Pentacyclic active compounds, conjugates and uses thereof

Abstract

The present application relates to pentacyclic active compounds and conjugates and uses thereof. The compounds and conjugates have excellent activity in inhibiting tumor cells, stability and efficacy in animals.

Description

Pentacyclic active compounds and conjugates thereof and uses thereof

[0001] This application claims priority to a prior application filed with the State Intellectual Property Office of China on August 18, 2023, with patent application number 202311046439.3, entitled “Pentacyclic Active Compounds, Conjugates Thereof, and Uses Thereof.” The entire text of the aforementioned application is incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of medical technology and relates to pentacyclic active compounds and conjugates thereof and uses thereof. Background Art

[0003] The concept of antibody-drug conjugates (ADCs) is a long-standing one. As early as the early 20th century, Nobel Prize winner Professor Paul Ehrlich first proposed the concept of a "magic bullet": a complex that conjugates a cytotoxic small molecule drug to an antibody via a rationally constructed linker, enabling the selective delivery of potent cytotoxic drugs into tumors. Currently available ADCs are based on this theory. With the continuous expansion of targets and indications, ADCs are ushering in a new era of targeted cancer therapy, potentially replacing traditional chemotherapy drugs.

[0004] ADC drugs use specific linkers to connect antibodies and small molecule cytotoxic drugs. Their main components include antibodies (mAbs), linkers, and small molecule cytotoxic drugs (payloads). After ADC drugs enter the bloodstream, their antibody components can recognize targets and bind to tumor cells that highly express cell surface antigens. When the ADC-antigen complex enters the tumor cell through endocytosis, it is degraded by lysosomes, and the cytotoxic payload (drug) is released, destroying DNA or preventing tumor cell division, thereby killing the tumor cells.

[0005] In 1958, attempts were made to use anti-mouse leukocyte immunoglobulin conjugated to methotrexate for the treatment of leukemia. In 2000, the US Food and Drug Administration (FDA) approved the first ADC, Mylotarg, for adult acute myeloid leukemia, marking the beginning of the era of ADC-targeted cancer therapy. Mylotarg, a first-generation ADC, was withdrawn from the market due to severe and fatal liver damage and a lack of significant survival benefit. The shortcomings of first-generation ADCs include the mouse origin of the antibody, insufficient cytotoxicity, and low expression of the target site.

[0006] The first approved ADC for breast cancer, trastuzumab emtansine, marked the starting point for the development of second-generation ADCs. Brentuximab is the most successful ADC in hematologic oncology, primarily targeting classical Hodgkin's lymphoma and anaplastic large cell lymphoma. The advantages of second-generation ADCs include diverse target antigen development and humanized antibodies. However, their disadvantages include low or high drug loading, a narrow therapeutic window, and low efficacy.

[0007] A typical representative of the third-generation ADC drug is Ennosumab, which is the second ADC drug targeting solid tumors. It is suitable for patients who are ineffective with PD-1 / PD-L1 antibody treatment, but it is insensitive to microtubule inhibitors.

[0008] At present, there is still a need to develop drug molecules and their conjugates with rational molecular structure design, improved pharmacodynamic activity and controllable safety.

[0009] Summary of the Invention

[0010] In order to improve the above technical problems, the present disclosure provides a ligand-drug conjugate represented by the following formula (C), its stereoisomers, racemates, tautomers, isotopomers, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts: Tp-L-G (C)

[0011] Among them, Tp is the targeting moiety;

[0012] L is selected from a chemical bond or a linker;

[0013] G is a group represented by the following formula (G):

[0014] wherein A is absent or selected from unsubstituted or optionally substituted with one, two or more R A The following groups substituted: alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heteroaryl, heterocyclic group or a combination of two or more thereof; the alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heteroaryl, heterocyclic group or a combination of two or more thereof may not be substituted or may be optionally substituted with one, two or more selected from -O-, -S-, -NR 5- 、-NR 6 C(=O)-, -C(=O)NR 6 -, -C(=O)-, -NR 7 C(=O)NR 8 - or -OC(=O)- group;

[0015] R 1 is selected from unsubstituted or optionally substituted with one, two or more R BSubstituted: cycloalkyl, cycloalkylalkyl, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy;

[0016] X is selected from O or S;

[0017] Z is selected from 0 or 1;

[0018] R 2 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted groups: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

[0019] R 22 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted groups: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

[0020] R 3 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted groups: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

[0021] R 4 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted groups: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

[0022] Or, R 2 、R 3 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D substituted ring structures;

[0023] Or, R 3 、R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D substituted ring structures;

[0024] Or, R 22 、R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D substituted ring structures;

[0025] B is absent or selected from -O-, -S-, -S(=O)-, -C(=O)-, -NR 5-、-C=N-NR 9 -, -C=NO- or -NR 10 -NR 11 -;

[0026] R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 are the same or different and are independently selected from hydrogen, deuterium, unsubstituted or optionally substituted by one, two or more R E Substituted: alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, HC(═O)—, alkyl C(═O)—, cycloalkyl C(═O)NH—, heterocyclyl C(═O)NH—, aryl C(═O)NH—, heteroaryl C(═O)NH—;

[0027] Every R A 、R B 、R C 、R D 、R E The same or different, independently selected from deuterated, halogen, hydroxy, cyano, nitro, oxo (=O), unsubstituted or optionally substituted by one, two or more R F Substituted: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH2, HC(=O)NH-, alkylC(=O)NH-, cycloalkylC(=O)NH-, heterocyclylC(=O)NH-, arylC(=O)NH-, heteroarylC(=O)NH-;

[0028] Every R F are the same or different and are independently selected from the group consisting of deuterated, halogen, hydroxy, cyano, nitro, oxo (=O), alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH2, HC(=O)NH-, alkylC(=O)NH-, cycloalkylC(=O)NH-, heterocyclylC(=O)NH-, arylC(=O)NH-, heteroarylC(=O)NH-;

[0029] The wavy line indicates the site connected to L;

[0030] According to an embodiment of the present disclosure, the groups in formula (G) may be independently selected from the following definitions:

[0031] wherein A is absent or selected from unsubstituted or optionally substituted with one, two or more R A Substituted with the following groups: C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof; said C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof may not be substituted or optionally substituted by one, two or more selected from -O-, -S-, -NR- 5- 、-NR 6 C(=O)-, -C(=O)NR 6 -, -C(=O)-, -NR 7 C(=O)NR 8 - or -OC(=O)-; preferably, A is absent or selected from unsubstituted or optionally substituted by one, two or more R A Substituted with the following groups: C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclic group or a combination of two or more thereof; the C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3- 6 cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclic group or a combination of two or more thereof may be substituted or optionally substituted by one, two or more selected from -O-, -S-, -NR- 5- 、-NR 6 C(=O)-, -C(=O)NR 6 -, -C(=O)-, -NR 7 C(=O)NR 8 - or -OC(=O)- group;

[0032] R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted with the following groups: C 3-10 Cycloalkyl, C3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy, 3-10 membered heterocyclic group, 3-10 membered heterocyclic group C 1-10 alkyl, 3-10 membered heterocyclyloxy; preferably, R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted with the following groups: C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy, 3-6 membered heterocyclic group, 3-6 membered heterocyclic group C 1-6 Alkyl, 3-6 membered heterocyclyloxy;

[0033] X is selected from O or S;

[0034] Z is selected from 0 or 1;

[0035] R 2 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted with the following groups: C 3-10 Alkyl, C 3-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 2 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted with the following groups: C 1- 6 alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 cycloalkyloxy;

[0036] R 22 Hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 3-10 Alkyl, C 3-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 2 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more RC Substituted with the following groups: C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 cycloalkyloxy;

[0037] R 3 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted with the following groups: C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 3 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted with the following groups: C 1- 6 alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 cycloalkyloxy;

[0038] R 4 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted with the following groups: C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 4 Selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted by one, two or more R C Substituted with the following groups: C 1- 6 alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 cycloalkyloxy;

[0039] Or, R 2 、R 3Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D A substituted 4-10 membered ring structure; wherein the 4-10 membered ring structure can be selected from, for example, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heteromonocyclic hydrocarbon groups, heterobicyclic hydrocarbon groups, monocyclic hydrocarbon groups, and bicyclic hydrocarbon groups; wherein the heteromonocyclic hydrocarbon groups and heterobicyclic hydrocarbon groups contain one, two, or more O, S, N, or carbonyl groups or any combination thereof;

[0040] Or, R 3 、R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D A substituted 4-10 membered ring structure; wherein the 4-10 membered ring structure can be selected from, for example, a 4-, 5-, 6-, 7-, 8-, 9- or 10-membered monocyclic hydrocarbon group, a bicyclic hydrocarbon group, a heteromonocyclic hydrocarbon group, or a heterobicyclic hydrocarbon group; wherein the heteromonocyclic hydrocarbon group and the heterobicyclic hydrocarbon group contain one, two or more O, S, N or carbonyl groups or any combination thereof;

[0041] Or, R 22 、R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D A substituted 4-10 membered ring structure; wherein the 4-10 membered ring structure can be selected from, for example, a 4-, 5-, 6-, 7-, 8-, 9- or 10-membered monocyclic hydrocarbon group, a bicyclic hydrocarbon group, a heteromonocyclic hydrocarbon group, or a heterobicyclic hydrocarbon group; wherein the heteromonocyclic hydrocarbon group and the heterobicyclic hydrocarbon group contain one, two or more O, S, N or carbonyl groups or any combination thereof;

[0042] B is absent or selected from -O-, -S-, -S(=O)-, -C(=O)-, -NR 5 -、-C=N-NR 9 -, -C=NO- or -NR 10 -NR 11 -;

[0043] R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 are the same or different and are independently selected from hydrogen, deuterium, unsubstituted or optionally substituted by one, two or more R E Substituted with the following groups: C 1-10 Alkyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 6-12 Aryl, C 6-12Aryl C 1-10 Alkyl, 5-12 membered heteroaryl, 5-12 membered heteroaryl C 1-10 Alkyl, 5-12 membered heterocyclic group, 5-12 membered heterocyclic group C 1-10 Alkyl, HC(=O)-, C 1-10 Alkyl C(=O)-, C 3-10 Cycloalkyl C(=O)NH-, 5-12 membered heterocyclic C(=O)NH-, C 6-12 Aryl C(=O)NH-, 5-12 membered heteroaryl C(=O)NH-; preferably, R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 are the same or different and are independently selected from hydrogen, deuterium, unsubstituted or optionally substituted by one, two or more R E Substituted with the following groups: C 1-6 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 6-10 Aryl, C 6-10 Aryl C 1-6 Alkyl, 5-6 membered heteroaryl, 5-6 membered heteroaryl C 1-6 Alkyl, 5-6 membered heterocyclic group, 5-6 membered heterocyclic group C 1-6 Alkyl, HC(=O)-, C 1-6 Alkyl C(=O)-, C 3-6 Cycloalkyl C(=O)NH-, 5-6 membered heterocyclic C(=O)NH-, C 6-10 Aryl C(=O)NH-, 5-6 membered heteroaryl C(=O)NH-;

[0044] Every R A 、R B 、R C 、R D 、R E The same or different, independently selected from deuterated, halogen, hydroxyl, cyano, nitro, unsubstituted or optionally substituted by one, two or more R F Substituted with the following groups: C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1- 10 Alkyl, C 3-10 Cycloalkyloxy, C 6-12 Aryl, C 6-12 Aryl C 1-10Alkyl, C 6-12 Aryloxy, 5-12 membered heteroaryl, 5-12 membered heteroaryl C 1- 10 alkyl, 5-12 membered heteroaryloxy, 5-12 membered heterocyclyl, 5-12 membered heterocyclyl C 1-10 Alkyl, 5-12 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-10 Alkyl C(=O)NH-, C 3-10 Cycloalkyl C(=O)NH-, 5-12 membered heterocyclic C(=O)NH-, C 6-12 Aryl C(=O)NH-, 5-12 membered heteroaryl C(=O)NH-; preferably, each R A 、R B 、R C 、R D 、R E The same or different, independently selected from deuterated, halogen, hydroxyl, cyano, nitro, unsubstituted or optionally substituted by one, two or more R F Substituted with the following groups: C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy, C 6-10 Aryl, C 6-10 Aryl C 1-6 Alkyl, C 6-10 Aryloxy, 5-6 membered heteroaryl, 5-6 membered heteroaryl C 1-6 alkyl, 5-6 membered heteroaryloxy, 5-6 membered heterocyclic group, 5-6 membered heterocyclic group C 1-6 Alkyl, 5-6 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-6 Alkyl C(=O)NH-, C 3-6 Cycloalkyl C(=O)NH-, 5-6 membered heterocyclic C(=O)NH-, C 6-10 Aryl C(=O)NH-, 5-6 membered heteroaryl C(=O)NH-;

[0045] Every R F the same or different, independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy, C 6-12 Aryl, C 6-12 Aryl C 1-10 Alkyl, C6-12 Aryloxy, 5-12 membered heteroaryl, 5-12 membered heteroaryl C 1-10 alkyl, 5-12 membered heteroaryloxy, 5-12 membered heterocyclyl, 5-12 membered heterocyclyl C 1-10 Alkyl, 5-12 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-10 Alkyl C(=O)NH-, C 3-10 Cycloalkyl C(=O)NH-, 5-12 membered heterocyclic C(=O)NH-, C 6-12 Aryl C(=O)NH-, 5-12 membered heteroaryl C(=O)NH-; preferably, each R F the same or different, independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy, C 6-10 Aryl, C 6-10 Aryl C 1-6 Alkyl, C 6-10 Aryloxy, 5-6 membered heteroaryl, 5-6 membered heteroaryl C 1-6 alkyl, 5-6 membered heteroaryloxy, 5-6 membered heterocyclic group, 5-6 membered heterocyclic group C 1-6 Alkyl, 5-6 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-6 Alkyl C(=O)NH-, C 3-6 Cycloalkyl C(=O)NH-, 5-6 membered heterocyclic C(=O)NH-, C 6-10 Aryl C(=O)NH-, 5-6 membered heteroaryl C(=O)NH-;

[0046] According to an embodiment of the present disclosure, the groups in formula (G) may be independently selected from the following definitions:

[0047] A is absent or selected from one of the following substructures, wherein the substructure may be unsubstituted or optionally substituted with one, two or more R A replace:

[0048] -(CH2) n1 -、-O(CH2) n2 -、-S(CH2) n3 -、-NR 5 (CH2) n4 -、-NR 6 C(=O)(CH2) n5 -, -C(=O)(CH2) n6 -、-NR 7C(=O)NR 8 (CH2) n7 -, -OC(=O)(CH2) n8 -, -C=C(CH2) n9 -、-C≡C(CH2) n10 -、

[0049] n1, n2, n3, n4, n5, n6, n7, n8, n9, and n10 are each independently selected from any integer between 0 and 6;

[0050] R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted with the following groups: C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl; for example, R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted groups:

[0051] R 2 selected from hydrogen;

[0052] R 22 selected from hydrogen;

[0053] R 3 is selected from unsubstituted or optionally substituted with one, two or more R C Substituted C 1-6 Alkyl; for example, R 3 Selected from methyl, ethyl, propyl;

[0054] R 4 selected from halogen;

[0055] B is absent or selected from -O-, -S-, NR 5 ; R 5 Selected from H, deuterium, C 1-6 Alkyl, C 3-6 Cycloalkyl;

[0056] According to an embodiment of the present disclosure, R 3 、R 4 Together with the atoms to which they are attached, they are unsubstituted or replaced by one, two or more R D The following ring structure is substituted:

[0057] According to an embodiment of the present disclosure, R 3 、R 4Together with the phenyl ring to which it is attached, it forms one of the following substructures, wherein the substructure may be unsubstituted or optionally substituted with one, two or more R D replace:

[0058] Among them, R D Has the definition given above.

[0059] According to an embodiment of the present disclosure, the groups in formula (G) may be independently selected from the following definitions:

[0060] A is absent or selected from one of the following substructures, wherein the substructure may be unsubstituted or optionally substituted with one, two or more R A replace:

[0061] B is absent or selected from -O-, -S-, NR 5 ; R 5 Selected from H, deuterium, C 1-6 Alkyl, C 3-6 Cycloalkyl;

[0062] In some embodiments, A is selected from unsubstituted or optionally substituted with one, two or more R A The following structures are replaced:

[0063] According to an embodiment of the present disclosure, in said G, Can be selected from the following groups:

[0064] According to an embodiment of the present disclosure, G is selected from the group represented by the following formula (G-1) or (G-2):

[0065] Among them, A, B, R 1 、R 2 、R 22 、R 3 、R 4 independently have the definitions set out above.

[0066] According to an embodiment of the present disclosure, G is selected from the group represented by formula (G-3), (G-4), (G-5) or (G-6):

[0067] Among them, A, B, R 1 independently have the definitions set out above.

[0068] According to an embodiment of the present disclosure, G is selected from the following groups:

[0069] Among them, A, B, R 2 、R 22 、R 3 、R 4 independently have the definitions set out above.

[0070] According to an embodiment of the present disclosure, G is selected from the following groups:

[0071] Among them, A, B, R 2 、R 22 、R 3 、R 4 independently have the definitions set out above.

[0072] According to an exemplary embodiment of the present disclosure, G is selected from the following groups:

[0073] In the above aspects of the present disclosure, wherein L is selected from a chemical bond or a linker represented by formula (L) as defined below: #L 1 —L 2 —L 3 —L 4 * (L)

[0074] Among them L 1 It is the connecting part with the targeting part Tp, composed of the reactive group L 1 ' and the targeting portion Tp, # represents the connection site with the targeting portion Tp;

[0075] For example, L 1 ' is a pyrimidine group or a maleimide group, then L 1 The structure is as follows:

[0076] or its open-loop form:

[0077] L 2 Does not exist or is L 1 and L 3 The interval part;

[0078] L 3 is the peptide portion;

[0079] L 4 is absent, or is the linking portion between the peptide portion and the bioactive molecule G, and is composed of the reactive group L 4 'Generated by reaction with bioactive molecule G or its intermediate, * represents the connection site with bioactive molecule G;

[0080] Optionally:

[0081] Between the above parts in L, preferably in L 1 With L 2 Between, or L 2 With L 3 Between or instead of L 2 or by inserting L 2 The method contains the following hydrophilic part:

[0082] The hydrophilic portion is a subunit substituted by one, two or more hydrophilic groups, such as a phenylene group substituted by one, two or more hydrophilic groups or an amino acid residue substituted by one, two or more hydrophilic groups, preferably: The hydrophilic part can also be the hydrophilic group itself forming a subunit such as or -(CH2CH2O) p -;

[0083] R 12 、R 12 'same or different, selected from hydrophilic groups or the following substituents: hydrogen, halogen, cyano, amino, nitro, unsubstituted or optionally substituted by one, two or more R zg Substituted C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; wherein R 12 、R 12’ At least one of them is selected from hydrophilic groups.

[0084] The hydrophilic group is selected from polyethylene glycol groups or polyethylene glycol subunits, C 1-10 Alkyl, a group containing a sugar ring or a heterocyclic subunit containing a nitrogen atom such as a piperidinyl or piperazinyl group, preferably a polyethylene glycol group, more preferably -(CH2CH2O) p -C 1-10 Alkyl, -C(=O)-NH-(CH2CH2O) p -C 1-10 Alkyl or -NH-(CH2CH2O) p -C 1-10 Alkyl, or preferably C substituted by 1 to 10 hydroxyl groups 1-10 Alkyl, more preferably

[0085] Each p is the same or different and is independently selected from an integer from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8;

[0086] Every R zg The same or different, independently selected from the following groups: halogen, hydroxy, amino, cyano, nitro, C 1-10 Alkyl, C 1- 10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 cycloalkyloxy;

[0087] Preferably, each R zg The same or different, independently selected from the following groups: halogen, hydroxy, amino, cyano, nitro, C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 cycloalkyloxy;

[0088] In certain embodiments of the present disclosure, the hydrophilic moiety defined above is absent from L.

[0089] In certain embodiments of the present disclosure, L contains a hydrophilic portion such as:

[0090] In a further preferred embodiment of the present disclosure, L 1 The group L that reacts with any targeting moiety Tp 1 'Form, L 1 'Preferred are sulfhydryl reactive groups, amino reactive groups, carboxyl reactive groups, dithiol bridging groups, and the like; for antibodies introduced with non-natural amino acids, click chemistry reactive groups such as ketones, hydrazines or hydrazides, azides, alkynes, cyclopropenes, or dienes can also be selected; when the Tp moiety is an antibody, the linking site includes any applicable amino acid residue or N297 sugar chain coupling of the CH2 domain, such as fucose, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (SA) introduced by glycoengineering; the linking reaction includes a chemical reaction or an enzymatic reaction, such as using transglutaminase (MTGase) to transfer an amine-containing drug linker or reactive spacer to a deglycosylated antibody.

[0091] L 1 'Preferably a thiol reactive group;

[0092] L 1 'Preferred sulfhydryl reactive groups of the following structure:

[0093] Hal-Het-

[0094] Hal is selected from halogen, OMs, OTs, OTf, nitro, and optionally substituted by one, two or more R z8 Substituted with the following groups: C 1-10 Alkyl sulfide group, C 6-12 Aryl sulfide group, 5-12 membered heteroaryl sulfide group, C 1-10 Alkyl sulfoxide, C 6-12 Arylsulfoxide, 5-12 membered heteroarylsulfoxide, C 1-10 Alkylsulfonyl, C 6-12 Arylsulfonyl, 5-12 membered heteroarylsulfonyl; wherein, R z8 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, C 6-12 aryl and 5-12 membered heteroaryl;

[0095] Het is selected from the group consisting of optionally one, two or more R z9 Substituted 5-12 membered heteroaryl; wherein R z9 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-10 Alkyl and halogenated C 1-10 Alkyl; preferably, Het is selected from optionally substituted by one, two or more R z9 substituted 5-10 membered heteroaryl; wherein R z9 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and halogenated C 1-4 alkyl;

[0096] In a preferred embodiment, Hal is a methanesulfonyl group and Het is a pyrimidine group;

[0097] In a preferred embodiment, Hal-Het- is:

[0098] The corresponding L 1 The structure is:

[0099] And L 1 '-L 2 The following structure is preferred:

[0100] q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8;

[0101] R z4 and R z5 The same or different, independently selected from H, C1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl;

[0102] In a further preferred embodiment of the present disclosure,

[0103] or L 1 'Further preferably a maleimide group or a substituted maleimide group, and L 1 -L 2 The following structure is preferred:

[0104] The fragment prepared from (N-maleimidomethyl)-carboxylic acid-N-hydroxysuccinimide ester has the structure:

[0105] (q is an integer from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8);

[0106] Or a fragment prepared from m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), the structure of which is:

[0107] The fragment prepared from succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) has the structure:

[0108] In a further embodiment of the present disclosure,

[0109] L 2 Select from non-existent, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof; said C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof may not be interrupted by or may be optionally interrupted by carbonyl, O, S or N atoms; the C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof may be optionally replaced by C 1-6 Alkyl, C 3-6 Cycloalkyl, halogen atom, halogenated C 1-6 Alkyl substituted; optionally, the C 1-6 Alkyl or halogenated C 1-6 The alkyl group can form a C 3-6 Cycloalkyl, L 2 Through any functional group or covalent bond with L 1 or L 3 fragment ligation;

[0110] Preferably, L 2 Selected from -(CH2) q -、-C(=O)-NH-(CH2) q -C(=O)-, -(CH2) q -C(=O)-, -(CH2) q -NH-C(=O)-, -(CH2) q -NCH3-C(=O)-、-(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -Cy-(CH2) q -C(R z4 R z5 )-C(=O)-, wherein q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8;

[0111] wherein Cy is a 5-12 membered heteroaryl or heterocyclic group optionally containing S, O or N heteroatoms, and optionally substituted by H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and halogenated C 1-4 Alkyl substituted, preferably, at least three atoms in Cy are N, more preferably three consecutive atoms in Cy are N, and further preferably Cy is 1,2,3-triazolyl;

[0112] Preferably, R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl;

[0113] Preferably, L2 Through -NHC(=O)-, -NCH3C(=O)- or -C(=O)- with peptide fragment L 3 The N-terminal connection.

[0114] In a further embodiment of the present disclosure,

[0115] Among them L 3 selected from a divalent peptide group comprising 2 to 8 optionally substituted natural or non-natural amino acid residues, L- or D-form, each of which is identical or different and is independently selected from the group consisting of alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine, demethylpyrrolysine, analogs of the above amino acids, or selected from the group consisting of AA 1 The indicated amino acid residues or stereoisomers thereof.

[0116] Among them, R G With R H are not H at the same time, and are each independently selected from H,

[0117] Or, R G With R H Together with the carbon atoms to which they are attached, they form an unsubstituted or optionally substituted group consisting of one, two or more R L Substituted C 3- 10 Cycloalkyl or 3-10 membered heterocyclic group;

[0118] r, r1 are each independently selected from any integer from 0 to 10;

[0119] R I 、R J 、R K Each independently selected from H, unsubstituted or optionally substituted with one, two or more R M Substituted C 1-6 Alkyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, ester group;

[0120] Or, R I With R J Together with the nitrogen atom to which they are attached, they form an unsubstituted or optionally substituted group consisting of one, two or more R L substituted 4-10 membered heterocyclic group;

[0121] R M 、R L are the same or different and are independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH2, alkylamino, dialkylamino acid, HC(=O)NH-, alkylC(=O)NH-, cycloalkylC(=O)NH-, heterocyclylC(=O)NH-, arylC(=O)NH-, heteroarylC(=O)NH-; optionally, two R attached to the same carbon atom M or R L Together they form C 3- 6-cycloalkyl.

[0122] Further preferred, L 3 selected from natural or non-natural, L- or D-type amino acid residues or AA 1 The peptide group is a divalent peptide group composed of a combination of amino acid residues, each of which is the same or different and is independently selected from the following amino acid residues: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine, demethylpyrrolysine or AA 1 ; For example -ValCit-; -ValAA 1-CitVal--AlaAla--AlaCit--CitAla--AsnCit--CitAsn--CitCit--ValGlu--GluVal--SerCit--CitSer--L ysCit-;-CitLys-;-AspCit-;-CitAsp-;-AlaVal-;-ValAla-;-PheAla-;-AlaPhe-;-PheLys-;-LysPhe-;-ValLys-;-GlyAA 1 -LysVal--AlaLys--LysAla--PheCit--CitPhe--LeuCit--CitLeu--IleCit--CitIle--PheArg--ArgPhe--CitTrp--TrpCit--AlaAlaAla--PhePheLys--ValAAA 1 Gly-;-AlaAA 1 Gly-;-GlyAA 1 -A laLeuAlaLeu-;-GlyGlyGly-;-GlyGlyGlyGly-;-GlyPheValGly-;-GlyValPheGly-;-GlyGlyPheGly-;-GlyGlyValGly-;

[0123] AA 1 Among the amino acid residues, preferably,

[0124] r, r1 are each independently selected from any integer from 0 to 5;

[0125] R G With R H Among them, any one is H, and the other is selected from:

[0126] Or, R G With R H Together with the carbon atoms to which they are attached, they form an unsubstituted or optionally substituted group consisting of one, two or more R L substituted 5-6 membered heterocyclic group;

[0127] R I 、R J 、R KEach independently selected from H, unsubstituted or optionally substituted with one, two or more R M Substituted methyl, ethyl, n-propyl, n-butyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, -COOCH3, COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3 and -COOCH2CH2CH2CH3;

[0128] Or, R I With R J Together with the nitrogen atom to which they are attached, they form an unsubstituted or optionally substituted group consisting of one, two or more R L substituted 5-6 membered heterocyclic group;

[0129] Most preferably,

[0130] L 3 Selected from -ValAA 1 Gly-;

[0131] Among them, AA 1 In the amino acid residues, r is 0 and r1 is 4;

[0132] R G With R H Among them, any one is H, and the other is selected from:

[0133] Or, R G With R H Together with the carbon atoms they are connected to, they form Where * indicates R G With R H Commonly linked carbon atoms;

[0134] R I 、R J 、R K Each independently selected from H, unsubstituted or optionally substituted with one, two or more R M Substituted methyl, ethyl, n-propyl, n-butyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-4 Alkyl or -COOC(CH3)3;

[0135] Or, R I With R J Together with the nitrogen atoms to which they are connected, they form

[0136] In a further embodiment of the invention, it is characterized in that:

[0137] AA 1 The amino acid residue is selected from one of the following substructures:

[0138] In a further embodiment of the invention,

[0139] L 4 Not present or selected from:

[0140] More preferably, L 4 for:

[0141] Most preferably, L 4 for: The corresponding L 4 'The reactive group is in the acetate form:

[0142] Its reaction with the compound of formula (GH) (B is an oxygen atom) is as follows:

[0143] When L 4 When it does not exist, the corresponding L 4 'Reactive group is L 3 The terminal amino acid forms an active ester, such as glycine.

[0144] In a further embodiment of the present disclosure,

[0145] L 1 It is generated by coupling Tp with a sulfhydryl reactive group selected from a maleimide group, a substituted maleimide group or Hal-Het-;

[0146] In L 1 With L 2 Between, or L 2 With L 3 Between or instead of L 2 In the form of or by inserting L 2 The form contains a hydrophilic portion as defined above.

[0147] In a further embodiment of the invention,

[0148] L 1 Generated by coupling Tp with a sulfhydryl reactive group selected from Hal-Het-;

[0149] L 4 for:

[0150] In a further embodiment of the invention,

[0151] L 1 -L 2 L is composed of the following structure 1 '-L 2 Coupling Tp generation:

[0152] q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8;

[0153] R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl, where R z4 or R z5 At least one is not H;

[0154] Further optimization of R z4 or R z5 Together they form C 3-6 Cycloalkyl;

[0155] L 1 -L 2 Most preferably, Coupled Tp generation.

[0156] Each segment L in the above-defined L 1 -L 4 Or the hydrophilic portion can be linked by any chemical bond, such as a direct bond, an ester bond (-CO-O-), an amide bond (-CO-NH-), an ether bond (-O-), a thioether bond (-S-), a carbamate (-N-CO-O-), or a urea group (-O-CO-O-);

[0157] Preferably, each H in the peptide bond or amide bond in L may be optionally substituted with a methyl group.

[0158] The present disclosure further provides a conjugate having the following structure: Tp-L-D (D)

[0159] Wherein Tp is the targeting moiety;

[0160] D is a biologically active molecular fragment, preferably a molecular fragment with anti-tumor biological activity;

[0161] L is selected from the linker represented by formula (L): #L 1 —L 2 —L 3 —L4 * (L)

[0162] Among them L 1 It is the connecting part with the targeting part Tp, composed of the reactive group L 1 ' and the targeting portion Tp, # represents the connection site with the Tp portion, L 1 Preferred

[0163] L 2 Selected from -(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -(CH2) q -C(R z4 R z5 )-C(=O)-;

[0164] Among them, L 1 '-L 2 The structure is as follows:

[0165] q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8;

[0166] R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form a C3-6 cycloalkyl group, wherein R z4 or R z5 At least one is not H;

[0167] or

[0168] L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing a hydrophilic portion as defined above:

[0169] And L 3 -ValAA 1 Gly-, AA 1 As defined above;

[0170] L 4 is absent or is the linking portion between the peptide portion and the bioactive molecule D, and is composed of the reactive group L4 ' is generated by reaction with biologically active molecules, and * represents the connection site with biologically active molecule D.

[0171] The present disclosure also provides an intermediate for the synthesis of a conjugate having the following structure: 1 '—L 2 —L 3 —L 4 -G(C')

[0172] Among them: G, L 1 '、L 2 , L 3 , L 4 As defined above.

[0173] The present disclosure also provides a conjugate of a linker and a drug: L 1 '—L 2 —L 3 —L 4 -D

[0174] Among them L 1 '、L 2 , L 3 , L 4 , D is as defined above, provided that:

[0175] L 1 ' is a reactive group connected to the targeting portion Tp,

[0176] L 1 'Preferred

[0177] L 2 Selected from -(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -(CH2) q -C(R z4 R z5 )-C(=O)-;

[0178] Among them, L 1 '-L 2 The structure is as follows:

[0179] q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8;

[0180] R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C1-4 Alkyl or R z4 or R z5 Together they form a C3-6 cycloalkyl group, wherein R z4 or R z5 At least one is not H;

[0181] or

[0182] L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing a hydrophilic portion as defined above:

[0183] And L 3 -ValAA 1 Gly-, AA 1 As defined above;

[0184] L 4 is absent or is the linking portion between the peptide portion and the bioactive molecule D, and is composed of the reactive group L 4 ' is generated by reaction with biologically active molecules, and * represents the connection site with biologically active molecule D.

[0185] Those skilled in the art will appreciate that the bioactive molecule D can be a compound with biological activity or potential biological activity recorded in the Chinese, American or European Pharmacopoeia or disclosed in other publications. As an example, the drug can be selected from cytotoxic drugs, cell growth inhibitory drugs or immunosuppressive drugs, for example, anti-tubulin agents, microtubule inhibitors, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of such cytotoxic drugs include, for example, auristatins, camptothecin, duocarmycin, etoposide, maytansine and maytansine alkaloids, taxanes, benzodiazepines, Benzodiazepines drugs and vinca alkaloids.

[0186] Tp is a targeting moiety (eg, a small molecule ligand, protein, peptide, non-protein agent such as a sugar, RNA, or DNA).

[0187] In some preferred embodiments, the target of Tp is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2. BMPR1B, Tyro7, c-Met, ApoE, CD1lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3 CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1Q A. C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1 C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and BCMA;.

[0188] In some preferred embodiments, Tp is a small molecule ligand, such as a folic acid derivative, a glutamic acid urea derivative, a somatostatin derivative, an arylsulfonamide derivative (such as a carbonic anhydrase IX inhibitor), an ICG dye, a cyanine dye, or a derivative thereof;

[0189] According to an embodiment of the present disclosure, the preferred ligand is selected from an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody; preferably a monoclonal antibody;

[0190] According to an exemplary embodiment of the present disclosure, the antibody or antigen-binding fragment thereof is selected from at least one of the following antibodies or antigen-binding fragments: anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody, and the antibody may be a bispecific antibody or a multispecific antibody;

[0191] As an example, the antibody or antigen-binding fragment thereof is selected from at least one of the following antibodies or antigen-binding fragments: Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glembatumumab.

[0192] According to an embodiment of the present disclosure, the conjugate, its linker or linker-drug may be selected from one of the following, wherein u is selected from an integer of 0 to 10, G has the definition described above, and LG has the definition of Tp described above;

[0193] According to an embodiment of the present disclosure, the conjugate may have a structure as shown in the following formula:

[0194] Among them, R 1 、R 2 、R 22 、R 3 、R 4 、R G、R H , A, B, X, Z, L 1 , L 2 , Tp has the definitions described above.

[0195] According to an embodiment of the present disclosure, the conjugate may have a structure as shown in the following formula:

[0196] Among them, A, B, R 3 、R 4 , L 1 , L 2 , Tp has the definitions described above.

[0197] According to an embodiment of the present disclosure, the conjugate may have a structure as shown in the following formula:

[0198] Among them, A, B, L 1 , L 2 , Tp has the definitions described above.

[0199] According to the embodiment of the present disclosure, the ligand drug conjugate represented by formula (C) can be further represented by the following formula (C A ) structure: Tp—(L—G)y (C A )

[0200] Wherein, Tp, L, and G are as defined above;

[0201] y represents the average number of small molecule drugs attached to each mAb (DAR), which can be selected from an integer or decimal, such as an integer or decimal of 1 to 50, an integer or decimal of 1 to 20, or an integer or decimal of 1 to 10.

[0202] According to an embodiment of the present disclosure, the conjugate may have a structure as shown in the following formula:

[0203] Among them, R 1 、R 2 、R 22 、R 3 、R 4 、R G 、R H , A, B, X, Z, L 1 , L 2 , Tp, and y have the definitions described above.

[0204] According to an embodiment of the present disclosure, the conjugate may have a structure as shown in the following formula:

[0205] Among them, A, B, R3 、R 4 , L 1 , L 2 , Tp, and y have the definitions described above.

[0206] According to an embodiment of the present disclosure, the conjugate may have a structure as shown in the following formula:

[0207] Among them, A, B, L 1 , L 2 , Tp, and y have the definitions described above.

[0208] According to an embodiment of the present disclosure, the conjugate may be selected from one of the following:

[0209] Among them, R 3 、R 4 has the meaning given above;

[0210] u is an integer selected from 0 to 10;

[0211] r2 is selected from an integer from 0 to 4, which may be 0, 1, 2, 3 or 4; r3 and r4 are independently selected from an integer from 0 to 3, which may be 0, 1, 2 or 3;

[0212] K is C or N;

[0213] R z6 、R z7 are identical or different and independently of one another are chosen from hydrogen, amino, alkylamino or dialkylamino, alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

[0214] Or, R z6 、R z7 Together with the atoms to which they are attached, they form a ring structure; preferably, they form a ring structure optionally substituted by C 1-4 an alkyl-substituted 5-6 membered heterocyclic group, wherein the 5-6 membered heterocyclic group is preferably a piperidinyl group or a piperazinyl group;

[0215] mAb stands for monoclonal antibody;

[0216] y represents the average number of small molecule drugs attached to each mAb (DAR), which can be selected from an integer or decimal, such as an integer or decimal of 1 to 50, an integer or decimal of 1 to 20, or an integer or decimal of 1 to 10.

[0217] According to the embodiments of the present disclosure, examples of the conjugate intermediate linker and drug conjugate can be selected from one of the following:

[0218] According to an embodiment of the present disclosure, the example of the conjugate may be selected from one of the following:

[0219] y represents the average number of small molecule drugs attached to each mAb (DAR), which can be selected from an integer or decimal, such as an integer or decimal of 1 to 50, an integer or decimal of 1 to 20, or an integer or decimal of 1 to 10;

[0220] The present disclosure also provides a method for preparing the ligand-drug conjugate, its stereoisomers, racemates, tautomers, isotopomers, isotope labels, nitrogen oxides, prodrugs, solvates, or pharmaceutically acceptable salts thereof, wherein the preparation method comprises the following steps:

[0221] Step 1: Provide L 1 '—L 2 —L 3 —L 4 '(L') represents a linker;

[0222] Preferably, in the above linker,

[0223] L 4 'for: The reaction form of is preferably acetate;

[0224] More preferably, L 4 'for: The reaction form is preferably acetate, with the following structure:

[0225] or L 4 If it does not exist, then L 4 'For L 3 The reaction form (such as L 3 The glycine gly at the C-terminus forms an active ester), L 4 ' Directly form an amide connection with GH where B is N.

[0226] Step 2: The linker is coupled with the compound of formula (GH) to obtain L 1 '—L 2 —L 3 —L 4 -G(C') coupling intermediate;

[0227] The structure of formula (GH) is:

[0228] Among them, A, B, Z, X, R 1 、R 2 、R 22 、R 3 、R 4 , L 1 '、L 1 , L 2 , L 3 , L 4 , L 4 ' independently have the definition as above.

[0229] In a specific embodiment of the present disclosure, the following preparation method is also provided:

[0230] Step 1: Provide L 4 'Linker fragment, the L 4 'As defined above;

[0231] Step 2: Include L 4 'The linker fragment is coupled with the compound of formula (GH) to obtain a compound comprising L 4 'Linker fragment-G intermediate;

[0232] Step 3: Provide another linker fragment to react with the above intermediate to form L containing the complete linker 1 '—L 2 —L 3 —L 4 -G(C') coupling intermediate;

[0233] Preferably, L 4 'The linker fragment is: It reacts with GH where B is -O- to form an ether bond;

[0234] Preferably, the other linker fragment is:

[0235] Preferably, the preparation method further comprises a fourth step of coupling the coupling intermediate of formula (C') with the targeting moiety Tp;

[0236] Optionally, if necessary, the functional groups of the reaction substrates can be protected using protecting groups known in the art to facilitate the reaction and remove the protecting groups after the reaction is completed.

[0237] The present disclosure also provides a coupling intermediate of the following structure:

[0238] Among them G N is H or an optional amino protecting group, A, R 1 、R 2 、R 22 、R 3 、R 4 , X, and Z independently have the definitions described above.

[0239] The present disclosure also provides a linker fragment compound with the following structure: L 1 '—L 2 —ValAA 1 '(L”)

[0240] in

[0241] Among them L 1 ' is a reactive group, preferably

[0242] L 2 Selected from -(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -(CH2) q -C(R z4 R z5 )-C(=O);

[0243] Among them, L 1 '-L 2 The structure is as follows:

[0244] L 1 '-L 2 for

[0245] q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8;

[0246] R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl, where R z4 or R z5 At least one is not H;

[0247] or

[0248] L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing a hydrophilic portion as defined above:

[0249] AA 1 ' for AA 1 itself or in its reactive form as an active ester, wherein AA 1 As defined above.

[0250] The present disclosure also provides a linker: L 1 '—L 2 —L 3 —L 4 '(L')

[0251] in

[0252] L 1 '-L 2 As defined above

[0253] or

[0254] L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing a hydrophilic portion as defined above:

[0255] and

[0256] L 3 For: -ValAA 1 Gly-, AA 1 As defined above.

[0257] L' is preferably:

[0258] The present disclosure also provides a compound obtained by connecting the above-mentioned group G to a hydrogen atom, wherein the group B is connected to the hydrogen atom.

[0259] The present disclosure also provides a compound selected from the group consisting of a compound represented by the following formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotope labelled substance, a nitrogen oxide, a prodrug, a solvate or a pharmaceutically acceptable salt thereof:

[0260] Among them, A, B, X, Z, R 1 、R 2 、R 22 、R 3 、R 4 Independently of each other have the definitions given above.

[0261] According to an embodiment of the present disclosure, in the formula (GH) described above and below, the hydrogen, carbon or other atoms in the structural formula are optionally replaced by their isotopes, regardless of whether the group is defined as selected from the above definitions or directly drawn in the structural formula, and regardless of whether the group is defined as a substituted group or an unsubstituted group. For example, the hydrogen atoms on any group in formula (GH) can be selected from 1 H. 2 H or 3 H. As an example, any hydrogen atom on an unsubstituted or substituted alkyl, alkylene, alkylidene, phenyl, pyridyl or lactone group or a ring-forming atom (if any) thereof in a compound of formula (GH) may be independently selected from 1 H. 2 H or 3 H.

[0262] According to an embodiment of the present disclosure, the compound represented by formula (GH) may be selected from the compound represented by the following formula (GH-1):

[0263] Among them, A, B, Z, R 1 、R 2 、R 22 、R 3 、R 4 Independently of each other, have the definitions given above.

[0264] According to an embodiment of the present disclosure, the compound represented by formula (GH) may be selected from the compound represented by the following formula (GH-2):

[0265] Among them, A, B, Z, R 1 、R 2 、R 22 、R 3 、R 4 Independently of each other, have the definitions given above.

[0266] According to an embodiment of the present disclosure, the compound represented by formula (GH) can be selected from the following compounds:

[0267] The present disclosure also provides a method for preparing a compound represented by formula (GH), comprising the steps of reacting a compound represented by formula (i) with a compound represented by formula (ii) to obtain a compound represented by formula (G'):

[0268] Where T is Or it can be converted into Those skilled in the art will appreciate that a group containing an active functional group such as an alkenyl group, an aldehyde group, a carbonyl group, a nitro group, etc. can usually be selected as T to introduce Specifically, when the active functional group is a carbon-containing structure such as an alkenyl group, an aldehyde group, or a carbonyl group, the residue after removing the active functional group from T is a structure in which A has one less carbon. When the active functional group is a nitro group, the residue after removing the nitro group from T is A, which can be selected from the following group forms: NO2-C(R 15 R 16 ) m -(CR 17 =CR 18 ) n -C(R 19 R 20 ) o -、(CR 17 =CR 18 ) n -C(R 19 R 20 ) o -、O=CR 21 -C(R 15 R 16 ) m -(CR 17 =CR 18 ) n -C(R 19 R 20 ) o -;

[0269] wherein m, n, and o are each independently selected from any integer from 0 to 6;

[0270] Every R 15 、R 16 、R 17 、R 18 、R 19 、R 20 、R 21 are identical or different, independently selected from hydrogen, halogen, cyano, unsubstituted or optionally substituted by one, two or more R A Substituted: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy;

[0271] Or, R 15 、R 16 Together with the atoms to which it is attached, it forms an unsubstituted or optionally substituted group consisting of one, two or more R A substituted 3-10 membered ring structures;

[0272] Or, R 19 、R 20 Together with the atoms to which it is attached, it forms an unsubstituted or optionally substituted group consisting of one, two or more R A substituted 3-10 membered ring structures;

[0273] Among them, R 1 、R 2 、R 22 、R 3 、R 4 , X, and Z independently have the definitions described above;

[0274] The present disclosure also provides a method for preparing a compound represented by formula (GH), comprising the steps of obtaining a compound represented by formula (GH) by reacting a compound represented by formula (G'):

[0275] Among them, A, B, R 1 、R 2 、R 22 、R 3 、R 4 , X, and Z independently have the definitions described above;

[0276] Wherein, T is a compound that can be converted into For example, those skilled in the art will appreciate that a group containing an active functional group such as an alkenyl group, an aldehyde group, a carbonyl group, a nitro group, etc. can usually be selected as T, thereby introducing Specifically, when the active functional group is a carbon-containing structure such as an alkenyl group, an aldehyde group, or a carbonyl group, the residue after removing the active functional group is a structure in which A has one less carbon. When the active functional group is a nitro group, the residue after removing the nitro group is A, which can be selected from the following group forms: preferably NO2-C(R 15 R 16 ) m -(CR 17 =CR 18 ) n -C(R 19 R 20 ) o -、(CR 17 =CR 18 ) n -C(R 19 R 20 ) o -、O=CR 21 -C(R 15 R 16 ) m -(CR 17 =CR 18 ) n -C(R 19 R 20 ) o -;

[0277] Among them, m, n, o, R 15 、R 16 、R 17 、R 18 、R 19 、R 20 、R 21 independently having the definitions set out above;

[0278] The present disclosure also provides a compound represented by formula (i):

[0279] Among them, R 1 , X, and Z independently have the definitions described above.

[0280] The present disclosure also provides a compound represented by formula (G'):

[0281] Among them, R 1 、R 2 、R 22 、R 3 、R 4 , X, Z, and T independently have the definitions described above.

[0282] The present disclosure also provides a pharmaceutical composition comprising at least one selected from the group consisting of: the ligand-drug conjugate of formula (C), its stereoisomers, racemates, tautomers, isotopomers, isotope labels, nitrogen oxides, prodrugs, solvates, or pharmaceutically acceptable salts thereof; the compound of formula (GH), its stereoisomers, racemates, tautomers, isotopomers, isotope labels, nitrogen oxides, prodrugs, solvates, or pharmaceutically acceptable salts thereof;

[0283] Preferably, the compound represented by formula (GH), its stereoisomers, racemates, tautomers, isotopologues, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts, and the ligand-drug conjugate represented by formula (C), its stereoisomers, racemates, tautomers, isotopologues, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts in the pharmaceutical composition are present in a therapeutically effective amount.

[0284] According to an embodiment of the present disclosure, the pharmaceutical composition comprises a therapeutically effective amount of at least one selected from the following: the compound represented by formula (GH), its stereoisomers, racemates, tautomers, isotopologues, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts thereof; the ligand-drug conjugate represented by formula (C), its stereoisomers, prodrugs or pharmaceutically acceptable salts or solvates thereof.

[0285] The present disclosure also provides uses of the compound represented by formula (GH), its stereoisomers, racemates, tautomers, isotopologues, isotope labels, nitrogen oxides, prodrugs, solvates, or pharmaceutically acceptable salts thereof, or the ligand-drug conjugate represented by formula (C), its stereoisomers, racemates, tautomers, isotopologues, isotope labels, nitrogen oxides, prodrugs, solvates, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, for preventing and / or treating diseases or conditions and / or preparing medicaments.

[0286] According to an embodiment of the present disclosure, the medicament is for preventing and / or treating a disease or disorder.

[0287] The present disclosure also provides a method for preventing and / or treating a disease or condition, comprising administering to a patient a therapeutically effective amount of at least one of the following: a compound represented by formula (GH), a stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate, or a pharmaceutically acceptable salt thereof; and a ligand-drug conjugate represented by formula (C), a stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate, or a pharmaceutically acceptable salt thereof.

[0288] According to an embodiment of the present disclosure, the disease or disorder may be selected from a tumor, such as a solid tumor or a hematological cancer.

[0289] The example of the solid tumor includes malignant tumors of various organ systems, for example, sarcoma, adenocarcinoma, blastoma, and cancer, such as those affecting the liver, lung, breast, lymph, gallbladder (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate and pharynx. Adenocarcinoma includes malignant tumors such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, small cell lung cancer, non-small cell lung cancer, small intestine cancer and esophageal cancer. In one embodiment, the cancer is a melanoma, for example, an advanced melanoma. Examples of other cancers that can be treated include: bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or inside the eye, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, cancer of the anal region, cancer of the peritoneum, stomach cancer, esophageal cancer, salivary gland cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, malignant glioma, neuroblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia), disease, acute lymphoblastic leukemia, chronic lymphocytic leukemia), solid tumors of childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesothelioma, schwannoma (including acoustic neuroma), meningioma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including cancers induced by asbestos), and combinations of said cancers.

[0290] The example of described hematological cancer includes leukemia, lymphoma and the malignant lymphoproliferative disorder that affects blood, bone marrow and lymphatic system.Leukemia can be classified as acute leukemia and chronic leukemia.Acute leukemia can be further classified as acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).Other related conditions include myelodysplastic syndrome (MDS, formerly known as "preleukemia"), which is a diversified set of hematological disorders combined with the invalid production (or dysplasia) of bone marrow blood cells and the risk of being converted into AML.Lymphoma is a group of blood cell tumors developed from lymphocytes.Exemplary lymphoma includes non-Hodgkin's lymphoma and Hodgkin's lymphoma.

[0291] According to an embodiment of the present disclosure, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, such as a carrier or excipient. The pharmaceutically acceptable excipient is preferably chemically non-reactive or inert to the active ingredient. For example, the pharmaceutically acceptable excipient is selected from at least one of the following excipients, including but not limited to: a filler, a disintegrant, a binder, a lubricant, a surfactant, a flavoring agent, a wetting agent, a matrix, and the like.

[0292] According to the embodiments of the present disclosure, the administration route of the pharmaceutical composition includes but is not limited to gastrointestinal administration or parenteral administration; wherein, the gastrointestinal administration can be oral administration; the parenteral administration can be topical administration, transdermal administration, injection administration, etc.

[0293] In one embodiment, the administration route of the pharmaceutical composition can be a combination of topical administration and oral administration.

[0294] According to an embodiment of the present disclosure, the dosage form of the pharmaceutical composition can be selected from capsules, tablets, patches, films, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, suppositories or injections.

[0295] Definitions and Explanations of Terms

[0296] Unless otherwise indicated, the definitions of terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with each other. Such combinations and couplings shall fall within the scope of this specification.

[0297] The term "antibody-drug conjugate" (ADC) refers to a targeting ligand, such as an antibody (e.g., a monoclonal antibody) or antibody fragment, linked to a biologically active small molecule drug via a stable chemical linker compound. Those skilled in the art will appreciate that in such ADCs, the antibody can be linked to a varying number of small molecule drugs. The average number of small molecule drugs attached to an antibody is typically expressed as DAR.

[0298] Unless otherwise stated, the numerical ranges recorded in this specification and claims are equivalent to recording at least each specific integer value therein. For example, the numerical range "0-10" is equivalent to recording each integer value in the numerical range "0-10", namely 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. It should be understood that when describing substituents herein, "one, two or more" should refer to integers ≥ 3, such as 3, 4, 5, 6, 7, 8, 9 or 10. When "one or more" is used to describe substituents herein, "more" can be understood as integers > 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10. In addition, when certain numerical ranges are defined as "numbers", it should be understood that the two endpoints of the range, each integer in the range and each decimal in the range are recorded. For example, "0-10" should be understood as describing not only each integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, but also at least the sum of each of these integers and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9, respectively. Similarly, the numerical ranges "0 to 5", "0 to 4", "0 to 3", "0 to 6", "an integer or decimal from 1 to 50", "an integer or decimal from 1 to 20", and "an integer or decimal from 1 to 10" should be understood as describing both endpoints of the numerical range, as well as each integer and each decimal within the range.

[0299] The term "isotopologue" includes isotopes in which an atom in a compound or conjugate of the present disclosure is replaced with another isotope having the same atomic number but a different atomic mass or mass number. For example, "hydrogen" in a compound or conjugate of the present disclosure can be selected from protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H); "carbon" can be selected from 12 C. 13 C and 14 C. Therefore, the compounds or conjugates of the present disclosure should at least be understood to include compounds or conjugates in various deuterated forms. For example, each available hydrogen atom attached to a carbon atom (e.g., C 1-10 One, two or more of the hydrogen atoms of the alkyl group (e.g., a alkyl group) may be independently replaced by a deuterium atom. Those skilled in the art will be able to synthesize deuterated compounds or conjugates with reference to relevant literature. When preparing deuterated compounds or conjugates, commercially available deuterated starting materials can be used, or deuterated reagents can be used for synthesis using conventional techniques. Optional deuterated reagents include, but are not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0300] The term "isotopically labeled" includes, but is not limited to, compounds or conjugates of the present disclosure labeled with at least one of the following elements: 111 In, 177 Lu, 212 Bi, 213 Bi, 211 At 62 Cu, 67 Cu, 90 Y. 125 I. 131 I. 32 P. 33 P. 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re、 188 Re、 189 Re、 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As、 89 Sr. 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Second, 194 Ir, 198 Au, 199 Au, 227 Th and 211 Pb.

[0301] The term "prodrug" refers to a compound that is converted into an active compound of the general formula in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or by enzymes in the blood or tissues to the parent structure. The prodrug compounds disclosed herein can be esters. In the prior art, esters that can be used as prodrugs include phenyl esters, aliphatic (C 1-24) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, a compound of the present disclosure containing a hydroxyl group can be acylated to produce a prodrug form of the compound. Other prodrug forms include phosphate esters, such as these phosphate ester compounds, which are obtained by phosphorylating a hydroxyl group on the parent compound. For a complete discussion of prodrugs, see T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, J. Rautio et al., Prodrugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270, and SJ Hecker et al., Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry, 2008, 51, 2328-2345.

[0302] The term "nitrogen oxide" refers to when a compound contains several amine functional groups, where one or more nitrogen atoms can be oxidized to form an N-oxide. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. Available oxidants such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids) can be used to treat the corresponding amine to form an N-oxide (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular, N-oxides can be prepared by the method of LW Deady (Syn. Comm. 1977, 7, 509-514), wherein, for example, in an inert solvent such as dichloromethane, an amine compound is reacted with meta-chloroperoxybenzoic acid (MCPBA).

[0303] The term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0304] The term "alkyl" is understood to mean preferably a linear or branched saturated monovalent hydrocarbon radical, preferably a "C 1-10 Alkyl". "C 1-10"Alkyl" is understood to mean preferably a straight-chain or branched saturated monovalent hydrocarbon radical having 1 to 10 carbon atoms. For example, "C 1-6 "Alkyl" means straight-chain and branched alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms. 1-4 The term "alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3 or 4 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof.

[0305] The term "cycloalkyl" is understood to mean a saturated monovalent monocyclic hydrocarbon group or a bicyclic (condensed, bridged or spiro) hydrocarbon group, preferably a "C 3-10 Cycloalkyl". The term "C 3-10 "Cycloalkyl" is understood to mean a saturated monovalent monocyclic, bicyclic (fused, bridged or spiro) hydrocarbon group having 3 to 10 carbon atoms, such as 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term "C 3-6 The "cycloalkyl" group has 3 to 6 carbon atoms, such as 3, 4, 5, or 6 carbon atoms. The cycloalkyl group may be a monocyclic hydrocarbon group, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl, or a bicyclic hydrocarbon group such as a decalin ring.

[0306] The term "ring structure" should be understood to mean a group having a monocyclic, bicyclic (fused, bridged or spiro) or more ring structure, such as a monocyclic hydrocarbon group, a bicyclic hydrocarbon group, a tricyclic hydrocarbon group, a heteromonocyclic hydrocarbon group, a heterobicyclic hydrocarbon group, a heterotricyclic hydrocarbon group, etc.; the ring structure may be saturated or unsaturated. The term "ring structure" may preferably be a 4-10 membered ring structure having 4, 5, 6, 7, 8, 9 or 10 ring atoms or preferably a 3-10 membered ring structure having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms. The heteromonocyclic hydrocarbon group, heterobicyclic hydrocarbon group, heterotricyclic hydrocarbon group may contain 1-10, preferably 1-5, heteroatoms independently selected from N, O and S, such as 1, 2, 3, 4, 5 heteroatoms independently selected from N, O and S. The bicyclic ring may be the following ring structures containing no heteroatoms or containing 1, 2, or 3 heteroatoms independently selected from N, O, and S: one or two of aryl, heteroaryl, cycloalkyl, heterocyclyl, spiro[2.5] ring, spiro[3.3] ring, spiro[4.2] ring, spiro[4.3] ring, spiro[5.2] ring, spiro[5.4] ring, bicyclo[2.1.1], bicyclo[2.2.1], bicyclo[2.2.2], bicyclo[3.2.1], bicyclo[4.1.0], etc., independently or fused to each other. Alternatively, the ring structure may have 1, 2, 3, 4, or 5 double bonds, such as carbon-carbon double bonds or carbon-nitrogen double bonds.

[0307] In various parts of this disclosure, linking substituents are described. When the structure clearly requires a linking group, the Markush variable listed for that group should be understood to be a linking group. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl", it should be understood that the "alkyl" or "aryl" represents a linking alkylene group or arylene group, respectively.

[0308] The term "alkylene" is understood to mean a saturated straight-chain or branched aliphatic hydrocarbon radical having two residues derived from the removal of two hydrogen atoms from the same carbon atom or from two different carbon atoms of a parent alkane, which is a straight-chain or branched radical containing 1 to 20 carbon atoms, preferably an alkylene radical containing 1 to 10 carbon atoms (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably an alkylene radical containing 1 to 6, or 1 to 4 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2-), 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment, and the substituents are independently preferably substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0309] The term "alkenylene" is understood to mean a straight or branched chain group of 1 to 20 carbon atoms and containing at least one carbon-carbon double bond, preferably containing 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and containing at least one carbon-carbon double bond, more preferably containing 2 to 6 carbon atoms and containing at least one carbon-carbon double bond. Non-limiting examples of alkenylene include, but are not limited to, -CH=CH-, -C(CH3)=CH-, -CH=CHCH2CH2-, -CH=CHCH2-, -CH=CHCH2CH=CH-, -CH=CHCH2CH2CH=CH-, -CH=CHCH2CH=CHCH2CH=CH-. The alkenylene group may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment, and the substituents are independently preferably substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0310] The term "alkynylene" is understood to mean a straight or branched chain group of 1 to 20 carbon atoms and comprising at least one carbon-carbon triple bond, preferably comprising 1 to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and comprising at least one carbon-carbon triple bond, more preferably comprising 1 to 6 carbon atoms or 1 to 4 carbon atoms and comprising at least one carbon-carbon triple bond. Non-limiting examples of alkynylene include, but are not limited to, -CH≡CH-, -C(CH3)≡CH-, -CH≡CHCH2CH2-, -CH≡CHCH2-, -CH≡CHCH2CH≡CH-, -CH≡CHCH2CH2CH≡CH-, -CH≡CHCH2CH2CH≡CH-, -CH≡CHCH2CH2CH≡CH-. Alkyne groups may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment, and the substituents are independently preferably substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0311] It should be understood that when the groups of the compounds described in the present disclosure form the above-mentioned ring structure and then form a fused ring, spiro ring or bicyclic ring with the original ring structure of the compound, and there is an unsaturated bond in the original ring structure of the compound, the ring structure formed by the groups preferably retains the unsaturated bond in the original ring structure.

[0312] The term "heterocyclyl" means a saturated monovalent monocyclic, bicyclic (fused, bridged or spirocyclic) hydrocarbon group containing 1 to 5 heteroatoms independently selected from N, O and S, preferably a "3-20 membered heterocyclyl". The term "3-20 membered heterocyclyl" means a saturated monovalent monocyclic, bicyclic (fused, bridged or spirocyclic) hydrocarbon group containing 1 to 5 heteroatoms independently selected from N, O and S, and a non-aromatic cyclic group with a total ring number of 3 to 20 (such as 3, 4, 5, 6, 7, 8, 9, 10, etc.) containing 1 to 5 heteroatoms independently selected from N, O and S, preferably a "3-10 membered heterocyclyl". The term "3-10 membered heterocyclyl" means a saturated monovalent monocyclic, bicyclic (fused, bridged or spirocyclic) hydrocarbon group containing 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S, such as 1, 2, or 3 heteroatoms independently selected from N, O and S. The heterocyclic group can be connected to the rest of the molecule through any one of the carbon atoms or nitrogen atom (if present). In particular, the heterocyclic group can include but is not limited to: 4-membered rings, such as azetidinyl, oxetanyl (such as azetidin-1-yl); 5-membered rings, such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or 6-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or 7-membered rings, such as diazepanyl. Optionally, the heterocyclic group can be benzo-fused. The heterocyclic group can be bicyclic, for example, but not limited to 5,5-membered rings, such as hexahydrocyclopenta [c] pyrrole -2 (1H) - base ring, or 5,6-membered bicyclic rings, such as hexahydropyrrolo [1,2-a] pyrazine -2 (1H) - base ring. The ring containing the nitrogen atom may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl, or it may be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. According to the present disclosure, the heterocyclic group is non-aromatic. When the heterocyclic group is connected to other groups to form the compounds of the present disclosure, the carbon atoms on the heterocyclic group may be connected to the other groups, or the heterocyclic atoms on the heterocyclic group may be connected to the other groups. For example, when the heterocyclic group is selected from piperazinyl, the nitrogen atom on the piperazinyl may be connected to the other groups. Or when the heterocyclic group is selected from piperidinyl, the nitrogen atom on the piperidinyl ring and the carbon atom at the para position thereof may be connected to the other groups.

[0313] The term "aryl" is understood to mean a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring group, preferably "C 6-20 Aryl". The term "C 6-20 "Aryl" is understood to mean preferably a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms, preferably "C 6-14 Aryl" or "C 6-12Aryl". The term "C 6-14 "Aryl" is understood to mean preferably a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring ("C 6-14 or a ring having 9 carbon atoms ("C9 aryl"), for example indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 aryl) such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C 13 aryl), such as fluorenyl, or a ring having 14 carbon atoms ("C 14 When the aryl group is substituted, it may be monosubstituted or polysubstituted. Furthermore, there is no limitation on the substitution site, for example, it may be ortho-, para- or meta-substituted.

[0314] The term "heteroaryl" is understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring system radical containing 1 to 5 heteroatoms independently selected from N, O and S, preferably a "5-20 membered heteroaryl". The term "5-20 membered heteroaryl" is understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring system radicals having 5 to 20 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O and S, for example, a "5-14 membered heteroaryl" or a "5-12 membered heteroaryl". The term "5-14 membered heteroaryl" is to be understood as including monovalent monocyclic, bicyclic or tricyclic aromatic ring system radicals having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and which contain 1 to 5, preferably 1, 2 or 3 heteroatoms independently selected from N, O and S and, in addition, in each case may be benzo-fused. In particular, the heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl and the like; or acininyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like. When the heteroaryl group is linked to other groups to form the compounds of the present disclosure, the linking may be to a carbon atom on the heteroaryl ring or to a heteroatom on the heteroaryl ring. When the heteroaryl group is substituted, it may be monosubstituted or polysubstituted. Furthermore, there is no limitation on the substitution sites; for example, a hydrogen atom linked to a carbon atom on the heteroaryl ring may be substituted, or a hydrogen atom linked to a heteroatom on the heteroaryl ring may be substituted.

[0315] Unless otherwise specified, a heterocyclic group, heteroaryl group, or heteroarylene group includes all possible isomeric forms thereof, such as positional isomers thereof. Thus, for some illustrative non-limiting examples, the following may be substituted or bonded to other groups at one, two, or more positions of the 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-position, etc. (if present): pyridin-2-yl, pyridin-2-ylene, pyridin-3-yl, pyridin-3-ylene, pyridin-4-ylene, and pyridin-4-ylene; thienyl or thienylene group includes thien-2-yl, thien-2-ylene, thien-3-ylene, and thien-3-ylene; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

[0316] The term "oxo" refers to a substituent wherein a carbon atom, nitrogen atom or sulfur atom is oxidized to form an oxy group (=O). It should be understood that when a carbon atom is substituted with an oxy group, a carbonyl group -C(=O)- is formed.

[0317] Unless otherwise specified, the definitions of terms herein also apply to the groups containing the terms. For example, the definition of alkyl also applies to the alkyl in alkyloxy or cycloalkylalkyl; the definition of cycloalkyl also applies to the cycloalkyl in cycloalkyloxy or cycloalkylalkyl; for example, C 1-10 The definition of alkyl also applies to C 1-10 Alkyloxy or C 3-10 Cycloalkyl C 1-10 C in the alkyl group 1-10 Alkyl; C 3-10 The definition of cycloalkyl also applies to C 3-10 Cycloalkyloxy or C 3-10 Cycloalkyl C 1-10 C in the alkyl group 3-10 Cycloalkyl.

[0318] The term "sulfhydryl-reactive group" refers to any group that can react with a sulfhydryl group contained in Tp (such as an antibody), such as a maleimide group, a substituted maleimide group, a halogen, OMs, OTs, OTf, a nitro group, an alkyl sulfide group, an aryl sulfide group, a heteroaryl sulfide group, an alkyl sulfoxide group, an aryl sulfoxide group, a heteroaryl sulfoxide group, an alkyl sulfonyl group, an aryl sulfonyl group, a heteroaryl sulfonyl group.

[0319] The term "amino-reactive group" refers to any group that can react with an amino group contained in Tp (such as an antibody), such as succinimidyl ester (NHS), nitrophenyl ester (NPC), pentafluorophenyl ester (PFP), aldehyde group (CHO) such as acetaldehyde or propionaldehyde, epoxy group (EPO), isothiocyanate (ISC).

[0320] The term "carboxyl reactive group" refers to any group that can react with a carboxyl group contained in Tp (such as an antibody), such as hydroxyl, amino, sulfhydryl, halogen, amidino, and guanidino.

[0321] The term "dithiol bridging group" refers to any bifunctional linker that can react with two free thiol / sulfhydryl groups contained in Tp (such as an antibody) to form a bridge, such as a bismaleimide reagent.

[0322] The term "click chemistry reactive group" refers to a group that can undergo a "click chemistry" reaction, such as a ketone, hydrazine or hydrazide, azide, alkyne, cyclopropene or diene. A typical click chemistry reaction is the reaction between azide and alkyne to form a 5-membered heteroatom ring. Those skilled in the art can introduce click chemistry groups into the antibody or other polypeptide targeting group Tp through techniques such as codon expansion.

[0323] It will be appreciated by those skilled in the art that the compounds of the present disclosure may exist in the form of various pharmaceutically acceptable salts. If these compounds have a basic center, they may form acid addition salts; if these compounds have an acidic center, they may form base addition salts; if these compounds contain both an acidic center (e.g., a carboxyl group) and a basic center (e.g., an amino group), they may also form internal salts.

[0324] The compounds of the present disclosure may exist in the form of solvates (e.g., hydrates), wherein the compounds of the present disclosure contain a polar solvent, such as water, methanol, or ethanol, as a structural element of the crystal lattice of the compound. The amount of the polar solvent, particularly water, may be present in a stoichiometric or non-stoichiometric ratio.

[0325] Depending on their molecular structure, the compounds or groups disclosed herein may be chiral or have chiral carbon atoms, and thus may exist in various enantiomeric forms. Thus, these compounds or groups may exist in racemic or optically active forms. For example, when the group A in the general formula (G), (G') or (GH) is CR A , and when the groups to which the carbon atom is attached are different, the carbon atom is a chiral carbon atom, which may have an R or S chiral configuration. The compounds disclosed herein or their intermediates can be separated into enantiomeric compounds by chemical or physical methods well known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers can be prepared from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids, such as R and S forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, appropriate N-protected amino acids (such as N-benzoylproline or N-phenylsulfonylproline) or various optically active camphorsulfonic acids. Chromatographic enantiomer resolution can also be advantageously performed with the aid of optically active resolving agents (such as dinitrobenzoylphenylglycine, cellulose triacetate or other carbohydrate derivatives or chiral derivatized methacrylate polymers immobilized on silica gel). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, for example hexane / isopropanol / acetonitrile.

[0326] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom between two positions in a molecule. Compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist as two or more interconvertible species. Prototropic tautomers arise from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present disclosure encompasses all tautomeric forms of the compounds.

[0327] The corresponding stable isomers can be separated according to known methods, for example by extraction, filtration or column chromatography.

[0328] The term "patient" refers to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

[0329] As used herein, the phrase "therapeutically effective amount" refers to that amount of an active compound or drug that will elicit the biological or medical response that a researcher, veterinarian, physician, or other clinician is seeking in a tissue, system, animal, individual, or human, and includes one or more of the following: (1) prevents disease, e.g., prevents a disease, disorder, or condition in an individual who is susceptible to the disease, disorder, or condition but who is not yet experiencing or developing the pathology or symptoms of the disease. (2) inhibits disease, e.g., inhibits the disease, disorder, or condition (i.e., prevents further development of the pathology and / or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder, or condition. (3) alleviates disease, e.g., alleviates the disease, disorder, or condition (i.e., reverses the pathology and / or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder, or condition.

[0330] The term "ligand" refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of a ligand is to present a drug to a target cell population bound to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells. In the embodiments of the present disclosure, the ligand is represented by LG. The ligand can form a linker bond with a linker through a heteroatom on the ligand, preferably an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody; preferably, a monoclonal antibody.

[0331] The term "drug" refers to any compound having a desired biological activity and a reactive functional group that can be used to incorporate the drug into the conjugates of the present disclosure. The desired biological activity includes diagnosing, curing, alleviating, treating, or preventing a disease in humans or other animals. The reactive functional group is in a state where the functional group L 3 or L 4 In some embodiments, the drug has a functional group L 3 or L 4 nitrogen atoms or hydroxyl groups that form bonds.

[0332] The term "antibody" refers to immunoglobulins, which consist of a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds (also known as "monoclonal antibody" or "monoepitope antibody"), or a tetrapeptide chain structure composed of two different heavy chains and two different light chains linked by interchain disulfide bonds (also known as "diabody" or "bi-epitope antibody"). Immunoglobulin heavy chain constant regions differ in their amino acid composition and arrangement, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class of Ig, there are different subclasses based on the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have a kappa chain or a lambda chain. The antibodies disclosed herein are preferably specific antibodies against cell surface antigens on target cells, and non-limiting examples include the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis antibody. Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody or more; preferably Trastuzumab (Trastuzumab, trade name Herceptin), Pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), Nimotuzumab (Nimotuzumab, trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 or Glembatumumab.

[0333] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

[0334] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0335] The term "murine antibody" as used herein refers to antibodies produced in mice according to the knowledge and skills in the art. During production, a test subject is injected with a specific antigen and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.

[0336] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0337] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the antibody variable region framework of a human, i.e., different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase), as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after CDR affinity maturation by phage display. References further describing methods for humanizing mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

[0338] The terms "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", are antibodies whose variable and constant regions are both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

[0339] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region, (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) a combination of two or more isolated CDRs, optionally connected by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0340] Fab is an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity, among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which approximately half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

[0341] F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and comprises two Fab regions linked at the hinge position.

[0342] Fab' is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.

[0343] Furthermore, the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.

[0344] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, using variants with 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0345] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3, or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3, or H2, H3) of the heavy chain variable domain.

[0346] The term "antibody framework" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0347] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0348] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0349] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0350] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, a vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, a vector is a viral vector, in which another DNA segment can be connected to a viral genome. Vectors disclosed herein can autonomously replicate in the host cell into which they have been introduced (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after introducing the host cell, thereby replicating (e.g., non-additional mammalian vectors) with the host genome.

[0351] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as those described in Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in the present invention utilize genetic engineering methods to add one or more human FR regions to non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website (http: / / imgt.cines.fr) by comparing the IMGT Human Antibody Variable Region Germline Gene Database with MOE software, or from the Journal of Immunoglobulins, 2001 ISBN 012441351.

[0352] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plants, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary) and NSO cells.

[0353] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended existing technology, mammalian expression systems will result in glycosylation of antibodies, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or freeze-dried.

[0354] The term "peptide" refers to a compound fragment between amino acids and proteins, which is composed of two or more amino acid molecules connected by peptide bonds. It is a structural and functional fragment of protein. Hormones, enzymes, etc. are essentially peptides.

[0355] The term "sugar" refers to biological macromolecules composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides, and polysaccharides.

[0356] The term "amino acid analog" (e.g., "arginine analog," "lysine analog," or "histidine analog") refers to amino acid variants that retain at least one function of the amino acid, such as variants that retain side chain polarity or electrostatic interactions. Such variants can have extended or shortened side chains, for example, by retaining side chain polarity or electrostatic interactions through one or more -CH2- groups. For example, an arginine analog can include an additional methylene or ethylene group between the backbone and the guanidine / guanidinium group. Beneficial effects

[0357] The compounds and conjugates disclosed herein have excellent tumor cell inhibition activity, stability, and in vivo efficacy in animals, and can be used as effective drugs for inhibiting tumor cells and preventing and / or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0358] FIG1 shows the test results of the bystander killing effect of the disclosed antibody drug conjugate on HER2 target tumor cells

[0359] FIG2 shows the test results of the bystander killing effect of the disclosed antibody drug conjugate on EGFR target tumor cells DETAILED DESCRIPTION

[0360] The technical solutions of the present disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present disclosure and should not be construed as limiting the scope of protection of the present disclosure. All technologies implemented based on the above content of the present disclosure are included within the scope of protection intended by the present disclosure.

[0361] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0362] I. Antibody Examples

[0363] The following antibodies were prepared according to conventional antibody methods, for example, after constructing the vector, they were transfected into eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019).

[0364] Exemplary antibody sequences are as follows:

[0365] The following is the sequence of Trastuzumab

[0366] light chain

[0367] heavy chain

[0368] The following is the sequence of Pertuzumab

[0369] light chain

[0370] heavy chain

[0371] The following is the sequence of Nimotuzumab

[0372] light chain

[0373] heavy chain

[0374] II. Compound Examples

[0375] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). -6 The unit of measurement is ppm. NMR measurements were performed using a Bruker NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0376] LCMS analysis was performed using an Agilent 1260 Infinity II (ESI) mass spectrometer, a Waters UPLC H Class plus (ESI), or a Shimadzu LCMS-2020 (ESI).

[0377] High performance liquid chromatography (HPLC) analysis was performed using Agilent 1260 or Shimadzu LC-20AD.

[0378] Preparative high performance liquid chromatography (pre-HPLC) was performed using a GILSON GX-281 or Agilent 1260 Infinity II preparative liquid phase.

[0379] Chiral preparation was performed using critical fluid chromatography (SFC) with a Shimadzu LC-30Adsf or Shimadzu LC-20AD instrument.

[0380] The thin layer chromatography silica gel plate used was GF254 acrylic adhesive silica gel plate produced by Anhui Liangchen Silicon Source Material Co., Ltd. The silica gel plate used in thin layer chromatography (TLC) was of 0.2 mm silica gel plate, and the specification used for thin layer chromatography separation and purification products was 0.5 mm silica gel plate.

[0381] Column chromatography generally uses 200-300 mesh silica gel produced by Anhui Liangchen Silicon Source Materials Co., Ltd. as a carrier.

[0382] Average kinase inhibition rate and IC 50 The values ​​were determined using SpectraMax i3X microplate reader (MD, USA).

[0383] The known starting materials disclosed herein can be synthesized using methods known in the art, or can be purchased from companies such as Bede Pharmaceuticals, Leyan, Shaoyuan Chemical Technology, and Anaiji Chemicals.

[0384] Unless otherwise specified in the following examples, all reactions were carried out under an argon or nitrogen atmosphere.

[0385] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

[0386] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0387] The hydrogenation reaction is usually carried out by evacuating the chamber and filling it with hydrogen, and the operation is repeated three times.

[0388] Oxygen atmosphere means that the reaction bottle is connected to an oxygen balloon with a capacity of about 1L.

[0389] In the following examples, unless otherwise specified, solution refers to aqueous solution, and the reaction temperature is room temperature, 20°C-30°C.

[0390] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reaction, the column chromatography eluent system used for the purified compounds, and the developing solvent system for thin layer chromatography included: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compounds, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0391] Example 2-1: Preparation of Compound 1

[0392] first step

[0393] Ethyl 2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolane]-7-yl)-3-cyclopropylpropanoate 1b

[0394] 1a (1.01 g, 3.31 mmol, prepared using the method disclosed in Example 30 on page 28 of patent application "WO2019238046") was dissolved in 15 mL of acetonitrile. Bromomethylcyclopropane (894.93 mg, 6.63 mmol) and potassium carbonate (916.16 mg, 6.63 mmol) were added, and the mixture was stirred at 80°C for 13 hours. 10 mL of water was added, and the diluted reaction solution was extracted with ethyl acetate (15 mL x 2). The organic phase was washed with saturated sodium chloride solution (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The resulting residue was purified by silica gel column chromatography using developing system A to obtain the title product 1b (1.12 g, 92% yield) as a yellow solid.

[0395] MS m / z(ESI):359.1[M+1].

[0396] Step 2

[0397] Ethyl 3-cyclopropyl-2-(6-formyl-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolane]-7-yl]propanoate 1c

[0398] 1b (1.12 g, 3.05 mmol) was dissolved in a mixed solvent of 5 mL of water, 5 mL of acetonitrile, and 5 mL of formic acid. Raney nickel (261.72 mg) was added under nitrogen protection. The atmosphere was replaced with hydrogen three times, and the reaction solution was stirred at 60°C under hydrogen (15 Psi) for 4 hours. The reaction solution was filtered through celite, and the filter cake was washed with dichloromethane (50 mL×3). The filtrate was washed with aqueous hydrochloric acid (4 M, 20 mL) and then with aqueous sodium carbonate (12 M, 50 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated by distillation under reduced pressure. The resulting residue was purified by reverse-phase liquid chromatography (separation conditions: chromatographic column: 120 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water: B-acetonitrile, gradient elution, flow rate: 85 mL / min, instrument: ISCO) to obtain the title product 1c (680 mg, yield: 60%) as a yellow solid.

[0399] MS m / z(ESI):362.1[M+1].

[0400] Step 3

[0401] 4-(Cyclopropylmethyl)-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4-f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1d

[0402] Dissolve 1c (680 mg, 1.85 mmol) in 10 mL of dichloromethane under nitrogen atmosphere. Cool to 0°C in an ice-water bath. Add sodium borohydride (108.02 mg, 2.86 mmol) portionwise. Stir the reaction mixture at 0°C for 30 minutes. Add acetic acid (133.15 mg, 2.22 mmol) dropwise at 25°C, generating gas. Stir for 2 hours. Add 30 mL of water dropwise at 15°C, generating gas. Stir at 15°C for 1.5 hours. Wash the reaction mixture with water (50 mL). Add p-toluenesulfonic acid monohydrate (35.15 mg, 184.78 μmol) to the washed organic phase at 15°C, and stir at 15°C for 12 hours. 50 mL of water was added, and the reaction solution was extracted with dichloromethane (45 mL×3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing system B to obtain the title product 1d (200 mg, yield: 32%) as a yellow oil.

[0403] MS m / z(ESI):318.1[M+1].

[0404] Step 4

[0405] 4-(Cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4-f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e

[0406] 1d (202.13 mg, 598.74 μmol) was dissolved in 0.5 mL of methanol and cooled to 0°C in an ice-water bath. Potassium carbonate (82.75 mg, 598.74 μmol) was added portionwise, and stirred at 0°C for 5 hours under oxygen bubbling (15 psi). The reaction solution was poured into 10 mL of saturated aqueous ammonium chloride and concentrated by distillation under reduced pressure to remove the methanol. The reaction solution was extracted with dichloromethane (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated by distillation under reduced pressure to obtain the crude title product 1e (140 mg) as a yellow oil, which was used directly in the next reaction without purification.

[0407] MS m / z(ESI):334.1[M+1].

[0408] Step 5

[0409] (S)-4-(Cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[3,4-f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e-1

[0410] (R)-4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[3,4-f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1e-2

[0411] 1e (1.30 g, 3.88 mmol) was separated by SFC (separation conditions: chromatographic column: DAICEL CHIRALPAK AS 250 mm × 50 mm, 10 μm; mobile phase: A-carbon dioxide: B-methanol (0.1% NH3·H2O), isocratic elution: B: 20%, flow rate: 120 mL / min, instrument: Shimadzu LC-30ADsf) to obtain the title product 1e-1 (301 mg, yield: 22.1%) as a yellow solid, and the title product 1e-2 (285 mg, yield: 19.4%) as a yellow solid.

[0412] Single configuration compound 1e-1

[0413] SFC analysis: retention time 1.392 minutes. (Chromatographic column: Chiralpak AS-3 50×4.6 mm ID, 3 μm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B%: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0414] MS m / z(ESI):334.0[M+1].

[0415] Single configuration compound 1e-2

[0416] SFC analysis: retention time 1.762 minutes. (Chromatographic column: Chiralpak AS-3 50×4.6 mm ID, 3 μm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B%: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0417] MS m / z(ESI):333.9[M+1].

[0418] Step 6

[0419] (S)-4-(Cyclopropylmethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione 1f-1

[0420] 1e-1 (301 mg, 857 μmol) was dissolved in a mixture of 2 mL of trifluoroacetic acid and 0.5 mL of water and stirred at 25°C for 4 hours. The reaction solution was concentrated by distillation under reduced pressure. The resulting residue was unpurified to give the crude title product 1f-1 (209 mg) as a yellow solid, which was used directly in the next reaction without purification.

[0421] MS m / z(ESI):290.1[M+1].

[0422] Step 7

[0423] Dissolve 1g (800mg, 5.83mmol) in 20mL of 1,2-dichloroethane. Cool to 0°C in an ice-water bath under nitrogen. Add a 1M dichloromethane solution of boron trichloride (4.08mL) and 4-pentenenitrile (709mg, 8.75mmol) dropwise. Stir at 80°C for 2 hours under nitrogen. Cool to room temperature, add aqueous hydrochloric acid (50mL, 2M), and stir at 80°C for 0.5 hours. Extract with dichloromethane (30mL x 3). Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography using developing system B to obtain the title product 1h (220mg, 17% yield) as a yellow oil.

[0424] MS m / z(ESI):220.2[M+1].

[0425] Step 8

[0426] 1f-1 (288 mg, 991 μmol) and 1h (220 mg, 991 μmol) were dissolved in 10 mL of toluene. 4-Methylbenzenesulfonate pyridinium chloride (124 mg, 495 μmol) and o-cresol (214 mg, 1.98 mmol) were added, and the mixture was stirred at 120°C for 2 hours under nitrogen. 50 mL of water was added to the reaction mixture, and the mixture was extracted with dichloromethane (45 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing system A to obtain the title product 1i (202 mg, 41% yield) as a yellow solid.

[0427] MS m / z(ESI):473.0[M+1].

[0428] Step 9

[0429] Dissolve 1i (200 mg, 406 μmol) in 10 mL of dichloromethane, cool to -78°C, and bubble ozone through for 0.5 hour (15 psi). Excess ozone is purged with nitrogen. Triphenylphosphine (213 mg, 812 μmol) is added at -78°C, and the mixture is stirred at -78°C for 0.5 hour. Add 50 mL of water to the reaction mixture, extract with dichloromethane (45 mL x 3), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography using dichloromethane / tetrahydrofuran to afford the title product 1j (70 mg, 36% yield) as a yellow solid.

[0430] MS m / z(ESI):475.3[M+1].

[0431] Step 10

[0432] 1j (70.0 mg, 147 μmol) was dissolved in 5 mL of tetrahydrofuran. Sodium borohydride (2.79 mg, 73.7 μmol) was added under nitrogen atmosphere, and the mixture was stirred at -78°C for 1 hour. 10 mL of ammonium chloride solution was added to the reaction solution, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (separation conditions: column: Phenomenex luna C18 150 × 25 mm × 10 μm; mobile phase: A-water (0.225% formic acid): B-acetonitrile, gradient elution: B%: 22%-42%) to obtain the title product 1 (8.95 mg, yield: 21%) as a yellow solid.

[0433] MS m / z(ESI):477.0[M+1].

[0434] 1 H NMR(400MHz,CD3OD)δ7.64–7.61(m,1H),7.55–7.51(m,1H),7.41–7.37(m,1H ),6.24–6.19(m,2H),5.62–5.55(m,1H),5.40–5.34(m,1H),5.26–5.21(m,2H) ,3.72–3.64(m,2H),3.25–3.19(m,2H),1.99–1.90(m,3H),1.89–1.81(m,1H) ,0.97–0.88(m,1H),0.52–0.38(m,2H),0.15–0.09(m,1H),0.07–0.00(m,1H).

[0435] Example 2-2: Preparation of Compound 2

[0436] first step

[0437] Dissolve 2a (1.00 g, 7.99 mmol) in 20 mL of 1,2-dichloroethane. Cool to 0°C in an ice-water bath under nitrogen. Add a 1 M dichloromethane solution of boron trichloride (10.4 mL) and 4-pentenenitrile (777 mg, 9.59 mmol) dropwise. Stir at 80°C for 5 hours under nitrogen. Cool to room temperature, add aqueous hydrochloric acid (2 M, 9 mL), and stir at 80°C for 0.5 hours. Extract with dichloromethane (30 mL x 3). Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography using developing system B to obtain the title product 2b (315 mg, 19% yield) as a yellow solid.

[0438] MS m / z(ESI):208.1[M+1].

[0439] Step 2

[0440] 1f-1 (400 mg, 1.37 mmol) and 2b (313 mg, 1.51 mmol) were dissolved in 50 mL of toluene. 4-Methylbenzenesulfonate pyridinium (207 mg, 823 μmol) and o-cresol (1.19 g, 11.0 mmol) were added, and the mixture was stirred at 135°C for 3 hours. The reaction solution was concentrated by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing system A to obtain the title product 2c (420 mg, 60% yield) as a yellow solid.

[0441] MS m / z(ESI):461.3[M+1].

[0442] Step 3

[0443] 2c (100 mg, 197 μmol) was dissolved in 2 mL of dichloromethane, cooled to -78°C, and ozone was bubbled through for 0.5 hour (15 psi). Excess ozone was purged with nitrogen. Triphenylphosphine (51.7 mg, 197 μmol) was added at -78°C, and stirred at -78°C for 0.5 hour. The reaction solution was concentrated by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing system A to obtain the title product 2d (53.0 mg, 54% yield) as a yellow solid.

[0444] MS m / z(ESI):463.2[M+1].

[0445] Step 4

[0446] 2d (40.0 mg, 80.1 μmol) was dissolved in a mixed solvent of 2 mL of tetrahydrofuran and 0.1 mL of N,N-dimethylacetamide under nitrogen protection. Sodium borohydride (1.52 mg, 40.1 μmol) was added and stirred at -78°C for 1 hour. 1 mL of ammonium chloride solution was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 mL×3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (separation conditions: chromatographic column: Phenomenex luna C18 The reaction mixture was purified by supercritical fluid chromatography on a column: Phenomenex-Cellulose-2 (250 mm × 30 mm, 10 μm); mobile phase: A-water (0.225% formic acid): B-acetonitrile, gradient elution: B%: 27%-57%), and then purified by supercritical fluid chromatography on a column: Phenomenex-Cellulose-2 (250 mm × 30 mm, 10 μm); mobile phase: A-carbon dioxide, B-methanol and acetonitrile, isocratic elution: B%: 55% to obtain the title product 2 (8.66 mg, yield: 45%) as a light yellow solid.

[0447] MS m / z(ESI):464.9[M+1].

[0448] 1 H NMR (400MHz, CD3OD) δ8.24–8.17(m,1H),7.80–7.73(m,1H),7.73–7.67(m,1H),5.64–5.57(m,1H),5.44–5.33(m,3H),3.75–3.66(m,2H),3.40–3. 34(m,2H),2.60–2.52(m,3H),2.04–1.96(m,2H),1.94–1.82(m,2H),0.93 –0.89(m,1H),0.52–0.38(m,2H),0.17–0.08(m,1H),0.07–-0.04(m,1H).

[0449] Example 2-3: Preparation of Compound 3

[0450] first step

[0451] Dissolve 1 g (1.5 g, 10.94 mmol) of the product in 20 mL of 1,2-dichloroethane. Cool to 0°C in an ice-water bath under nitrogen. Add a 1 M dichloromethane solution of boron trichloride (8.75 mL) and 5-hexenonitrile (1.56 g, 16.4 mmol) dropwise. Stir at 80°C for 2 hours under nitrogen. Cool to room temperature, add aqueous hydrochloric acid (50 mL, 2 M), and stir at 80°C for 0.5 hours. Extract with dichloromethane (50 mL x 3). Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography using developing system B to obtain the title product 3a (502 mg, 19% yield) as a yellow oil.

[0452] MS m / z(ESI):234.0[M+1].

[0453] Step 2

[0454] 1f-1 (249 mg, 857 μmol) and 3a (200 mg, 780 μmol) were dissolved in 5 mL of toluene. 4-Methylbenzenesulfonate pyridinium chloride (150 mg, 600 μmol) and o-cresol (185 mg, 1.71 mmol) were added, and the mixture was stirred at 120°C for 2 hours under nitrogen. 50 mL of water was added to the reaction solution, which was then extracted with dichloromethane (45 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing system A to obtain the title product 3b (310 mg, 53% yield) as a yellow solid.

[0455] MS m / z(ESI):487.3[M+1].

[0456] Step 3

[0457] 3b (250 mg, 513 μmol) was dissolved in 5 mL of dichloromethane, cooled to -78°C, and ozone was bubbled through for 0.5 hour (15 psi). Excess ozone was purged with nitrogen. Triphenylphosphine (674 mg, 2.57 mmol) was added at -78°C, and the mixture was stirred at -78°C for 0.5 hour. The reaction solution was concentrated by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography using dichloromethane / tetrahydrofuran to obtain the title product 3c (65.0 mg, 26% yield) as a yellow solid.

[0458] MS m / z(ESI):489.2[M+1].

[0459] Step 4

[0460] 3c (65.0 mg, 133 μmol) was dissolved in 15 mL of tetrahydrofuran under nitrogen atmosphere. Sodium borohydride (2.5 mg, 66 μmol) was added and stirred at -78°C for 1 hour. 20 mL of ammonium chloride solution was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (separation conditions: column: Phenomenex luna C18 150 × 25 mm × 10 μm; mobile phase: A-water (0.225% formic acid): B-acetonitrile, gradient elution: B%: 25%-45%) to obtain the title product 3 (5.69 mg, yield: 9%) as a yellow solid.

[0461] MS m / z(ESI):491.0[M+1].

[0462] 1 H NMR (400MHz, CD3OD) δ7.62–7.58(m,1H),7.50–7.46(m,1H),7.37–7.34(m,1H),6. 24–6.18(m,2H),5.62–5.54(m,1H),5.42–5.32(m,1H),5.21–5.16(m,2H),3.69–3. 62(m,2H),3.17–3.10(m,2H),1.98–1.90(m,1H),1.88–1.79(m,3H),1.78–1.70(m, 2H),0.96–0.85(m,1H),0.52–0.40(m,2H),0.18–0.08(m,1H),0.07–-0.00(m,1H).

[0463] Example 2-4: Preparation of Compound 4

[0464] Example 2-5: Preparation of Compound 5

[0465] Example 2-6: Preparation of Compound 6

[0466] Example 2-7: Preparation of Compound 7

[0467] Example 2-8: Preparation of Compound 8

[0468] Example 2-9: Preparation of Compound 9

[0469] Example 2-10: Preparation of Compound 10

[0470] Example 2-11: Preparation of Compound 11

[0471] Example 2-12: Preparation of Compound 12

[0472] Example 2-13: Preparation of Compound 13

[0473] Example 2-14: Preparation of Compound 14

[0474] Example 2-15: Preparation of Compound 15

[0475] Example 2-16: Preparation of Compound 16

[0476] Example 2-17: Preparation of Compound 17

[0477] Example 2-18: Preparation of Compound 18

[0478] Example 2-19: Preparation of Compound 19

[0479] Example 2-20: Preparation of Compound 20

[0480] Example 2-21: Preparation of Compound 21

[0481] Example 2-22: Preparation of Compound 22

[0482] Example 2-23: Preparation of Compound 23

[0483] Example 2-24: Preparation of Compound 24

[0484] Example 2-25: Preparation of Compound 25

[0485] Example 2-26: Preparation of Reference Material 1

[0486] It was prepared using the method disclosed in Example A1.9 on page 207 of patent application "WO2022170971 Al".

[0487] III. Example of Preparation of Conjugate Intermediate Linker-Drug

[0488] Example 3-1 LD-1

[0489] first step

[0490] LD-1a (2.20 g, 12.1 mmol, prepared using the method disclosed in Example 4 on page 62 of patent application "WO2020077038 A1"), silver nitrite (111 mg, 724 μmol), bis(benzonitrile)palladium(II) chloride (555 mg, 1.45 mmol), nitromethane (4.90 g, 80.3 mmol), and copper(II) chloride dihydrate (247 mg, 1.45 mmol) were dissolved in 44 mL of tert-butanol. The mixture was purged with oxygen three times and stirred at 25°C under oxygen for 12 hours. At 25°C, 150 mL of water was added to the reaction solution, which was then extracted with dichloromethane (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The resulting residue was purified by silica gel column chromatography using developing system B to obtain the title product, LD-1b, as a white solid (800 mg, 33% yield).

[0491] Step 2

[0492] LD-1b (800 mg, 4.04 mmol) was dissolved in 24 mL of methanol, followed by the addition of potassium carbonate (1.12 g, 8.07 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (930 mg, 4.84 mmol), and the mixture was stirred at 25°C for 12 hours. 50 mL of water was added to the reaction solution, which was then extracted with ethyl acetate (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The resulting residue was purified by silica gel column chromatography using developing system B to obtain the title product, LD-1c (650 mg, 83% yield), as a white solid.

[0493] Step 3

[0494] LD-1c (650 mg, 3.35 mmol), 5-bromo-2-methylthiopyrimidine (686 mg, 3.35 mmol), triethylamine (3.39 g, 33.4 mmol), cuprous iodide (63.7 mg, 334 μmol), and bistriphenylphosphine palladium dichloride (244 mg, 349 μmol) were dissolved in 10 mL of tetrahydrofuran. The atmosphere was replaced with nitrogen three times and stirred at 60°C under nitrogen for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing system B to obtain the title product, LD-1d, as a white solid (770 mg, 68% yield).

[0495] MS m / z(ESI):319.6[M+1].

[0496] Step 4

[0497] LD-1d (1.80 g, 5.65 mmol) was dissolved in 20 mL of dichloromethane, followed by the addition of m-chloroperbenzoic acid (2.87 g, 14.1 mmol, 85.0% purity). The mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing system B to obtain the title product, LD-1e (1.70 g, 85% yield), as a white solid.

[0498] MS m / z(ESI):351.1[M+1].

[0499] Step 5

[0500] Dissolve LD-1e (500 mg, 1.43 mmol) in 6 mL of dichloromethane, then add trifluoroacetic acid (2.30 g, 20.2 mmol) and stir at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude title product LD-1f (418 mg) as a white solid. This product was used directly in the next reaction without purification.

[0501] MS m / z(ESI):295.2[M+1].

[0502] Step 6

[0503] LD-1f (10 mg, 34.0 μmol) was dissolved in 1 mL of N,N-dimethylformamide, and then 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (13 mg, 34.0 μmol) and N,N-diisopropylethylamine (13 mg, 100.9 μmol) were added in sequence, and stirred at 25 ° C for 0.5 hours. LD-1g (14.5 mg, 44.0 μmol, prepared by the method disclosed in Example C1.22 on page 241 of patent application "WO2022170971 A1") was added and stirred at 25 ° C for 3 hours. The reaction solution was purified without treatment by pre-HPLC (chromatographic column: InfinityLab Poroshell 120SB-C18 21.2×150 mm, 4 μm; mobile phase: A-water (0.1% formic acid), B-acetonitrile; gradient elution: B%: 10%-70%) to obtain the title product LD-1h (8.10 mg, yield: 39%) as a white solid.

[0504] MS m / z(ESI):606.5[M+1].

[0505] Step 7

[0506] LD-1i (325 mg, 882 μmol) and 1 (42 mg, 88.1 μmol) were dissolved in 4 mL of N,N-dimethylformamide. A solution of hydrochloric acid in ethyl acetate (4 mol / L, 100 μL) was then added and stirred at 25°C for 15 hours. The reaction mixture was purified by reverse-phase liquid chromatography (mobile phase: A-water (0.1% formic acid), B-acetonitrile; gradient elution: B%: 20%-80%) to obtain the title product, LD-1j (34 mg, 48% yield), as a yellow solid.

[0507] MS m / z(ESI):785.6[M+1].

[0508] Step 8

[0509] LD-1j (38 mg, 48.4 μmol) was dissolved in 1 mL of N,N-dimethylformamide, followed by the addition of diethylamine (142 mg, 1.93 mmol) and stirring at 25°C for 0.5 h. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by pre-HPLC (column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A - water (0.1% formic acid), B - acetonitrile; gradient elution: B%: 5%-50%) to afford the title product, LD-1k (11 mg, 40% yield), as a yellow solid.

[0510] MS m / z(ESI):282.2[1 / 2(M+2)].

[0511] Step 9

[0512] LD-1k (11 mg, 19.6 μmol), LD-1h (12 mg, 19.8 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (9 mg, 23.6 μmol) were dissolved in 1 mL of N,N-dimethylformamide. N,N-Diisopropylethylamine (8 mg, 61.9 μmol) was then added and stirred at 25°C for 1 hour. The reaction mixture was purified by pre-HPLC (column: InfinityLab Poroshell 120SB-C18 2 1.2 × 150 mm, 4 μm; mobile phase: A - water (0.1% formic acid), B - acetonitrile; gradient elution: B%: 20%-55%) to obtain the title product LD-1 (4.3 mg, 19% yield) as an off-white solid.

[0513] MS m / z(ESI):576.2[1 / 2(M+2)].

[0514] 1 H NMR(400MHz,DMSO-d6)δ9.10–9.00(m,2H),8.69–8.58(m,1H),8.29–8.20(m, 1H),8.02–7.93(m,1H),7.63–7.55(m,1H),7.53–7.47(m,1H),7.36–7.24(m,2 H),6.59–6.52(m,1H),6.31–6.21(m,2H),5.49–5.35(m,2H),5.31–5.20(m,2H ),4.66–4.53(m,2H),4.48–4.36(m,4H),4.27–4.18(m,1H),4.11–4.03(m,1H) ,3.79–3.67(m,2H),3.54–3.47(m,2H),3.41–3.39(m,3H),3.14–3.05(m,2H), 2.64–2.56(m,2H),2.35–2.31(m,1H),2.05–1.82(m,7H),1.76–1.48(m,4H),1 .44–1.36(m,5H),1.30–1.20(m,2H),1.03–0.93(m,2H),0.87–0.76(m,13H),0 .75–0.68(m,2H),0.39–0.28(m,2H),0.11–0.03(m,1H),-0.03–-0.10(m,1H).

[0515] Example 3-2 LD-2

[0516] Example 3-3 LD-3

[0517] Example 3-4 LD-4

[0518] Example 3-5 LD-5

[0519] Example 3-6 LD-6

[0520] Example 3-7 LD-7

[0521] Example 3-8 LD-8

[0522] Example 3-9 LD-9

[0523] Example 3-10 LD-10

[0524] first step

[0525] LD-1i (333 mg, 904 μmol) and 2 (42 mg, 90.4 μmol) were dissolved in 4 mL of N,N-dimethylformamide. A 4 mol / L solution of hydrochloric acid in ethyl acetate (100 μL) was added and stirred at 25°C for 15 hours. The reaction mixture was purified by pre-HPLC (column: InfinityLab Poroshell 120SB-C18 21.2 × 150 mm, 4 μm; mobile phase: A-water (0.1% formic acid), B-acetonitrile; gradient elution: B%: 20%-90%) to obtain the title product LD-10a (35 mg, 49% yield) as a yellow solid.

[0526] MS m / z(ESI):773.6[M+1].

[0527] Step 2

[0528] LD-10a (35 mg, 44.6 μmol) was dissolved in 1 mL of N,N-dimethylformamide, followed by the addition of diethylamine (142 mg, 1.94 mmol) and stirring at 25°C for 0.5 h. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by pre-HPLC (column: InfinityLab Poroshell 120 SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A - water (0.1% formic acid), B - acetonitrile; gradient elution: B%: 5%-70%) to afford the title product, LD-10b, as a yellow solid (17 mg, 66% yield).

[0529] MS m / z(ESI):551.4[M+1].

[0530] Step 3

[0531] LD-10b (17 mg, 30.0 μmol), LD-1h (19 mg, 30.5 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (13 mg, 30.5 μmol) were dissolved in 1 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (12 mg, 92.8 μmol) was then added and stirred at 25°C for 1 hour. The reaction mixture was purified by pre-HPLC (column: InfinityLab Poroshell 120SB-C18 21.2 × 150 mm, 4 μm; mobile phase: A - water (0.1% formic acid), B - acetonitrile; gradient elution: B%: 20%-60%) to obtain the title product LD-10 (7.2 mg, 20% yield) as a white solid.

[0532] MS m / z(ESI):570.3[1 / 2(M+2)].

[0533] 1 H NMR(400MHz,DMSO-d6)δ9.10–9.02(m,2H),8.67–8.61(m,1H),8.26–8.18(m,2H ),8.02–7.96(m,1H),7.91–7.85(m,1H),7.40–7.35(m,1H),7.33–7.26(m,1H),6 .62–6.57(m,1H),5.48–5.38(m,2H),5.34–5.26(m,2H),4.64–4.55(m,2H),4.26 –4.19(m,1H),4.09–4.04(m,1H),3.75–3.70(m,2H),3.54–3.46(m,2H),3.42–3. 37(m,3H),3.23–3.17(m,2H),2.69–2.65(m,1H),2.63–2.57(m,2H),2.54–2.51( m,3H),2.34–2.31(m,1H),2.03–1.83(m,8H),1.77–1.64(m,3H),1.57–1.49(m,2 H),1.46–1.37(m,5H),1.31–1.19(m,3H),1.01–0.95(m,2H),0.87–0.75(m,13H) ,0.73–0.68(m,2H),0.38–0.30(m,2H),0.11–0.05(m,1H),-0.04–-0.09(m,1H).

[0534] Example 3-11 LD-11

[0535] Example 3-12 LD-12

[0536] Example 3-13 LD-13

[0537] Example 3-14 LD-14

[0538] Example 3-15 LD-15

[0539] Example 3-16 LD-16

[0540] Example 3-17 LD-17

[0541] Example 3-18 LD-18

[0542] Example 3-19 LD-19

[0543] Example 3-20 LD-20

[0544] Example 3-21 LD-21

[0545] Example 3-22 LD-22

[0546] Example 3-23 LD-23

[0547] Example 3-24 LD-24

[0548] Example 3-25 LD-25

[0549] Example 3-26 LD-26

[0550] Example 3-27 LD-27

[0551] Example 3-28 LD-28

[0552] Example 3-29 LD-29

[0553] Example 3-30 LD-30

[0554] Example 3-31 LD-31

[0555] Example 3-32 LD-32

[0556] Example 3-33 LD-33

[0557] Example 3-34 LD-34

[0558] Example 3-35 LD-35

[0559] Example 3-36 LD-36

[0560] Example 3-37 LD-37

[0561] Example 3-38 LD-38

[0562] Example 3-39 LD-39

[0563] Example 3-40 LD-40

[0564] Example 3-41 LD-41

[0565] Example 3-42 LD-42

[0566] Example 3-43 LD-43

[0567] Example 3-44 LD-44

[0568] Example 3-45 LD-45

[0569] Example 3-46 LD-46

[0570] Example 3-47 LD-47

[0571] Example 3-48 LD-48

[0572] Example 3-49 LD-49

[0573] Example 3-50 LD-50

[0574] Example 3-51 LD-51

[0575] Example 3-52 LD-52

[0576] Example 3-53 LD-53

[0577] Example 3-54 LD-54

[0578] Example 3-55 LD-55

[0579] Example 3-56 LD-56

[0580] Example 3-57 LD-57

[0581] Example 3-58 LD-58

[0582] Example 3-59 LD-59

[0583] Example 3-60 LD-60

[0584] Example 3-61 LD-61

[0585] Example 3-62 LD-62

[0586] Example 3-63 LD-63

[0587] Example 3-64 LD-64

[0588] Example 3-65 LD-65

[0589] Example 3-66 LD-66

[0590] Example 3-67 LD-67

[0591] Example 3-68 LD-68

[0592] Example 3-69 LD-69

[0593] Example 3-70 LD-70

[0594] Example 3-71 LD-71

[0595] 4. ADC Conjugate Examples

[0596] Preparation steps of antibody conjugates

[0597] A: The antibody was replaced with 50mM PBS / 1.0mM EDTA buffer (pH 7.4 adjusted with sodium hydroxide solution) using an ultrafiltration tube with a molecular weight cutoff of 50kD. 5-10 equivalents of 10mM TCEP aqueous solution were added and shaken at 25°C for 3 hours. The linker-drug conjugate was dissolved in DMSO, and 10-20 equivalents of the linker-drug conjugate were added to the reduced antibody solution. Vortex and shake at 25°C for 2 hours. After the reaction was complete, 40 equivalents of 100mM NAC aqueous solution were added and shaken at 25°C for 20 minutes to terminate the linker reaction. Excess small molecules were removed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 50kD or a Sephadex G-25 desalting column. The antibody-drug conjugate was replaced with 50mM PBS buffer (pH 6.0) and the sample was filtered through a 0.22μm filter membrane to obtain the antibody-drug conjugate, which was stored in a refrigerator at 4°C. Determine the DAR value of the conjugate using reverse-phase high-performance liquid chromatography or mass spectrometry. B: Replace the antibody into 50 mM PBS / 1.0 mM EDTA buffer (pH 6.5 adjusted with sodium hydroxide solution) using a 50 kD ultrafiltration tube. Add 5-10 equivalents of 10 mM TCEP aqueous solution and shake at 25°C for 3 hours. Dissolve the linker-drug conjugate in DMSO, take 10-20 equivalents of the linker-drug conjugate, and add it dropwise to the reduced antibody solution. Vortex and shake to mix evenly. Shake at 25°C for 2 hours. Once the reaction is complete, add 40 equivalents of 100mM NAC aqueous solution and shake at 25°C for 20 minutes to terminate the linker reaction. Use a 50kD molecular weight cutoff ultrafiltration tube or Sephadex G-25 desalting column to remove excess small molecules, and replace the antibody-drug conjugate with 50mM PBS buffer (pH 6.0). After ultrafiltration purification, filter the sample with a 0.22μm filter membrane to obtain the antibody-drug conjugate, which is stored in a refrigerator at 4°C. Determine the DAR value of the conjugate using reversed-phase high-performance liquid chromatography or mass spectrometry.

[0598] Example 4-1 ADC-1

[0599] Trastuzumab and LD-1 were used as raw materials and prepared by the method of step A. The exemplary product ADC-1 of the coupling mixture FADC-1 was obtained in PBS buffer and stored at 4° C., which is Example 4-1.

[0600] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0601] Example 4-2 ADC-2

[0602] Trastuzumab and LD-2 were used as raw materials and prepared by the method of step A. The exemplary product ADC-2 of the coupling mixture FADC-2 was obtained in PBS buffer and stored at 4° C., which is Example 4-2.

[0603] Example 4-3 ADC-3

[0604] Trastuzumab and LD-3 were used as raw materials and prepared by the method of step A. The exemplary product ADC-3 of the coupling mixture FADC-3 was obtained in PBS buffer and stored at 4° C., which is Example 4-3.

[0605] Example 4-4 ADC-4

[0606] Trastuzumab and LD-4 were used as raw materials and prepared by the method of step A. The exemplary product ADC-4 of the coupling mixture FADC-4 was obtained in PBS buffer and stored at 4° C., which is Example 4-4.

[0607] Example 4-5 ADC-5

[0608] Trastuzumab and LD-5 were used as raw materials and prepared by the method of step A. The exemplary product ADC-5 of the coupling mixture FADC-5 was obtained in PBS buffer and stored at 4° C., which is Example 4-5.

[0609] Example 4-6 ADC-6

[0610] Trastuzumab and LD-6 were used as raw materials and prepared by the method of step A. The exemplary product ADC-6 of the coupling mixture FADC-6 was obtained in PBS buffer and stored at 4° C., which is Example 4-6.

[0611] Example 4-7 ADC-7

[0612] The antibody Trastuzumab and LD-7 were used as raw materials and prepared by the method of step B. The PBS buffer solution of the exemplary product ADC-7 of the coupling mixture (mixture of FADC-7A and / or FADC-7B and / or FADC-7C) was obtained and stored at 4°C.

[0613] Example 4-8 ADC-8

[0614] Trastuzumab and LD-8 were used as raw materials and prepared by the method of step A. The exemplary product ADC-8 of the coupling mixture FADC-8 was obtained in PBS buffer and stored at 4° C., which is Example 4-8.

[0615] Example 4-9 ADC-9

[0616] Trastuzumab and LD-9 were used as raw materials and prepared by the method of step A. The exemplary product ADC-9 of the coupling mixture FADC-9 was obtained in PBS buffer and stored at 4° C., which is Example 4-9.

[0617] Example 4-10 ADC-10

[0618] Trastuzumab and LD-10 were used as raw materials and prepared by the method of step A. The exemplary product ADC-10 of the coupling mixture FADC-10 was obtained in PBS buffer and stored at 4° C., which is Example 4-10.

[0619] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0620] Example 4-11 ADC-11

[0621] Trastuzumab and LD-11 were used as raw materials and prepared by the method of step A. The exemplary product ADC-11 of the coupling mixture FADC-11 was obtained in PBS buffer and stored at 4° C., which is Example 4-11.

[0622] Example 4-12 ADC-12

[0623] Trastuzumab and LD-12 were used as raw materials and prepared by the method of step A. The exemplary product ADC-12 of the coupling mixture FADC-12 was obtained in PBS buffer and stored at 4° C., which is Example 4-12.

[0624] Example 4-13 ADC-13

[0625] Trastuzumab and LD-13 were used as raw materials and prepared by the method of step A. The exemplary product ADC-13 of the coupling mixture FADC-13 was obtained in PBS buffer and stored at 4° C., which is Example 4-13.

[0626] Example 4-14 ADC-14

[0627] The antibody trastuzumab and LD-14 were used as raw materials and prepared by the method of step B. The PBS buffer solution of the exemplary product ADC-14, a coupling mixture (mixture of FADC-14 A and / or FADC-14B and / or FADC-14C), was obtained and stored at 4°C.

[0628] Example 4-15 ADC-15

[0629] Trastuzumab and LD-15 were used as raw materials and prepared by the method of step A. The exemplary product ADC-15 of the coupling mixture FADC-15 was obtained in PBS buffer and stored at 4° C., which is Example 4-15.

[0630] Example 4-16 ADC-16

[0631] Trastuzumab and LD-16 were used as raw materials and prepared by the method of step A. The exemplary product ADC-16 of the coupling mixture FADC-16 was obtained in PBS buffer and stored at 4° C., which is Example 4-16.

[0632] Example 4-17 ADC-17

[0633] Trastuzumab and LD-17 were used as raw materials and prepared by the method of step A. The exemplary product ADC-17 of the coupling mixture FADC-17 was obtained in PBS buffer and stored at 4° C., which is Example 4-17.

[0634] Example 4-18 ADC-18

[0635] Trastuzumab and LD-18 were used as raw materials and prepared by the method of step A. The exemplary product ADC-18 of the coupling mixture FADC-18 was obtained in PBS buffer and stored at 4° C., which is Example 4-18.

[0636] Example 4-19 ADC-19

[0637] The antibody Trastuzumab and LD-19 were used as raw materials and prepared by the method of step A. The exemplary product ADC-19 of the coupling mixture FADC-19 was obtained in PBS buffer and stored at 4° C., which is Example 4-19.

[0638] Example 4-20 ADC-20

[0639] Trastuzumab and LD-20 were used as raw materials and prepared by the method of step A. The exemplary product ADC-20 of the coupling mixture FADC-20 was obtained in PBS buffer and stored at 4° C., which is Example 4-20.

[0640] Example 4-21 ADC-21

[0641] The antibody trastuzumab and LD-21 were used as raw materials and prepared by the method of step B. The PBS buffer solution of the exemplary product ADC-21 of the coupling mixture (mixture of FADC-21A and / or FADC-21B and / or FADC-21C) was obtained and stored at 4°C.

[0642] Example 4-22 ADC-22

[0643] The antibody Trastuzumab and LD-22 were used as raw materials and prepared by the method of step A. The exemplary product ADC-22 of the coupling mixture FADC-22 was obtained in PBS buffer and stored at 4° C., which is Example 4-22.

[0644] Example 4-23 ADC-23

[0645] Trastuzumab and LD-23 were used as raw materials and prepared by the method of step A. The exemplary product ADC-23 of the coupling mixture FADC-23 was obtained in PBS buffer and stored at 4° C., which is Example 4-23.

[0646] Example 4-24 ADC-24

[0647] Trastuzumab and LD-24 were used as raw materials and prepared by the method of step A. The exemplary product ADC-24 of the coupling mixture FADC-24 was obtained in PBS buffer and stored at 4° C., which is Example 4-24.

[0648] Example 4-25 ADC-25

[0649] Trastuzumab and LD-25 were used as raw materials and prepared by the method of step A. The exemplary product ADC-25 of the coupling mixture FADC-25 was obtained in PBS buffer and stored at 4° C., which is Example 4-25.

[0650] Example 4-26 ADC-26

[0651] The antibody Trastuzumab and LD-26 were used as raw materials and prepared by the method of step A. The exemplary product ADC-26 of the coupling mixture FADC-26 was obtained in PBS buffer and stored at 4° C., which is Example 4-26.

[0652] Example 4-27 ADC-27

[0653] Trastuzumab and LD-27 were used as raw materials and prepared by the method of step A. The exemplary product ADC-27 of the coupling mixture FADC-27 was obtained in PBS buffer and stored at 4° C., which is Example 4-27.

[0654] Example 4-28 ADC-28

[0655] The antibody Trastuzumab and LD-28 were used as raw materials and prepared by the method of step B. The PBS buffer solution of the exemplary product ADC-28, a coupling mixture (a mixture of FADC-28A and / or FADC-28B and / or FADC-28C), was obtained and stored at 4°C.

[0656] Example 4-29 ADC-29

[0657] Trastuzumab and LD-29 were used as raw materials and prepared by the method of step A. The exemplary product ADC-29 of the coupling mixture FADC-29 was obtained in PBS buffer and stored at 4° C., which is Example 4-29.

[0658] Example 4-30 ADC-30

[0659] Trastuzumab and LD-30 were used as raw materials and prepared by the method of step A. The exemplary product ADC-30 of the coupling mixture FADC-30 was obtained in PBS buffer and stored at 4° C., which is Example 4-30.

[0660] Example 4-31 ADC-31

[0661] Trastuzumab and LD-31 were used as raw materials and prepared by the method of step A. The exemplary product ADC-31 of the coupling mixture FADC-31 was obtained in PBS buffer and stored at 4° C., which is Example 4-31.

[0662] Example 4-32 ADC-32

[0663] Trastuzumab and LD-32 were used as raw materials and prepared by the method of step A. The exemplary product ADC-32 of the coupling mixture FADC-32 was obtained in PBS buffer and stored at 4° C., which is Example 4-32.

[0664] Example 4-33 ADC-33

[0665] Trastuzumab and LD-33 were used as raw materials and prepared by the method of step A. The exemplary product ADC-33 of the coupling mixture FADC-33 was obtained in PBS buffer and stored at 4° C., which is Example 4-33.

[0666] Example 4-34 ADC-34

[0667] Trastuzumab and LD-34 were used as raw materials and prepared by the method of step A. The exemplary product ADC-34 of the coupling mixture FADC-34 was obtained in PBS buffer and stored at 4° C., which is Example 4-34.

[0668] Example 4-35 ADC-35

[0669] Trastuzumab and LD-35 were used as raw materials and prepared by the method of step A. The exemplary product ADC-35 of the coupling mixture FADC-35 was obtained in PBS buffer and stored at 4° C., which is Example 4-35.

[0670] Example 4-36 ADC-36

[0671] Trastuzumab and LD-36 were used as raw materials and prepared by the method of step A. The exemplary product ADC-36 of the coupling mixture FADC-36 was obtained in PBS buffer and stored at 4° C., which is Example 4-36.

[0672] Example 4-37 ADC-37

[0673] The antibody Trastuzumab and LD-37 were used as raw materials and prepared by the method of step A. The exemplary product ADC-37 of the coupling mixture FADC-37 was obtained in PBS buffer and stored at 4° C., which is Example 4-37.

[0674] Example 4-38 ADC-38

[0675] The antibody Trastuzumab and LD-38 were used as raw materials and prepared by the method of step A. The exemplary product ADC-38 of the coupling mixture FADC-38 was obtained in PBS buffer and stored at 4° C., which is Example 4-38.

[0676] Example 4-39 ADC-39

[0677] Trastuzumab and LD-39 were used as raw materials and prepared by the method of step A. The exemplary product ADC-39 of the coupling mixture FADC-39 was obtained in PBS buffer and stored at 4° C., which is Example 4-39.

[0678] Example 4-40 ADC-40

[0679] Trastuzumab and LD-40 were used as raw materials and prepared by the method of step A. The exemplary product ADC-40 of the coupling mixture FADC-40 was obtained in PBS buffer and stored at 4° C., which is Example 4-40.

[0680] Example 4-41 ADC-41

[0681] Trastuzumab and LD-41 were used as raw materials and prepared by the method of step A. The exemplary product ADC-41 of the coupling mixture FADC-41 was obtained in PBS buffer and stored at 4° C., which is Example 4-41.

[0682] Example 4-42 ADC-42

[0683] Trastuzumab and LD-42 were used as raw materials and prepared by the method of step A. The exemplary product ADC-42 of the coupling mixture FADC-42 was obtained in PBS buffer and stored at 4° C., which is Example 4-42.

[0684] Example 4-43 ADC-43

[0685] Trastuzumab and LD-43 were used as raw materials and prepared by the method of step A. The exemplary product ADC-43 of the coupling mixture FADC-43 was obtained in PBS buffer and stored at 4° C., which is Example 4-43.

[0686] Example 4-44 ADC-44

[0687] Trastuzumab and LD-44 were used as raw materials and prepared by the method of step A. The exemplary product ADC-44 of the coupling mixture FADC-44 was obtained in PBS buffer and stored at 4° C., which is Example 4-44.

[0688] Example 4-45 ADC-45

[0689] Trastuzumab and LD-45 were used as raw materials and prepared by the method of step A. The exemplary product ADC-45 of the coupling mixture FADC-45 was obtained in PBS buffer and stored at 4° C., which is Example 4-45.

[0690] Example 4-46 ADC-46

[0691] The antibody Trastuzumab and LD-46 were used as raw materials and prepared by the method of step A. The exemplary product ADC-46 of the coupling mixture FADC-46 was obtained in PBS buffer and stored at 4° C., which is Example 4-46.

[0692] Example 4-47 ADC-47

[0693] The antibody Trastuzumab and LD-47 were used as raw materials and prepared by the method of step A. The exemplary product ADC-47 of the coupling mixture FADC-47 was obtained in PBS buffer and stored at 4° C., which is Example 4-47.

[0694] Example 4-48 ADC-48

[0695] Trastuzumab and LD-48 were used as raw materials and prepared by the method of step A. The exemplary product ADC-48 of the coupling mixture FADC-48 was obtained in PBS buffer and stored at 4° C., which is Example 4-48.

[0696] Example 4-49 ADC-49

[0697] The antibody Trastuzumab and LD-49 were used as raw materials and prepared by the method of step A. The exemplary product ADC-49 of the coupling mixture FADC-49 was obtained in PBS buffer and stored at 4° C., which is Example 4-49.

[0698] Example 4-50 ADC-50

[0699] Trastuzumab and LD-50 were used as raw materials and prepared by the method of step A. The exemplary product ADC-50 of the coupling mixture FADC-50 was obtained in PBS buffer and stored at 4° C., which is Example 4-50.

[0700] Example 4-51 ADC-51

[0701] Trastuzumab and LD-51 were used as raw materials and prepared by the method of step A. The exemplary product ADC-51 of the coupling mixture FADC-51 was obtained in PBS buffer and stored at 4° C., which is Example 4-51.

[0702] Example 4-52 ADC-52

[0703] Trastuzumab and LD-52 were used as raw materials and prepared by the method of step A. The exemplary product ADC-52 of the coupling mixture FADC-52 was obtained in PBS buffer and stored at 4° C., which is Example 4-52.

[0704] Example 4-53 ADC-53

[0705] Trastuzumab and LD-53 were used as raw materials and prepared by the method of step A. The exemplary product ADC-53 of the coupling mixture FADC-53 was obtained in PBS buffer and stored at 4° C., which is Example 4-53.

[0706] Example 4-54 ADC-54

[0707] Trastuzumab and LD-54 were used as raw materials and prepared by the method of step A. The exemplary product ADC-54 of the coupling mixture FADC-54 was obtained in PBS buffer and stored at 4° C., which is Example 4-54.

[0708] Example 4-55 ADC-55

[0709] Trastuzumab and LD-55 were used as raw materials and prepared by the method of step A. The exemplary product ADC-55 of the coupling mixture FADC-55 was obtained in PBS buffer and stored at 4° C., which is Example 4-55.

[0710] Example 4-56 ADC-56

[0711] The antibody Trastuzumab and LD-56 were used as raw materials and prepared by the method of step A. The exemplary product ADC-56 of the coupling mixture FADC-56 was obtained in PBS buffer and stored at 4° C., which is Example 4-56.

[0712] Example 4-57 ADC-57

[0713] Trastuzumab and LD-57 were used as raw materials and prepared by the method of step A. The exemplary product ADC-57 of the coupling mixture FADC-57 was obtained in PBS buffer and stored at 4° C., which is Example 4-57.

[0714] Example 4-58 ADC-58

[0715] The antibody Trastuzumab and LD-58 were used as raw materials and prepared by the method of step A. The exemplary product ADC-58 of the coupling mixture FADC-58 was obtained in PBS buffer and stored at 4° C., which is Example 4-58.

[0716] Example 4-59 ADC-59

[0717] Trastuzumab and LD-59 were used as raw materials and prepared by the method of step A. The exemplary product ADC-59 of the coupling mixture FADC-59 was obtained in PBS buffer and stored at 4° C., which is Example 4-59.

[0718] Example 4-60 ADC-60

[0719] Trastuzumab and LD-60 were used as raw materials and prepared by the method of step A. The exemplary product ADC-60 of the coupling mixture FADC-60 was obtained in PBS buffer and stored at 4° C., which is Example 4-60.

[0720] Example 4-61 ADC-61

[0721] Trastuzumab and LD-61 were used as raw materials and prepared by the method of step A. The exemplary product ADC-61 of the coupling mixture FADC-61 was obtained in PBS buffer and stored at 4° C., which is Example 4-61.

[0722] Example 4-62 ADC-62

[0723] Trastuzumab and LD-62 were used as raw materials and prepared by the method of step A. The exemplary product ADC-62 of the coupling mixture FADC-62 was obtained in PBS buffer and stored at 4° C., which is Example 4-62.

[0724] Example 4-63 ADC-63

[0725] Trastuzumab and LD-63 were used as raw materials and prepared by the method of step A. The exemplary product ADC-63 of the coupling mixture FADC-63 was obtained in PBS buffer and stored at 4° C., which is Example 4-63.

[0726] Example 4-64 ADC-64

[0727] Trastuzumab and LD-64 were used as raw materials and prepared by the method of step A. The exemplary product ADC-64 of the coupling mixture FADC-64 was obtained in PBS buffer and stored at 4° C., which is Example 4-64.

[0728] Example 4-65 ADC-65

[0729] Trastuzumab and LD-65 were used as raw materials and prepared by the method of step A. The exemplary product ADC-65 of the coupling mixture FADC-65 was obtained in PBS buffer and stored at 4° C., which is Example 4-65.

[0730] Example 4-66 ADC-66

[0731] The antibody Trastuzumab and LD-66 were used as raw materials and prepared by the method of step A. The exemplary product ADC-66 of the coupling mixture FADC-66 was obtained in PBS buffer and stored at 4° C., which is Example 4-66.

[0732] Example 4-67 ADC-67

[0733] Trastuzumab and LD-67 were used as raw materials and prepared by the method of step A. The exemplary product ADC-67 of the coupling mixture FADC-67 was obtained in PBS buffer and stored at 4° C., which is Example 4-67.

[0734] Example 4-68 ADC-68

[0735] The antibody Trastuzumab and LD-68 were used as raw materials and prepared by the method of step A. The exemplary product ADC-68 of the coupling mixture FADC-68 was obtained in PBS buffer and stored at 4° C., which is Example 4-68.

[0736] Example 4-69 ADC-69

[0737] Trastuzumab and LD-69 were used as raw materials and prepared by the method of step A. The exemplary product ADC-69 of the coupling mixture FADC-69 was obtained in PBS buffer and stored at 4° C., which is Example 4-69.

[0738] Example 4-70 ADC-70

[0739] Trastuzumab and LD-70 were used as raw materials and prepared by the method of step A. The exemplary product ADC-70 of the coupling mixture FADC-70 was obtained in PBS buffer and stored at 4° C., which is Example 4-70.

[0740] Example 4-71 ADC-71

[0741] The antibody Trastuzumab and LD-71 were used as raw materials and prepared by the method of step A. The PBS buffer solution of the exemplary product ADC-71 of the coupling mixture FADC-71 was obtained and stored at 4° C., which is Example 4-71.

[0742] Example 4-72 ADC-72

[0743] Nimotuzumab and LD-1 were used as raw materials and prepared by the method of step A. The exemplary product of the coupling mixture FADC-72 was obtained in PBS buffer solution of ADC-72 and stored at 4° C., which is Example 4-72.

[0744] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0745] Example 4-73 ADC-73

[0746] Nimotuzumab and LD-10 were used as raw materials and prepared by the method of step A. The exemplary product ADC-73 of the coupling mixture FADC-73 was obtained in PBS buffer and stored at 4° C., which is Example 4-73.

[0747] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0748] Example 4-74 ADC-74

[0749] Pertuzumab and LD-1 were used as raw materials and prepared by the method of step A. The exemplary product ADC-74 of the coupling mixture FADC-74 was obtained in PBS buffer and stored at 4° C., which is Example 4-74.

[0750] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0751] Example 4-75 ADC-75

[0752] The antibody Pertuzumab and LD-10 were used as raw materials and prepared by the method of step A. The exemplary product ADC-75 of the coupling mixture FADC-75 was obtained in PBS buffer and stored at 4° C., which is Example 4-75.

[0753] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0754] Example 4-76 Reference 2

[0755] The antibody Trastuzumab and N-((11S,14S)-11-(4-di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (prepared by the method disclosed in Example 2.37 on page 273 of the patent application "WO2022170971 Al") are used as raw materials and prepared by the method of step A. An exemplary product of the coupling mixture FADC-reference 2 was obtained in PBS buffer and stored at 4° C. as Example 4-76.

[0756] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0757] Example 4-77 Reference 3

[0758] The antibody Nimotuzumab and N-((11S,14S)-11-(4-di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (prepared by the method disclosed in Example 2.37 on page 273 of the patent application "WO2022170971 Al") are used as raw materials and prepared by the method of step A. An exemplary product of the coupling mixture FADC-reference 3 was obtained in PBS buffer and stored at 4° C. as Example 4-77.

[0759] The average drug loading value calculated by mass spectrometry was: y=8.00.

[0760] Example 4-78 Reference 4

[0761] The antibody Pertuzumab and N-((11S,14S)-11-(4-di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (prepared by the method disclosed in Example 2.37 on page 273 of the patent application "WO2022170971 Al") are used as raw materials and prepared by the method of step A. An exemplary product of the coupling mixture FADC-reference 4 was obtained in PBS buffer and stored at 4° C. as Example 4-78.

[0762] The average drug loading value calculated by mass spectrometry was: y=7.60.

[0763] ADC stock solution drug loading analysis

[0764] Experimental purpose and principle

[0765] ADC bulk solutions are antibody-based crosslinked drugs. Their therapeutic mechanism relies on the targeted delivery of toxin molecules into cells, leading to cell killing. Drug loading plays a crucial role in drug efficacy. The drug loading of ADC bulk solutions was determined using mass spectrometry and reversed-phase high-performance liquid chromatography.

[0766] Method 1: Reversed-Phase High-Performance Liquid Chromatography:

[0767] (1) Experimental method: The test sample was diluted to 0.5 mg / mL with ultrapure water, reduced with dithiothreitol at a final concentration of 25 mM at 37°C for 30 min, and then injected directly with a sample volume of 5 μL. The chromatographic column was a reverse phase chromatography column (Agilent PLRP-S (5μm) 50×2.1mm), gradient elution was performed with mobile phases A and B (A: 0.1% formic acid-0.025% trifluoroacetic acid-water, B: 0.1% formic acid-0.025% trifluoroacetic acid-acetonitrile). The flow rate was 0.25mL / min, the detection wavelength was 280nm, and the column temperature was 70℃. (2) Data analysis: By comparing the spectra of the sample with the naked antibody, the positions of the light and heavy chains were distinguished, and then the spectra of the test sample were integrated to calculate the DAR value. The calculation formula is as follows: LC: 0 (number of connected drugs), LC+1: 2 (number of connected drugs), HC: 0 (number of connected drugs), HC+1: 2 (number of connected drugs), HC+2: 4 (number of connected drugs), HC+3: 6 (number of connected drugs). LC peak area sum = LC peak area + LC+1 peak area; HC peak area sum = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area; LC DAR = Σ(number of connected drugs × peak area percentage) / LC peak area sum; HC DAR = Σ(number of connected drugs × peak area percentage) / HC peak area sum; DAR = LC DAR + HC DAR

[0768] Method 2: Mass spectrometry: (1) Experimental method: Take 10 μL of the test sample with a concentration of 1 mg / mL into a 1.5 mL centrifuge tube, add 0.5 μL of rapid PNGase F (Adamas), vortex mix, and incubate at 37°C for 60 min. After the N-sugar is cut, add 6 μL of ultrapure water and 4 μL of 0.5 M dithiothreitol aqueous solution to the centrifuge tube, vortex mix, and incubate at 37°C for 30 min. After the reaction is completed, centrifuge and take the supernatant into the injection bottle, and inject 2 μL. The chromatographic column is a reverse phase chromatography column (Agilent PLRP-S The column was eluted with a gradient of mobile phases A and B (A: 0.1% formic acid-water, B: 0.1% formic acid-acetonitrile) at a flow rate of 0.5 mL / min, a column temperature of 60°C, and a detection wavelength of 280 nm. ESI-Tof (LC-MS) was used to collect m / z values ​​in the positive ion mode over the m / z range of 200 to 3200. The original mass spectra were then deconvoluted using software. (2) Data analysis: The DAR value was calculated using peak height values ​​using a method similar to that described above for reversed-phase HPLC.

[0769] III. Test Case

[0770] Biological evaluation

[0771] Test Example 1: Determination of the in vitro proliferation inhibitory activity of the compound of formula (GH) on A549, SK-BR-3, and NCI-N87 tumor cells

[0772] 1. Test purpose:

[0773] The purpose of this experiment was to detect the in vitro cytotoxicity of the compound of the disclosed formula (GH) against A549 tumor cells (human lung cancer cells, Beijing Tongren Hospital), SK-BR-3 tumor cells (human breast cancer cells, Wuhan Punosai, Catalog No. CL-0211), and NCI-N87 tumor cells (human gastric cancer cells, Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences, Catalog No. 3111C0001CCC000481). Cells were treated with different concentrations of the compound in vitro. After 6 days of culture, the number of viable cells was detected using CCK8 (Cell Counting Kit-8, Catalog No. TS547). According to the IC 50 The in vitro activity of the test compounds was evaluated.

[0774] 2. Experimental methods:

[0775] Taking the in vitro cytotoxicity test method for A549 cells, SK-BR-3 cells, and NCI-N87 cells as an example, the following test was performed. It should be understood that this method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells (HCC1569, JIMT-1, DIFI, etc.).

[0776] (1) Cell culture: SK-BR-3 cells and NCI-N87 cells were cultured in RPMI 1640 medium containing 10% FBS (Shanghai Yuanpei Biotechnology Co., Ltd., Catalog No. G211018); A549 cells were cultured in DMEM / F12 medium containing 10% FBS (Shanghai Yuanpei Biotechnology Co., Ltd., Catalog No. L310KJ).

[0777] (2) Cell preparation: A549 cells, SK-BR-3 cells and NCI-N87 cells in the logarithmic growth phase were taken respectively, washed once with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd., product number E211004), and then 2-3 mL of trypsin (0.25% Trypsin-EDTA (1x), Shanghai Yuanpei Biotechnology Co., Ltd., product number A121002) was added to digest for 2-3 minutes. After the cells were completely digested, 10 mL of cell culture medium containing 10% FBS was added to elute the digested cells, and the cells were centrifuged at 300 g for 5 minutes. The supernatant was discarded, and then 10-20 mL of cell culture medium containing 2% FBS was added to resuspend the cells to prepare a single-cell suspension.

[0778] (3) Cell plating: The single cell suspension was mixed and the cell density of live cells was adjusted with cell culture medium containing 2% FBS (the plating density of SK-BR-3 was 3×10 4 cells / mL, and the A549 plating density was 2×10 4cells / mL, and the NCI-N87 plating density was 4×10 4 cells / mL), mix the density-adjusted cell suspension and add 100 μL / well to a 96-well cell culture plate. Add only 200 μL of PBS to the peripheral wells of the 96-well plate. Incubate the plate in an incubator (37°C, 5% CO2) for 24 hours.

[0779] (4) Compound preparation: Dissolve the compound in DMSO (dimethyl sulfoxide, SIGMA) to prepare a 2 mM stock solution.

[0780] Dilute a 2mM stock solution of small molecule compounds to 600nM using cell culture medium containing 2% FBS. Filter sterilize using a 0.22μM filter. Add 300μL of each sample to the first column of a 96-well U-bottom plate at a 600nM concentration. Add 210μL of cell culture medium containing 2% FBS to each well in columns 2 through 10. Transfer 70μL of the sample from column 1 to 210μL of cell culture medium in column 2, mix thoroughly, and then transfer 70μL to column 3, and so on through column 9.

[0781] (5) Sample addition: Add 100 μL of the sample to be tested at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37° C., 5% CO 2 ).

[0782] (6) Color development: Take out the 96-well cell culture plate, remove the cell culture supernatant, add 100 μL of basal medium containing 10% CCK8 (without FBS), and then place in an incubator for about 2 hours (37°C, 5% CO2).

[0783] (7) Plate reading: Take out the 96-well cell culture plate and place it in a microplate reader (MD SpectraMax i3X). 450 .

[0784] (8) Data analysis: Data were processed and analyzed using Microsoft Excel and Graphpad Prism 5.

[0785] The results of the above tests are summarized in Table 1:

[0786] Table 1: IC values ​​of the test compounds for inhibition of proliferation of A549, SK-BR-3 and NCI-N87 cells in vitro 50 value.

[0787] Conclusion: The small molecule fragments disclosed herein exhibit significant antiproliferative activity against A549, SK-BR-3, and NCI-N87 cells. Compounds 1, 2, and 3 exhibited significantly superior in vitro antiproliferative effects on A549, SK-BR-3, and NCI-N87 cells compared to reference compound 1 at equivalent doses.

[0788] Test Example 2: In vitro proliferation inhibition test of HER2-targeted tumor cells by the disclosed antibody-drug conjugate

[0789] 1. Test purpose:

[0790] The purpose of this experiment is to detect the inhibitory activity of the disclosed antibody-drug conjugate targeting HER2 on the in vitro proliferation of SK-BR-3 cells (human breast cancer cells, Wuhan Punosai, Catalog No. CL-0211) and NCI-N87 cells (human gastric cancer cells, Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences, Catalog No. 3111C0001CCC000481). The cells were treated with different concentrations of the conjugate in vitro. After 6 days of culture, the cell proliferation was detected using CCK8 (Cell Counting Kit-8, Catalog No.: TS547). According to the IC 50 The values ​​were used to evaluate its in vitro activity.

[0791] 2. Experimental methods:

[0792] Taking the in vitro proliferation activity test method for SK-BR-3 cells and NCI-N87 cells as an example, the following test was performed. It should be understood that this method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells (HCC1569, JIMT-1, DIFI, etc.).

[0793] (1) Cell culture: SK-BR-3 cells and NCI-N87 cells were cultured in RPMI 1640 medium containing 10% FBS (Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: L210KJ).

[0794] (2) Cell preparation: Take cells in the logarithmic growth phase, wash them once with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: E211004), and then add 1 mL of trypsin (0.25% Trypsin-EDTA (1x), Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: A121002) to digest for 2-3 minutes. After the cells are completely digested, add 10 mL of cell culture medium to elute the digested cells, centrifuge at 300g for 5 minutes, discard the supernatant, and then add 10 mL of cell culture medium to resuspend the cells to make a single-cell suspension.

[0795] (3) Cell plating: Mix the single cell suspension and adjust the viable cell density to 3×10 4 cells / mL, mix the density-adjusted cell suspension and add 100 μL / well to a 96-well cell culture plate. Add only 200 μL of PBS to the outer wells of the 96-well plate and incubate the plate in an incubator (37°C, 5% CO2) for 24 hours.

[0796] (4) Preparation of samples to be tested:

[0797] Dilute the sample to be tested to 400 nM in cell culture medium containing 2% FBS. Add 300 μL of sample to the first well of a 96-well U-bottom plate for a sample concentration of 400 nM. Add 210 μL of cell culture medium containing 2% FBS to each well in columns 2 through 10. Add 70 μL of sample from the first column to 210 μL of cell culture medium in the second column, mix thoroughly, and then add 70 μL to the third column, and so on through column 9.

[0798] (5) Sample addition: Add 100 μL of the sample to be tested at different concentrations to the culture plate, with two replicate wells for each sample. Incubate the culture plate in an incubator for 6 days (37° C., 5% CO 2 ).

[0799] (6) Color development: Take out the 96-well cell culture plate, remove the culture medium supernatant, add 100 μL of 10% CCK8 basal culture medium (without FBS) to each well, and incubate at room temperature for 2 h.

[0800] (7) Plate reading: Take out the 96-well cell culture plate and place it in a microplate reader (SpectraMax i3X). 450 .

[0801] (8) Data analysis: Data were processed and analyzed using Microsoft Excel and Graphpad Prism 5.

[0802] The results of the above tests are summarized in Table 2:

[0803] Table 2: IC values ​​of the disclosed antibody drug conjugates for inhibition of HER2-targeted tumor cell proliferation in vitro 50 value.

[0804] Conclusion: The antibody-drug conjugate targeting HER2 disclosed in the present invention has significant proliferation inhibitory activity on HER2-positive cells SK-BR-3 and NCI-N87.

[0805] Test Example 3 In vitro proliferation inhibition test of EGFR-targeted tumor cells by the disclosed antibody-drug conjugate

[0806] 1. Test purpose:

[0807] The purpose of this experiment is to detect the inhibitory activity of the disclosed antibody drug conjugate against EGFR target on the in vitro proliferation of MDA-MB-468 cells (human breast cancer cells, Cell Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Catalog No. 3131C0001000700120), SK-BR-3 cells (human breast cancer cells, Wuhan Pu Nuo Sai, Catalog No. CL-0211) and SW620 cells (human colon cancer cells, Biofeng, bc067). The cells were treated with different concentrations of the conjugate in vitro. After 6 days of culture, the cell proliferation was detected using CCK8 (Cell Counting Kit-8, Catalog No.: TS547). According to the IC 50 The values ​​were used to evaluate its in vitro activity.

[0808] 2. Experimental methods:

[0809] Taking the in vitro proliferation activity test method for MDA-MB-468 cells, SK-BR-3 cells, and SW620 cells as an example, the following test was performed. It should be understood that this method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells (HCC1569, JIMT-1, DIFI, etc.).

[0810] (1) Cell culture: MDA-MB-468 cells and SW620 cells were cultured in L-15 medium (Gibco, Catalog No. 11415-064) supplemented with 10% FBS, respectively; SK-BR-3 cells were cultured in RPMI 1640 medium (Shanghai Yuanpei Biotechnology Co., Ltd., Catalog No. L210KJ) supplemented with 10% FBS.

[0811] (2) Cell preparation: Take cells in the logarithmic growth phase, wash them once with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: E211004), and then add 1 mL of trypsin (0.25% Trypsin-EDTA (1x), Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: A121002) to digest for 2-3 minutes. After the cells are completely digested, add 10 mL of the corresponding cell culture medium containing 10% FBS to elute the digested cells, centrifuge at 300g for 5 minutes, discard the supernatant, and then add 10 mL of cell culture medium to resuspend the cells to make a single-cell suspension.

[0812] (3) Cell plating: Mix the single-cell suspension and adjust the viable cell density to 3 × 104 cells / mL using the corresponding cell culture medium containing 2% FBS. Mix the cell suspension after density adjustment and add 100 μL / well to a 96-well cell culture plate. Add only 200 μL of PBS to the peripheral wells of the 96-well plate. Incubate the culture plates of MDA-MB-468 and SW620 cells in a non-CO2 incubator for 24 hours (37°C). Incubate the culture plates of SK-BR-3 cells in a 5% CO2 incubator for 24 hours (37°C).

[0813] (4) Preparation of samples to be tested:

[0814] Dilute the sample to be tested to 400 nM using the appropriate cell culture medium containing 2% FBS for the cells to be added. Add 300 μL of sample to the first well of a 96-well U-bottom plate for a sample concentration of 400 nM. Add 210 μL of the appropriate cell culture medium containing FBS to each well in columns 2 through 10. Add 70 μL of the sample from column 1 to 210 μL of cell culture medium from column 2, mix thoroughly, and then add 70 μL to column 3, and so on through column 9.

[0815] (5) Sample addition: Add 100 μL of the sample to be tested at different concentrations to the culture plate, with two replicates for each sample. The MDA-MB-468 and SW620 cell culture plates were incubated in a CO2-free incubator for 6 days (37°C), and the SK-BR-3 cell culture plates were incubated in a 5% CO2 incubator for 6 days (37°C).

[0816] (6) Color development: Take out the 96-well cell culture plate, remove the culture medium supernatant, add 100 μL of 10% CCK8 (prepared in RPMI 1640 basal medium, without FBS) solution to each well, and incubate at room temperature for 2 h.

[0817] (7) Plate reading operation: Take out the 96-well cell culture plate, place it in a microplate reader (SpectraMax i3X), and measure A450 using the microplate reader.

[0818] (8) Data analysis: Data were processed and analyzed using Microsoft Excel and Graphpad Prism 5.

[0819] The results of the above tests are summarized in Table 3:

[0820] Table 3: IC values ​​of the disclosed antibody drug conjugates for inhibition of EGFR-targeted tumor cell proliferation in vitro 50 value.

[0821] Conclusion: The antibody-drug conjugates targeting EGFR disclosed in the present invention have significant proliferation inhibitory activity on EGFR-positive cells MDA-MB-468; at the same time, they have weak proliferation inhibitory activity on EGFR-negative cells SW620 and have good selectivity.

[0822] Test Example 4: In vitro proliferation inhibition test of HER2-targeted tumor cells by the disclosed antibody-drug conjugates 2

[0823] 1. Test purpose:

[0824] The purpose of this experiment is to detect the inhibitory activity of the disclosed antibody-drug conjugate against HER2 target on the in vitro proliferation of SK-BR-3 cells (human breast cancer cells, Wuhan Punosai, Catalog No. CL-0211) and MDA-MB-468 cells (human breast cancer cells, Cell Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Catalog No. 3131C0001000700120). The cells were treated with different concentrations of the conjugate in vitro. After 6 days of culture, the cell proliferation was detected using CCK8 (Cell Counting Kit-8, Catalog No.: TS547). According to the IC 50 The values ​​were used to evaluate its in vitro activity.

[0825] 2. Experimental methods:

[0826] Taking the in vitro proliferation activity test method for SK-BR-3 cells and MDA-MB-468 cells as an example, the following test was performed. It should be understood that this method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells (HCC1569, JIMT-1, DIFI, etc.).

[0827] (1) Cell culture: SK-BR-3 cells and MDA-MB-468 cells were cultured in RPMI 1640 medium containing 2% FBS (Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: L210KJ).

[0828] (2) Cell preparation: Take cells in the logarithmic growth phase, wash them once with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: E211004), and then add 1 mL of trypsin (0.25% Trypsin-EDTA (1x), Shanghai Yuanpei Biotechnology Co., Ltd., catalog number: A121002) to digest for 2-3 minutes. After the cells are completely digested, add 10 mL of cell culture medium to wash the digested cells, centrifuge at 300g for 5 minutes, discard the supernatant, and then add 10 mL of cell culture medium to resuspend the cells to make a single-cell suspension.

[0829] (3) Cell plating: Mix the single cell suspension and adjust the viable cell density to 3×10 4 cells / mL. Mix the density-adjusted cell suspension and add 100 μL / well to a 96-well cell culture plate. Add only 200 μL of PBS to the outer wells of the 96-well plate. Incubate the plate in an incubator (37°C, 5% CO2) for 24 hours.

[0830] (4) Preparation of samples to be tested:

[0831] Dilute the sample to be tested to 400 nM in cell culture medium containing 2% FBS. Add 300 μL of sample to the first well of a 96-well U-bottom plate for a sample concentration of 400 nM. Add 210 μL of cell culture medium containing 2% FBS to each well in columns 2 through 10. Add 70 μL of sample from the first column to 210 μL of cell culture medium in the second column, mix thoroughly, and then add 70 μL to the third column, and so on through column 9.

[0832] (5) Sample addition: Add 100 μL of the sample to be tested at different concentrations to the culture plate, with two replicate wells for each sample. Incubate the culture plate in an incubator for 6 days (37° C., 5% CO 2 ).

[0833] (6) Color development: Take out the 96-well cell culture plate, remove the culture medium supernatant, add 100 μL of 10% CCK8 (prepared in RPMI 1640 basal medium, without FBS) solution to each well, and incubate at room temperature for 2 h.

[0834] (7) Plate reading: Take out the 96-well cell culture plate and place it in a microplate reader (SpectraMax i3X). 450 .

[0835] (8) Data analysis: Data were processed and analyzed using Microsoft Excel and Graphpad Prism 5.

[0836] The results of the above tests are summarized in Table 4:

[0837] Table 4: IC values ​​of the disclosed antibody drug conjugates for inhibition of HER2-targeted tumor cell proliferation in vitro 50 value.

[0838] Conclusion: The antibody-drug conjugates targeting HER2 disclosed herein have significant proliferation inhibitory activity on HER2-positive SK-BR-3 cells. ADC-1 and ADC-74 have better in vitro proliferation inhibitory effects on SK-BR-3 cells than reference substances 2 and 4 at the same dose. At the same time, they have weak proliferation inhibitory activity on HER2-negative MDA-MB-468 cells and have good selectivity.

[0839] Test Example 5: Testing the bystander killing effect of the disclosed antibody drug conjugate on HER2-targeted tumor cells

[0840] HER2-positive SK-BR-3 cells (human breast cancer cells, Wuhan Punosai, catalog number CL-0211) and HER2-negative MDA-MB-468 cells (human breast cancer cells, Cell Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number 3131C0001000700120) were cultured with RPMI-1640 + 10% FBS to adjust the cell density to 1.2 × 10 5 cells / ml and 4×10 4 cells / ml, 1 mL each of SK-BR-3 and MDA-MB-468 cells were added to a 6-well plate and mixed and cultured overnight at 37°C and 5% CO2. The sample ADC-113 was prepared at a concentration of 4.5 μg / mL (30 nM), and 1 mL was added to the cells to make a total volume of 3 mL and a final concentration of 10 nM. A solvent control group was set up and cultured at 37°C and 5% CO2 for 5 days. Cells were digested by adding trypsin (0.25% Trypsin-EDTA (1x), Shanghai Yuanpei Biotechnology Co., Ltd., Catalog No.: A121002) to the 6-well plate, and 1 mL of FACS buffer (PBS + 4% FBS) was added to resuspend, the cells were stained with trypan blue, and counted. The remaining cells were centrifuged, and trastuzumab was diluted to 10 μg / mL in FACS buffer. The cells were resuspended in 200 μL of antibody solution, incubated on ice for 30 min, centrifuged and the supernatant was discarded, and the cells were washed once with 1 mL of FACS buffer. 200 μL of AF647-labeled goat anti-human Fc antibody (Jackson, Catalog No.: 109-606-170) solution was added and incubated on ice for 15 min. A 5 μg / mL PI (Sigma, Catalog No.: 31845) solution was added and incubated on ice for 5 min. The cells were centrifuged and the supernatant was discarded. The cells were washed once with 1 mL of FACS buffer, and the cells were resuspended in 400 μL of PBS. The cells were analyzed by BD Celesta flow cytometer, and the ratio of the two cell types and the cell number were determined based on the flow cytometry and counting results.

[0841] The results of the above tests are summarized in Figure 1, which demonstrates that the antibody-drug conjugates disclosed herein have excellent bystander killing effects. ADC-1, ADC-10, ADC-74, and ADC-75 exhibited superior bystander killing effects on HER2-targeted tumor cells compared to Reference 2 and Reference 4 at equivalent doses.

[0842] Test Example 6: Testing the bystander killing effect of the disclosed antibody drug conjugate on EGFR-targeted tumor cells

[0843] EGFR-positive MDA-MB-468 cells (human breast cancer cells, Cell Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number 3131C0001000700120) and EGFR-negative SW620 cells (human colon cancer cells, Biofeng, bc067) were cultured with RPMI-1640 + 10% FBS to adjust the cell density to 1.2 × 10 5 cells / ml and 4×10 4 cells / ml, 1 mL each of MDA-MB-468 and SW620 cells were added to a 6-well plate and mixed and cultured overnight at 37°C, 5% CO2. The ADC sample was prepared at a concentration of 4.5 μg / mL (30 nM), and 1 mL was added to the cells to make a total volume of 3 mL and a final concentration of 10 nM. A solvent control group was set up and cultured at 37°C, 5% CO2 for 5 days. Cells were digested with trypsin (0.25% Trypsin-EDTA (1x), Shanghai Yuanpei Biotechnology Co., Ltd., Catalog No.: A121002) in a 6-well plate, and resuspended in 1 mL of FACS buffer (PBS + 4% FBS). The cells were stained with trypan blue and counted. The remaining cells were centrifuged, Nimotuzumab was diluted to 10 μg / mL in FACS buffer, the cells were resuspended in 200 μL of antibody solution, incubated on ice for 30 min, centrifuged and the supernatant was discarded, and the cells were washed once with 1 mL of FACS buffer. 200 μL of AF647-labeled goat anti-human Fc antibody (Jackson, Catalog No.: 109-606-170) solution was added and incubated on ice for 15 min. A 5 μg / mL PI (Sigma, Catalog No.: 31845) solution was added and incubated on ice for 5 min. The cells were centrifuged and the supernatant was discarded. The cells were washed once with 1 mL of FACS buffer, and the cells were resuspended in 400 μL of PBS. The cells were analyzed by BD Celesta flow cytometer, and the ratio of the two cell types and the cell number were determined based on the flow cytometry and counting results.

[0844] The results of the above tests are summarized in Figure 2, which shows that the Nimotuzumab antibody drug conjugates disclosed herein have excellent bystander killing effects. ADC-72 and ADC-73 have significantly better bystander killing effects on EGFR-targeted tumor cells than reference compound 3 at the same dose.

[0845] The above is an exemplary description of the implementation methods of the technical solutions disclosed herein. It should be understood that the scope of protection of the present disclosure is not limited to the above-mentioned implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of this application.

Claims

1. A ligand-drug conjugate represented by the following formula (C), its stereoisomers, racemates, tautomers, isotopomers, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts thereof: Tp-LG (C) in, Tp is the targeting moiety; L is selected from a chemical bond or a linker; G is a group represented by the following formula (G): wherein A is absent or selected from unsubstituted or optionally substituted with one, two or more R A The following groups substituted: alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heteroaryl, heterocyclic group or a combination of two or more thereof; the alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heteroaryl, heterocyclic group or a combination of two or more thereof may not be substituted or may be substituted with one, two or more selected from -O-, -S-, -NR 5 -、-NR 6 C(=O)-, -C(=O)NR 6 -, -C(=O)-, -NR 7 C(=O)NR 8 - or -OC(=O)-; R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted: cycloalkyl, cycloalkylalkyl, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy; X is selected from O or S; Z is selected from 0 or 1; R 2 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy; R 22 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy; R 3 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy; R 4 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy; Or, R 2 , R 3 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D substituted ring structures; Or, R 3 , R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D substituted ring structures; Or, R 22 , R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D substituted ring structures; B is absent or selected from -O-, -S-, -S(=O)-, -C(=O)-, -NR 5 -、-C=N-NR 9 -、-C=NO- or -NR 10 -NR 11 -; R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 are the same or different and are independently selected from hydrogen, deuterium, unsubstituted or optionally substituted by one, two or more R E Substituted: alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, HC(=O)-, alkylC(=O)-, cycloalkylC(=O)NH-, heterocyclylC(=O)NH-, arylC(=O)NH-, heteroarylC(=O)NH-; Every R A , R B , R C , R D , R E are the same or different and are independently selected from deuterated, halogen, hydroxy, cyano, nitro, oxo (=O), unsubstituted or optionally substituted by one, two or more R F Substituted: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH2, HC(=O)NH-, alkylC(=O)NH-, cycloalkylC(=O)NH-, heterocyclylC(=O)NH-, arylC(=O)NH-, heteroarylC(=O)NH-; Every R F are the same or different and are independently selected from the group consisting of deuterated, halogen, hydroxy, cyano, nitro, oxo (=O), alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH2, HC(=O)NH-, alkylC(=O)NH-, cycloalkylC(=O)NH-, heterocyclylC(=O)NH-, arylC(=O)NH-, heteroarylC(=O)NH-; The wavy line indicates the site to which L is attached.

2. The ligand-drug conjugate according to claim 1, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein: The groups in formula (G) are independently selected from the following definitions: wherein A is absent or selected from unsubstituted or optionally substituted with one, two or more R A Substituted with the following groups: C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof; the C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof may be substituted or optionally substituted by one, two or more selected from -O-, -S-, -NR 5- 、-NR 6 C(=O)-, -C(=O)NR 6 、-C(=O)-、-NR 7 C(=O)NR 8 - or -OC(=O)-; preferably, A is absent or selected from unsubstituted or optionally substituted by one, two or more R A Substituted with the following groups: C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 Cycloalkyl, C 6-10 aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclic group or a combination of two or more thereof; the C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclic group or a combination of two or more thereof may be substituted or optionally substituted by one, two or more selected from -O-, -S-, -NR- 5- 、-NR 6 C(=O)-, -C(=O)NR 6 -, -C(=O)-, -NR 7 C(=O)NR 8 - or -OC(=O)-; R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted with the following groups: C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy, 3-10 membered heterocyclic group, 3-10 membered heterocyclic group C 1-10 alkyl, 3-10 membered heterocyclyloxy; preferably, R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted with the following groups: C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy, 3-6 membered heterocyclic group, 3-6 membered heterocyclic group C 1-6 Alkyl, 3-6 membered heterocyclyloxy; X is selected from O or S; Z is selected from 0 or 1; R 2 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 3-10 Alkyl, C 3-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 2 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy; R 22 hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 3-10 Alkyl, C 3-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 2 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy; R 3 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 3 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy; R 4 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; preferably, R 4 is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, amino, cyano, unsubstituted or optionally substituted with one, two or more R C Substituted with the following groups: C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy; Or, R 2 , R 3 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D A substituted 4-10 membered ring structure; wherein the 4-10 membered ring structure can be selected from, for example, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered heteromonocyclic hydrocarbon groups, heterobicyclic hydrocarbon groups, monocyclic hydrocarbon groups, and bicyclic hydrocarbon groups; wherein the heteromonocyclic hydrocarbon groups and heterobicyclic hydrocarbon groups contain one, two or more O, S, N or carbonyl groups or any combination thereof; Or, R 3 , R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D A substituted 4-10 membered ring structure; wherein the 4-10 membered ring structure can be selected from, for example, a 4-, 5-, 6-, 7-, 8-, 9- or 10-membered monocyclic hydrocarbon group, a bicyclic hydrocarbon group, a heteromonocyclic hydrocarbon group, a heterobicyclic hydrocarbon group; wherein the heteromonocyclic hydrocarbon group and the heterobicyclic hydrocarbon group contain one, two or more O, S, N or carbonyl groups or any combination thereof; Or, R 22 , R 4 Together with the atoms to which they are attached, they are unsubstituted or optionally substituted with one, two or more R D A substituted 4-10 membered ring structure; wherein the 4-10 membered ring structure can be selected from, for example, a 4-, 5-, 6-, 7-, 8-, 9- or 10-membered monocyclic hydrocarbon group, a bicyclic hydrocarbon group, a heteromonocyclic hydrocarbon group, a heterobicyclic hydrocarbon group; wherein the heteromonocyclic hydrocarbon group and the heterobicyclic hydrocarbon group contain one, two or more O, S, N or carbonyl groups or any combination thereof; B is absent or selected from -O-, -S-, -S(=O)-, -C(=O)-, -NR 5 、-C=N-NR 9 、-C=NO- or -NR 10 -NR 11 -; R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 are the same or different and are independently selected from hydrogen, deuterium, unsubstituted or optionally substituted by one, two or more R E Substituted with the following groups: C 1-10 Alkyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 6-12 Aryl, C 6-12 Aryl C 1-10 Alkyl, 5-12 membered heteroaryl, 5-12 membered heteroarylC 1-10 Alkyl, 5-12 membered heterocyclic group, 5-12 membered heterocyclic group C 1-10 Alkyl, HC(=O)-, C 1-10 Alkyl C(=O)-, C 3-10 Cycloalkyl C(=O)NH-, 5-12 membered heterocyclic C(=O)NH-, C 6-12 Aryl C(=O)NH-, 5-12 membered heteroaryl C(=O)NH-; preferably, R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 are the same or different and are independently selected from hydrogen, deuterium, unsubstituted or optionally substituted by one, two or more R E Substituted with the following groups: C 1-6 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 6-10 Aryl, C 6-10 Aryl C 1-6 Alkyl, 5-6 membered heteroaryl, 5-6 membered heteroarylC 1-6 Alkyl, 5-6 membered heterocyclic group, 5-6 membered heterocyclic group C 1-6 Alkyl, HC(=O)-, C 1-6 Alkyl C(=O)-, C 3-6 Cycloalkyl C(=O)NH-, 5-6 membered heterocyclic C(=O)NH-, C 6-10 Aryl C(=O)NH-, 5-6 membered heteroaryl C(=O)NH-; Every R A , R B , R C , R D , R E are the same or different, independently selected from deuterated, halogen, hydroxyl, cyano, nitro, unsubstituted or optionally substituted with one, two or more R F Substituted with the following groups: C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy, C 6-12 Aryl, C 6-12 Aryl C 1-10 Alkyl, C 6-12 Aryloxy, 5-12 membered heteroaryl, 5-12 membered heteroaryl C 1-10 alkyl, 5-12 membered heteroaryloxy, 5-12 membered heterocyclyl, 5-12 membered heterocyclylC 1-10 alkyl, 5-12 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-10 Alkyl C(=O)NH-, C 3-10 Cycloalkyl C(=O)NH-, 5-12 membered heterocyclic C(=O)NH-, C 6-12 Aryl C(=O)NH-, 5-12 membered heteroaryl C(=O)NH-; preferably, each R A , R B , R C , R D , R E are the same or different, independently selected from deuterated, halogen, hydroxyl, cyano, nitro, unsubstituted or optionally substituted with one, two or more R F Substituted with the following groups: C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy, C 6-10 Aryl, C 6-10 Aryl C 1-6 Alkyl, C 6-10 Aryloxy, 5-6 membered heteroaryl, 5-6 membered heteroaryl C 1-6 alkyl, 5-6 membered heteroaryloxy, 5-6 membered heterocyclyl, 5-6 membered heterocyclylC 1-6 Alkyl, 5-6 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-6 Alkyl C(=O)NH-, C 3-6 Cycloalkyl C(=O)NH-, 5-6 membered heterocyclic C(=O)NH-, C 6-10 Aryl C(=O)NH-, 5-6 membered heteroaryl C(=O)NH-; Every R F are the same or different and are independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy, C 6-12 Aryl, C 6-12 Aryl C 1-10 Alkyl, C 6-12 Aryloxy, 5-12 membered heteroaryl, 5-12 membered heteroaryl C 1-10 alkyl, 5-12 membered heteroaryloxy, 5-12 membered heterocyclyl, 5-12 membered heterocyclylC 1-10 alkyl, 5-12 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-10 Alkyl C(=O)NH-, C 3-10 Cycloalkyl C(=O)NH-, 5-12 membered heterocyclic C(=O)NH-, C 6-12 Aryl C(=O)NH-, 5-12 membered heteroaryl C(=O)NH-; preferably, each R F are the same or different and are independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy, C 6-10 Aryl, C 6-10 Aryl C 1-6 Alkyl, C 6-10 Aryloxy, 5-6 membered heteroaryl, 5-6 membered heteroaryl C 1-6 alkyl, 5-6 membered heteroaryloxy, 5-6 membered heterocyclyl, 5-6 membered heterocyclylC 1-6 Alkyl, 5-6 membered heterocyclyloxy, NH2, HC(=O)NH-, C 1-6 Alkyl C(=O)NH-, C 3-6 Cycloalkyl C(=O)NH-, 5-6 membered heterocyclic C(=O)NH-, C 6-10 Aryl C(=O)NH-, 5-6 membered heteroaryl C(=O)NH-.

3. The ligand-drug conjugate according to any one of claims 1 to 2, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein: A is absent or selected from one of the following substructures, wherein the substructure may be unsubstituted or optionally substituted with one, two or more R A replace: -(CH2) n1 -、-O(CH2) n2 -、-S(CH2) n3 -、-NR 5 (CH2) n4 -、-NR 6 C(=O)(CH2) n5 -、-C(=O)(CH2) n6 -、-NR 7 C(=O)NR 8 (CH2) n7 -、-OC(=O)(CH2) n8 -、-C=C(CH2) n9 -、-C≡C(CH2) n10 -、 n1, n2, n3, n4, n5, n6, n7, n8, n9, n10 are each independently selected from any integer between 0 and 6; R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted with the following groups: C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl; for example, R 1 is selected from unsubstituted or optionally substituted with one, two or more R B Substituted with the following groups: R 2 Selected from hydrogen; R 22 Selected from hydrogen; R 3 is selected from unsubstituted or optionally substituted with one, two or more R C Substituted C 1-6 Alkyl; for example, R 3 Selected from methyl, ethyl, propyl; R 4 is selected from halogen; B is absent or selected from -O-, -S-, NR 5 ; R 5 Selected from H, deuterium, C 1-6 Alkyl, C 3-6 Cycloalkyl; Or, R 3 , R 4 Together with the benzene ring to which it is attached, it forms one of the following substructures, wherein the substructure may be unsubstituted or optionally substituted with one, two or more R D replace: Where R D Independently has the definition of any one of claims 1-2.

4. The ligand-drug conjugate according to any one of claims 1 to 3, its stereoisomers, racemates, tautomers, isotopologues, isotope-labeled substances, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts thereof, characterized in that: A is absent or selected from one of the following substructures, wherein the substructure may be unsubstituted or optionally substituted with one, two or more R A replace: B is absent or selected from -O-, -S-, NR 5 ; R 5 Selected from H, deuterium, C 1-6 Alkyl, C 3-6 Cycloalkyl.

5. The ligand-drug conjugate according to any one of claims 1 to 4, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein: G is selected from the group represented by the following formula (G-1) or (G-2): Among them, A, B, R 1 , R 2 , R 22 , R 3 , R 4 Independently has the definition as described in any one of claims 1 to 4.

6. The ligand-drug conjugate according to any one of claims 1 to 4, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein: G is selected from the group represented by formula (G-3), (G-4), (G-5) or (G-6): Among them, A, B, R 1 Having the definition as described in any one of claims 1 to 4.

7. The ligand-drug conjugate according to any one of claims 1 to 4, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein: G is selected from the following groups: Among them, A, B, R 2 , R 22 , R 3 , R 4 Independently has the definition as described in any one of claims 1 to 4.

8. The ligand-drug conjugate according to any one of claims 1 to 4, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein: G is selected from the following groups: Among them, A, B, R 2 , R 22 , R 3 , R 4 Independently has the definition as described in any one of claims 1 to 4.

9. The ligand-drug conjugate according to any one of claims 1 to 4, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein: G is selected from the following groups:

10. The ligand drug conjugate according to any one of claims 1 to 4, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, Wherein L is selected from a chemical bond or a linker represented by formula (L) as defined below: #L 1 -L 2 -L 3 -L 4 * (L) Where L 1 It is the linking part with the targeting part Tp, composed of the reactive group L 1 ' and the targeting moiety Tp, # represents the connection site with the targeting moiety Tp; For example, L 1 ' is a pyrimidine group or a maleimide group, then L 1 The structure is as follows: or its open loop form: L 2 Does not exist or is L 1 and L 3 The interval part of L 3 is the peptide portion; L 4 is absent, or is the linking portion between the peptide portion and the biologically active molecule G, and is composed of the reactive group L 4 'Generated by reaction with biologically active molecule G or its intermediate, * represents the connection site with biologically active molecule G; Optionally: Between the above parts in L, preferably in L 1 With L 2 Between or L 2 With L 3 between or to replace L 2 or by inserting L 2 The method contains the following hydrophilic part: The hydrophilic part is a subunit substituted by one, two or more hydrophilic groups, such as a phenylene group substituted by one, two or more hydrophilic groups or an amino acid residue substituted by one, two or more hydrophilic groups, preferably: The hydrophilic part can also be a hydrophilic group itself forming a subunit such as or -(CH2CH2O) p -; R 12 , R 12 'same or different, at least one selected from a hydrophilic group, the other selected from the following substituents: hydrogen, halogen, cyano, amino, nitro, unsubstituted or optionally substituted with one, two or more R zg Substituted C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; The hydrophilic group is selected from polyethylene glycol or polyethylene glycol subunit, C 1-10 An alkyl group, a group containing a sugar ring, or a heterocyclic subunit containing a nitrogen atom such as a piperidinyl group or a piperazinyl group, preferably a polyethylene glycol group, and more preferably -(CH2CH2O) p -C 1-10 Alkyl, -C(=O)-NH-(CH2CH2O) p -C 1-10 Alkyl or -NH-(CH2CH2O) p -C 1-10 Alkyl, or preferably C 1-10 Alkyl, more preferably Each p is the same or different and is independently selected from an integer of 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; Every R zg The same or different, independently selected from the following groups: halogen, hydroxyl, amino, cyano, nitro, C 1-10 Alkyl, C 1-10 Alkyloxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, C 3-10 Cycloalkyloxy; Preferably, each R zg The same or different, independently selected from the following groups: halogen, hydroxyl, amino, cyano, nitro, C 1-6 Alkyl, C 1-6 Alkyloxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, C 3-6 Cycloalkyloxy; The hydrophilic part defined above is present or absent; L 1 -L 4 Or the hydrophilic part can be connected by any chemical bond, such as a direct bond, an ester bond (—CO—O—), an amide bond (—CO—NH—), an ether bond (—O—), a thioether bond (—S—), a carbamate (—N—CO—O—), a urea group (—O—CO—O—); Preferably, each H in the peptide bond or amide bond in L may be optionally substituted with a methyl group.

11. [Corrected 29.08.2024 in accordance with Rule 26] The ligand drug conjugate according to claim 10, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, L 1 Any group L that reacts with the targeting moiety Tp 1 'Forming, L 1 'Preferred are sulfhydryl reactive groups, amino reactive groups, carboxyl reactive groups, dithiol bridging groups, etc.; for antibodies introduced with non-natural amino acids, click chemistry reactive groups such as ketones, hydrazines or hydrazides, azides, alkynes, cyclopropenes or dienes can also be selected; when the Tp moiety is an antibody, the connection site includes any applicable amino acid residue or N297 sugar chain coupling of the CH2 domain, such as fucose, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (SA) introduced by sugar engineering; the connection reaction includes a chemical reaction or an enzymatic reaction, such as using transglutaminase (MTGase) to transfer an amine-containing drug linker or a reactive spacer to a deglycosylated antibody; L 1 'Preferably a thiol reactive group; L 1 'Preferred thiol reactive groups are of the following structure: Hal-Het- Hal is selected from halogen, OMs, OTs, OTf, nitro, and optionally substituted by one, two or more R z8 Substituted with the following groups: C 1-10 Alkyl sulfide group, C 6-12 Aryl sulfide group, 5-12 membered heteroaryl sulfide group, C 1-10 Alkyl sulfoxide, C 6-12 Aryl sulfoxide, 5-12 membered heteroaryl sulfoxide, C 1-10 Alkylsulfonyl, C 6-12 Arylsulfonyl, 5-12 membered heteroarylsulfonyl; wherein, R z8 are independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, C 6-12 5- to 12-membered aryl and 5- to 12-membered heteroaryl; Het is selected from the group consisting of optionally one, two or more R z9 substituted 5-12 membered heteroaryl; wherein R z9 are independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-10 Alkyl and halogenated C 1-10 Alkyl; preferably, Het is selected from optionally substituted by one, two or more R z9 substituted 5-10 membered heteroaryl; wherein R z9 are independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and halogenated C 1-4 alkyl; In the preferred embodiment, Hal is preferably methanesulfonyl, and Het is preferably pyrimidine; In a preferred embodiment, Hal-Het- is: The corresponding L 1 The structure is: And L 1 '-L 2 The following structure is preferred: q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl; Further preferred: or L 1 'Further preferably a maleimide group or a substituted maleimide group, and L 1 -L 2 The following structure is preferred: The fragment prepared from (N-maleimidomethyl)-carboxylic acid-N-hydroxysuccinimide ester has the structure: (q is an integer from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8); Or a fragment prepared from m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), the structure is: The fragment prepared from 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) has the structure:

12. The ligand-drug conjugate according to claim 10, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, L 2 Select from non-existent, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof; the C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 The aryl group, the 5-12 membered heteroaryl group, the 5-12 membered heterocyclic group or a combination of two or more thereof may not be interrupted by or may be optionally interrupted by a carbonyl group, an O, a S or a N atom; the C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof may be optionally replaced by C 1-6 Alkyl, C 3-6 Cycloalkyl, halogen atom, halogenated C 1-6 Alkyl substituted; optionally, the C 1-6 Alkyl or halogenated C 1-6 The alkyl group can form a C 3-6 Cycloalkyl, L 2 Through any functional group or covalent bond with L 1 or L 3 fragment ligation; Preferably, L 2 Selected from -(CH2) q -、-C(=O)-NH-(CH2) q -C(=O)-, -(CH2) q -C(=O)-, -(CH2) q -NH-C(=O)-, -(CH2) q -NCH3-C(=O)-、-(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -Cy-(CH2) q -C(R z4 R z5 )-C(=O)-, wherein q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; wherein Cy is a 5-12 membered heteroaryl or heterocyclic group optionally containing S, O or N heteroatoms and optionally substituted by H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and halogenated C 1-4 Alkyl substituted, preferably, at least three atoms in Cy are N, more preferably three consecutive atoms in Cy are N, and more preferably Cy is 1,2,3-triazolyl; Preferably, R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl. Preferably, L 2 Through -NHC(=O)-, -NCH3C(=O)- or -C(=O)- 3 The N-terminal connection.

13. The ligand drug conjugate according to claim 10, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, Where L 3 The invention is selected from a divalent peptide group comprising 2 to 8 optionally substituted natural or non-natural, L- or D-type amino acid residues, each of which is identical or different and is independently selected from the following amino acid residues: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine, demethylpyrrolysine, analogs of the above amino acids or selected from AA 1 The indicated amino acid residues or their stereoisomers. in, R G With R H are not H at the same time, and are each independently selected from H, Or, R G With R H Together with the carbon atoms to which they are attached, they form an unsubstituted or optionally substituted with one, two or more R L Substituted C 3-10 Cycloalkyl or 3-10 membered heterocyclic group; r, r1 are each independently selected from any integer from 0 to 10; R I , R J , R K Each independently selected from H, unsubstituted or optionally substituted with one, two or more R M Substituted C 1-6 Alkyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, ester group; Or, R I With R J Together with the nitrogen atom to which they are attached, they form an unsubstituted or optionally substituted with one, two or more R L substituted 4-10 membered heterocyclyl; R M , R L are the same or different and are independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH2, alkylamino, dialkylaminoacid, HC(=O)NH-, alkylC(=O)NH-, cycloalkylC(=O)NH-, heterocyclylC(=O)NH-, arylC(=O)NH-, heteroarylC(=O)NH-; optionally, two R attached to the same carbon atom M or R L Together they form C 3-6 Cycloalkyl; Further preferred, L 3 selected from a group consisting of 2, 3, 4, 5 or 6 optionally substituted or non-natural, L- or D-form amino acid residues or AA 1 A divalent peptide group comprising a combination of amino acid residues, each of which is the same or different and is independently selected from the following amino acid residues: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine, demethylpyrrolysine, analogs of the above amino acids or AA 1 ; For example -ValCit-; -ValAA 1 -CitVal--AlaAla--AlaCit--CitAla--AsnCit--CitAsn--CitCit--ValGlu--GluVal--SerCit--CitSer--L ysCit-;-CitLys-;-AspCit-;-CitAsp-;-AlaVal-;-ValAla-;-PheAla-;-AlaPhe-;-PheLys-;-LysPhe-;-ValLys-;-GlyAA 1 -LysVal--AlaLys--LysAla--PheCit--CitPhe--LeuCit--CitLeu--IleCit--CitIle--PheArg--ArgPhe--CitTrp--TrpCit--AlaAlaAla--PhePheLys--ValAAA 1 Gly-;-AlaAA 1 Gly-;-GlyAA 1 Gly-;-LysPhePhe-;-DPhePheLys-;-DLysPhePhe-;-GlyPheLys-;-LysPheGly-;-GlyPheLeuGly-;-GlyLeuPheGly-;-AlaLeuAlaLeu-;-GlyGlyGly-;-GlyGlyGlyGly-;-GlyPheValGly-;-GlyValPheGly-;-GlyGlyPheGly-;-GlyGlyValGly-; AA 1 Among the amino acid residues, preferably, r, r1 are each independently selected from any integer from 0 to 5; R G With R H Among them, either one is H, and the other is selected from: Or, R G With R H Together with the carbon atoms to which they are attached, they form an unsubstituted or optionally substituted with one, two or more R L substituted 5-6 membered heterocyclic group; R I , R J , R K Each independently selected from H, unsubstituted or optionally substituted with one, two or more R M Substituted methyl, ethyl, n-propyl, n-butyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-6 Alkyl, -COOCH3, COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3 and -COOCH2CH2CH2CH3; Or, R I With R J Together with the nitrogen atom to which they are attached, they form an unsubstituted or optionally substituted with one, two or more R L substituted 5-6 membered heterocyclic group; Most preferably, L 3 Selected from -ValAA 1 Gly-; Among them, AA 1 In the amino acid residues, r is 0 and r1 is 4; R G With R H Among them, either one is H, and the other is selected from: Or, R G With R H Together with the carbon atoms they are connected to, they form * indicates R G With R H carbon atoms that are linked together; R I , R J , R K Each independently selected from H, unsubstituted or optionally substituted with one, two or more R M Substituted methyl, ethyl, n-propyl, n-butyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl C 1-4 alkyl; Or, R I With R J Together with the nitrogen atoms to which they are attached, they form 14. The ligand-drug conjugate according to any one of claims 10 to 13, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, characterized in that: AA 1 The amino acid residues are selected from one of the following substructures:

15. The ligand drug conjugate according to claim 10, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, L 4 Not present or selected from: More preferably, L 4 for: Most preferably, L 4 for:

16. The ligand drug conjugate according to any one of claims 10 to 15, its stereoisomer, racemate, tautomer, isotopologue, isotope labelled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, L 1 It is generated by coupling Tp with a thiol reactive group selected from a maleimide group, a substituted maleimide group or Hal-Het-; In L 1 With L 2 Between or L 2 With L 3 between or replace or insert L 2 The hydrophilic part is contained in the form of.

17. The ligand drug conjugate according to any one of claims 10 to 15, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, L 1 It is generated by coupling Tp with a thiol reactive group selected from a maleimide group or a substituted maleimide group.

18. The ligand drug conjugate according to any one of claims 10 to 15, its stereoisomer, racemate, tautomer, isotopologue, isotope labelled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, L 1 Generated by coupling Tp with a thiol reactive group selected from Hal-Het-; L 4 for:

19. The ligand drug conjugate according to any one of claims 10 to 15, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, L 1 -L 2 The following structure L 1 '-L 2 Coupling Tp generation: q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl, where R z4 or R z5 At least one is not H; Further optimization of R z4 or R z5 Together they form C 3-6 Cycloalkyl; L 1 -L 2 The most preferred Coupled Tp generation.

20. The ligand drug conjugate according to any one of claims 1 to 19, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, Tp is a targeting moiety selected from a small molecule ligand, a protein, a peptide, a non-protein agent (e.g., a sugar, RNA, or DNA); Preferably, the target of Tp is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2, BMPR1B, Tyro7, c-Met, ApoE, CD1lc, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINE1, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFl6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD1lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and BCMA;. Preferably, Tp is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an arylsulfonamide derivative (such as a carbonic anhydrase IX inhibitor), an ICG dye, a cyanine dye or a derivative thereof; Preferably, Tp is selected from an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody; preferably a monoclonal antibody; Preferably, the antibody or antigen-binding fragment thereof is selected from at least one of the following antibodies or antigen-binding fragments thereof: anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody, and the antibody may be a bispecific antibody or a multispecific antibody; Further preferably, the antibody or antigen-binding fragment thereof is selected from at least one of the following antibodies or antigen-binding fragments thereof: Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glembatumumab.

21. The ligand drug conjugate according to any one of claims 1 to 20, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the conjugate, its linker or linker-drug is selected from one of the following: in, u is an integer selected from 0 to 10, G has the definition as described in any one of claims 1 to 20, and LG has the definition as described in any one of claims 1 to 20; 22. The ligand-drug conjugate according to any one of claims 1 to 20, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the conjugate has a structure as shown in the following formula: in, R 1 , R 2 , R 22 , R 3 , R 4 , R G , R H , A, B, X, Z, L 1 , L 2 , Tp has the definition as described in any one of claims 1-20.

23. The ligand-drug conjugate according to any one of claims 1 to 20, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the conjugate has a structure as shown in the following formula: in, A, B, R 3 , R 4 , L 1 , L 2 , Tp has the definition as described in any one of claims 1-20.

24. The ligand drug conjugate according to any one of claims 1 to 20, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the conjugate has a structure as shown in the following formula: in, A, B, L 1 , L 2 , Tp has the definition as described in any one of claims 1-20.

25. The ligand drug conjugate according to any one of claims 1 to 20, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the conjugate is selected from one of the following: in, R 3 , R 4 Independently of each other, have the definitions as described in any one of claims 1 to 20; u is an integer selected from 0 to 10; r2 is selected from an integer from 0 to 4; r3 and r4 are independently selected from an integer from 0 to 3; K is C or N; R z6 , R z7 are identical or different and independently of one another are chosen from hydrogen, amino, alkylamino or dialkylamino, alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy; Or, R z6 , R z7 Together with the atoms to which they are attached, they form a ring structure; preferably, they form a ring structure optionally substituted by C 1-4 An alkyl-substituted 5-6-membered heterocyclic group, wherein the 5-6-membered heterocyclic group is preferably a piperidinyl group or a piperazinyl group; mAb stands for monoclonal antibody; y represents the average number of small molecule drugs attached to each mAb (DAR), which can be selected from an integer or a decimal, such as an integer or a decimal of 1 to 50, an integer or a decimal of 1 to 20, or an integer or a decimal of 1 to 10.

26. The ligand drug conjugate according to any one of claims 1 to 25, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the conjugate is selected from one of the following: y represents the average number of small molecule drugs attached to each monoclonal antibody (DAR), which can be selected from an integer or a decimal, such as an integer or a decimal of 1 to 50, an integer or a decimal of 1 to 20, or an integer or a decimal of 1 to 10; 27. A method for preparing the ligand drug conjugate according to any one of claims 1 to 26, its stereoisomers, racemates, tautomers, isotopologues, isotope-labeled substances, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts thereof, characterized in that: The method is method A, comprising the following steps: Step 1: Provide L 1 '-L 2 -L 3 -L 4 '(L') represents a linker; Preferably, in the above linker, L 4 'for: acetate; More preferably, L 4 'for: acetate; Step 2: The linker is coupled with the compound of formula (GH) to obtain L 1 '-L 2 -L 3 -L 4 -G(C') coupling intermediate; The structure of formula (GH) is: Among them, A, B, Z, X, R 1 , R 2 , R 3 , R 4 , L 1 ', L 1 , L 2 , L 3 , L 4 , L 4 'Independently having the definition as described in any one of claims 1-26; Preferably, the preparation method further comprises a third step of coupling the coupling intermediate of formula (C') with the targeting moiety Tp; Or method B, comprising the following steps: Step 1: Provide L 4 'Linker fragment, the L 4 'As defined above; Step 2: Include L 4 The linker fragment of ' is coupled with the compound of formula (GH) to obtain a compound comprising L 4 'Linker fragment-G intermediate; Step 3: Provide another linker fragment to react with the above intermediate to form L containing the complete linker 1 '-L 2 -L 3 -L 4 -G(C') coupling intermediate; Preferably, L 4 The linker fragment is: It reacts with GH where B is -O- to form an ether bond. Preferably, the other linker fragment is: Preferably, the preparation method further comprises a fourth step of coupling the coupling intermediate of formula (C') with the targeting moiety Tp; Optionally, if necessary, the functional groups of the reaction substrates can be protected using protecting groups known in the art to facilitate the reaction and remove the protecting groups after the reaction is completed.

28. A compound represented by the following formula (GH), its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or a pharmaceutically acceptable salt thereof: in, A, B, X, Z, R 1 , R 2 , R 22 , R 3 , R 4 Independently of each other, they have the meanings as set forth in any one of claims 1 to 26.

29. The compound according to claim 28, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the compound represented by formula (GH) can be selected from the compound represented by the following formula (GH-1): in, A, B, Z, R 1 , R 2 , R 22 , R 3 , R 4 Independently of each other, they have the meaning as defined in claim 28.

30. The compound according to claim 28, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the compound represented by formula (GH) can be selected from the compound represented by the following formula (GH-2): in, A, B, Z, R 1 , R 2 , R 22 , R 3 , R 4 Independently of each other, they have the meaning as set forth in any one of claims 28.

31. The compound of claim 28, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, wherein the compound is selected from the following compounds:

32. A method for preparing a compound of formula (GH) according to claim 28, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotope-labeled substance, a nitrogen oxide, a prodrug, a solvate or a pharmaceutically acceptable salt thereof, characterized in that: The method comprises the steps of reacting a compound of formula (i) with a compound of formula (ii) to obtain a compound of formula (G'): Where T is Or it can be converted into Groups; Preferably, a group containing an active functional group such as an olefinic group, an aldehyde group, a carbonyl group, a nitro group, etc. is selected as T; Preferably, when the active functional group is a carbon-containing structure such as alkenyl, aldehyde, carbonyl, etc., the residue after removing the active functional group from T is a structure with one carbon less than A; when the active functional group is a nitro group, the residue after removing the nitro group from T is A.

33. A method for preparing a compound of formula (GH) according to claim 28, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotope-labeled substance, a nitrogen oxide, a prodrug, a solvate or a pharmaceutically acceptable salt thereof, characterized in that: The method comprises the steps of obtaining a compound of formula (GH) by reacting a compound of formula (G'): Among them, A, B, R 1 , R 2 , R 22 , R 3 , R 4 , X, Z independently have the definitions as described in any one of claims 28-31; Wherein, T is a compound that can be converted into Groups; Preferably, a group containing an active functional group such as an olefinic group, an aldehyde group, a carbonyl group, a nitro group, etc. can be selected as T; Preferably, when the active functional group is a carbon-containing structure such as alkenyl, aldehyde, carbonyl, etc., the residue after removing the active functional group from T is a structure with one carbon less than A; when the active functional group is a nitro group, the residue after removing the nitro group from T is A.

34. The compound represented by formula (i): in, R 1 , X, and Z independently have the definitions as described in any one of claims 28-31.

35. The compound represented by formula (G'): in, R 1 , R 2 , R 22 , R 3 , R 4 , X, Z, and T independently have the definitions as described in any one of claims 28-32.

36. Conjugate with the following structure: Tp-LD (D) Wherein Tp is the targeting moiety; D is a biologically active molecular fragment, preferably a molecular fragment having anti-tumor biological activity; The conditions are: L is selected from the linker represented by formula (L): #L 1 -L 2 -L 3 -L 4 * (L) Where L 1 It is the linking part with the targeting part Tp, composed of the reactive group L 1 ' and the targeting part Tp, # represents the connection site with the Tp part, L 1 Best L 2 Selected from -(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -(CH2) q -C(R z4 R z5 )-C(=O)-; in, L 1 '-L 2 The structure is as follows: q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl, where R z4 or R z5 At least one is not H; or L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing a hydrophilic portion as defined in claim 10: L 3 -ValAA 1 Gly-, AA 1 As defined in any one of claims 13-14; L 4 is absent or is the linking portion between the peptide portion and the biologically active molecule D, and is composed of the reactive group L 4 ' is generated by reaction with biologically active molecules, and * represents the connection site with biologically active molecule D.

37. The conjugate of claim 36, wherein the bioactive molecule D is a compound having biological activity or potential biological activity recorded in the Chinese, American or European Pharmacopoeia or disclosed in other publications; The drug can be selected from cytotoxic drugs, cytostatic drugs or immunosuppressive drugs, preferably anti-tubulin agents, tubulin inhibitors, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc.; Further preferred are auristatin, camptothecin, duocarmycin, etoposide, maytansine and maytansine alkaloids, taxanes, benzodiazepines Benzodiazepine drugs and vinca alkaloids.

38. A pharmaceutical composition comprising at least one selected from the following: The ligand-drug conjugate of formula (C) according to any one of claims 1 to 26, its stereoisomers, racemates, tautomers, isotopomers, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts thereof; and / or The compound of formula (GH) according to any one of claims 28 to 31, its stereoisomer, racemate, tautomer, isotopologue, isotope-labeled substance, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof; and / or The ligand-drug conjugate of formula (D) according to any one of claims 36 to 37, its stereoisomers, racemates, tautomers, isotopomers, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts thereof; Preferably, the compound represented by formula (GH), its stereoisomers, racemates, tautomers, isotopologues, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts, and the ligand-drug conjugate represented by formula (C) or (D), its stereoisomers, racemates, tautomers, isotopologues, isotope labels, nitrogen oxides, prodrugs, solvates or pharmaceutically acceptable salts in the pharmaceutical composition are present in a therapeutically effective amount.

39. Use of the compound of formula (GH) according to any one of claims 28 to 31, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, or the ligand-drug conjugate of formula (C) according to any one of claims 1 to 26, or the ligand-drug conjugate of formula (D) according to any one of claims 36 to 37, its stereoisomer, racemate, tautomer, isotopologue, isotope label, nitrogen oxide, prodrug, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 38 for preventing and / or treating a disease or condition and / or preparing a medicament; Preferably, the disease or disorder may be selected from a tumor, such as a solid tumor or a hematological cancer.

40. The use according to claim 39, wherein the medicament is for preventing and / or treating a disease or condition; Preferably, the disease or disorder may be selected from a tumor, such as a solid tumor or a hematological cancer; The solid tumor is selected from malignancies of various organ systems, e.g., sarcomas, adenocarcinomas, blastomas, and carcinomas, such as those affecting the liver, lung, breast, lymph, biliary tract (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, hepatocarcinomas, small cell lung cancers, non-small cell lung cancers, small intestinal cancers, and esophageal cancers; The hematological cancer is selected from the group consisting of leukemias, lymphomas, and malignant lymphoproliferative disorders affecting the blood, bone marrow, and lymphatic system.

41. The conjugate of the linker and the drug has the following structure: L 1 '-L 2 -L 3 -L 4 -G (C') in: G is defined in any one of claims 1 to 9; L 1 ',L 2 , L 3 , L 4 As defined in any one of claims 10 to 19.

42. The conjugate of a linker and a drug as claimed in claim 2, wherein the conjugate is selected from one of the following:

43. The conjugate of the linker and the drug has the following structure: L 1 '-L 2 -L 3 -L 4 -D in, D as defined in claims 36-37, L 1 ' is a reactive group, preferably L 2 Selected from -(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -(CH2) q -C(R z4 R z5 )-C(=O); Among them, L 1 '-L 2 The structure is as follows: q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl, where R z4 or R z5 At least one is not H; or L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing the hydrophilic part defined in claim 10: L 3 -ValAA 1 Gly-, AA 1 As defined above; L 4 is absent or is the linking portion between the peptide portion and the biologically active molecule D, and is composed of the reactive group L 4 'Generated by reaction with biologically active molecules, * represents the connection site with biologically active molecule D; Optionally, in L 1 With L 2 Between or L 2 With L 3 between or to replace L 2 or by inserting L 2 The form contains the hydrophilic part defined in claim 10.

44. The linker of the following structure: L 1 '-L 2 -L 3 -L 4 ' (L') in Where L 1 ' is a reactive group, preferably L 2 Selected from -(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -(CH2) q -C(R z4 R z5 )-C(=O); in, L 1 '-L 2 The structure is as follows: L 1 '-L 2 for R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl, where R z4 or R z5 At least one is not H, q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; or L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing the hydrophilic part defined in claim 10: and L3 is: -ValAA 1 Gly-, AA 1 It has the definition as described in any one of claims 13-14. L' is preferably:

45. The coupling intermediate of the following structure: Among them G N is H or an optional amino protecting group, A, R 1 , R 2 , R 22 , R 3 , R 4 , X, and Z independently have the definitions as described in any one of claims 28-31.

46. ​​Linker fragment compound: L 1 '-L 2 -ValAA 1 ' (L”) in Where L 1 ' is a reactive group, preferably L 2 Selected from -(C≡C)-(CH2) q -C(R z4 R z5 )-C(=O)- or -(CH2) q -C(R z4 R z5 )-C(=O); in, L 1 '-L 2 The structure is as follows: L 1 '-L 2 for R z4 and R z5 The same or different, independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl or R z4 or R z5 Together they form C 3-6 Cycloalkyl, where R z4 or R z5 At least one is not H; q is an integer selected from 0 to 10, preferably 1, 2, 3, 4, 5, 6, 7 or 8; or L 2 Selected from -(C≡C)-(CH2) q -C(=O)-, in L 2 With L 3 Containing the hydrophilic part defined in claim 10: AA 1 ' is AA 1 itself or in its reactive form as an active ester, wherein AA 1 It has the definition as described in any one of claims 13-14.

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