atoms (O), sulfur atoms (S), phosphorus atoms (P), or boron atoms (B). The heteroatoms such as nitrogen atoms and sulfur atoms may be oxidized, and the nitrogen atoms may be quaternized; examples of the heteroatom groups include, but are not limited to, -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH-, or -NHC(=O)NH-. 3-6 membered heterocycloalkyl refers to a 3-, 4-, 5- or 6-membered heterocycloalkyl group, specific examples of which include but are not limited to aziridine, oxirane, thiirane, azetidinyl, oxetanyl, thietanyl, azolanyl, oxolanyl, thiolanyl, piperazinyl, piperidinyl, oxhexyl, morpholinyl, and 1,4-dioxanyl.

[0148] In the present invention, the letters used to represent amino acids or amino acid residues are G for glycine (Gly), F for phenylalanine (Phe), V for valine (Val), A for alanine (Ala), and Cit for citrulline.

[0149] The term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product resulting, directly or indirectly, from combination of the specified ingredients in the specified amounts. Those skilled in the art can modify the actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure so as to achieve the desired therapeutic response in the amount of the active compound obtained, tailored to the specific patient, composition, and route of administration.

[0150] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0151] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0152] The term "excipient" generally refers to a carrier, diluent, and / or vehicle required to formulate an effective pharmaceutical composition.

[0153] The term "effective amount" refers to an amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, to treat a disease or condition at a reasonable benefit / risk ratio applicable to any medical treatment. The amount of a compound of the present disclosure that constitutes an "effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and this disclosure.

[0154] In the present disclosure, the substituent Indicates the position of attachment of the substituent to the parent structure or other fragment. A dash "-" in a substituent structure is used to indicate the point of attachment of the substituent, for example, -CH3 indicates that the group is attached to the parent structure or other fragment through a C atom. Indicates the absolute configuration of a stereocenter, i.e., R or S configuration.

[0155] In this disclosure, chemical bonds depicted by solid and dashed lines Whether it represents a single bond or a double bond can be determined based on the valence of the atoms at both ends of the chemical bond within a range known to those skilled in the art.

[0156] In the present disclosure, the "isomers" include geometric isomers and stereoisomers, such as atropisomers, cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic mixtures and other mixtures thereof, all of which fall within the scope of the present disclosure. The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to a type of functional group isomer that has different hydrogen attachment points due to one or more double bond displacements, for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to a stereoisomer in which the molecule has two or more chiral centers and is not a mirror image of the other molecules. The term "cis-trans isomer" refers to different spatial configurations in which double bonds or single bonds of ring carbon atoms in a molecule cannot rotate freely. The term "atropisomer" refers to stereoisomers that can be separated because single bond rotation is hindered or rotates very slowly.

[0157] Stereoisomers of the disclosed compounds can be prepared through chiral synthesis, chiral reagents, or other conventional techniques. For example, one enantiomer of a compound disclosed herein can be prepared through asymmetric catalysis or chiral auxiliary derivatization techniques. Alternatively, a single stereoisomer of the compound can be obtained from a mixture using chiral resolution techniques. Alternatively, the enantiomers can be prepared directly using chiral starting materials. Separation of the optically pure compounds disclosed herein is typically accomplished using preparative chromatography, employing chiral chromatographic columns to achieve the purpose of separating chiral compounds.

[0158] In this disclosure, the solution concentration unit "M" represents mol / L, "mM" represents mmol / L, and "nM" represents nmol / L. The solution concentration unit "N" represents normality, which is expressed as the number of gram equivalents of solute contained in 1 liter of solution and is represented by the symbol N. For example, if 1 liter of concentrated hydrochloric acid contains 12.0 gram equivalents of hydrochloric acid (HCl), the concentration is 12.0N. Normality = gram equivalents of solute / solution volume (liters).

[0159] Trastuzumab used in the present disclosure was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. Those skilled in the art can also prepare it by referring to published literature.

[0160] The chemical abbreviations used in this disclosure and the chemical names they refer to are as follows: Example

[0161] The present disclosure is further described in detail below through specific preparation examples and biological experiments. However, it should be understood that these examples and biological experiments are only for specific illustration purposes and should not be understood as limiting the present disclosure in any form. It is clear to those skilled in the art that, hereinafter, unless otherwise specified, the materials used are well known in the art and can be purchased from the market or obtained by those skilled in the art according to published literature or conventional methods. Unless otherwise stated, all reactions of the present disclosure are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, and the solvent is a dry solvent, wherein: (i) the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature, which generally refers to 15-35°C, preferably 20-30°C, and more preferably 20-25°C; (ii) the removal of the solvent is carried out by reduced pressure evaporation on a rotary evaporator, and the bath temperature is not higher than 60°C; (iii) the reaction process is tracked by thin layer chromatography (TLC); (iv) the final product has a satisfactory hydrogen nuclear magnetic resonance spectrum ( 1 H-NMR) and / or mass spectrometry (MS) data.

[0162] Example 1 Preparation of Compound 1

[0163] Step 1:

[0164] 86.4 mL of 2,2,6,6-tetramethylpiperidine was added to a 3 L three-necked flask, followed by 1 L of anhydrous tetrahydrofuran. The system was cooled to -78°C and allowed to react for 15 minutes. 272 ​​mL of a 2.5 M solution of n-butyllithium in petroleum ether was added and allowed to react for 30 minutes. 32 g of compound L-1 was dissolved in 200 mL of anhydrous tetrahydrofuran and added to the system, allowing to react for 1 hour. 50 mL of 1-penten-3-one was dissolved in 200 mL of anhydrous tetrahydrofuran and added to the system, allowing to react for 1 hour. After TLC monitoring of the reaction, 640 mL of 4N hydrochloric acid was added and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium bicarbonate aqueous solution, saturated brine, and dried over anhydrous sodium sulfate. The system was concentrated in vacuo and purified by column chromatography to obtain 21 g of compound L-2 in a 49% yield. ESI-MS m / z: 253.68, [M+H] + .

[0165] 1 H NMR(400MHz, CDCl3) δ6.99(s,1H),6.13(dd,J=17.3,10.6Hz,1H),5.29(d,J=7.0Hz,1H),5.26(s,1H ),3.99(s,3H),2.73(s,2H),2.04(dt,J=14.8,7.3Hz,1H),1.98–1.87(m,1H),0.91(t,J=7.3Hz,3H).

[0166] Step 2

[0167] 21g of compound L-2 was added to a 1L three-necked flask, followed by 400mL of anhydrous ethanol. After cooling the system to 0°C, 14.21g of sodium borodeuteride was added, stirred for 15 minutes, and then warmed to room temperature for 12 hours. TLC monitored the reaction completion, and 30mL of 1N hydrochloric acid was added. The mixture was extracted with dichloromethane, and the organic phases were combined and washed sequentially with saturated sodium bicarbonate aqueous solution and saturated brine, and dried over anhydrous sodium sulfate. After vacuum concentration, the system was purified by column chromatography to obtain 17g of compound L-3 as a transparent viscous liquid, with a yield of 80%. ESI-MS m / z: 259.73, [M+H] + . 1 H NMR(400MHz, CDCl3) δ6.99(s,1H),6.13(dd,J=17.3,10.6Hz,1H),5.29(d,J=7.0Hz,1H),5.26(s,1H ),3.99(s,3H),2.73(s,2H),2.04(dt,J=14.8,7.3Hz,1H),1.98–1.87(m,1H),0.91(t,J=7.3Hz,3H).

[0168] Step 3:

[0169] 2 g of compound L-3 was added to a 250 mL round-bottom flask, along with 100 mL of dichloromethane. The system was cooled to -78°C and ozone was introduced. After TLC monitoring of the reaction, 0.5 mL of dimethyl sulfide was added, and the mixture was warmed to room temperature and stirred for 30 minutes. The system was concentrated in vacuo and purified by column chromatography to obtain 1.9 g of compound L-4 in a 95% yield. The product was used directly in the next reaction without further purification. ESI-MS m / z: 261.70, [M+H] + .

[0170] 1 H NMR (400MHz, CDCl3) δ7.15 (s, 1H), 5.19 (d, J = 4.8Hz, 1H), 3.96 (s, 3H), 3.21 (d, J = 4.8Hz, 1H), 2.64 (s, 1H), 1.80 (q, J = 7.5Hz, 2H), 0.92 (t, J = 7.5Hz, 3H).

[0171] Step 4:

[0172] 12g of compound L-4 was added to a 1L single-necked flask, followed by 240mL of dichloromethane. The mixture was stirred for 30 minutes and the temperature was lowered to 0°C. 286mg of 2,2,6,6-tetramethylpiperidinyl oxide, 616mg of sodium bicarbonate, 654mg of potassium bromide, and 18mL of water were added and stirred for 15 minutes. 120mL of aqueous sodium hypochlorite solution (>7.5wt%) was added and the mixture was allowed to react for 30 minutes. TLC monitored the reaction for completion, and 12g of sodium bisulfite was added. The mixture was extracted with dichloromethane. The organic phases were combined, washed with water, saturated brine, and dried over anhydrous sodium sulfate. The mixture was concentrated in vacuo and purified by column chromatography to yield 6.6g of compound L-5 in a 55% yield. ESI-MS m / z: 259.68, [M+H] + .

[0173] 1 H NMR (400MHz, CDCl3) δ7.20 (s, 1H), 4.00 (s, 3H), 3.64 (s, 1H), 1.79 (q, J = 7.4Hz, 2H), 0.97 (t, J = 7.4Hz, 3H).

[0174] Step 5:

[0175] Under a carbon monoxide atmosphere, 3g of compound L-5 was added to a 100mL three-necked flask. 286mg of 1,3-bis(diphenylphosphino)propane, 2.4g of potassium carbonate, 129mg of palladium acetate, 15mL of N,N-dimethylformamide, and 30mL of deuterated methanol were also added. The system was heated to 65°C and reacted for 12 hours. TLC monitored the reaction completion. 20mL of 1N hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed sequentially with saturated sodium bicarbonate aqueous solution and saturated brine, and dried over anhydrous sodium sulfate. The system was concentrated in vacuo and purified by column chromatography to obtain 1.33g of compound L-6 in a 40% yield. ESI-MS m / z: 286.29, [M+H] + .

[0176] 1 H NMR (400MHz, CDCl3) δ7.98 (d, J = 2.0 Hz, 1H), 4.09 (d, J = 2.0 Hz, 3H), 3.71 (d, J = 2.6 Hz, 1H), 1.88–1.75 (m, 2H), 0.96 (td, J = 7.3, 1.9 Hz, 3H).

[0177] Step 6:

[0178] 1 g of compound L-6 was added to a 50 mL two-necked flask, along with 1.05 g of sodium iodide and 10 mL of acetonitrile. The system was cooled to 0°C and stirred for 30 minutes. 0.89 mL of trimethylsilyl chloride was added, and the system was warmed to room temperature for 12 hours. TLC monitored the reaction for completion. 40 mL of water and 1 mL of saturated sodium bisulfite aqueous solution were added, and the mixture was stirred for 1 hour. The mixture was extracted with dichloromethane and dried over anhydrous sodium sulfate. The system was concentrated in vacuo and purified by column chromatography to yield 800 mg of compound L-7 in an 84% yield. ESI-MS m / z: 272.27, [M+H] + .

[0179] 1 H NMR (400MHz, CDCl3) δ10.03 (s, 1H), 7.32 (s, 1H), 3.90 (s, 1H), 1.81 (dd, J = 14.5, 7.2Hz, 2H), 0.99 (t, J = 7.4Hz, 3H).

[0180] Step 7:

[0181] 620 mg of compound L-7 was added to a 25 mL two-necked flask, along with 1.48 g of cesium carbonate, 8 mL of dimethyl sulfoxide, and 1.67 mL of tert-butyl acrylate. The system was heated to 50°C and reacted for 12 hours. TLC monitored the reaction for completion. 1 mL of concentrated hydrochloric acid was added, followed by 40 mL of water. The mixture was extracted with dichloromethane and dried over anhydrous sodium sulfate. After vacuum concentration, preparative SPC chiral separation (SFC-350 (Waters), column: AS 25*250 mm, 10 μm, mobile phase: CO2 / MEOH = 65 / 35, flow rate: 200 mL / min) afforded 468 mg of compound L-8 in a 55% yield. ESI-MS m / z: 365.38, [M+H] + .

[0182] Step 8:

[0183] 200 mg of compound L-8 was added to a 10 mL single-necked bottle, followed by 5 mL of toluene and 0.5 mL of trifluoroacetic acid. The system was heated to 110°C and reacted for 2 hours. TLC monitored the reaction completion. The system was concentrated in vacuo and purified by column chromatography to obtain 140 mg of compound L-9 in a 98% yield. ESI-MS m / z: 265.26, [M+H] + .

[0184] Step 9:

[0185] 300 mg of compound L-9 was added to a 25 mL single-necked bottle, along with 266 mg of N-(5-methyl-6-fluoro-8-amino-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide, 9 mL of toluene, 770 mL of o-cresol, and 133 mg of pyridinium p-toluenesulfonate. The system was heated to 110°C and reacted for 18 hours. TLC monitored the reaction for completion. The system was concentrated in vacuo and purified by column chromatography (dichloromethane:methanol = 10:1) to afford 443 mg of compound L-10, in an 81% yield.

[0186] Step 10:

[0187] 443 mg of intermediate compound L-10 was added to a 25 mL single-necked flask, followed by 7 mL of purified water and 2.2 mL of methanesulfonic acid. The atmosphere was replaced with nitrogen. The system was heated to 85°C and reacted for 10 hours. TLC monitored the reaction completion. The system was concentrated under vacuum and purified by column chromatography (dichloromethane:methanol) to afford 141 mg of compound 1 or its mesylate salt in a 35% yield. ESI-MS m / z: 437.47, [M+H] + .

[0188] 1 H NMR(400MHz,D2O)δ7.20–7.06(m,2H),5.42–5.14(m,5H),3.30(dd,J=18.1,4.0Hz,1H),3.06–2.91 (m,1H),2.69(s,2H),2.62–2.47(m,1H),2.18(s,3H),1.80(q,J=7.3Hz,2H),0.79(t,J=7.4Hz,3H).

[0189] Example 2 Preparation of Compound 2

[0190] To a mixture of anhydrous sodium sulfate (2.25 eq), ethyl 2-oximecyanoacetate (2.25 eq), 2-hydroxyacetic acid (1.45 eq), and the methanesulfonate salt of compound 1 (100 mg) was added 2.4 mL of water and 3.0 mL of tetrahydrofuran. After stirring at room temperature for 15 minutes, N-methylmorpholine (1.10 eq) was added. After stirring at room temperature for 15 minutes, EDCI (2 eq) was added and stirred at room temperature for 3 hours. The reaction of compound 1 was monitored by HPLC for completion. The mixture was filtered, the filter cake was air-dried, and then purified by silica gel column chromatography (DCM:MeOH) to obtain the title compound.

[0191] 1H NMR (400MHz, DMSO) δ8.41(d,J=8.9Hz,1H),7.77(d,J=11.0Hz,1H),7.30(s,1H),6.52(s,1H),5.58(d,J=5.4Hz,1H),5.49(t,J=5.7Hz,1H),5.20(t,J =13.9Hz,2H),3.96(d,J=5.5Hz,2H),3.28–3.05(m,2H),2.39(s,3H),2.29 –2.11(m,2H),1.96–1.78(m,2H),0.87(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :496.1.

[0192] Example 3 Preparation of Compound 3

[0193] Referring to the preparation method of Example 2, using (R)-2-cyclopropyl-2-hydroxyacetic acid as raw material, 52 mg of compound 3 was prepared with a yield of 52%.

[0194] 1 H NMR (400MHz, DMSO-d6) δ8.36(d,J=8.4Hz,1H),7.78(d,J=10.9Hz,1H),7.31(s,1H),6.52(s, 0H),5.58–5.48(m,1H),5.38(d,J=5.3Hz,1H),5.21(q,J=19.0Hz,2H),3.62(d,J=5.2Hz,1H), 3.16(dd,J=11.2,5.7Hz,3H),2.39(s,4H),2.22–2.12(m,3H),1.86(dt,J=14.3,7.1Hz,2H), 1.14(q,J=7.3Hz,2H),0.87(t,J=7.2Hz,3H),0.38(dd,J=20.9,5.6Hz,4H).MS(ESI)m / z[M+H] + :536.2.

[0195] Example 4 Preparation of Compound 4

[0196] Referring to the preparation method of Example 2, (S)-2-cyclopropyl-2-hydroxyacetic acid was used as the starting material to prepare 54 mg of compound 4 in a yield of 54%.

[0197] 1H NMR (400MHz, DMSO-d6) δ8.37(d,J=9.0Hz,1H),7.77(d,J=11.0Hz,1H),7.30(s,1H),6.51 (s,1H),5.58(q,J=6.7Hz,1H),5.49(d,J=5.2Hz,1H),5.35–5.06(m,2H),3.60(t,J=5.7Hz ,1H),3.17(q,J=17.2Hz,2H),2.39(s,3H),2.16(d,J=6.1Hz,2H),1.85(dq,J=14.2,7.1H z,2H),1.24(q,J=8.1Hz,1H),0.87(t,J=7.3Hz,3H),0.59–0.32(m,4H).MS(ESI)m / z[M+H] + :536.2.

[0198] Example 5 Preparation of Compound 5

[0199] Referring to the preparation method of Example 2, (R)-3-hydroxybutyric acid was used as the starting material to prepare 55 mg of compound 5 with a yield of 56%.

[0200] 1 H NMR (400MHz, DMSO-d6) δ8.42(d,J=8.7Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H),6.52(s, 1H),5.55(dt,J=9.3,5.0Hz,1H),5.29–5.12(m,2H),4.66(d,J=4.7Hz,1H),4.05(dt,J=12.1 ,6.0Hz,1H),3.25–3.09(m,2H),2.39(s,3H),2.33–2.17(m,2H),2.13(dd,J=10.7,5.4Hz,2H ),1.86(hept,J=7.1Hz,2H),1.09(d,J=6.2Hz,3H),0.87(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :524.2.

[0201] Example 6 Preparation of Compound 6

[0202] Referring to the preparation method of Example 2, (S)-3-hydroxybutyric acid was used as the starting material to prepare 51 mg of compound 6 with a yield of 52%.

[0203] 1H NMR (400MHz, DMSO-d6) δ8.41(d,J=8.7Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H),6.53(s ,1H),5.57(dt,J=8.8,4.5Hz,1H),5.22(d,J=2.7Hz,2H),4.64(d,J=4.5Hz,1H),4.05(dt,J =11.9,6.1Hz,1H),3.16(t,J=5.7Hz,2H),2.39(s,3H),2.32–2.17(m,2H),2.17–2.04(m,2H ),1.86(hept,J=7.1Hz,2H),1.08(d,J=6.1Hz,3H),0.87(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :524.2.

[0204] Example 7 Preparation of Compound 7

[0205] Compound 7-1 (87 mg, 1 eq), compound 7-2 (144 mg, 1.1 eq), pyridinium p-toluenesulfonate (50 mg, 0.4 eq), acetic acid (2.6 mL, 30 V), and toluene (2.6 mL, 30 V) were placed in a reaction flask and reacted at 110° C. for 18 hours. The reaction was complete as monitored by TLC. The solvent was removed by rotary evaporation, and the mixture was separated and purified by silica gel column chromatography (dichloromethane:methanol) to obtain 123 mg of compound 7 with a yield of 61%.

[0206] 1 H NMR (400MHz, DMSO-d6) δ7.72(d,J=9.0Hz,1H),7.29(d,J=9.0Hz,1H),7.17(s,1H),6.45(s,1H),5.65(s,2H),5.16(s,2H),3.04 (t,J=6.1Hz,2H),2.75(t,J=6.1Hz,2H),2.08–1.95(m,2H),1.85(hept,J=7.1Hz,2H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :406.1.

[0207] Example 8 Preparation of Compound 8

[0208] Referring to the preparation method of Example 7, 127 mg of compound 8 was prepared using compounds 8-1 and 7-2 as starting materials with a yield of 60%.

[0209] 1H NMR (400MHz, DMSO-d6) δ8.41(d,J=8.7Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H),6.53(s ,1H),5.57(dt,J=8.8,4.5Hz,1H),5.22(d,J=2.7Hz,2H),4.64(d,J=4.5Hz,1H),4.05(dt,J =11.9,6.1Hz,1H),3.16(t,J=5.7Hz,2H),2.39(s,3H),2.32–2.17(m,2H),2.17–2.04(m,2H ),1.86(hept,J=7.1Hz,2H),1.08(d,J=6.1Hz,3H),0.87(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :424.1.

[0210] Example 9 Preparation of Compound 9

[0211] Step 1:

[0212] 250 mg of compound 9-1, 300 mg of compound 7-2, 7.5 mL of acetic acid, 7.5 mL of toluene, and 68 mg of pyridinium p-toluenesulfonate were added to a 50 mL single-necked flask and reacted at 110°C for 18 hours. The reaction was monitored for completion by TLC. The solvent was evaporated under reduced pressure and the product was purified by silica gel column chromatography (DCM:MeOH) to obtain 386 mg of compound 9-3 in an 81% yield. MS (ESI) m / z [M+H] + :480.2.

[0213] Step 2:

[0214] 380 mg of compound 9-3 was added to a 100 mL single-necked bottle, along with 7.6 mL of purified water and 3.8 mL of methanesulfonic acid. The mixture was reacted at 85°C under nitrogen for 10 hours. TLC confirmed the reaction was complete, and the mixture was cooled to room temperature. 22.8 mL of methanol was added, stirred at room temperature for 2 hours, and filtered to obtain the crude product. This was then separated and purified using silica gel column chromatography (dichloromethane:methanol) to afford 148 mg of compound 9, in a 35% yield.

[0215] 1H NMR(400MHz,D2O)δ7.20–7.06(m,2H),5.42–5.14(m,5H),3.30(dd,J=18.1,4.0Hz,1H),3.06–2.91 (m,1H),2.69(s,2H),2.62–2.47(m,1H),2.18(s,3H),1.80(q,J=7.3Hz,2H),0.79(t,J=7.4Hz,3H). MS(ESI)m / z[M+H]+:438.2.

[0216] Example 10 Preparation of Compound 10

[0217] The preparation method of Example 9 was followed, and compound 10-1 and compound 7-2 were used as raw materials to prepare 70 mg of compound 10 with a yield of 34%.

[0218] 1 H NMR(400MHz,DMSO-d6)δ8.32(d,J=8.4Hz,1H),8.20(dd,J=8.4,1.2Hz,1H),7.93 -7.84(m,1H),7.79(t,J=7.6Hz,1H),7.35(s,1H),6.56(s,1H),5.42(s,2H),3.98 (p,J=6.7Hz,1H),3.50(t,J=8.0Hz,2H),3.42-3.35(m,2H),3.00(s,3H),1.88(he pt,J=7.3Hz,2H),1.15(d,J=6.7Hz,6H),0.88(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :514.2.

[0219] Example 11 Preparation of Compound 11

[0220] The preparation method of Example 9 was followed, and compound 11-1 and compound 7-2 were used as raw materials to prepare 80 mg of compound 11 with a yield of 37%.

[0221] 1H NMR (500MHz, DMSO-d6) δ0.88(t,J=7.25Hz,3H),1.32(t,J=7.5Hz,3H),1.85(m,2H),3.11(q,J=7.5Hz,2 H), 5.26 (s, 2H), 6.48 (s, 1H), 7.23 (s, 1H), 7.41 (d, J = 10.0Hz, 2H), 8.01 (d, J = 10.0Hz, 1H), 10.3 (s, 1H). MS(ESI)m / z[M+H] + :395.1.

[0222] Example 12 Preparation of Compound 12

[0223] 255 mg of compound 12-1, 300 mg of compound 7-2, 7.5 mL of acetic acid, 7.5 mL of toluene, and 68 mg of pyridinium p-toluenesulfonate were added to a 50 mL single-necked flask and reacted at 110°C for 18 hours. The reaction was monitored for completion by TLC. The solvent was evaporated under reduced pressure, and the mixture was purified by silica gel column chromatography (DCM:MeOH) to obtain 330 mg of compound 12-3, in a 70% yield.

[0224] 1 H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.30(s,2H),5.26(s,2H ),3.81(d,J=5.9Hz,2H),3.22(s,2H),1.98(d,J=6.7Hz,4H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :471.1.

[0225] 300 mg of intermediate compound 12-3 was dissolved in 15 mL of 10% sulfuric acid and reacted at 110°C for 48 hours. After TLC monitoring, the reaction was adjusted to neutrality by adding saturated aqueous sodium carbonate. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by silica gel column chromatography (DCM:MeOH) to obtain 89 mg of compound 12, a yield of 31%.

[0226] 1H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.32-5.19(m ,2H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.92–1.76(m,4H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :453.2.

[0227] Example 13 Preparation of Compound 13

[0228] Referring to the preparation method of Example 12, compound 13-1 and compound 7-2 were used as raw materials to prepare 74 mg of compound 13 with a two-step yield of 16%.

[0229] 1 H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.32-5.19(m ,2H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.94–1.76(m,6H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :467.2.

[0230] Example 14 Preparation of Compound 14

[0231] Step 1:

[0232] 213 mg of compound 14-1, 300 mg of compound 7-2, 7.5 mL of acetic acid, 7.5 mL of toluene, and 68 mg of pyridinium p-toluenesulfonate were added to a 50 mL single-necked flask and reacted at 110°C for 18 hours. The reaction was monitored for completion by TLC. The solvent was removed by evaporation, and the product was purified by silica gel column chromatography (DCM:MeOH) to obtain 300 mg of compound 14-3 in a 68% yield. MS (ESI) m / z [M+H]+: 443.1.

[0233] Step 2:

[0234] 300 mg of compound 14-3 was dissolved in 3 mL of N,N-dimethylformamide, and sodium azide (48 mg, 1.1 eq) was added. The mixture was allowed to react at 80°C for 16 hours. The reaction was monitored by TLC until complete. The mixture was cooled to room temperature, and 18 mL of water was added. The mixture was extracted with ethyl acetate. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and dried to dryness. This afforded 186 mg of crude compound 14-4, a 63% yield. MS (ESI) m / z [M+H]+: 436.1.

[0235] Step 3:

[0236] 186 mg of compound 14-4 was dissolved in 4 mL of tetrahydrofuran, and triphenylphosphine (134 mg, 1.2 eq) was added. The mixture was stirred at room temperature for 4 hours. The reaction was completed after monitoring by TLC. Hydrochloric acid (4 M, 1 mL) was added to the reaction system, and the mixture was reacted at 55° C. for 16 hours. The mixture was concentrated under reduced pressure and purified by reverse phase column (SEPA FLASH SW025, Spherical C18, 20-45 μm, The residue was separated and purified by mobile phase A: 0.05% formic acid / water, mobile phase B: acetonitrile; mobile phase A: mobile phase B = 40:60, flow rate 20 mL / min) to obtain 63 mg of compound 14, yield: 35%.

[0237] 1 H NMR(400MHz,DMSO-d6)δ8.62(s,2H),8.45(s,1H),7.50(s,1H),7.25(s,1H),6.48(s,1H),6.28(s,2H),5.41(s, 2H),5.20(s,2H),4.70(d,J=6.0Hz,2H),1.86(tt,J=14.2,6.1Hz,2H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :424.1.

[0238] Example 15 Preparation of Compound 15

[0239] Referring to the preparation method of Example 14, 50 mg of compound 15 was prepared using compounds 15-1 and 7-2 as starting materials with a three-step yield of 11%.

[0240] 1H NMR (400MHz, DMSO-d6) δ8.62(s,2H),8.45(s,1H),7.50(s,1H),7.25(s,1H),6.48(s,1H),6.28(s,2H),5.41(s,2H),5. 20(s,2H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.86(tt,J=14.2,6.1Hz,2H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :438.2.

[0241] Example 16 Preparation of Compound 16

[0242] 1 g of compound 16-1, 1.5 g of compound 7-2, 30 mL of acetic acid, 30 mL of toluene, and 340 mg of pyridinium p-toluenesulfonate were added to a 250 mL single-necked flask and reacted at 110°C for 18 hours. The reaction was monitored for completion by TLC. The solvent was removed and the product was purified by silica gel column chromatography (dichloromethane:methanol) to obtain 1.6 g of the target intermediate compound 16-3 in a 75% yield. MS (ESI) m / z [M+H]+: 431.1.

[0243] Compound 16-3 (1.6 g) was dissolved in 16 mL of N,N-dimethylformamide, and sodium azide (256 mg, 1.1 eq) was added. The mixture was allowed to react at 80°C for 16 hours. TLC confirmed the reaction was complete. The mixture was cooled to room temperature, and 96 mL of water was added. The mixture was extracted with ethyl acetate. The combined organic phases were washed with water, dried over anhydrous sodium sulfate, filtered, and dried to dryness. This afforded 1.1 g of crude compound 16-4 in a 68% yield. MS (ESI) m / z [M+H]+: 438.2.

[0244] 1.1 g of compound 16-4 was dissolved in 22 mL of tetrahydrofuran, and triphenylphosphine (0.79 g, 1.2 eq) was added. The mixture was stirred at room temperature for 4 hours. The reaction was completed after monitoring by TLC. Hydrochloric acid (4 M, 5 mL) was added to the reaction system, and the mixture was reacted at 55° C. for 16 hours. The mixture was concentrated under reduced pressure and separated and purified using a reverse phase column (instrument model SEPA FLASH SW025, chromatographic column: Spherical C18, 20-45 μm, Mobile phase A: 0.05% formic acid / water, mobile phase B: acetonitrile; mobile phase A: mobile phase B = 45:55, flow rate 20 mL / min), to obtain 0.58 g of compound 16, yield: 56%.

[0245] 1H NMR(400MHz, DMSO-d6)δ8.62(s,3H),8.43(d,J=8.0Hz,1H),7.99(d,J=10.7Hz,1H),7.35(s,1H),5.59(s,2H),4 .70(d,J=6.0Hz,2H),2.55(s,3H),1.88(hept,J=7.1Hz,2H),0.88(t,J=7.3Hz,3H).MS(ESI)m / z[M+H]+:412.2.

[0246] Example 17 Preparation of Compound 17

[0247] To a mixture of anhydrous sodium sulfate (2.25 eq), 2-hydroxyacetic acid (1.45 eq), and compound 16 (100 mg) were added 2.4 mL of water and 3.0 mL of tetrahydrofuran. After stirring at room temperature for 15 minutes, N-methylmorpholine (1.10 eq) was added. After stirring at room temperature for 15 minutes, EDCI (2 eq) was added and stirred at room temperature for 3 hours. The reaction of compound 16 was monitored by HPLC for completion. The product was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by spin drying. The product was then purified by silica gel column chromatography (DCM:MeOH) to obtain 82 mg of compound 17, a yield of 72%.

[0248] 1 H NMR (400MHz, DMSO) δ8.41(d,J=8.9Hz,1H),7.77(d,J=11.0Hz,1H),7.30(s,1H),6.52(s,1H),5.58(d,J=5.4Hz,1H),5.49(t,J=5. 7Hz,1H),5.20(t,J=13.9Hz,2H),3.96(d,J=5.5Hz,2H),2.55(s,3H),1.96–1.78(m,2H),0.87(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :470.2.

[0249] Example 18 Preparation of Compound 18

[0250] Referring to the preparation method of Example 17, using compound 3-1 and compound 16 as starting materials, 78 mg of compound 18 was prepared with a yield of 63%.

[0251] 1H NMR (400MHz, DMSO-d6) δ8.36(d,J=8.4Hz,1H),7.78(d,J=10.9Hz,1H),7.31(s,1 H),6.52(s,0H),5.58–5.48(m,1H),5.38(d,J=5.3Hz,1H),5.21(q,J=19.0Hz,2H ),3.62(d,J=5.2Hz,1H),2.39(s,4H),1.86(dt,J=14.3,7.1Hz,2H),1.14(q,J=7 .3Hz,2H),0.87(t,J=7.2Hz,3H),0.38(dd,J=20.9,5.6Hz,4H).MS(ESI)m / z[M+H] + :510.2.

[0252] Example 19 Preparation of Compound 19

[0253] Referring to the preparation method of Example 17, using compound 4-1 and compound 16 as starting materials, 74 mg of compound 19 was prepared with a yield of 60%.

[0254] 1 H NMR (400MHz, DMSO-d6) δ8.37(d,J=9.0Hz,1H),7.77(d,J=11.0Hz,1H),7.30( s,1H),6.51(s,1H),5.58(q,J=6.7Hz,1H),5.49(d,J=5.2Hz,1H),5.35–5.06( m,2H),3.60(t,J=5.7Hz,1H),2.39(s,3H),1.85(dq,J=14.2,7.1Hz,2H),1.24 (q,J=8.1Hz,1H),0.87(t,J=7.3Hz,3H),0.59-0.32(m,4H).MS(ESI)m / z[M+H] + :510.2.

[0255] Example 20 Preparation of Compound 20

[0256] Referring to the preparation method of Example 17, using compound 5-1 and compound 16 as starting materials, 70 mg of compound 20 was prepared with a yield of 58%.

[0257] 1H NMR(400MHz,DMSO-d6)δ8.42(d,J=8.7Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H) ,6.52(s,1H),5.55(dt,J=9.3,5.0Hz,1H),5.29–5.12(m,2H),4.66(d,J=4.7Hz,1H ),4.05(dt,J=12.1,6.0Hz,1H),2.39(s,3H),2.13(dd,J=10.7,5.4Hz,2H),1.86(h ept,J=7.1Hz,2H),1.09(d,J=6.2Hz,3H),0.87(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :498.2.

[0258] Example 21 Preparation of Compound 21

[0259] Referring to the preparation method of Example 17, using compound 6-1 and compound 16 as starting materials, 75 mg of compound 21 was prepared with a yield of 62%.

[0260] 1 H NMR (400MHz, DMSO-d6) δ8.41(d,J=8.7Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1 H), 6.53 (s, 1H), 5.57 (dt, J = 8.8, 4.5Hz, 1H), 5.22 (d, J = 2.7Hz, 2H), 4.64 (d, J = 4. 5Hz,1H),4.05(dt,J=11.9,6.1Hz,1H),2.39(s,3H),2.17–2.04(m,2H),1.86(he pt,J=7.1Hz,2H),1.08(d,J=6.1Hz,3H),0.87(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :498.2.

[0261] Example 22 Preparation of Compound LK1-1

[0262] Step 1:

[0263] Compound 22-2 (160 g, 1.0 eq) and 2.4 L of ethylene glycol dimethyl ether were added to a 10 L four-necked flask with mechanical stirring. Compound 22-1 (123.37 mL, 2.0 eq) was then added, stirring at 0°C. 10 mol / L sodium hydroxide (43.43 mL, 1.0 eq) was added dropwise using a constant pressure dropping funnel and stirred for 1 h. After the reaction was complete, as monitored by TLC, acetic acid (12.42 mL) was added, stirring for 1 h, followed by water (660 mL) and purified water (1.54 L). The mixture was stirred for 1 h and filtered. The filter cake was washed with 50% (v / v) ethylene glycol dimethyl ether aqueous solution (640 mL) and dried in an oven at 40°C to obtain 204.3 g of compound 22-3, a 99% yield. MS (ESI) m / z: 475.2 [M+H] + .

[0264] Step 2:

[0265] Compound 22-3 (180 g, 90%, 1.0 eq) was added to a 10 L four-necked flask, followed by 5.4 L of acetonitrile and mechanical stirring. DBU (25.5 mL, 0.5 eq) was added and stirred at room temperature for 4 h. HOBT (101.5 g, 2.2 eq) was added and stirred at room temperature for 0.5 h. The temperature was lowered to 0 ° C and stirred overnight. The mixture was filtered and the filter cake was washed with acetonitrile (800 mL) and dried in an oven at 40 ° C to obtain 119.0 g of compound 22-4 with a yield of 81%. 1 H NMR (400MHz, MeOD) δ7.69(dd,J=12.9,8.6Hz,2H),7.40–7.24(m,7H),5.19(s,2H),4.80(s,2H),4.21(s,2H),3.66(s,2H).

[0266] Step 3:

[0267] Compound 22-5 (55 g, 1.0 eq) was added to a 5L three-necked flask, dissolved in 600 mL of acetonitrile and 100 mL of water, and then compound 22-4 (50 g, 1.0 eq) was added. EDCI (24 g, 1.0 eq) was added with stirring at 0°C and stirred for 4 h. After the reaction was complete as monitored by TLC, 500 mL of ethanol and 750 mL of water were added, and the mixture was stirred at room temperature overnight. Subsequently, 1450 mL of water was added, stirred for 2 h, and filtered. The filter cake was dried in an oven at 40°C to obtain 80.0 g of compound 22-6, with a yield of 96%. MS (ESI) m / z: 648.3 [M+H] + .

[0268] Step 4:

[0269] Under nitrogen, compound 22-6 (40.0 g, 1.0 eq) was added with 840 mL of tetrahydrofuran and 540 mL of water. Pd (5%) / C (8.8 g, 0.07 eq) was then added to displace the hydrogen atmosphere. The mixture was stirred at room temperature overnight. After HPLC analysis of the reaction, the Pd / C was filtered out using celite and washed with 300 mL of water. The combined filtrates were concentrated under reduced pressure and then concentrated under reduced pressure with 400 mL of ethanol. This process was repeated three times. The mixture was then magnetically stirred with 800 mL of ethanol, filtered, and the filter cake dried in an oven at 40°C to yield 19.6 g of compound 22-7, with a yield of 75%. MS (ESI) m / z: 424.2 [M+H] + .

[0270] 1 H NMR (400MHz, D2O) δ7.31(ddd,J=22.4,14.6,7.3Hz,5H),4.72–4.56(m,3H),3.94(s,2H),3.93–3.73(m,6H),3.08(ddd,J=22.1,13.7,7.7Hz,2H).

[0271] Step 5:

[0272] To a 250 mL single-necked flask, succinimidyl 6-(maleimido)hexanoate (5.4 g, 1.5 eq) was added and dissolved in 45 mL of acetonitrile. Compound 22-7 (5.0 g, 1.0 eq), 105 mL of water, and DIPEA (1.56 mL, 0.8 eq) were added and stirred overnight at room temperature. After completion of the reaction as monitored by HPLC, 50 mL of isopropyl acetate, 10 g of anhydrous sodium dihydrogen phosphate, and 0.65 g of disodium hydrogen phosphate were added to the reaction mixture. The mixture was stirred for 0.5 h, and the organic phase was separated and removed. 50 mL of isopropyl acetate was added, and the organic phase was separated and removed. To the aqueous phase, 25 mL of ethylene glycol dimethyl ether, 25 mL of ethyl acetate, 2.5 mL of acetonitrile, and 40 g of anhydrous sodium dihydrogen phosphate were added. The mixture was stirred for 1 h, and the aqueous phase was separated. Repeat twice, add 75 mL of acetonitrile, 11.3 mL of water, 3 g of sodium chloride, and 750 mg of anhydrous sodium dihydrogen phosphate to the organic phase, stir, and then separate and remove the aqueous phase. The organic phase is concentrated under reduced pressure to 50 mL, and 75 mL of ethylene glycol dimethyl ether is added and concentrated to 50 mL. 1 mL of water and 100 mL of ethylene glycol dimethyl ether are then added, and the mixture is stirred at room temperature overnight. The filter cake is then filtered, and 200 mL of ethylene glycol dimethyl ether and 6.5 mL of water are added. Stir at 45°C for 0.5 h, rinse with 15 mL of a mixture of ethylene glycol dimethyl ether and water (v / v: 97 / 3), and the combined filtrates are concentrated to 100 mL. 25 mL of ethylene glycol dimethyl ether is added, and the mixture is stirred at room temperature overnight. The precipitated solid is rinsed with 25 mL of ethylene glycol dimethyl ether and dried under reduced pressure at 25°C to obtain compound 22-9 with a yield of 58%. MS (ESI) m / z: 617.3 [M+H]+ .

[0273] 1 H NMR (400MHz, DMSO) δ12.59(s,1H),8.56(t,J=6.5Hz,1H),8.31(t,J=5.5Hz,1H),8.13(d,J=8.0Hz,1H),8.08(t,J=5.2Hz,1 H),8.01(t,J=5.4Hz,1H),7.21(dt,J=23.3,6.0Hz,5H),7.00(s,2H),4.61(d,J=6.6Hz,2H),4.49(dd,J=12.4,8.7Hz,1H), 3.98(s,2H),3.78–3.69(m,3H),3.66(d,J=5.4Hz,2H),3.59(dd,J=16.8,5.3Hz,1H),3.42(s,4H),3.36(t,J=7.2Hz,3H),3 .23(s,6H),3.08–3.02(m,1H),2.84–2.76(m,1H),2.10(t,J=7.4Hz,2H),1.51–1.42(m,4H),1.20(dd,J=15.1,7.7Hz,2H).

[0274] Step 6:

[0275] In a 10 mL single-necked flask, anhydrous sodium sulfate (60 mg, 2.25 eq), ethyl 2-oximecyanoacetate (60 mg, 2.25 eq), compound 22-9 (20 mg, 1.45 eq), 0.8 mL of water, and 0.6 mL of tetrahydrofuran were added and stirred at room temperature for 30 min. Compound 22-10 (100 mg, 1.0 eq), 0.3 mL of water, and 0.5 mL of tetrahydrofuran were then added. After stirring for 15 minutes, N-methylmorpholine (23 uL, 1.1 eq) and 0.3 mL of tetrahydrofuran were added. After stirring for 15 minutes, EDCI (72 mg, 2.0 eq), 0.5 mL of water, and 0.5 mL of tetrahydrofuran were added. The mixture was stirred at room temperature for 3 hours, and the reaction was monitored for completion by HPLC. The mixture was extracted with dichloromethane and dried. The organic phases were combined and dried by rotary evaporation. Reverse phase preparative separation was performed using a Santai C18 column (20 g), 35-40% acetonitrile / water to obtain 99 mg of compound LK1-1, with a yield of 51%. MS (ESI) m / z: 1036.4 [M+H] + .

[0276] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J= 5.7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7 .75(d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2 H),6.51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz, 2H),4.53–4.42(m,1H),4.02(s,2H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3 .17(q,J=17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H) ,2.37(s,3H),2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1 Hz,2H),1.42(tt,J=14.3,7.4Hz,4H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0277] Example 23 Preparation of Compound LK1-2

[0278] Referring to the preparation method of Example 22, compound LK1-2 was prepared by replacing compound 22-1 (i.e., benzyl glycolate) with (R)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester. MS (ESI) m / z: 1076.4 [M+H] + .

[0279] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5. 7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75 (d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6. 51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.5 3–4.42(m,1H),3.79–3.53(m,7H),3.35(q,J=6.3Hz,2H),3.17(q,J=17.5Hz,2H),3 .02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H),2.18(dt,J= 12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1Hz,2H),1.42(tt,J=14.3,7 .4Hz,4H),1.17(m,3H),0.85(dt,J=14.7,7.3Hz,3H),0.38(dd,J=20.9,5.6Hz,4H).

[0280] Example 24 Preparation of Compound LK1-3

[0281] Referring to the preparation method of Example 22, (S)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester was substituted for compound 22-1 (i.e., benzyl glycolate) to prepare compound LK1-3. MS (ESI) m / z: 1076.4 [M+H] + .

[0282] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7 Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75(d, J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s ,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.4 2(m,1H),3.79–3.53(m,7H),3.35(q,J=6.3Hz,2H),3.17(q,J=17.5Hz,2H),3.02(dd ,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H),2.18(dt,J=12.0,6. 9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1Hz,2H),1.42(tt,J=14.3,7.4Hz,4H), 1.24(q,J=8.1Hz,1H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H),0.59–0.32(m,4H).

[0283] Example 25 Preparation of Compound LK1-4

[0284] Referring to the preparation method of Example 22, compound LK1-4 was prepared by replacing compound 22-1 (i.e., benzyl glycolate) with (R)-3-hydroxybutyrate. MS (ESI) m / z: 1064.4 [M+H] + .

[0285] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7H z,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75(d,J= 10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s,1H) ,5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m,1 H),4.05(dt,J=12.1,6.0Hz,1H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3.17(q,J =17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H) ,2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,4H),1.85(hept,J=7.1Hz,2H),1.42(tt, J=14.3,7.4Hz,4H),1.17(m,2H),1.09(d,J=6.2Hz,3H),0.85(dt,J=14.7,7.3Hz,3H).

[0286] Example 26 Preparation of Compound LK1-5

[0287] Referring to the preparation method of Example 22, (S)-3-hydroxybutyric acid benzyl ester was substituted for compound 22-1 (i.e., benzyl glycolate) to prepare compound LK1-5. MS (ESI) m / z: 1064.4 [M+H] + .

[0288] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7H z,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75(d,J= 10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s,1H) ,5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m,1 H),4.05(dt,J=11.9,6.0Hz,1H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3.17(q,J =17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H) ,2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,4H),1.85(hept,J=7.1Hz,2H),1.42(tt, J=14.3,7.4Hz,4H),1.17(m,2H),1.08(d,J=6.1Hz,3H),0.85(dt,J=14.7,7.3Hz,3H).

[0289] Example 27 Preparation of Compound LK1a-1

[0290] Step 1:

[0291] To a 250mL four-necked flask, add glycine (3g, 1.00eq), 90mL of tetrahydrofuran, and 30mL of purified water. Add sodium bicarbonate (3.70g, 1.10eq) and mechanically stir until the solution is clear. Dissolve compound 27-1 (12.8g, 1.00eq) in DME (30mL) and slowly add it using a constant pressure dropping funnel. Stir overnight at room temperature. Add 100mL of saturated sodium bicarbonate and 100mL of purified water, wash three times with 100mL of ethyl acetate, and slowly add 12M hydrochloric acid dropwise to adjust the pH to 4. Filter, rinse the filter cake with water, and dry to obtain 10.1g of compound 27-2 in a 98% yield. MS (ESI) m / z: 281.1[M+H] + .

[0292] Step 2:

[0293] To a 500mL four-necked flask, add L-phenylalanine tert-butyl ester hydrochloride (6.5g, 1.00eq) and compound 27-2 (7.8g, 1.1eq) to 200mL of N,N-dimethylformamide, triethylamine (3.5mL, 1.00eq), and DMTMM (8.9g, 1.20eq). Stir at room temperature for 2 hours. The reaction mixture is added to 1L of purified water, filtered, and the filter cake is rinsed with water to obtain 11g of compound 27-3 in a 90% yield. MS (ESI) m / z: 484.2 [M+H] + .

[0294] Step 3:

[0295] Compound 27-3 (10 g, 1.0 eq), 100 mL of dichloromethane, and trifluoroacetic acid (50 mL, 30.00 eq) were added to a 250 mL four-necked flask and stirred at room temperature for 2 hours. The reaction mixture was concentrated, slurried with MTBE, filtered, and the filter cake rinsed with water to obtain 8.5 g of compound 27-4, with a yield of 96%. MS (ESI) m / z: 428.2 [M+H] + .

[0296] Step 4:

[0297] Referring to the preparation method of Example 22, N-((benzyloxy)carbonyl)-N-methylglycylglycyl-L-phenylalanine (i.e., compound 27-4) was used instead of N-(benzyloxy)carbonylglycylglycyl-L-phenylalanine (i.e., compound 22-5) to prepare LK1a-1. MS (ESI) m / z: 1050.4 [M+H] + .

[0298] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J =5.7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),7.75(d,J=10.9Hz,1H ),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s,1H),5. 59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m ,1H),4.02(s,2H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3.17(q,J=17.5Hz ,2H),3.02(dd,J=13.7,4.3Hz,1H),2.97(s,3H),2.77(dd,J=13.7,9.7Hz,1H),2 .37(s,3H),2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1H z,2H),1.42(tt,J=14.3,7.4Hz,4H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0299] Example 28 Preparation of Compound LK1b-1

[0300] Referring to the preparation method of Example 22, 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (CAS No.: 2813270-39-0) was used instead of compound 22-8 to prepare LK1b-1. MS (ESI) m / z: 1093.4 [M+H] + .

[0301] 1H NMR(400MHz, DMSO-d6)δ9.10(s,2H),8.64(t,J=6.6Hz,1H),8.51(d,J=8.8Hz,1H),8.31(t,J=5.7Hz,1H),8.26–8.00(m,3H),7.77(d,J=10.9Hz, 1H),7.36–7.10(m,6H),6.52(s,1H),5.67–5.52(m,1H),5.19(s,2H),4. 64(d,J=6.5Hz,2H),4.47(dt,J=12.9,6.7Hz,1H),4.02(s,2H),3.77–3.6 8(m,4H),3.63–3.57(m,1H),3.40(s,3H),3.17(td,J=12.8,11.5,6.7Hz,2H),3.02(dd,J=13.7,4.2Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.38( s,3H),2.32(t,J=7.3Hz,2H),2.17(dd,J=13.4,6.8Hz,2H),2.08(s,1H) ,1.84(dq,J=18.9,7.0Hz,4H),1.28–1.22(m,2H),0.87(t,J=7.2Hz,3H).

[0302] Example 29 Preparation of Compound LK1c-1

[0303] Referring to the preparation method of Example 22, LK1c-1 was prepared by replacing compound 22-8 with 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycine ester (CAS No.: 1956326-21-8). MS (ESI) m / z: 1093.4 [M+H] + .

[0304] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7 Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.96(t ,J=5.5Hz,1H),7.75(d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5 H),6.99(s,2H),6.51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d ,J=6.5Hz,2H),4.53–4.42(m,1H),4.02(s,2H),3.80–3.53(m,8H),3.35(q,J=6.3Hz ,2H),3.17(q,J=17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz ,1H),2.37(s,3H),2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7 .1Hz,2H),1.42(tt,J=14.3,7.4Hz,4H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0305] Example 30 Preparation of Compound LK1d-1

[0306] Step 1;

[0307] Step 2:

[0308] Referring to the preparation method of Example 22, LK1d-1 was prepared by replacing compound 22-8 with 2,5-dioxo-1-pyrrolidinyl 3-[2-[2-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)ethoxy]ethoxy]propanoate (CAS: 1433997-01-3). MS (ESI) m / z: 1082.4 [M+H] + .

[0309] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t ,J=5.5Hz,1H),7.75(d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7. 2,6.7Hz,5H),6.99(s,2H),6.51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27 –5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m,1H),4.02(s,2H),3.7 9–3.53(m,16H),3.17(q,J=17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2. 77(dd,J=13.7,9.7Hz,1H),2.37(s,3H),2.28(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1Hz,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0310] Example 31 Preparation of Compound LK1b-1

[0311] Step 1:

[0312] To a 250 mL single-necked flask, 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (10.0 g, 1.0 eq) and N-hydroxysuccinimide (5.15 g, 1.2 eq) were added and dissolved in 100 mL of dichloromethane. N,N'-dicyclohexylcarbodiimide (10.0 g, 1.3 eq) was added and stirred at room temperature overnight. After TLC monitoring, the reaction was complete, filtered, and the filtrate was concentrated under reduced pressure to obtain 14.0 g of compound 31-2. This was used directly in the next step without further isolation and purification. MS (ESI) m / z: 366.36 [M+H] + .

[0313] To a 250 mL single-necked flask, 31-2 (13.6 g, 1.0 eq) was added and dissolved in 135 mL of acetonitrile. Compound 31-3 (18.9 g, 1.2 eq), 270 mL of water, and DIPEA (5.3 mL, 0.8 eq) were then added and stirred at room temperature overnight. After completion of the reaction as monitored by HPLC, the reaction solution was extracted three times with 300 mL of ethyl acetate. 10 g of anhydrous sodium dihydrogen phosphate was added to the aqueous phase, and the aqueous phase was extracted three times with 300 mL of ethylene glycol dimethyl ether. The ethylene glycol dimethyl ether was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the initial product. The crude product was separated using reverse phase preparative separation (acetonitrile / water) to obtain 18.6 g of compound 31-4, with a yield of 74%.

[0314] In a 250 mL single-necked flask, compound 31-4 (8.3 g, 1.4 eq), compound 31-5 (4.9 g, 1.0 eq), and 175 mL of DMF were added and stirred at room temperature. Triethylamine (1.9 mL, 1.6 eq) and DMTMM (3.87 g, 1.5 eq) were then added. Stirring was continued at room temperature for 1 hour. The reaction was monitored for completion by HPLC. The reaction mixture was added to 500 mL of water, filtered, and dried to obtain 12 g of crude product. The crude product was purified by column chromatography to obtain 8.9 g of compound LK1b-1 in an 85% yield.

[0315] MS (ESI) m / z: 1093.4 [M+H] +.

[0316] 1H NMR(400MHz, DMSO-d6)δ9.10(s,2H),8.64(t,J=6.6Hz,1H),8.51(d,J=8.8Hz,1H),8.31(t,J=5.7Hz,1H),8.26–8.00(m,3H),7.77(d,J=10.9Hz, 1H),7.36–7.10(m,6H),6.52(s,1H),5.67–5.52(m,1H),5.19(s,2H),4. 64(d,J=6.5Hz,2H),4.47(dt,J=12.9,6.7Hz,1H),4.02(s,2H),3.77–3.6 8(m,4H),3.63–3.57(m,1H),3.40(s,3H),3.17(td,J=12.8,11.5,6.7Hz,2H),3.02(dd,J=13.7,4.2Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.38( s,3H),2.32(t,J=7.3Hz,2H),2.17(dd,J=13.4,6.8Hz,2H),2.08(s,1H) ,1.84(dq,J=18.9,7.0Hz,4H),1.28–1.22(m,2H),0.87(t,J=7.2Hz,3H).

[0317] Example 32 General Preparation Method for Antibody Drug Conjugates

[0318] The antibody was exchanged into 10mM phosphate buffer at pH 7.0 by desalting chromatography or ultrafiltration, and the antibody concentration was determined using UV method. 10mM TCEP solution (6-10 equivalents) was added to the antibody solution, mixed, and reduced at 37°C for 3 hours. The reaction solution was then cooled to 15-25°C in a water bath, and a 10mM dimethyl sulfoxide solution (10-20 equivalents) of the drug-linker conjugate was added, mixed, and reacted for 1.5-2 hours. After the coupling reaction is complete, 100mM N-acetyl-cysteine ​​solution was added at a quenching ratio of 20:1 (quencher: antibody), shaken, and quenched at room temperature for 20 minutes to terminate the coupling reaction. Use desalting chromatography or ultrafiltration to replace the ADC buffer with 20 mM His-HAc buffer at pH 5.5. Filter through a 0.22 μm PES filter to obtain the ADC stock solution. Aliquot according to experimental requirements and store frozen at -20°C or -40°C. Determine the antibody concentration using either UV or Lowry methods, depending on the specific properties of the ADC. UV absorbance was measured at 280 nm and 370 nm, respectively, to calculate the DAR value, n. The prepared compounds and their DAR results are shown in the table below.

[0319] The antibody drug conjugates and payload-linker conjugates provided in the present disclosure can be prepared based on the relevant compound preparation methods described in the present disclosure with reference to the relevant preparation methods described in WO2014057687A, WO2020063676A, WO2022068878, etc.

[0320] Biological activity test

[0321] 1. In vitro anti-tumor activity assay of the compounds disclosed herein

[0322] (1) In vitro anti-tumor activity detection of the loaded compound disclosed herein

[0323] Purpose of the experiment: To detect the in vitro inhibitory activity of the loaded compounds disclosed herein against NCI-N87 (human gastric cancer cells), Calu-3 (human lung adenocarcinoma cells), MDA-MB-453 (human breast cancer cells), KPL-4 (human breast cancer cells), or MDA-MB-468 (human breast cancer cells).

[0324] Take tumor cells NCI-N87 (source: ATCC, catalog number: CRL-5822), Calu-3 (source: ATCC, catalog number: HTB-55), MDA-MB-453 (source: ATCC, catalog number: HTB-131), KPL-4 (source: Nanjing Kebai Biological, catalog number: CBP60379) or MDA-MB-468 (source: ATCC, catalog number: HTB-132) in the logarithmic growth phase and add them to the cell plate at a number of 5000 cells / well. Place the cell plate in a 37°C, 5% CO2 cell culture incubator and incubate for 12-16 hours. Add 100 μl of sample to each well (starting at 10 μM, 4-fold dilution, 10 concentrations), shake gently and place in the incubator for incubation. After incubation for 48 hours, add 70 μL CellTiter-Glo TM (Promega, Cat. No. G7572) working solution was added, gently shaken to lyse the cells, and the plate was read on a microplate reader. The cell proliferation inhibition rate was calculated as follows: Cell proliferation inhibition rate = (1 - sample well / control well) × 100%. GraphPad Prism 8.0 software was used to plot the logarithm of sample concentration against cytotoxicity % on the ordinate. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC value for each sample. 50 The specific results are shown in Table 1-1 and Table 1-2.

[0325] Table 1-1 In vitro activity test results of the loaded compounds disclosed herein

[0326] Table 1-2 In vitro activity test results of the loaded compounds disclosed herein

[0327] The experimental results show that the loaded compound disclosed herein has significant proliferation inhibitory activity against NCI-N87, Calu-3, MDA-MB-453, KPL-4 or MDA-MB-468.

[0328] (2) In vitro anti-tumor activity detection of the ADC compounds disclosed herein

[0329] Experimental purpose: To detect the in vitro inhibitory activity of the ADC compounds disclosed herein against NCI-N87 (human gastric cancer cells), KPL-4 (human breast cancer cells) or MDA-MB-468 (human breast cancer cells).

[0330] Take tumor cells NCI-N87, KPL-4 (source: ATCC, catalog number: ATCC-477) or MDA-MB-468 in the logarithmic growth phase and add 2000 cells / well to the cell plate. Incubate the cell plate in a 37°C, 5% CO2 cell culture incubator for 12-16 hours. Add 100ul of sample (starting at 100μg / ml, 5-fold dilution, 9 concentrations) to each well, shake gently, and place in the incubator for incubation. After incubation for 144 hours, add 70μL CellTiter-Glo TM (Promega, Cat. No. G7572) working solution was added, gently shaken to lyse the cells, and the plate was read on a microplate reader. The cell proliferation inhibition rate was calculated as follows: Cell proliferation inhibition rate = (1 - sample well / control well) × 100%. GraphPad Prism 8.0 software was used to plot the logarithm of sample concentration against cytotoxicity % on the ordinate. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC value of each test sample. 50 The specific results are shown in Table 1-3.

[0331] Table 1-3 In vitro anti-tumor activity test of the ADC compounds disclosed herein

[0332] Conclusion: The ADC drugs disclosed in the present invention have obvious proliferation inhibitory activity against HER2-positive cells KPL-4 and NCI-N87; at the same time, they have weak proliferation inhibitory activity against Her2-negative cells MDA-MB-468, showing good selectivity.

[0333] 2. Pharmacokinetics of the payload compounds disclosed herein in mice

[0334] Experimental purpose: To evaluate the metabolic kinetics of the cargo compound in the present disclosure in mice.

[0335] Balb / c mice were randomly divided into 12 groups (half male and half female) and injected once with the sample (Formulation A: 5% DMSO + 95% (15% sulfobutyl-β-cyclodextrin-normal saline); Formulation B: 5% DMSO-10% Solutol HS15-85% normal saline, diluted according to the dose). Plasma was collected at different points before administration and 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 24 hours after administration. The plasma sample concentrations were detected by LC-MS, and the pharmacokinetic parameters were calculated as shown in Table 2-1.

[0336] Table 2-1 PK parameters of the disclosed payload compounds in mice

[0337] The experimental results show that the payload compound of the present disclosure has a shorter in vivo half-life in mice. When it is used as a payload compound for ADC, the ADC can be cleared more quickly when the payload is shed in the body, and has better in vivo safety.

[0338] 3. Pharmacokinetics Experiment - Rats

[0339] (1) Pharmacokinetics of the Loaded Compounds of the Present Disclosure in Rats

[0340] Experimental purpose: To evaluate the metabolic kinetics of the cargo compound in the present disclosure in rats.

[0341] SD rats (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups of 6 rats per group, half male and half female, and injected once with the sample (preparation A is: 5% DMSO + 95% (15% sulfobutyl-β-cyclodextrin-normal saline), diluted according to the dose). Plasma was collected at cross points before administration and 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 24 hours after administration. The plasma sample concentrations were detected by LC-MS, and the pharmacokinetic parameters were calculated. The results are shown in Table 3-1.

[0342] Table 3-1 PK parameters of the disclosed loading compounds in rats

[0343] The experimental results show that the cargo compound disclosed in the present invention also has a shorter in vivo half-life and a faster in vivo clearance rate in rats.

[0344] (2) Pharmacokinetic study of the ADC drug disclosed in the present invention in rats

[0345] The purpose of the experiment was to detect the pharmacokinetic characteristics of the ADC drug in the present disclosure in SD rats.

[0346] SD rats (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups of 4 rats / group, half male and half female, and injected once with the sample (the test solution was prepared with an appropriate volume of normal saline). Blood samples were collected before administration and 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 336 hours, 504 hours, and 672 hours after administration. The concentrations of total antibody and conjugated mode in plasma samples were determined by ELISA; the concentration of free toxin in plasma samples was determined by LC-MS / MS. Pharmacokinetic parameters for different detection formats were calculated using WinNonlin software. Specific results are shown in Tables 3-2, 3-3, and 3-4.

[0347] Table 3-2 Pharmacokinetic properties of ADC compounds disclosed herein in rats

[0348] Table 3-3 Pharmacokinetic properties of ADC compounds disclosed herein in rats

[0349] Table 3-4 Pharmacokinetic properties of ADC compounds disclosed herein in rats

[0350] The experimental results show that the ADC compound disclosed in the present invention has a higher exposure amount and a lower in vivo clearance rate in rats.

[0351] 4. In vitro plasma stability

[0352] (1) In vitro plasma stability of the cargo compound disclosed herein

[0353] Take 398 μL of plasma, preheat to 37°C, and incubate for 15 minutes. Add 2 μL of sample solution to the plasma to a final concentration of 5 μM. Incubate the solution at 37°C. At 0, 15, 30, 60, and 120 minutes, take 50 μL of the solution and add it to 450 μL of ice-cold acetonitrile (containing the internal standard). Vortex the solution for 10 minutes, centrifuge, and determine the remaining amount of unchanged substrate by LC-MS / MS. See Table 4-1 for detailed results.

[0354] Table 4-1 Plasma stability of the cargo compounds disclosed herein

[0355] The experimental results show that the loaded compound disclosed in the present invention has good plasma stability.

[0356] (2) Human plasma stability of the ADC drug disclosed herein

[0357] The purpose of the experiment is to detect the stability of the ADC drug disclosed in the present invention in human plasma.

[0358] ADC samples, healthy human plasma, and 1% BSA (prepared in the laboratory using BSA powder purchased from Shanghai Sangon Biotechnology Co., Ltd.) were sterilized by filtration using a 0.22 μm filter. The ADC samples were added to the sterile plasma at a final concentration of 100 μg / ml and incubated in a 37°C cell culture incubator. The day of incubation was designated as day 0. Samples were then removed on day 21 for toxin release.

[0359] Free toxin assay: Add 15 μL of sample and 60 μL of acetonitrile to a centrifuge tube, vortex to mix, and centrifuge at 13,000 rpm for 15 minutes. Add 30 μL of supernatant and 30 μL of deionized water to a new centrifuge tube, vortex to mix, and perform LC-MS / MS analysis on a Shimadzu LC-MS 8050 triple quadrupole liquid chromatography-mass spectrometry (LC-MS / MS). Specific experimental results are shown in Table 4-2.

[0360] Table 4-2 Free toxin release rate of the ADC compounds disclosed in the present invention in human plasma on day 21

[0361] The results showed that the ADC drugs disclosed in the present invention had good stability in human plasma.

[0362] 5. Efficacy evaluation of the ADC compounds disclosed herein in NCI-N87 tumor-bearing mice

[0363] BALB / c Nude mice (purchased from Beijing Weitonglihua) were used as test animals to evaluate the efficacy of anti-Her2-ADC after tail vein injection on nude mice bearing human gastric cancer cell NCI-N87 xenografts.

[0364] Mice were subcutaneously inoculated with NCI-N87 cells (source: ATCC, catalog number: CRL-5822) (5×10 6 The tumors grew for 11 days and reached a size of 160.1±24.7 mm. 3 The animals were then randomly divided into groups (d1), 5 animals per group.

[0365] A single dose was administered via tail vein injection. Tumor volume and body weight were measured twice weekly and recorded. Tumor inhibition rate (%) = (Crtv - Trtv) / Crtv (%), where Crtv and Trtv represent the relative tumor volumes of the blank control group (vehicle, PBS) and the experimental group at the end of the experiment, respectively. For detailed experimental results at the end of the experiment on day 28, see 5-1.

[0366] Table 5-1 In vivo efficacy evaluation of the ADC compounds disclosed herein

[0367] 6. Repeated-dose toxicity study in rats

[0368] (1) Repeated administration toxicity test of the loaded compound of the present disclosure in rats

[0369] Preparation of test sample: Weigh the test sample according to the predetermined weight into a glass bottle; first add 1 mL of DMSO and mix thoroughly. After the test sample is dissolved, add 1 mL of Solutol HS-15 and mix thoroughly. After that, add 8 mL of sodium chloride injection and vortex to mix thoroughly to obtain a clear and transparent solution.

[0370] SD rats (purchased from Beijing Weitonglihua) were used as test animals and were injected with compound 2 and positive control Dxd via tail vein for 7 consecutive days. Both compound 2 and positive control Dxd were set at two doses of 0.3 mg / kg and 1.0 mg / kg. During the adaptation period, cage-side observation was performed once a day; during the dosing period, all animals were clinically observed once in the morning and once in the afternoon. The reactions of the skin, fur, eyes (sclera), ears, nose, mouth, chest, abdomen, urogenital area, limbs, as well as breathing, movement, urination, defecation (urine and feces color, etc.) and behavioral changes were recorded. The daily weighing data of the animals in the main test group were recorded; for animals planned for dissection, the final body weight was measured before dissection (fasting). The food intake of the test animals was also recorded. The specific results are shown in Table 6-1.

[0371] Table 6-1 Results of repeated administration toxicity experiments of the disclosed loaded compounds in rats

[0372] Among them, ♂ represents male and ♀ represents female;

[0373] After the administration, gross autopsy was performed on D8 after the administration, and focal / punctate dark red discoloration was found in the lungs of some rats that died in the Dxd control group, while this symptom was not found in the groups loaded with compound 2.

[0374] The above experimental data show that the loading compound 2 of the present disclosure has lower in vivo toxicity and better in vivo safety than the positive control Dxd.

[0375] (2) Repeated-dose toxicity study of ADC compounds in rats

[0376] SD rats were used as test animals to study the toxicity of the ADC compounds disclosed herein after repeated administration to SD rats (5 females and 5 males / group). The dosing regimen was: intravenous injection at a dose of 200 mg / kg, once every three weeks, for a total of three doses. The status of the animals was observed on the day of administration, including whether they were dead, dying, feces, appearance, respiration, fur, activity, etc. Body weight and food intake were measured twice a week after administration. Approximately 0.2 mL of blood was collected from the jugular sinus of the animals on the 7th, 16th, 28th, and 50th days after administration into anticoagulant tubes containing an anticoagulant (EDTA-K2). All animals scheduled for autopsy were fasted the night before the autopsy on the 50th day for approximately 12 hours. Moribund animals were euthanized, and approximately 2 mL of blood was collected from the abdominal aorta into anticoagulant tubes containing an anticoagulant (EDTA-K2) during autopsy. Hematological indicators were measured using a Sysmex XN-1000 hematology analyzer. The changes in some hematological indicators measured on the 7th day are shown in Table 6-2.

[0377] Table 6-2 Changes in hematological parameters on day 7;

[0378] Note: ↓ indicates the degree of decrease compared with the control group rats; / indicates no significant change compared with the control group rats;

[0379] The experimental results showed that at the same dose, the degree of decrease in WBC (white blood cell count), #LYMPH (lymphocyte percentage), #MONO (monocyte count), #NEUT (neutrophil count), and #EO (eosinophil percentage) of animals in the ADC1-LK1b-1 dose group was lower than that of ADC1-LK1-Dxd, indicating that the ADC compound disclosed herein has lower hematological toxicity and better safety.

[0380] In the present disclosure, the comparative compound LK1-Dxd refers to the following compound, which can be prepared according to the published document WO2014057687A:

Claims

1. The compound represented by formula I or its pharmaceutically acceptable salt, or its stereoisomer: in, Ab represents an antibody or antigen-binding fragment; L represents a linker connecting Ab and the warhead drug molecule; n is selected from 1-12; R1 is selected from H, halogen, OH, SH, NH2, C 1-4 Alkyl, C 1-4 Haloalkyl or C 1-4 Alkoxy; R2 is selected from H, halogen, C 1-4 Alkyl or C 1-4 Alkoxy; Or R1 and R2 are cyclized to -O-(CH2) m -O-, wherein m is selected from 1, 2, 3; R3 and R4 are each independently selected from H, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Haloalkyl or C 1-4 Haloalkoxy; Or R3 and R4 cyclize to -(CH2) k -, wherein k is selected from 1, 2, 3, 4; X is selected from H, OH, HO-CH(R5)-(CH2) p -CO-NH- or -N(R6)(R7), wherein p is selected from 0, 1, 2; R5 is selected from H, C 1-4 Alkyl, C 1-4 Haloalkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl; R6 is selected from H, C 1-4 Alkyl or C 1-4 Haloalkyl; R7 is selected from H or R8-S(O)2-; R8 is selected from C 1-4 Alkyl; and t is selected from 0, 1, 2, 3, 4, 5.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R1 is C 1-4 alkyl, and R2 is halogen; or R1 is methyl, and R2 is F; or R1 is H, R2 is H; or R1 and R2 are cyclized to -O-CH2-O-; or R1 is NH2, and R2 is H or halogen; or R1 is NH2, and R2 is H or F.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R3 is H and R4 is H; or R3 is H and R4 is C 1-4 Alkyl; R3 is H, R4 is methyl; or R3 and R4 are cyclized to form -CH2-CH2-.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein X is HO-CH(R5)-(CH2) p -CO-NH-, wherein p is selected from 0, 1, 2; or X is selected from OH or NH2; or X is H, and t is 0; or X is -N(R6)(R7).

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein n is selected from 4-9; or n is selected from 6-8, or 7-8, or 7.4-8.0, or 7.5-8.5; or n is selected from 6, 7, 8; or n is selected from 7-8, or 7.3-7.9, or 7.4-7.8, or 7.5-7.7, or 7.5-7.6, or 7.5-7.8, or 7.4-7.

7.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, which is a compound of formula Ia, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; in, R1, R2, R3, R4 are as defined in any one of claims 1 to 3; X1 is selected from a chemical bond, -O-CH(R5)-(CH2) p -CO-, wherein the -CO- terminal is connected to -NH-; p is selected from 0, 1, 2; R5 is selected from H, C 1-4 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl.

7. The compound according to claim 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R1 is selected from methyl or methoxy; or, R2 is selected from F or Cl; or, R1 is methyl and R2 is F; or, R3 and R4 are cyclized to -(CH2) k -, wherein k is 2; or, R3 is H; and R4 is H.

8. The compound according to any one of claims 6 to 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein X1 is -O-CH(R5)-(CH2) p -CO-, p is selected from 0 or 1, R5 is selected from H, C 1-4 alkyl or 3-6 membered cycloalkyl; or, X1 is -O-CH(R5)-CO-, R5 is selected from H or 3-6 membered cycloalkyl; or, X1 is -O-CH(R5)-CO-, R5 is selected from H or cyclopropyl; or, X1 is -O-CH(R5)-CH2-CO-; R5 is C 1-4 Alkyl, or, X1 is -O-CH(R5)-CH2-CO-, R5 is methyl; or, X1 is selected from the following groups: in, The -CO- terminal of X1 is connected to NH.

9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, which is a compound of formula Ia-1 or formula Ia-2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; in, Ab, L, R1, R2, X1, and n are as defined in any one of claims 1 to 8.

10. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein Selected from the following compound fragments: in Indicates the position connected to the linker L through a chemical bond.

11. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, which is a compound of formula Ib, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; in, Ab, L, n as defined in claim 1; R1 is selected from H, OH, halogen, NH2, C 1-4 Alkyl, C 1-4 Haloalkyl or C 1-4 Alkoxy; R2 is selected from H, halogen, C 1-4 Alkyl or C 1-4 Alkoxy; Or R1 and R2 are cyclized to -O-(CH2) m -O-, wherein m is selected from 1, 2, 3; R3 and R4 are each independently selected from H, C 1-4 Alkyl or C 1-4 Alkoxy; Or R3 and R4 cyclize to -(CH2) k -, wherein k is selected from 1, 2, 3; X2 is selected from O or -N(R6)-; R6 is selected from H or C 1-4 alkyl; q is selected from 0, 1, 2, 3, 4.

12. [Corrected 10.01.2025 according to Rule 91] A compound according to claim 11 or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R1 is selected from methyl and R2 is selected from H, Cl, F; or R1 and R2 are cyclized to -O-(CH2) m -O-, wherein m is selected from 1 or 2.

13. [Corrected 10.01.2025 according to Rule 91] A compound according to claim 11 or 12, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R3 and R4 are both H; or, R3 is H and R4 is methyl.

14. [Corrected on 10.01.2025 according to Rule 91] A compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 11 to 13, wherein X2 is selected from -O-, -NH-; or, X2 is selected from -O-, -NH-, and q is 0, 1, 2, 3 or 4; or, X2 is selected from -O-, -NH-, and q is 1, 2, 3, 4.

15. [Corrected 10.01.2025 according to Rule 91] A compound according to any one of claims 11 to 14, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the compound of formula Ib is a compound of formula Ib-1: in, Ab, L, n are as defined in claim 1, and X2 and q are as defined in any one of claims 12-15.

16. [Corrected 10.01.2025 according to Rule 91] A compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 11 to 15, wherein: Selected from the following compound fragments; in Indicates the position connected to the linker L through a chemical bond.

17. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, which is a compound of formula Ic, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: in, X3 is selected from NH, O, S, or X3 is selected from NH, O; R2, R3, R4 are as defined in any one of claims 1-3; and Ab, L, n are as defined in claim 1.

18. [Corrected 10.01.2025 according to Rule 91] A compound according to claim 17 or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R2 is selected from H or F; or, R3 is selected from H, and R4 is selected from methyl; or, R3 and R4 are cyclized to -(CH2) k -, k is selected from 1, 2, 3; or, R3 and R4 are cyclized to -(CH2)2-; or, X3 is selected from NH, or, X3 is selected from O.

19. [Corrected 10.01.2025 in accordance with Rule 91] A compound according to claim 17, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: Selected from the following compound fragments:

20. The compound or pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 5, wherein the compound is of formula Id or a pharmaceutically acceptable salt thereof: in, Ab, L, n, R6 and R7 are as defined in claim 1; R1, R2, R3, R4 are as defined in any one of claims 1-3, and r is selected from 0, 1, 2, 3.

21. The compound according to claim 20 or its pharmaceutically acceptable salt, or its stereoisomer, wherein R1 and R2 are both H; or, R3 and R4 are both H; or, r is 1; or, R6 is selected from methyl, ethyl, isopropyl; or, R6 is isopropyl; or, R7 is selected from H; or, R7 is selected from R8-S(O)2-, wherein R8 is selected from methyl, ethyl, or, R8 is selected from methyl.

22. The compound according to claim 20 or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein Selected from the following compound fragments:

23. The compound or pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 22, wherein the Ab is selected from anti-Her2 antibody, anti-Trop2 antibody, and anti-Claudin18.2 antibody.

24. The compound or pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 22, wherein the Ab is an anti-Her2 antibody having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. 25 . The compound or pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 24 , wherein the Ab is an anti-Her2 antibody having a heavy chain variable region shown in SEQ ID NO: 4 and a light chain variable region shown in SEQ ID NO:

9.

26. The compound or pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 25, wherein the Ab is an anti-Her2 antibody having a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10; 27. The compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the Ab is selected from Trastuzumab, Pertuzumab, Sacituzumab, zolbetuximab.

28. The compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein said L is -L1-L2-L3-L4-, wherein, One end of L1 is connected to the Ab, and one end of L4 is connected to the warhead drug molecule; L1 is selected from the following groups, and the end of L1 marked with an asterisk * is connected to the Ab: L2 is selected from a chemical bond, -N(R 10 )-CH2-CO-, One end of CO of L2 is connected to L3, and the other end is connected to L1; R 10 Selected from C 1-4 Alkyl, C 1-4 L3 is selected from a polypeptide residue consisting of 2-6 amino acids, and the C-terminus of the polypeptide residue is connected to L4; and L4 is selected from a chemical bond, -NH-CH-, One end of CH is connected to the warhead drug molecule, and one end of -NH- is connected to L3.

29. The compound according to claim 28, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein L2 is a chemical bond; or L2 is -N(CH3)-CH2-CO-; or / and L3 is selected from the following polypeptide residues: GFG, GGFG, GGGFG, GGVA, V-Cit, VA; or, L1 is selected from L2 is selected from a chemical bond or -N(CH3)-CH2-CO-, and L3 is selected from GFG, GGFG, GGGFG, and L4 is -NH-CH-.

30. The compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein L is selected from the following fragments: in, "*" indicates the position where the linker is connected to the warhead drug molecule, and the other end indicates the position where the linker is connected to the Ab.

31. A compound according to any one of claims 1 or 23-27, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: Selected from the following structural fragments:

32. The compound or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1 or 23 to 27, which is a compound or pharmaceutically acceptable salt or stereoisomer thereof selected from the following: in, n is 7.0-8.0, or n is 7.4-7.8, or n is 7.4-8.0, or n is 7.5-8.

5.

33. A compound selected from one of the following compounds or a pharmaceutically acceptable salt or a stereoisomer thereof:

34. A structural fragment selected from the following: The end with the asterisk * is connected to the warhead drug molecule.

35. A pharmaceutical composition comprising the compound according to any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and a pharmaceutically acceptable excipient.

36. Use of the compound according to any one of claims 1 to 32 or its pharmaceutically acceptable salt, its stereoisomer, and the pharmaceutical composition according to claim 35 in the preparation of a medicament for treating tumors.

37. A method for treating a tumor, comprising administering an effective amount of the compound according to any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or the pharmaceutical composition according to claim 35 to a patient in need thereof.

38. The compound according to any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof, a stereoisomer thereof or the pharmaceutical composition according to claim 35 for use in treating a tumor.