CN118079013A

CN118079013A - Condensed ring compound, conjugate and application thereof

Info

Publication number: CN118079013A
Application number: CN202211545125.3A
Authority: CN
Inventors: 陈博
Original assignee: Connaught Biomedical Technology Chengdu Co ltd
Current assignee: Connaught Biomedical Technology Chengdu Co ltd
Priority date: 2022-11-22
Filing date: 2022-11-22
Publication date: 2024-05-28

Abstract

The present disclosure relates to fused ring compounds and conjugates and uses thereof. The compound and the conjugate have excellent activity and stability for inhibiting tumor cells and in-vivo efficacy of animals.

Description

Condensed ring compound, conjugate and application thereof

Technical Field

The present disclosure relates to fused ring compounds, conjugates thereof and uses thereof, and belongs to the field of medicine.

Background

The concept of Antibody-Drug conjugates (ADCs) or ADC drugs has long been known. As early as 1913, the professor of neuter prize Paul Ehrlich first proposed the concept of magic bullets (magic bullet), namely, the installation of cytotoxic drugs on specific monoclonal antibodies, to achieve directional killing of tumor cells. The ADC drugs that have been marketed at present are based on this theory by linking cytotoxic drugs to monoclonal antibodies, which are used as carriers to efficiently transport small molecule cytotoxic drugs into target tumor cells in a targeted manner.

The ADC medicine adopts a specific connector to connect the antibody and the small molecular cytotoxic medicine, and the main components of the ADC medicine comprise the antibody, the connector and the small molecular cytotoxic medicine. After the ADC medicine enters blood, the antibody component can identify a target spot and be combined with tumor cells which highly express cell surface antigens, when the ADC-antigen complex enters the tumor cells through endocytosis, the complex can be released under the degradation effect of lysosomes, and DNA or tissue tumor cell division is destroyed, so that the effect of killing the tumor cells is achieved.

The first generation of ADC drugs was the 1958 attempt to treat leukemia using anti-murine leukocyte immunoglobulins coupled with methotrexate. Mylotarg is the first ADC drug marketed, approved by FDA in 2000, for the treatment of acute myelogenous leukemia, but is returned to market without significant survival benefit due to severe fatal liver injury by Mylotarg. The disadvantages of the first generation ADC drugs are: the antibody is a murine antibody, cytotoxin deficiency and site low expression.

The second generation of ADC drug development starting point is enmetrastuzumab, which is the first ADC drug obtained targeting breast cancer. The most successful ADC drug in the hematological oncology field is rituximab, which is directed mainly against classical hodgkin's lymphoma and anaplastic large cell lymphoma. The second generation ADC medicine has the advantages that: the target antigen develops various humanized antibodies, and the defects are as follows: too low or too high a drug loading rate, narrow therapeutic window and low effectiveness.

The third generation of ADC drugs typically represent enrolment mab, which is the 2 nd ADC drug targeting solid tumors, suitable for PD-1/PD-L1 antibody treatment in null patients, but which is insensitive to tubulin inhibitors.

At present, there is still a need to develop drug molecules and conjugates thereof with reasonable molecular structure design and improved pharmacodynamic activity.

Disclosure of Invention

In order to improve the above technical problems, the present disclosure provides a conjugate, a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitrogen oxide or a pharmaceutically acceptable salt thereof, represented by the following formula (C):

Tp—L—G(C)

wherein Tp is a targeting moiety;

l is selected from a bond or a linker;

g is a group of the formula (G):

wherein A is selected from N or C-R ^A;

When a is N, R ¹ is selected from hydrogen, hydroxy, the following groups unsubstituted or substituted with one, two or more R ^B: alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, NH ₂, HC (=o) NH-, alkyl C (=o) NH-, cycloalkyl C (=o) NH-, heterocyclyl C (=o) NH-, aryl C (=o) NH-, heteroaryl C (=o) NH-;

When a is C-R ^A, R ^A and R ¹ are the same or different and are independently selected from hydrogen, halogen, hydroxy, mercapto, cyano, nitro, the following groups, unsubstituted or substituted with one, two or more R ^C: alkyl, cycloalkyl, cycloalkylalkyl, alkyloxy, cycloalkyloxy, alkyloxyalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, NH ₂, HC (=o) NH-, alkyl C (=o) NH-, cycloalkyl C (=o) NH-, heterocyclyl C (=o) NH-, aryl C (=o) NH-, heteroaryl C (=o) NH-;

or R ^A、R¹ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^D;

z is selected from 0 or 1;

r ² is selected from hydrogen, halogen, the following groups, unsubstituted or substituted with one, two or more R ^E: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

R ³ is selected from hydrogen, halogen, the following groups, unsubstituted or substituted with one, two or more R ^E: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

Or R ¹、R² taken together with the atoms to which it is attached form a ring structure that is unsubstituted or substituted with one, two or more R ^E, for example a 5-10 membered ring structure; wherein the 5-10 membered ring structure may be selected from, for example, 5, 6, 7,8, 9 or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon groups;

Or R ²、R³ taken together with the atoms to which it is attached form a ring structure that is unsubstituted or substituted with one, two or more R ^F, for example a 4-10 membered ring structure; wherein the 4-10 membered ring structure may be selected from, for example, 4, 5, 6, 7, 8, 9 or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon groups;

r ⁴ is selected from alkyl, alkyloxy, halogen, hydroxy, mercapto, amino, cyano;

r ⁵ is selected from hydroxy;

R ⁶ is selected from the following groups, unsubstituted or substituted with one, two or more R ^G: cycloalkyl, cycloalkylalkyl, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, alkyl, alkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, amino;

b is selected from the group consisting of absent, triazolyl, -NR ⁷ -or-N (NR ⁷R⁸) -;

R ⁷、R⁸, identical or different, are independently of one another selected from the following radicals which are hydrogen, unsubstituted or substituted by one, two or more R ^H: 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-;

T ^L is selected from the group consisting of bond 、#O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(＝O)*、#O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n*、#N(R¹³)-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)* 、 #S-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)* 、#O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(NR¹³)*、 #O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-P(＝O)(OR¹⁴)*、*O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(＝O)# 、 *O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n# 、*N(R¹³)-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)# 、 *S-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)# 、*O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(NR¹³)#、*O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-P(＝O)(OR¹⁴)#, wherein # represents the site of attachment to L and x represents the site of attachment to B;

Each R ⁹、R¹⁰、R¹¹、R¹², identical or different, is independently selected from hydrogen, halogen, cyano, the following groups, unsubstituted or substituted with one, two or more R ^I: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy;

Or R ⁹、R¹⁰ taken together with the atoms to which it is attached form a ring structure that is unsubstituted or substituted with one, two or more R ^I, for example a 3-10 membered ring structure; or R ¹¹、R¹² taken together with the atoms to which it is attached form a ring structure that is unsubstituted or substituted with one, two or more R ^I, for example a 3-10 membered ring structure; or R ⁹、R¹¹ taken together with the atoms to which it is attached form a ring structure that is unsubstituted or substituted with one, two or more R ^I, for example a 3-10 membered ring structure; wherein any of the ring structures described may be monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbyl, unsubstituted or substituted with one, two or more R ^I; for example, any of the ring structures described may be the following groups, unsubstituted or substituted with one, two or more R ^I: 3.4, 5, 6,7, 8, 9, or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon;

Each R ¹³, identical or different, is independently selected from the following groups, unsubstituted or substituted by one, two or more R ^J: alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl;

each m is the same or different and is independently selected from integers from 0 to 10;

Each n is the same or different and is independently selected from integers from 0 to 10;

Each R ^B、R^C、R^D、R^E、R^F、R^G、R^H、R^I、R^J, identical or different, is independently selected from deuterated, halogen, hydroxy, cyano, nitro, the following groups, unsubstituted or substituted by one, two or more R ^K: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH ₂, HC (=o) NH-, alkyl C (=o) NH-, cycloalkyl C (=o) NH-, heterocyclyl C (=o) NH-, aryl C (=o) NH-, heteroaryl C (=o) NH-;

Each R ^K, identical or different, is independently selected from deuterated, halogen, hydroxy, cyano, nitro, the following groups, unsubstituted or substituted by one, two or more R ^L: alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH ₂, HC (=o) NH-, alkyl C (=o) NH-, cycloalkyl C (=o) NH-, heterocyclyl C (=o) NH-, aryl C (=o) NH-, heteroaryl C (=o) NH-;

Each R ^L is the same or different and is independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH ₂, HC (=o) NH-, alkyl C (=o) NH-, cycloalkyl C (=o) NH-, heterocyclyl C (=o) NH-, aryl C (=o) NH-, heteroaryl C (=o) NH-;

Wavy lines indicate the site of attachment to L;

Provided that when R ⁶ is selected from alkyl, alkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, or amino that is unsubstituted or substituted with one, two, or more R ^G:

A is N, R ¹、R² is taken together with the atoms to which it is attached to form a 5-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^E, and T ^L is not a bond when R ⁷ is hydrogen;

Or R ¹、R² taken together with the atoms to which it is attached form a 5-10 membered ring structure which is unsubstituted or substituted by one, two or more R ^E, which 5-10 membered ring structure may be selected from, for example, 5, 6, 7, 8, 9 or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon; and, R ^A is the following group, unsubstituted or substituted with one, two or more R ^C: cycloalkyl, cycloalkylalkyl, cycloalkyloxy;

Or B is-N (NR ⁷R⁸) -;

Or R ²、R³ taken together with the atoms to which it is attached form a 4-10 membered ring structure which is unsubstituted or substituted by one, two or more R ^F, which 4-10 membered ring structure may be selected from, for example, 4, 5,6, 7, 8, 9 or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon groups.

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

a is selected from N or C-R ^A;

z is selected from 0 or 1;

Or R ¹、R² taken together with the atoms to which it is attached form a 5-10 membered ring structure which is unsubstituted or substituted by one, two or more R ^E, which 5-10 membered ring structure may be selected from, for example, 5, 6, 7, 8, 9 or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon;

Or R ²、R³ taken together with the atoms to which it is attached form a 4-10 membered ring structure which is unsubstituted or substituted by one, two or more R ^F, which 4-10 membered ring structure may be selected from, for example, 4, 5, 6, 7, 8, 9 or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon;

r ⁵ is selected from hydroxy;

r ⁶ is selected from the following groups, unsubstituted or substituted with one, two or more R ^G: cycloalkyl, cycloalkylalkyl, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy;

Or R ⁹、R¹⁰ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ¹¹、R¹² taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ⁹、R¹¹ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; wherein any of the 3-10 membered ring structures described may be the following groups, unsubstituted or substituted with one, two or more R ^I: 3. 4,5,6, 7, 8, 9, or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon;

m and n are the same or different and are independently selected from integers from 0 to 10;

Each R ^L is the same or different and is independently selected from deuterated, halogen, hydroxy, cyano, nitro, alkyl, alkyloxy, cycloalkyl, cycloalkylalkyl, cycloalkyloxy, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, NH ₂, HC (=o) NH-, alkyl C (=o) NH-, cycloalkyl C (=o) NH-, heterocyclyl C (=o) NH-, aryl C (=o) NH-, heteroaryl C (=o) NH-.

a is selected from N or C-R ^A;

z is selected from 0 or 1;

R ⁴ is selected from alkyl, alkyloxy, halogen, hydroxy, amino, cyano;

r ⁵ is selected from hydroxy;

b is selected from the absence, -NR ⁷ -or-N (NR ⁷R⁸) -;

Or R ⁹、R¹⁰ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ¹¹、R¹² taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ⁹、R¹¹ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; wherein any of the 3-10 membered ring structures described may be, for example, the following groups which are unsubstituted or substituted with one, two or more R ^I: 3. 4,5,6, 7,8,9, or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon;

According to an embodiment of the invention, in formula (G) as described in the context of the specification, the hydrogen, carbon or other atom in the formula is optionally replaced by its isotope, whether the group is defined as selected from the above definitions or is drawn directly in the formula, and whether the group is defined as a substituted group or an unsubstituted group. For example, the hydrogen atom on any of the groups in formula (G) may be selected from ¹H、² H or ³ H. As an example, the substituents on the unsubstituted or substituted alkyl, alkylene, phenyl, pyridinyl or lactone groups in the compound of formula (G) or any hydrogen atom on its ring-forming atoms, if any, may be independently selected from ¹H、² H or ³ H.

a is selected from N or C-R ^A;

When a is N, R ¹ is selected from ¹H、² H, hydroxy, deuterated hydroxy, the following groups unsubstituted or substituted with one, two or more R ^B: c _1-10 alkyl, C _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, 3-10 membered heterocyclyl C _1-10 alkyl, C _6-10 aryl, C _6-10 aryl C _1-10 alkyl, 5-10 membered heteroaryl or 5-10 membered heteroaryl C _1-10 alkyl, NH ₂、HC(＝O)NH-、C_1-10 alkyl C (=o) NH-, C _3-10 cycloalkyl C (=o) NH-, 3-10 membered heterocyclyl C (=o) NH-, C _6-10 aryl C (=o) NH-, 5-10 membered heteroaryl C (=o) NH-; preferably, when a is N, R ¹ is selected from ¹H、² H, hydroxy, deuterated hydroxy, the following groups unsubstituted or substituted with one, two or more R ^B: c _1-6 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl C _1-6 alkyl, 3-6 membered heterocyclyl C _1-6 alkyl, phenyl C _1-6 alkyl, 5-6 membered heteroaryl or 5-6 membered heteroaryl C _1-6 alkyl, NH ₂、HC(＝O)NH-、C_1-6 alkyl C (=O) NH-, C _3-6 cycloalkyl C (=o) NH-, 3-6 membered heterocyclyl C (=o) NH-, phenyl C (=o) NH-, 5-6 membered heteroaryl C (=o) NH-;

When a is C-R ^A, R ^A and R ¹ are the same or different and are independently selected from ¹H、² H, halogen, hydroxy, mercapto, deuterated hydroxy, deuterated mercapto, cyano, nitro, the following groups, unsubstituted or substituted with one, two or more R ^C: c _1-10 alkyl, C _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, C _1-10 alkyloxy, C _3-10 cycloalkyloxy, C _1-10 Alkyloxy C _1-10 alkyl, 3-10 membered heterocyclyl C _1-10 alkyl, C _6-10 aryl, C _6-10 aryl C _1-10 alkyl, 5-10 membered heteroaryl or 5-10 membered heteroaryl C _1-10 alkyl, NH ₂、HC(＝O)NH-、C_1-10 alkyl C (=O) NH-, C _3-10 cycloalkyl C (=O) NH-, 3-10 membered heterocyclyl C (=O) NH-, C _6-10 aryl C (=O) NH-, 5-10 membered heteroaryl C (=o) NH-; preferably, when a is C-R ^A, R ^A and R ¹ are the same or different and are independently selected from ¹H、² H, halogen, hydroxy, mercapto, deuterated hydroxy, deuterated mercapto, cyano, nitro, the following groups, unsubstituted or substituted with one, two or more R ^C: c _1-6 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl C _1-6 alkyl, C _1-6 alkyloxy, C _3-6 cycloalkyloxy, C _1-6 Alkyloxy C _1-6 alkyl, 3-6 membered heterocyclyl C _1-6 alkyl, phenyl C _1-6 alkyl, 5-6 membered heteroaryl or 5-6 membered heteroaryl C _1-6 alkyl, NH ₂、HC(＝O)NH-、C_1-6 alkyl C (=O) NH-, C _3-6 cycloalkyl C (=O) NH-, 3-6 membered heterocyclyl C (=O) NH-, phenyl C (=O) NH-, 5-6 membered heteroaryl C (=O) NH-;

Or R ^A、R¹ taken together with the atoms to which it is attached form a 3-8 membered ring structure, e.g., a 3, 4, 5,6, 7, or 8 membered ring structure, which is unsubstituted or substituted with one, two, or more R ^D;

z is selected from 0 or 1;

R ² is selected from ¹H、² H, halogen, the following groups, unsubstituted or substituted with one, two or more R ^E: 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 ² is selected from ¹H、² H, hydrogen, halogen, the following groups, unsubstituted or substituted with one, two or more R ^E: 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 ¹H、² H, halogen, the following groups, unsubstituted or substituted with one, two or more R ^E: 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 ¹H、² H, halogen, the following groups, unsubstituted or substituted with one, two or more R ^E: c _1-6 alkyl, C _1-6 alkyloxy, C _3-6 cycloalkyl, C _3-6 cycloalkyl C _1-10 alkyl, C _3-6 cycloalkyloxy;

R ⁴ is selected from C _1-10 alkyl, C _1-10 alkyloxy, halogen, hydroxy, mercapto, amino, cyano; preferably, R ⁴ is selected from C _1-6 alkyl, C _1-6 alkyloxy, halogen, hydroxy, mercapto, amino, cyano;

r ⁵ is selected from hydroxy;

R ⁶ is selected from the following groups, unsubstituted or substituted with one, two or more R ^G: c _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, C _3-10 cycloalkyloxy, 3-10 membered heterocyclyl, 3-10 membered heterocyclylC _1-10 alkyl, 3-10 membered heterocyclyloxy; preferably, R ⁶ is selected from the following groups, unsubstituted or substituted with one, two or more R ^G: c _3-6 cycloalkyl, C _3-6 cycloalkyl C _1-6 alkyl, C _3-6 cycloalkyloxy, 3-6 membered heterocyclyl, 3-6 membered heterocyclylC _1-6 alkyl, 3-6 membered heterocyclyloxy

R ⁷、R⁸ are identical or different and are selected, independently of one another, from ¹H、² H, the following radicals which are unsubstituted or substituted by one, two or more R ^H: c _1-10 alkyl, C _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, C _6-10 aryl, C _6-10 aryl C _1-10 alkyl, 5-10 membered heteroaryl C _1-10 alkyl, 3-10 membered heterocyclyl C _1-10 alkyl, and, HC (=o) -, C _1-10 alkyl C (=o) -, C _3-10 cycloalkyl C (=o) NH-, 3-10 membered heterocyclyl C (=o) NH-, C _6-10 aryl C (=o) NH-, 5-10 membered heteroaryl C (=o) NH-; preferably, R ⁷、R⁸, identical or different, are independently selected from hydrogen, the following groups unsubstituted or substituted by one, two or more R ^H: 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 C _1-6 alkyl, 3-6 membered heterocyclyl C _1-6 alkyl, and, HC (=o) -, C _1-6 alkyl C (=o) -, C _3-6 cycloalkyl C (=o) NH-, 3-6 membered heterocyclyl C (=o) NH-, C _6-10 aryl C (=o) NH-, 5-6 membered heteroaryl C (=o) NH-;

Each R ⁹、R¹⁰、R¹¹、R¹², which is identical or different, is independently selected from ¹H、² H, halogen, cyano, the following groups, unsubstituted or substituted by one, two or more R ^I: 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 _6-10 aryl, C _6-10 aryl C _1-10 alkyl, C _6-10 aryloxy, 5-10 membered heteroaryl, 5-10 membered heteroaryl C _1-10 alkyl, 5-10 membered heteroaryloxy, 3-10 membered heterocyclyl, 3-10 membered heterocyclylc _1-10 alkyl, 3-10 membered heterocyclyloxy; preferably, each R ⁹、R¹⁰、R¹¹、R¹², identical or different, is independently selected from hydrogen, unsubstituted or substituted by one, two or more R ^I, from 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, 3-6 membered heterocyclyl, 3-6 membered heterocyclylc _1-10 alkyl, 3-6 membered heterocyclyloxy;

Or R ⁹、R¹⁰ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ¹¹、R¹² taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ⁹、R¹¹ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ⁹、R¹¹ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; wherein any of the 3-10 membered ring structures described may be, for example, the following groups which are unsubstituted or substituted with one, two or more R ^I: 3. 4,5, 6,7, 8, 9, or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon;

Each R ¹³, identical or different, is independently selected from the following groups, unsubstituted or substituted by one, two or more R ^J: c _1-10 alkyl, C _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, C _6-10 aryl, C _6-10 aryl C _1-10 alkyl, 5-10 membered heteroaryl C _1-10 alkyl, 3-10 membered heterocyclyl C _1-10 alkyl; preferably, each R ¹³, identical or different, is independently selected from the following groups, unsubstituted or substituted by one, two or more R ^J: 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 C _1-6 alkyl, 3-6 membered heterocyclyl C _1-6 alkyl;

m and n are identical or different and are independently selected from integers from 0 to 10, for example 0,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Each R ^B、R^C、R^D、R^E、R^F、R^G、R^H、R^I、R^J, identical or different, is independently selected from deuterated, halogen, hydroxy, cyano, nitro, the following groups, unsubstituted or substituted by one, two or more R ^K: 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 _6-10 aryl, C _6-10 aryl C _1-10 alkyl, C _6-10 aryloxy, 5-10 membered heteroaryl, 5-10 membered heteroaryl C _1-10 alkyl, 5-10 membered heteroaryloxy, 3-10 membered heterocyclyl, 3-10 membered heterocyclylc _1-10 alkyl, 3-10 membered heterocyclyloxy, NH ₂、HC(＝O)NH-、C_1-10 alkylc (=o) NH-, C _3-10 cycloalkylc (=o) NH-, 3-10 membered heterocyclyl C (=o) NH-, C _6-10 arylc (=o) NH-, 5-10 membered heteroaryl C (=o) NH-; preferably, each R ^B、R^C、R^D、R^E、R^F、R^G、R^H、R^I、R^J, identical or different, is independently selected from deuterated, halogen, hydroxy, cyano, nitro, the following groups, unsubstituted or substituted by one, two or more R ^K: 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, 3-6 membered heterocyclyl, 3-6 membered heterocyclyloxy C _1-6 alkyl, 3-6 membered heterocyclyloxy, NH ₂、HC(＝O)NH-、C_1-6 alkyl C (=o) NH-, C _3-6 cycloalkyl C (=o) NH-, 3-6 membered heterocyclyl C (=o) NH-, C _6-10 aryl C (=o) NH-, 5-6 membered heteroaryl C (=o) NH-;

Each R ^K, identical or different, is independently selected from deuterated, halogen, hydroxy, cyano, nitro, the following groups, unsubstituted or substituted by one, two or more R ^L: 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 _6-10 aryl, C _6-10 aryl C _1-10 alkyl, C _6-10 aryloxy, 5-10 membered heteroaryl, 5-10 membered heteroaryl C _1-10 alkyl, 5-10 membered heteroaryloxy, 3-10 membered heterocyclyl, 3-10 membered heterocyclylc _1-10 alkyl, 3-10 membered heterocyclyloxy, NH ₂、HC(＝O)NH-、C_1-10 alkylc (=o) NH-, C _3-10 cycloalkylc (=o) NH-, 3-10 membered heterocyclyl C (=o) NH-, C _6-10 arylc (=o) NH-, 5-10 membered heteroaryl C (=o) NH-; preferably, each R ^K, identical or different, is independently selected from deuterated, halogen, hydroxy, cyano, nitro, the following groups, unsubstituted or substituted by one, two or more R ^L: 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, 3-6 membered heterocyclyl, 3-6 membered heterocyclyloxy C _1-6 alkyl, 3-6 membered heterocyclyloxy, NH ₂、HC(＝O)NH-、C_1-6 alkyl C (=o) NH-, C _3-6 cycloalkyl C (=o) NH-, 3-6 membered heterocyclyl C (=o) NH-, C _6-10 aryl C (=o) NH-, 5-6 membered heteroaryl C (=o) NH-;

Each R ^L is the same or different and is independently selected from deuterated, halogen, hydroxy, 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, C _6-10 aryl, C _6-10 aryl C _1-10 alkyl, C _6-10 aryloxy, 5-10 membered heteroaryl C _1-10 alkyl, 5-10 membered heteroaryloxy, 3-10 membered heterocyclyl C _1-10 alkyl, 3-10 membered heterocyclyloxy, NH ₂、HC(＝O)NH-、C_1-10 alkyl C (=O) NH-, C _3-10 cycloalkyl C (=O) NH-, 3-10 membered heterocyclyl C (=O) NH-, C _6-10 aryl C (=O) NH-, 5-10 membered heteroaryl C (=O) NH-.

a is selected from N or C-R ^A;

z is selected from 0 or 1;

Provided that R ¹、R² together with the atom to which it is attached or R ²、R³ together with at least one group of atoms to which it is attached form a 4-10 membered ring structure which is unsubstituted or substituted by one, two or more R ^E, which 4-10 membered ring structure may be selected from, for example, 4, 5,6,7,8,9 or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon groups;

r ⁵ is selected from hydroxy;

R ⁶ is selected from the following groups, unsubstituted or substituted with one, two or more R ^G: c _1-10 alkyl, C _1-10 alkyloxy, C _6-10 aryl, C _6-10 arylalkyl, C _6-10 aryloxy, 5-6 membered heteroaryl, 5-6 membered heteroarylalkyl, 5-6 membered heteroaryloxy;

Each R ⁹、R¹⁰、R¹¹、R¹², which is identical or different, is independently selected from ¹H、² H, halogen, cyano, the following groups, unsubstituted or substituted by one, two or more R ^I: 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 _6-10 aryl, C _6-10 aryl C _1-10 alkyl, C _6-10 aryloxy, 5-10 membered heteroaryl, 5-10 membered heteroaryl C _1-10 alkyl, 5-10 membered heteroaryloxy, 3-10 membered heterocyclyl, 3-10 membered heterocyclylc _1-10 alkyl, 3-10 membered heterocyclyloxy; preferably, each R ⁹、R¹⁰、R¹¹、R¹², identical or different, is independently selected from hydrogen, halogen, cyano, the following groups, unsubstituted or substituted by one, two or more R ^I: 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, 3-6 membered heterocyclyl, 3-6 membered heterocyclylc _1-10 alkyl, 3-6 membered heterocyclyloxy;

Each R ^B、R^C、R^D、R^E、R^F、R^G、R^H、R^I、R^J, identical or different, is independently selected from deuterated, halogen, hydroxy, amino, nitro, the following groups, unsubstituted or substituted by one, two or more R ^K: 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 _6-10 aryl, C _6-10 aryl C _1-10 alkyl, C _6-10 aryloxy, 5-10 membered heteroaryl, 5-10 membered heteroaryl C _1-10 alkyl, 5-10 membered heteroaryloxy, 3-10 membered heterocyclyl, 3-10 membered heterocyclylc _1-10 alkyl, 3-10 membered heterocyclyloxy, NH ₂、HC(＝O)NH-、C_1-10 alkylc (=o) NH-, C _3-10 cycloalkylc (=o) NH-, 3-10 membered heterocyclyl C (=o) NH-, C _6-10 arylc (=o) NH-, 5-10 membered heteroaryl C (=o) NH-; preferably, each R ^B、R^C、R^D、R^E、R^F、R^G、R^H、R^I、R^J, identical or different, is independently selected from deuterated, halogen, hydroxy, cyano, nitro, the following groups, unsubstituted or substituted by one, two or more R ^K: 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, 3-6 membered heterocyclyl, 3-6 membered heterocyclyloxy C _1-6 alkyl, 3-6 membered heterocyclyloxy, NH ₂、HC(＝O)NH-、C_1-6 alkyl C (=o) NH-, C _3-6 cycloalkyl C (=o) NH-, 3-6 membered heterocyclyl C (=o) NH-, C _6-10 aryl C (=o) NH-, 5-6 membered heteroaryl C (=o) NH-;

A is selected from N;

R ¹ is selected from ¹H、² H, hydroxy, deuterated hydroxy, the following groups unsubstituted or substituted with one, two or more R ^B: c _1-10 alkyl, C _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, 3-10 membered heterocyclyl C _1-10 alkyl, C _6-10 aryl, C _6-10 aryl C _1-10 alkyl, 5-10 membered heteroaryl or 5-10 membered heteroaryl C _1-10 alkyl, NH ₂、HC(＝O)NH-、C_1-10 alkyl C (=o) NH-, C _3-10 cycloalkyl C (=o) NH-, 3-10 membered heterocyclyl C (=o) NH-, C _6-10 aryl C (=o) NH-, 5-10 membered heteroaryl C (=o) NH-; preferably, when a is N, R ¹ is selected from ¹H、² H, hydroxy, deuterated hydroxy, the following groups unsubstituted or substituted with one, two or more R ^B: c _1-6 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl C _1-6 alkyl, 3-6 membered heterocyclyl C _1-6 alkyl, phenyl C _1-6 alkyl, 5-6 membered heteroaryl or 5-6 membered heteroaryl C _1-6 alkyl, NH ₂、HC(＝O)NH-、C_1-6 alkyl C (=O) NH-, C _3-6 cycloalkyl C (=o) NH-, 3-6 membered heterocyclyl C (=o) NH-, phenyl C (=o) NH-, 5-6 membered heteroaryl C (=o) NH-;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

R ⁶ is selected from the following groups, unsubstituted or substituted with one, two or more R ^G: c _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, C _3-10 cycloalkyloxy, 3-10 membered heterocyclyl, 3-10 membered heterocyclylC _1-10 alkyl, 3-10 membered heterocyclyloxy; preferably, R ⁶ is selected from the following groups, unsubstituted or substituted with one, two or more R ^G: c _3-6 cycloalkyl, C _3-6 cycloalkyl C _1-6 alkyl, C _3-6 cycloalkyloxy, 3-6 membered heterocyclyl, 3-6 membered heterocyclylC _1-6 alkyl, 3-6 membered heterocyclyloxy;

B is selected from-NR ⁷ -or-N (NR ⁷R⁸) -;

Or R ⁹、R¹⁰ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ¹¹、R¹² taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; or R ⁹、R¹¹ taken together with the atoms to which it is attached form a 3-10 membered ring structure that is unsubstituted or substituted with one, two or more R ^I; any of the 3-10 membered ring structures described may be, for example, the following groups which are unsubstituted or substituted with one, two or more R ^I: 3. 4,5, 6, 7,8, 9, or 10 membered monocyclic, bicyclic, heteromonocyclic, heterobicyclic hydrocarbon;

According to embodiments of the present disclosure, R ¹、R² together with the quinolinyl to which it is attached form one of the following substructures, wherein the substructures may be unsubstituted or substituted with one, two or more R ^E:

Or in accordance with embodiments of the present disclosure, R ²、R³ together with the quinolinyl to which it is attached form one of the following substructures, wherein the substructures may be unsubstituted or substituted with one, two or more R ^F:

According to an embodiment of the present disclosure, B is selected from the group consisting of absent, -NH-or-N (NR ⁷R⁸) -, wherein R ⁷、R⁸ independently of one another have the above-described definition.

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

Wherein A, B, T ^L、R¹、R²、R³、R⁶ has the definition described above.

According to an embodiment of the present disclosure, G is selected from the group represented by formula (G-2) or (G-3):

/>

wherein R ^A、B、T^L、R¹、R²、R³、R⁶ has the definition described above.

According to an embodiment of the disclosure, G is selected from the following groups or groups following-T ^L -linkage at the wavy line:

Wherein A, R ¹、R²、R³、R⁴、R⁶、R⁷, z have the definitions described above.

wherein A, R ¹、R²、R³、R⁴、R⁷ has the definition described above.

wherein A, R ¹、R⁴、R⁶、R⁷, z have the definitions described above.

According to an exemplary embodiment of the disclosure, G is selected from the following groups or groups following-T ^L -linkage at the wavy line:

In the above aspect of the invention, wherein L is selected from a bond or a linker represented by formula (L) as defined below:

#L¹—L²—L³—L⁴—L⁵*(L)

Wherein L ¹ is a linking moiety to the targeting moiety Tp, resulting from coupling of the reactive group L ¹' and the targeting moiety Tp, # represents a linking site to the Tp moiety;

For example, L ¹' is a maleimide group, then L ¹ is of the structure:

or an open-loop version thereof: /(I)

L ² is absent or a spacer of L ¹ and L ³;

l ³ is a peptide moiety;

L ⁴ is absent or is a spacer to L ⁵ for the peptide moiety;

L ⁵ is the linking moiety of L ⁴ with the bioactive molecule G, generated by the reaction of the reactive group L ⁵' with the bioactive molecule G or its intermediate;

Optionally:

Between the above moieties in L, preferably between L ¹ and L ², or between L ² and L ³, or in the alternative to L ², contain hydrophilic or sterically hindered moieties as follows:

hydrophilic moiety:

Sterically hindered moiety:

r ¹⁶、R¹⁶' are identical or different and at least one is selected from hydrophilic groups and the other is selected from the following substituents: hydrogen, halogen, cyano, amino, nitro, C _1-10 alkyl unsubstituted or substituted with one, two or more R ^zg, 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 groups, C _1-10 alkyl substituted with 1 to 10 hydroxyl groups or sugar ring-containing groups, preferably polyethylene glycol groups, more preferably-C (=O) -NH- (CH ₂CH₂O)_p-C_1-10 alkyl or-NH- (CH ₂CH₂O)_p-C_1-10 alkyl), or preferably C _1-10 alkyl substituted with 1 to 10 hydroxyl groups, more preferably

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

Y is selected from O, S, C _1-10 alkylene groups, wherein 1,2 or 3 methylene groups of the alkylene groups may optionally be replaced by O or S;

R ¹⁴、R¹⁵, which are identical or different, are independently of one another selected from hydrogen, halogen, cyano, amino, nitro, C _1-10 alkyl which is unsubstituted or substituted by one, two or more R ^zh, C _1-10 alkyloxy, C _3-10 cycloalkyl, C _3-10 cycloalkyl C _1-10 alkyl, C _3-10 cycloalkyloxy;

Or R ¹⁴、R¹⁵ together with the atoms to which it is attached form C _3-10 cycloalkyl which is unsubstituted or substituted by one, two or more R ^zh;

Each R ^zg、R^zh, identical or different, is selected independently of the others 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;

In certain embodiments of the invention, no hydrophilic or sterically hindered moiety as defined above is present in L.

In a further preferred embodiment of the present invention,

L ¹ is formed by any group L ¹ 'which reacts with the targeting moiety Tp, L ¹' can be a sulfhydryl reactive group, an amino reactive group, a carboxyl reactive group, a proline residue reactive group, a tyrosine residue reactive group, a disulfide bridge group, etc.; antibodies directed against the introduction of unnatural amino acids can also be selected from click chemistry reactive groups such as ketones, azides, alkynes, cyclopropenes, or dienes.

L ¹' is preferably a thiol-reactive group;

L ¹' is further preferably a maleimide group or a substituted maleimide group, and L ¹-L² is preferably of the structure:

fragments prepared from (N-maleimidomethyl) -carboxylic acid-N-hydroxysuccinimide ester having the structure:

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

or fragments prepared from m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), having the structure:

A fragment prepared from 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) having the structure:

Or L ^1' is preferably a thiol-reactive group of the structure:

Hal-Het-

Hal is selected from halogen, OMs, OTs, OTf, nitro, the following groups optionally substituted with one or more R ^z6: alkyl sulfide, aryl sulfide, heteroaryl sulfide, alkyl sulfoxide, aryl sulfoxide, heteroaryl sulfoxide, alkyl sulfonyl, aryl sulfonyl, heteroaryl sulfonyl; wherein R ^z6 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C _1-6 alkyl, halogenated C _1-6 alkyl, C _1-6 alkoxy, 6-10 membered aryl and 5-12 membered heteroaryl;

Het is selected from 5-10 membered heteroaryl optionally substituted with one or more R ^z7; wherein R ^z7 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C _1-4 alkyl, and halogenated C _1-4 alkyl;

in a preferred embodiment Hal is preferably methanesulfonyl and Het is preferably pyrimidine;

In a preferred embodiment Hal-Het-is:

The corresponding L ¹ structure is as follows:

and L ¹'-L² is preferably of the structure:

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

R ^z4 or R ^z5 are independently H, C _1-4 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl-C _1-4 alkyl or R ^z4 or R ^z5 together form C _3-6 cycloalkyl.

Further preferred is:

In a further preferred embodiment of the present invention,

L ² is selected from the group consisting of absent, C _1-10 alkylene, C _2-10 alkenylene, C _2-10 alkynylene, C _3-10 cycloalkyl, C _6-12 aryl, or a 6-12 membered heteroaryl, or a combination of the foregoing fragments, and is optionally interrupted by a carbonyl group, O, S, N atom, optionally substituted with a C _1-6 alkyl group, a C _3-6 cycloalkyl group, a halogen atom, a halogenated C _1-6 alkyl group, and the C atom to which the alkyl or haloalkyl group is optionally attached forms a C _3-6 cycloalkyl group, L ² being attached to either L ¹ or L ³ fragments by any functional or covalent bond;

Preferably, L ² is linked to the N-terminus of peptide fragment L ³ by-C (R ^z4R^z5) -CO-, wherein

L ² is preferably -(CH₂)_q-、-(CH₂)_q-C(＝O)-、-(C≡C)-(CH₂)_q-C(R^z4R^z5)-C(＝O)-, wherein q is an integer selected from 0 to 10, preferably 1,2, 3, 4, 5, 6, 7 or 8;

In a further embodiment of the present invention,

Wherein L ³ is selected from a divalent peptide group comprising 2 to 8 optionally substituted natural or unnatural amino acid residues, each of which is the same or different, independently of the other selected from the residues of amino acids: 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 (gin), 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, norpyrrolysine.

Further preferred is:

L ³ is selected from a divalent peptide group comprising 2, 3, 4, 5 or 6 optionally substituted natural amino acid residues or a combination of non-natural amino acid residues, each of which is the same or different, independently of the other selected from the residues of the amino acids: 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 (gin), 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, norpyrrolysine; for example -ValCit-;-CitVal-;-AlaAla-;-AlaCit-;-CitAla-;-AsnCit-;-CitAsn-;-CitCit-;-ValGlu-;-GluVal-;-SerCit-;-CitSer-;-LysCit-;-CitLys-;-AspCit-;-CitAsp-;-AlaVal-;-ValAla-;-PheAla-;-AlaPhe-;-PheLys-;-LysPhe-;-ValLys-;-LysVal-;-AlaLys-;-LysAla-;-PheCit-;-CitPhe-;-LeuCit-;-CitLeu-;-IleCit-;-CitIle-;-PheArg-;-ArgPhe-;-CitTrp-;-TrpCit-;-PhePheLys-;-LysPhePhe-;-DPhePheLys-;-DLysPhePhe-;-GlyPheLys-;-LysPheGly-;-GlyPheLeuGly-;-GlyLeuPheGly-;-AlaLeuAlaLeu-,-GlyGlyGly-;-GlyGlyGlyGly-;-GlyPheValGly-;-GlyValPheGly-;-GlyGlyPheGly-;-AlaAlaAla-.

Most preferably, L ³ is-GLYGLYPHEGLY-.

In a further embodiment of the present invention,

L ⁴ is preferably a group with self-cleaving properties, a self-cleaving group or a self-sacrificing group (self-immolative group) is used to initiate drug release by intramolecular reactions such as 1, 4-elimination, 1, 6-elimination or cyclization elimination, independent of the enzyme.

L ⁵ is formed by reacting any reactive group L ⁵ 'with a bioactive molecule or an intermediate thereof, L ⁵' is preferably a carboxylic acid group or an active ester group which reacts with OH, SH or NH ₂ in the bioactive molecule to form an L ⁵ structure of: -C (O) O-, -C (O) S-, -C (O) N-, or-C (O) NH- (containing O, S, N atoms in the biologically active molecule);

L ⁴-L⁵ is preferably:

PABC spacer arm, the structure is:

GABA interval arm, the structure is:

the alpha, alpha-dimethyl GABA spacer has the structure:

Or a beta, beta-dimethyl GABA spacer arm.

Most preferably, L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

r ^z1 is H or C1-4 alkyl;

R ^z2 or R ^z3 are independently H or C1-4 alkyl;

T ^L is as defined above, and one of T ^L in L ⁴-L⁵ or G is a bond.

In the most preferred aspect of the invention:

L ¹ is generated by coupling Tp with a thiol-reactive group selected from the group consisting of a maleimide group, a substituted maleimide group, or Hal-Het-;

Between L ¹ and L ², or between L ² and L ³, or in the form of substitution L ², contains the following hydrophilic moieties:

Hydrophilic moiety: :

In the most preferred aspect of the invention:

L ¹ is formed by coupling Tp with a thiol-reactive group selected from a maleimide group or a substituted maleimide group;

between L ¹ and L ², or between L ² and L ³, or in the form of substitution for L ², contains a sterically hindered moiety as follows:

Sterically hindered moiety:

In the most preferred aspect of the invention:

L ¹ is formed by coupling Tp with a thiol-reactive group selected from Hal-Het-;

L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

R ^z1 is H or C _1-4 alkyl;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

T ^L is as defined above; and one of T ^L in L ⁴-L⁵ and G is a bond.

Further preferred is:

T ^L as defined above in G is a bond, L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-O-(CH₂)_m-C(R^z4R^z5) -CO-;

Wherein:

R ^z1 is H or C _1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

The invention further provides conjugates of the structure:

Tp—L—D(D)

wherein Tp is a targeting moiety as defined above;

d is a biologically active molecular fragment, preferably a molecular fragment having anti-tumor biological activity;

Wherein L is selected from the group consisting of linkers represented by formula (L):

#L¹—L²—L³—L⁴—L⁵*(L)

Wherein L ¹ is a linking moiety to the targeting moiety Tp, formed by the reactive group L ¹' and the targeting moiety Tp, # represents a linking site to the Tp moiety; ;

l ² is absent or a spacer of L ¹ and L ³;

l ³ is a peptide moiety;

L ⁴ is absent or is a spacer to L ⁵ for the peptide moiety;

l ⁵ is the linking moiety of L ⁴ with the bioactive molecule D, generated by the reaction of the reactive group L ⁵' with the bioactive molecule D or its intermediate;

The conditions were as follows:

Condition I:

l is as defined above, with the following hydrophilic moiety interposed between each fragment:

hydrophilic moiety:

Or condition II:

Sterically hindered moiety:

or condition III:

L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

R ^z1 is H or C _1-4 alkyl;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

T ^L is as defined above.

In a further embodiment of the present invention,

L ¹ is formed by coupling Tp with a thiol-reactive group selected from maleimide groups or Hal-Het-;

hydrophilic moiety:

In a further embodiment of the present invention,

In the most preferred aspect of the invention:

l ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-O-(CH₂)_m-C(R^z4R^z5) -CO-;

Wherein:

R ^z1 is H or C _1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

Or condition IV:

L ¹-L² is generated by coupling Tp from a structural reactive group as follows:

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

R ^z4 or R ^z5 are independently H, C _1-4 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl-C _1-4 alkyl or R ^z4 or R ^z5 together form C _3-6 cycloalkyl, wherein at least one of R ^z4 or R ^z5 is not H;

Further preferably R ^z4 or R ^z5 together form C _3-6 cycloalkyl;

l ¹-L² is most preferably composed of Coupling Tp.

The invention also provides intermediates for conjugate synthesis:

L¹'—L²—L³—L⁴—L⁵-G

Wherein L ¹'、L²、L³、L⁴、L⁵, G are as defined above.

The invention also provides intermediates for conjugate synthesis:

L¹'—L²—L³—L⁴—L⁵-D

Wherein L ¹'、L²、L³、L⁴、L⁵, D are as defined above, provided that:

Condition I:

hydrophilic moiety:

Or condition II:

l ¹' is selected from a thiol-reactive group of a maleimide group or a substituted maleimide group;

Between L1 and L2, or between L2 and L3, or in the form of substitution for L2, contains a sterically hindered moiety as follows:

Sterically hindered moiety:

or condition III:

l ¹' is selected from the mercapto-reactive group of Hal-Het-;

L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

R ^z1 is H or C _1-4 alkyl;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

T ^L is as defined above.

Or condition IV:

L ¹'-L² is the following structure:

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

Further preferably R ^z4 or R ^z5 together form C _3-6 cycloalkyl;

L ¹'-L² is most preferably

In a further embodiment of the present invention,

L ¹' is selected from maleimide groups, thiol-reactive groups substituted with maleimide groups or Hal-Het-;

Between L ¹' and L ², or between L ² and L ³, or in the form of substitution L ², contains the following hydrophilic moiety:

hydrophilic moiety:

In a further embodiment of the present invention,

In the most preferred aspect of the invention:

l ¹' is selected from the mercapto-reactive group of Hal-Het-;

l ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-O-(CH₂)_m-C(R^z4R^z5) -CO-;

Wherein:

R ^z1 is H or C _1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

It will be appreciated by those skilled in the art that the bioactive molecule D may be a biologically active or potentially biologically active compound as described in the chinese, us or european pharmacopoeias or as disclosed in other publications. As examples, the drug may be selected from cytotoxic drugs, cytostatic drugs or immunosuppressive drugs, for example, anti-tubulin agents, tubulin inhibitors, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of such cytotoxic drugs include, for example, auristatin, camptothecin, docamicin, etoposide, maytansine and maytansinoids, taxanes, benzodiazepinesClass or benzene-containing dinitrogen/>Class i drugs and vinca alkaloids.

Tp is a targeting moiety (e.g., a small molecule ligand, protein, polypeptide, non-protein agent (e.g., sugar, RNA, or DNA)).

In some preferred embodiments, the target of Tp is selected from the group consisting of 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、LY64、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、CD1 lc、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、SERPINEl、STAT1、TGFBR1、CTSS、PGF、VEGFA、C1QA、C1QB、ANGPTL4、EGLN、EGLN3、BNIP3、AIF1、CCL5、CXCL10、CXCL11、IFI6、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、CDl lb、ADGRE1、EMR2、TNFRSF21、UPK1B、TNFSF9、MMP16、MFI2、IGF-1R、RNF43、NaPi2b and BCMA.

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 (e.g., carbonic anhydrase IX inhibitor), an ICG dye, a cyanine dye, or a derivative thereof.

According to an embodiment of the present disclosure, the preferred ligand is selected from an antibody or antigen binding fragment thereof, said antibody being selected from a chimeric, humanized or fully human antibody; monoclonal antibodies are preferred.

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: anti-CD 20, anti-CD 22, anti-CD 30, anti-CD 33, anti-CD 44, anti-CD 56, anti-CD 70, anti-CD 73, anti-CD 105, anti-CEA, anti-a 33, anti-Cripto, anti-EphA 2, anti-G250, anti-HER 2 (ErbB 2), anti-EGFR, anti-B7-H3, anti-C-Met, anti-HER 3 (ErbB 3), anti-HER 4 (ErbB 4), anti-MUCl, anti-Lewis Y, anti-VEGFR, anti-GPNMB, anti-Integrin, anti-PSMA, anti-Tenascin-C, anti-SLC 44A4, or anti-Mesothelin antibodies, which may be bispecific or multispecific.

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 Glematumamab thereof.

According to an embodiment of the present disclosure, the conjugate, linker or linker-drug thereof may be selected from one of the following, wherein u, v, w are independently selected from integers from 0 to 10, G has the definition as described above, LG has the definition of Tp as described above; r ²⁰、R^20', which are identical or different, are independently of one another selected from H, C _1-4 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl-C _1-4 alkyl, or R ²⁰、R^20' together with the carbon atom to which they are attached form C _3-6 cycloalkyl:

/>

According to embodiments of the present disclosure, the conjugate may have a structure represented by the formula:

Wherein ,A、R¹、R²、R³、R⁴、R⁶、R⁷、R¹¹、R¹²、L¹、L²、Tp has the definition described above. According to embodiments of the present disclosure, the conjugate may have a structure represented by the formula:

wherein ,A、R¹、R²、R³、R⁴、R⁷、R¹¹、R¹²、L¹、L²、Tp has the definition described above.

Wherein R ¹¹、R¹²、L¹、L², tp have the definitions described above.

According to embodiments of the present disclosure, the conjugate may be selected from one of the following:

/>

Wherein R ¹¹、R¹²、R¹⁶、R^16' has the definition described above;

mAb represents a monoclonal antibody;

y represents the average number of small molecule Drugs (DAR) attached to each mab, which may be selected from integers or fractions, such as from integers or fractions of 1 to 50, 1 to 20, or 1 to 10.

Wherein R ¹¹、R¹²、L¹、L², tp have the definitions described above.

/>

Wherein R ¹¹、R¹²、R¹⁶、R^16' independently of each other have the definition described above;

mAb represents a monoclonal antibody;

Wherein R ¹¹、R¹²、L¹、L², tp have the definitions described above.

/>

Wherein R ¹¹、R¹²、R¹⁶、R^16' has the definition described above;

mAb represents a monoclonal antibody;

Examples of the conjugate intermediate linker and drug conjugate according to embodiments of the present disclosure may be selected from one of the following:

/>

According to embodiments of the present disclosure, an example of the conjugate may be selected from one of the following, wherein y represents an average number of small molecule drugs attached per mab (DAR), which may be selected from integers or fractions, such as integers or fractions selected from 1-50, 1-20, or 1-10:

/>

The invention also provides a preparation method of the conjugate, the stereoisomer, the racemate, the tautomer, the isotopologue, the isotope label, the nitrogen oxide or the pharmaceutically acceptable salt thereof, wherein the preparation method comprises the following steps of:

The first step: providing a linker as shown in L ¹'—L²—L³—L⁴—L⁵ '(L');

preferably, in the above-mentioned linker,

L ⁴-L⁵' is: -NR ^z1-C(R^z2R^z3)-T^L -carboxylic acid form or active ester form;

Wherein:

R ^z1 is H or C _1-4 alkyl;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

Further preferred is:

l ⁴-L⁵' is: -NR ^z1-C(R^z2R^z3)-O-(CH₂)_m-C(R^z4R^z5) -CO-in the form of a carboxylic acid or an active ester;

Wherein:

R ^z1 is H or C _1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

R ^z4 or R ^z5 are independently H, C _1-4 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl-C _1-4 alkyl or R ^z4 or R ^z5 together form C _3-6 cycloalkyl;

And a second step of: coupling the linker with an intermediate compound of formula (G ') to obtain a coupled intermediate of L ¹'—L²—L³—L⁴—L⁵ -G (C');

Wherein the intermediate structure of formula (G') is:

Wherein ,A、B、T^L、R¹、R²、R³、R⁴、R⁵、R⁶、L¹'、L¹、L²、L³、L⁴、L⁵、L⁵' independently have the definitions as described above;

preferably, the preparation method further comprises a third step of: coupling the coupling intermediate of formula (C') with a targeting moiety Tp;

Optionally, if desired, the functional groups of the reaction substrate may also be protected with protecting groups known in the art, so that the reaction proceeds and the protecting groups are removed after the reaction has ended.

According to an embodiment of the present invention, the protecting group may be selected from protecting groups known in the art, such as a hydroxyl protecting group or an amino protecting group, to allow the reaction to proceed.

The present disclosure also provides compounds obtained by linking the above group G to a hydrogen atom, wherein T ^L of the group G is linked to a hydrogen atom.

The present disclosure also provides a compound selected from the group consisting of a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, of formula (GH):

wherein T is selected from H-T ^L;

a is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

Or B is-N (NR ⁷R⁸) -;

According to an embodiment of the invention, the groups in formula (GH) may be independently selected from the following definitions:

a is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

a is selected from N or C-R ^A;

z is selected from 0 or 1;

R ⁴ is selected from alkyl, alkyloxy, halogen, hydroxy, amino, cyano;

r ⁵ is selected from hydroxy;

b is selected from the absence, -NR ⁷ -or-N (NR ⁷R⁸) -;

According to an embodiment of the invention, in the formula (GH) described in the context of the description, the hydrogen, carbon or other atoms in the formula are optionally replaced by their isotopes, whether the group is defined as selected from the above definitions or is drawn directly in the formula, and whether the group is defined as a substituted group or an unsubstituted group. For example, the hydrogen atom on any of the groups in formula (GH) may be selected from ¹H、² H or ³ H. As an example, the substituents on the unsubstituted or substituted alkyl, alkylene, phenyl, pyridinyl or lactone groups or any hydrogen atom on the ring-forming atoms thereof (if any) in the compound of formula (GH) may be independently selected from ¹H、² H or ³ H.

a is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

a is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

A is selected from N;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

B is selected from-NR ⁷ -or-N (NR ⁷R⁸) -;

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

/>

Wherein R ^A、B、T、R¹、R²、R³、R⁶ independently has the definition as described above.

According to an embodiment of the present disclosure, the compound represented by formula (GH) may be selected from compounds represented by the following formula (GH-2) or (GH-3):

According to embodiments of the present disclosure, the compound represented by formula (GH) may be selected from the following compounds:

/>

The present disclosure also provides a process for preparing a compound of formula (GH), a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, comprising the step of reacting a compound of formula (i) with a compound of formula (ii):

Wherein A, B, T, R ¹、R²、R³、R⁴、R⁵、R⁶, z independently have the definitions described above;

LE represents a group that is removed or left after the reaction.

The present invention also provides a process for the preparation of a compound of formula (ii), comprising the step of reacting a compound of formula (iii) with a compound of formula (iv) to give a compound of formula (v):

Wherein A, B, R ¹、R²、R³、R⁴、R⁵、R⁶, z independently have the definitions described above;

r ²¹ is selected from H or a protecting group.

According to an embodiment of the invention, when R ²¹ is selected from H, the compound of formula (v) is a compound of formula (ii).

According to an embodiment of the present invention, when R ²¹ is selected from protecting groups, the compound of formula (v) may be reacted under deprotected conditions to give the compound of formula (ii) after the compound of formula (v) is obtained.

According to an embodiment of the invention, the protecting group is selected from amino protecting groups.

According to embodiments of the present invention, the kind of protecting group (e.g., amino protecting group) and the conditions under which the protecting group is removed may be selected from the kinds or conditions known to those skilled in the art.

The present disclosure also provides compounds of formula (iii):

Wherein R ⁵、R⁶, z independently have the definitions described above.

The present disclosure also provides compounds of formula (v):

Wherein A, B, R ¹、R²、R³、R⁴、R⁵、R⁶、R²¹, z independently have the definitions described above.

The present disclosure also provides a pharmaceutical composition comprising at least one selected from the group consisting of: the compound shown in the formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitrogen oxide or a pharmaceutically acceptable salt thereof, and the conjugate shown in the formula (C), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitrogen oxide or a pharmaceutically acceptable salt thereof.

Preferably, when present, the compound of formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof in the pharmaceutical composition, and the conjugate of formula (C), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof is present in a therapeutically effective amount.

According to an embodiment of the present disclosure, the pharmaceutical composition comprises a therapeutically effective amount of at least one selected from the group consisting of: the compound shown in the formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitrogen oxide or a pharmaceutically acceptable salt thereof, and the conjugate shown in the formula (C), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitrogen oxide or a pharmaceutically acceptable salt thereof.

The present disclosure also provides the use of the compound represented by formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitroxide or a pharmaceutically acceptable salt thereof, or the conjugate represented by formula (C), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitroxide or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of a disease or disorder and/or for the preparation of a medicament.

According to embodiments of the present disclosure, the medicament is for the prevention and/or treatment of a disease or disorder.

The present disclosure also provides a method of preventing and/or treating a disease or disorder, the method comprising administering to a patient a therapeutically effective amount of at least one of: the compound shown in the formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitrogen oxide or a pharmaceutically acceptable salt thereof, and the conjugate shown in the formula (C), a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitrogen oxide or a pharmaceutically acceptable salt thereof.

According to embodiments of the present disclosure, the disease or disorder may be selected from a tumor, such as a solid tumor or hematological cancer.

Examples of such solid tumors include malignant tumors of various organ systems, e.g., sarcomas, adenocarcinomas, blastomas, and carcinomas such as those affecting the liver, lung, breast, lymph, biliary intestine (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon, rectum, renal cell carcinoma, liver, small cell lung, non-small cell lung, small intestine and esophagus cancers. In one embodiment, the cancer is melanoma, e.g., advanced melanoma. Examples of other cancers that may be treated include: bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, anal region cancer, peritoneal cancer, gastric cancer, esophageal cancer, salivary gland cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, glioblastoma, neuroblastoma, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, chronic or acute leukemia (including acute myelogenous leukemia, chronic myelogenous leukemia acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphomas, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal cord axis tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, neurosheath tumors (including acoustic neuroma), meningioma, epidermoid cancer, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers (including cancers induced by asbestos), and combinations of said cancers.

Examples of such hematological cancers include leukemia, lymphoma, and malignant lymphoproliferative disorders affecting the blood, bone marrow, and lymphatic systems. Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemias can be further classified as Acute Myelogenous Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL). Chronic leukemias include Chronic Myelogenous Leukemia (CML) and Chronic Lymphocytic Leukemia (CLL). Other related disorders include myelodysplastic syndrome (MDS, previously referred to as "pre-leukemia"), which is a diverse collection of hematological disorders that result from the combined risk of ineffective production (or dysplasia) of bone marrow blood cells and conversion to AML. Lymphomas are a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-hodgkin lymphomas and hodgkin lymphomas.

According to embodiments of the present disclosure, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients, such as carriers or excipients. The pharmaceutically acceptable excipients are preferably chemically non-reactive or inert to the active ingredient. For example, the pharmaceutically acceptable excipients are selected from at least one of the following excipients including, but not limited to: fillers, disintegrants, binders, lubricants, surfactants, flavoring agents, wetting agents, matrices, and the like.

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

In one embodiment, the route of administration of the pharmaceutical composition may be coordinated with topical administration and oral administration.

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

Definition and description of terms

Unless otherwise indicated, the definitions of terms set forth in the specification and claims of the present application, including as examples, exemplary definitions, preferred definitions, definitions set forth in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. Such combinations and combinations are intended to be within the scope of the present disclosure.

The numerical ranges recited in the specification and claims are equivalent to at least each specific integer number recited therein unless otherwise stated. For example, a numerical range "1-10" corresponds to each of the integer numbers 1,2, 3,4, 5, 6, 7, 8, 9, 10 in the numerical range "1-10". It is to be understood that "more" in one, two or more as used herein in describing substituents shall mean an integer of ≡3, such as 3,4, 5, 6, 7, 8, 9 or 10. Furthermore, when certain numerical ranges are defined as "numbers," it is to be understood that both endpoints of the range, each integer within the range, and each fraction within the range are delineated. For example, a "number of 0 to 10" should be understood to describe 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 integer with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, respectively. Likewise, "an integer or fraction of 1-50", "an integer or fraction of 1-20", "an integer or fraction of 1-10" should be understood to describe both endpoints of the above numerical ranges, as well as each integer within the range and each fraction within the range.

The term "isotopologue" includes the replacement of an atom in a compound or conjugate of the present disclosure with another isotope having the same atomic number but a different atomic mass or mass number. For example, the "hydrogen" in a compound or conjugate of the present disclosure may be selected from protium (¹ H), deuterium (² H) and tritium (³ H); "carbon" may be selected from ¹²C、¹³ C and ¹⁴ C. Accordingly, a compound or conjugate of the present disclosure should be understood to include at least various deuterated forms of the compound or conjugate. For example, one, two or more of the individual available hydrogen atoms attached to a carbon atom (e.g., hydrogen atoms of a C _1-10 alkyl group) may be independently replaced with deuterium atoms. One skilled in the art can refer to the relevant literature for the synthesis of deuterated forms of a compound or conjugate. In preparing the deuterated forms of the compound or conjugate, commercially available deuterated starting materials may be used, or synthesized using conventional techniques with deuterated reagents. Alternative deuterated reagents include, but are not limited to, deuterated borane, trideutero borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

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

The term "halogen" means fluorine, chlorine, bromine and iodine.

The term "alkyl" is understood to mean preferably a straight-chain or branched saturated monovalent hydrocarbon radical, preferably a "C _1-10 alkyl radical". "C _1-10 alkyl" is understood to mean preferably a straight-chain or branched saturated monovalent hydrocarbon radical having from 1 to 10 carbon atoms. For example, "C _1-6 alkyl" means straight and branched alkyl groups having 1,2, 3,4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, or the like, or an isomer thereof.

The term "cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic (fused, bridged or spiro) hydrocarbon radical, preferably "C _3-10 cycloalkyl". The term "C _3-10 cycloalkyl" is understood to mean a saturated monovalent monocyclic, bicyclic (fused, bridged or spiro) hydrocarbon radical having 3 to 10 carbon atoms, such as 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The C _3-10 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.

The term "cyclic structure" is understood to mean a group having a monocyclic, bicyclic (condensed, bridged or spiro) or more cyclic structure, such as monocyclic hydrocarbon, bicyclic hydrocarbon, tricyclic hydrocarbon, heteromonocyclic hydrocarbon, heterobicyclic hydrocarbon, heterotricyclic hydrocarbon, and the like; the ring structure may be saturated or unsaturated. The heteromonocyclic, heterobicyclic, and heterotricyclic hydrocarbyl groups may contain 1 to 10, preferably 1 to 5 heteroatoms independently selected from N, O and S, for example 1,2, 3,4,5,6, 7, 8,9, or 10 heteroatoms independently selected from N, O and S. The bicyclic ring may be of the following ring structure containing no heteroatoms or 1,2, 3 heteroatoms independently selected from N, O and S: 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 [ 2.2.2.2 ], bicyclo [3.2.1], bicyclo [4.1.0], and the like, independently or each other. Or alternatively the ring structure may have 1,2, 3,4 or 5 double bonds, for example carbon-carbon double bonds or carbon-nitrogen double bonds.

The term "3-10 membered ring structure" is understood to mean a group having a monocyclic or bicyclic (fused, bridged or spiro) structure of 3,4, 5, 6, 7, 8, 9 or 10 ring-forming atoms, such as monocyclic hydrocarbon, bicyclic hydrocarbon, heteromonocyclic hydrocarbon or heterobicyclic hydrocarbon; the ring structure may be saturated or unsaturated. The heteromonocyclic or heterobicyclic hydrocarbon groups may contain 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S, for example 1,2, 3 heteroatoms independently selected from N, O and S. The bicyclic ring may be of the following ring structure containing no heteroatoms or 1,2, 3 heteroatoms independently selected from N, O and S: 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 [ 2.2.2.2 ], bicyclo [3.2.1], bicyclo [4.1.0], and the like, independently or each other. Or alternatively the ring structure may have 1,2, 3,4 or 5 double bonds, for example carbon-carbon double bonds or carbon-nitrogen double bonds.

The term "4-10 membered ring structure" is understood to mean a group having a monocyclic or bicyclic (fused, bridged or spiro) structure of 4,5, 6,7, 8,9 or 10 ring-forming atoms, for example monocyclic hydrocarbon, bicyclic hydrocarbon, heteromonocyclic hydrocarbon or heterobicyclic hydrocarbon; the ring structure may be saturated or unsaturated. The heteromonocyclic or heterobicyclic hydrocarbon groups may contain 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S, for example 1, 2,3 heteroatoms independently selected from N, O and S. The bicyclic ring may be of the following ring structure containing no heteroatoms or 1, 2,3 heteroatoms independently selected from N, O and S: 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 [ 2.2.2.2 ], bicyclo [3.2.1], bicyclo [4.1.0], and the like, independently or each other. Or alternatively the ring structure may have 1, 2,3, 4 or 5 double bonds, for example carbon-carbon double bonds or carbon-nitrogen double bonds.

The term "5-10 membered ring structure" is understood to mean a group having a monocyclic or bicyclic (fused, bridged or spiro) structure of 5, 6, 7, 8, 9 or 10 ring-forming atoms, such as monocyclic hydrocarbon, bicyclic hydrocarbon, heteromonocyclic hydrocarbon or heterobicyclic hydrocarbon; the ring structure may be saturated or unsaturated. The heteromonocyclic or heterobicyclic hydrocarbon groups may contain 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S, for example 1,2,3 heteroatoms independently selected from N, O and S. The bicyclic ring may be of the following ring structure containing no heteroatoms or 1,2,3 heteroatoms independently selected from N, O and S: 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 [ 2.2.2.2 ], bicyclo [3.2.1], bicyclo [4.1.0], and the like, independently or each other.

Or alternatively, the "4-10 membered ring structure" and "5-10 membered ring structure" may have 1,2, 3, 4 or 5 double bonds, for example carbon-carbon double bonds or carbon-nitrogen double bonds.

It is to be understood that when the groups of the compounds described herein form a condensed ring, spiro ring or bicyclic ring with the original ring structure of the compounds after forming the above-mentioned ring structure, and an unsaturated bond exists in the original ring structure of the compounds, the ring structure formed by the groups preferably retains the unsaturated bond in the original ring structure.

The term "heterocyclyl" means a saturated monovalent monocyclic, bicyclic (fused, bridged or spiro) 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 heterocyclic group" means a saturated monovalent monocyclic, bicyclic (fused, bridged or spiro) hydrocarbon group containing 1 to 5 non-aromatic cyclic groups having 3 to 20 (e.g., 3,4, 5,6, 7, 8, 9, 10, etc. atoms) total ring atoms of heteroatoms independently selected from N, O and S, preferably "3-10 membered heterocyclic groups". The term "3-10 membered heterocyclyl" means a saturated monovalent monocyclic, bicyclic (fused, bridged or spiro) hydrocarbon group containing 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S, for example 1,2, 3 heteroatoms independently selected from N, O and S. The heterocyclic group may be attached to the remainder of the molecule through any of the carbon atoms or a nitrogen atom, if present. In particular, the heterocyclic groups may include, but are not limited to: a 4-membered ring such as azetidinyl, oxetanyl (e.g., azetidin-1-yl); a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6 membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclyl may be benzo-fused. The heterocyclyl may be bicyclic, such as, but not limited to, a 5,5 membered ring, such as hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl ring, or a 5,6 membered bicyclic ring, such as hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl ring. The ring containing nitrogen atoms 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-dihydro-oxazolyl or 4H- [1,4] thiazinyl, or it may be benzo-fused, such as but not limited to dihydroisoquinolinyl. According to the present disclosure, the heterocyclyl is non-aromatic. When the 3-20 membered heterocyclic group is connected with other groups to form the compound disclosed in the invention, the carbon atom on the 3-20 membered heterocyclic group can be connected with other groups, or the heterocyclic atom on the 3-20 membered heterocyclic group ring can be connected with other groups. For example, when the 3-20 membered heterocyclic group is selected from piperazinyl, it may be that the nitrogen atom on the piperazinyl group is attached to other groups. Or when the 3-20 membered heterocyclic group is selected from piperidyl, it may be that the nitrogen atom on the piperidyl ring and the carbon atom at the para position thereof are attached to other groups.

The term "aryl" is understood to mean a monovalent aromatic or partially aromatic, mono-, bi-or tricyclic hydrocarbon ring radical, preferably "C _6-20 aryl". The term "C _6-20 aryl" is understood to mean preferably a mono-, bi-or tricyclic hydrocarbon ring of monovalent aromatic or partly aromatic nature having from 6 to 20 carbon atoms, preferably "C _6-14 aryl". The term "C _6-14 aryl" is understood to mean preferably a mono-, bi-or tricyclic hydrocarbon ring ("C _6-14 aryl") having a monovalent aromatic or partially aromatic nature of 6,7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms, in particular a ring having 6 carbon atoms ("C ₆ aryl"), for example phenyl; or biphenyl, or a ring having 9 carbon atoms ("C ₉ aryl"), such as 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 aryl"), such as anthracenyl. When the C _6-20 aryl group is substituted, it may be mono-substituted or poly-substituted. The substitution site is not limited, and may be, for example, ortho, para or meta substitution.

The term "heteroaryl" is understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring system group containing 1 to 5 heteroatoms independently selected from N, O and S, preferably "5-20 membered heteroaryl". The term "5-20 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring system groups: having 5 to 20 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O and S, for example "5-14 membered heteroaryl". The term "5-14 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring system groups: it has 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 it contains 1 to 5, preferably 1 to 3 heteroatoms each independently selected from N, O and S and, in addition, can be benzo-fused in each case. 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 their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazole, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, and the like; or an axcinyl group, an indolizinyl group, a purinyl group, etc., and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like. When the 5-20 membered heteroaryl is attached to other groups to form the disclosed compounds, the carbon atoms on the 5-20 membered heteroaryl ring may be attached to other groups, or the heteroatoms on the 5-20 membered heteroaryl ring may be attached to other groups. When the 5-20 membered heteroaryl is substituted, it may be mono-substituted or poly-substituted. And, the substitution site thereof is not limited, and for example, hydrogen attached to a carbon atom on a heteroaryl ring may be substituted, or hydrogen attached to a heteroatom on a heteroaryl ring may be substituted.

Unless otherwise indicated, heterocyclyl, heteroaryl or heteroarylene include all possible isomeric forms thereof, e.g. positional isomers thereof. Thus, for some illustrative non-limiting examples, forms that may include substitution at one, two, or more of its 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-positions, etc. (if present) or bonding to other groups include pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, and pyridin-4-yl; thienyl or thienylene include thiophen-2-yl, thienylene-2-yl, thiophen-3-yl and thienylene-3-yl; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

The term "oxo" refers to the substitution of a carbon atom, nitrogen atom or sulfur atom in a substituent with an oxo group (=o) formed after oxidation. It is understood that when a carbon atom is substituted with an oxy group, a carbonyl group-C (=o) -, is formed.

Unless otherwise indicated, the definition of a term herein applies equally to a group containing the term, e.g., the definition of alkyl applies to alkyl in alkyloxy or cycloalkylalkyl; the definition of cycloalkyl also applies to cycloalkyl in cycloalkyloxy or cycloalkylalkyl; as another example, the definition of C _1-10 alkyl applies to C _1-10 alkyl in C _1-10 alkyloxy or C _3-10 cycloalkyl C _1-10 alkyl; the definition of C _3-10 cycloalkyl also applies to C _3-10 cycloalkyl in C _3-10 cycloalkyloxy or C _3-10 cycloalkyl C _1-10 alkyl.

Those skilled in the art will appreciate that the compounds of the present disclosure may exist in various pharmaceutically acceptable salt forms. 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; these compounds may also form internal salts if they contain both acidic (e.g., carboxyl) and basic (e.g., amino) centers.

The compounds of the present disclosure may exist in the form of solvates (e.g., hydrates) wherein the compounds of the present disclosure comprise a polar solvent as a structural element of the compound lattice, such as, inter alia, water, methanol, or ethanol. The polar solvent, in particular water, may be present in stoichiometric or non-stoichiometric amounts.

Depending on its molecular structure, a compound or group of the present disclosure may be chiral or have chiral carbon atoms present, and thus various enantiomeric forms may be present. These compounds or groups may thus be present in racemic or optically active form. For example, when the group A in the general formula (G), (G') or (GH) is C-R ^A and the groups to which the carbon atoms are attached are not identical, the carbon atom is a chiral carbon atom, which may have a chiral configuration of R or S. The compounds of the present disclosure or intermediates thereof may be isolated as enantiomer 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 tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g.N-benzoylproline or N-benzenesulfonylproline) or various optically active camphorsulfonic acids in R and S form. The chromatographic resolution can also advantageously be carried out 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. Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, for example hexane/isopropanol/acetonitrile.

The term "tautomer" refers to a functional group isomer that results from the rapid movement of an atom in a molecule at two positions. Compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Proton-mobile tautomers result from the migration of a hydrogen atom covalently bonded between two atoms. Tautomers generally exist in equilibrium and attempts to isolate individual tautomers often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The location of the equilibrium depends on the chemical nature of the molecule. For example, among many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type predominates; whereas, among phenols, the enol form is dominant. The present disclosure encompasses all tautomeric forms of the compounds.

The corresponding stable isomer may be isolated according to known methods, for example by extraction, filtration or column chromatography.

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

The phrase "therapeutically effective amount" as used herein refers to the amount of active compound or drug substance that elicits the biological or medicinal response that is being sought by a researcher, veterinarian, medical doctor or other clinician in a tissue, system, animal, individual or human, which includes one or more of the following: (1) prevention of disease: for example, preventing a disease, disorder or condition in an individual who is susceptible to the disease, disorder or condition but has not experienced or developed a pathology or symptomatology of the disease. (2) inhibition of disease: for example, inhibiting a disease, disorder or condition (i.e., preventing further development of pathology and/or symptoms) in an individual experiencing or presenting with the pathology or symptoms of the disease, disorder or condition. (3) alleviation of disease: for example, alleviating a disease, disorder or condition (i.e., reversing the pathology and/or symptoms) in an individual experiencing or presenting with the pathology or symptoms of the disease, disorder or condition.

The term "ligand" is a macromolecular compound that is capable of recognizing and binding to an antigen or receptor associated with a target cell. The ligand functions to present the drug to the target cell population to which the ligand binds, including but not limited to, a protein hormone, lectin, growth factor, antibody or other molecule capable of binding to the cell. In embodiments of the present disclosure, the ligand is denoted LG, and the ligand may form a bond with a linking unit through a heteroatom on the ligand, preferably an antibody or antigen-binding fragment thereof, selected from chimeric, humanized, fully human or murine; monoclonal antibodies are preferred.

The term "drug" refers to any compound having the desired biological activity and reactive functionality that can be used to incorporate a drug into a conjugate of the present disclosure. Desirable biological activities include diagnosing, curing, alleviating, treating, or preventing a disease in a human or other animal. The reactive functional group forms a bond with functional group L ⁴ or L ⁵. In some embodiments, the drug has a nitrogen atom or a hydroxyl group that can form a bond with functional group L ⁴ or L ⁵.

The term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed by joining two identical heavy chains and two identical light chains via interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be categorized into five classes, or isotypes, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, and epsilon chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The antibodies described in the present disclosure are preferably specific antibodies to cell surface antigens on target cells, non-limiting examples being the following antibodies: one or more of an anti-HER 2 (ErbB 2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3 (ErbB 3) antibody, an anti-HER 4 (ErbB 4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, or an anti-Mesothelin antibody; trastuzumab (trade name Herceptin), pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), nituzumab (Nimotuzumab, trade names tenascin )、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96 and Glematumamab) are preferred.

The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

Antibodies of the present disclosure include murine, chimeric, humanized and fully human antibodies, with humanized and fully human antibodies being preferred.

The term "murine antibody" is used in this disclosure to refer to antibodies prepared by murine methods according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with a specific antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce the immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., into framework sequences of different types of human germline antibodies. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/VBase) and in Kabat, E.A. et al, 1991, sequences ofProteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured for CDRs by phage display. Further references describing methods of using mouse antibodies for the humanization involved include, for example, queen et al, proc., natl. Acad. Sci. USA,88, 2869, 1991 and methods of Winter and co-workers [ Jones et al, nature,321, 522 (1986), riechmann et al, nature,332, 323-327 (1988), verhoeyen et al, science,239, 1534 (1988) ].

The terms "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", are used to eliminate immunogenicity and toxic side effects by making both the variable and constant regions of the antibody human. Monoclonal antibody development has undergone 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 related 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), single B cell antibody preparation technology, and the like.

The term "antigen binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments contained in the "antigen-binding fragment" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of the single arm of the antibody; (v) Single domain or dAb fragments (Ward et al, (1989) Nature 341:544-546) consisting of VH domains; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, so that they can produce a single protein chain (known as a single chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, e.g., bird et al (1988) Science242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity in a fragment obtained by treating an IgG antibody molecule with protease papain (cleavage of amino acid residue at position 224 of the H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds.

F (ab') 2 is an antibody fragment having a molecular weight of about 100,000 and having antigen binding activity and comprising two Fab regions linked at hinge positions, obtained by digestion of the lower part of the two disulfide bonds in the IgG hinge region with the enzyme pepsin.

Fab 'is an antibody fragment having a molecular weight of about 50,000 and antigen binding activity obtained by cleavage of disulfide bonds in the hinge region of the above F (ab') 2.

In addition, the Fab ' may be produced by inserting DNA encoding a Fab ' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryotic organism to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" means 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 may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (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.

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat e.a. et al, (1991) Sequences of proteins of immunological interface. As used herein, the Kabat definition of a 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.

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 that does not have CDRs.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes generally comprise at least 3,4,5,6,7,8,9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epitope Mapping Protocols in Methods in Molecular B iology, volume 66, g.e.Morris, ed. (1996).

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of about less than 10 ^-7 M, e.g., about less than 10 ^-8M、10^-9 M or 10 ^-10 M or less.

The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is 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.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the genome of a host cell upon introduction into the host cell so as to replicate with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions into the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http:// IMGT. Cines. FR, or from the journal of immunoglobulins, 2001ISBN012441351 by aligning IMGT human antibody variable region germline gene databases with MOE software.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are susceptible to transformation include members of the enterobacteriaceae (enterobacteriaceae), such as strains of escherichia coli (ESCHERICHIA COLI) or Salmonella (Salmonella); the family of Bacillaceae (baciliaceae) such as bacillus subtilis (Bacillus subtilis); pneumococci (Pneumococcus); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

The engineered antibodies or antigen binding fragments of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The term "peptide" refers to a fragment of a compound between an amino acid and a protein, wherein 2 or more amino acid molecules are connected to each other through peptide bonds, and the peptide is a structural and functional fragment of the protein, such as hormone, enzyme, etc., and is essentially a peptide.

The term "sugar" refers to a biological macromolecule composed of C, H, O elements, which can be classified into monosaccharides, disaccharides, polysaccharides, and the like.

Advantageous effects

The compound and the conjugate have excellent activity and stability for inhibiting tumor cells and in-vivo efficacy of animals, and can be used as an effective drug for inhibiting tumor cells and preventing and/or treating cancers.

Detailed Description

The technical scheme of the present disclosure will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the present disclosure. All techniques implemented based on the foregoing disclosure are intended to be within the scope of the disclosure.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

I. antibody examples

The following antibodies are prepared according to conventional methods of antibodies, for example, eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019) may be transfected after vector construction

Exemplary antibody sequences are as follows:

the sequence of Trastuzumab is as follows

Light chain

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:1

Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:2

The following is the sequence of Pertuzumab

Light chain

DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.3 heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Examples of compounds of SEQ ID NO.4II

The structure of the compound is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). The chemical shift delta is given in units of 10 ^-6 (ppm). NMR was performed using a Bruker nuclear magnetic resonance apparatus with deuterated dimethyl sulfoxide (DMSO-d ₆), deuterated chloroform (CDCl ₃), deuterated methanol (CD ₃ OD) as the solvent and Tetramethylsilane (TMS) as the internal standard.

Determination of LCMS: an Agilent 1260 affinity II (ESI) mass spectrometer or Shimadzu LCMS-2020 (ESI).

High Performance Liquid Chromatography (HPLC) analysis used Agilent 1260 or Shimadzu LC-20AD.

Preparation-type high performance liquid chromatography (pre-HPLC) uses GILSON GX-281.

Chiral preparation was performed using critical fluid chromatography (SFC) and the instrument was performed using Shimadzu LC-30Adsf.

The thin layer chromatography silica gel plate uses GF254 acrylic acid adhesive silica gel plate of Anhui ministerial silicon source material Co., ltd., the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.2mm silica gel plate, and the specification of the thin layer chromatography separation and purification product is 0.5mm silica gel plate.

Column chromatography generally uses 200-300 mesh silica gel of Anhui Liangjia silicon source material limited company as a carrier.

The average inhibition rate of kinase and IC ₅₀ value were measured using a SpectraMax i3X microplate reader (MD Co., USA).

Known starting materials of the present disclosure may be synthesized using or following methods known in the art, or may be purchased from companies such as pichia, music research, shaoshan chemical technology, an Naiji chemicals, and the like.

In the examples below, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

An argon or nitrogen atmosphere means that the reactor flask is connected to a balloon of argon or nitrogen of about 1L volume.

The hydrogen atmosphere is defined as the reaction flask being connected to a balloon of hydrogen gas of about 1L volume.

The hydrogenation reaction is usually vacuumized, filled with hydrogen and repeatedly operated for 3 times.

The oxygen atmosphere is defined as the reaction flask being connected to an oxygen balloon of about 1L volume.

The examples below refer to aqueous solutions, where the reaction temperature is from 20℃to 30℃at room temperature unless otherwise specified.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), the developing reagent used for the reaction, the system of eluent for column chromatography employed for purifying the compound and the developing reagent system of thin layer chromatography included: a: dichloromethane/methanol system, B: the volume ratio of the petroleum ether to the ethyl acetate is adjusted according to the polarity of the compound, and small amount of alkaline or acidic reagents such as triethylamine, acetic acid and the like can be added for adjustment.

Example 2-1: preparation of isomers 1-A, 1-B, 1-C and 1-D of Compound 1

N- ((1R, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) -2-hydroxyacetamide 1-A

N- ((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-B ] quinolin-1-yl) -2-hydroxyacetamide 1-B

N- ((1S, 9R) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) -2-hydroxyacetamide 1-C

N- ((1R, 9R) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) -2-hydroxyacetamide 1-D

First step

2- (6-Cyano-5-oxo-2, 3-dihydro-5H-spiro [ indolizine-1, 2' - [1,3] dioxolan ] -7-yl) -3-cyclopropylpropionic acid ethyl ester

1b

1A (1.01 g,3.31mmol, prepared as disclosed in example 30 on page 28 of patent application "WO 2019238046") was dissolved in 15mL of acetonitrile, bromomethylcyclopropane (894.93 mg,6.63 mmol) was added, and potassium carbonate (916.16 mg,6.63 mmol) was stirred at 80℃for 13 hours. 10mL of water was added, the diluted reaction solution was extracted with ethyl acetate (15 mL. Times.2), the organic phase was washed with saturated sodium chloride solution (10 mL. Times.2), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography to give the title product 1b (1.12 g, yield: 92%) as a yellow solid. MS m/z (ESI): 359.1[ M+1].

Second step

3-Cyclopropyl-2- (6-formyl-5-oxo-2, 3-dihydro-5H-spiro [ indolizine-1, 2' - [1,3] dioxolan ] -7-yl ] propionic acid ethyl ester

1c

1B (1.12 g,3.05 mmol) was dissolved in a mixed solvent of 5mL of water, 5mL of acetonitrile and 5mL of formic acid, raney nickel (261.72 mg) was added under nitrogen, the hydrogen was replaced three times, and the reaction solution was stirred under hydrogen (15 Psi) at 60℃for 4 hours. The reaction solution was filtered with celite, the filter cake (50 mL. Times.3) was washed with methylene chloride, the filtrate was washed with aqueous hydrochloric acid (4M, 20 mL), then with aqueous sodium carbonate (12M, 50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by reverse phase liquid chromatography (separation conditions: column: 120g Flash Coulmn Welch Ultimate XB_C1820-40 μm; mobile phase: A-water: B-acetonitrile, gradient elution, flow rate: 85mL/min, instrument: ISCO) to give the title product 1c (680 mg, yield: 60%) as a yellow solid.

MS m/z(ESI):362.1[M+1]。

Third step

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

Diketone 1d

1C (680 mg,1.85 mmol) was dissolved in 10mL of dichloromethane, the ice-water bath was cooled to 0℃under nitrogen, sodium borohydride (108.02 mg,2.86 mmol) was added in portions, and the reaction was stirred for 30 minutes at 0 ℃. Acetic acid (133.15 mg,2.22 mmol) was added dropwise at 25℃to give a gas, and stirring was continued for 2 hours. 30mL of water was added dropwise at 15℃to generate a gas, and the mixture was stirred at 15℃for 1.5 hours. The reaction mixture was washed with water (50 mL), and p-toluenesulfonic acid monohydrate (35.15 mg, 184.78. Mu. Mol) was added to the washed organic phase at 15℃and stirred at 15℃for 12 hours. 50mL of water was added, the reaction solution was extracted with methylene chloride (45 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography to give the title product 1d (200 mg, yield: 32%) as a yellow oil.

MS m/z(ESI):318.1[M+1]。

Fourth step

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

1D (202.13 mg, 598.74. Mu. Mol) was dissolved in 0.5mL of methanol, cooled to 0℃in an ice-water bath, potassium carbonate (82.75 mg, 598.74. Mu. Mol) was added in portions, and stirred at 0℃for 5 hours under bubbling of oxygen (15 psi). The reaction solution was poured into 10mL of saturated aqueous ammonium chloride, methanol was removed by distillation under reduced pressure, the reaction solution (20 mL. Times.3) was extracted with methylene chloride, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure to give the crude title product 1e (140 mg) as a yellow oil, which was used in the next reaction without purification.

MS m/z(ESI):334.1[M+1]。

Fifth step

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

1E (64.52 mg, 180.00. Mu. Mol) was dissolved in a mixed solution of 0.5mL of trifluoroacetic acid and 0.125mL of water, and stirred at 25℃for 1 hour. The reaction solution was concentrated by distillation under the reduced pressure, and the obtained residue was used as a yellow solid to give the crude title product 1f (40 mg), which was used in the next reaction without purification.

MS m/z(ESI):290.1[M+1]。

Sixth step

N- (9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) acetamide for 1h

1F (40 mg, 128.59. Mu. Mol) and 1g (38.62 mg, 154.31. Mu. Mol, prepared by the method disclosed in example 5-1 on page 61 of patent application "CN 111065621A"), pyridine 4-methylbenzenesulfonate (6.46 mg, 25.72. Mu. Mol) were dissolved in 0.5mL of toluene and stirred at 130℃for 2 hours under nitrogen. The reaction solution was filtered through celite, 10mL of water was added to the filtrate, extracted with methylene chloride (8 ml×3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by preparative thin layer chromatography to develop system a to give the title product as a brown solid for 1h (15 mg, yield: 23%).

MS m/z(ESI):504.2[M+1]。

Seventh step

1-Amino-9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10 h,13 h-benzo [ de ] pyrano [3',4':6, 7-Indolizino [1,2-b ] quinoline-10, 13-dione mesylate 1i

1H (50 mg, 96.5. Mu. Mol) was dissolved in 1mL of ethylene glycol dimethyl ether, 0.5mL of methanesulfonic acid and 0.5mL of water were added, and the mixture was stirred at 85℃for 18 hours. The reaction solution was poured into 10mL of water, the pH was adjusted to 7-8 with saturated sodium bicarbonate solution, extracted with dichloromethane (10 ml×5), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude title product 1i (40 mg) as a brown solid, which was used in the next reaction without purification.

MS m/z(ESI):462.3[M+1]。

Eighth step

1I methanesulfonate (35 mg, 62.2. Mu. Mol) and glycolic acid (5.7 mg, 74.6. Mu. Mol) were dissolved in 1.5mL of N, N-dimethylformamide, followed by the sequential addition of 1-hydroxybenzotriazole (12.6 mg, 93.2. Mu. Mol), N-diisopropylethylamine (16.1 mg, 124. Mu. Mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (17.9 mg, 93.2. Mu. Mol) and stirring at 25℃for 1 hour. To the reaction solution was added 20mL of water, the diluted reaction solution was extracted with methylene chloride (10 mL. Times.5), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by supercritical fluid chromatography to give four single configuration title products 1-A, 1-B, 1-C and 1-D:

Single configuration of Compound 1 Compound one (1.02 mg)

SFC analysis: retention time 1.472 min, purity: 98%. (chromatography column: CHIRALPAK IC-350 x 4.6mm I.D.,3um, mobile phase: A-carbon dioxide, B-methanol and acetonitrile (0.05% diethylamine), isocratic elution: B:60%, flow rate: 3mL/min, instrument: shimadzu LC-30 ADsf).

MS m/z(ESI):520.5[M+1]。

¹HNMR(400MHz,CD₃OD)δ7.71–7.61(m,2H),5.73–5.66(m,1H),5.63–5.56(m,1H),5.40–5.32(m,2H),5.12–5.06(m,1H),4.28–4.22(m,1H),4.18–4.12(m,1H),3.32–3.32(m,1H),3.20–3.11(m,1H),2.42(s,3H),2.39–2.30(m,1H),2.29–2.19(m,1H),1.97–1.89(m,1H),1.88–1.80(m,1H),0.94–0.86(m,1H),0.51–0.38(m,2H),0.14–0.07(m,1H),0.04–-0.01(m,1H).

Single configuration Compound II of Compound 1 (1.02 mg)

SFC analysis: retention time 2.075 min, purity: 93%. (chromatographic column: CHIRALPAKAS-350X 4.6mm I.D.,3um, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B:5% -40%, flow rate: 3mL/min, instrument: shimadzu LC-30 ADsf).

MS m/z(ESI):520.4[M+1]。

¹HNMR(400MHz,CD₃OD)δ7.66–7.53(m,2H),5.70–5.63(m,1H),5.60–5.54(m,1H),5.41–5.31(m,2H),5.22–5.14(m,1H),4.34–4.23(m,1H),4.22–4.11(m,1H),3.43–3.36(m,1H),3.22–3.13(m,1H),2.43–2.37(m,3H),2.35–2.26(m,2H),1.95–1.80(m,2H),0.93–0.86(m,1H),0.55–0.35(m,2H),0.17–0.08(m,1H),0.07–-0.03(m,1H).

Single configuration Compound III of Compound 1 (1.21 mg)

SFC analysis: retention time 0.468 min, purity: 98%. (chromatographic column: CHIRALCEL OD-350 x 4.6mm I.D.,3um, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B%:5% -40%, flow rate: 3mL/min, instrument: shimadzu LC-30 ADsf).

MS m/z(ESI):520.1[M+1]。

¹HNMR(400MHz,CD₃OD)δ7.71–7.64(m,2H),5.73–5.67(m,1H),5.62–5.56(m,1H),5.43–5.40(m,1H),5.38–5.35(m,1H),5.18–5.16(m,1H),4.28–4.21(m,1H),4.18–4.12(m,1H),3.50–3.45(m,1H),3.20–3.12(m,1H),2.49–2.40(m,3H),2.36–2.23(m,2H),1.96–1.82(m,2H),0.93–0.88(m,1H),0.52–0.39(m,2H),0.15–0.08(m,1H),0.06–-0.02(m,1H).

Single configuration Compound IV of Compound 1 (0.27 mg)

SFC analysis: retention time 0.464 min, purity: 98%. (chromatographic column: CHIRALCEL OD-350 x 4.6mm I.D.,3um, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B%:5% -40%, flow rate: 3mL/min, instrument: shimadzu LC-30 ADsf).

MS m/z(ESI):520.1[M+1]。

¹HNMR(400MHz,CD₃OD)δ7.71–7.62(m,2H),5.73–5.67(m,1H),5.62–5.56(m,1H),5.41–5.35(m,2H),5.23–5.20(m,1H),4.33–4.23(m,1H),4.21–4.12(m,1H),3.38–3.36(m,1H),3.22–3.13(m,1H),2.44(s,3H),2.37–2.28(m,2H),1.96–1.83(m,2H),0.91–0.86(m,1H),0.50–0.38(m,2H),0.14–0.09(m,1H),0.06–-0.01(m,1H).

Examples 2-2 to 2-13: preparation of Compounds 2 to 13

With reference to the procedure of example 2-1, the corresponding substrates were selected to prepare the following compounds 2 to 13:

Examples 2 to 14: preparation of Compound 14

N ' -acetyl-N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) -2-hydroxyacetylhydrazine 14

First step

2- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) hydrazine-1-carboxylic acid tert-butyl ester 14b

14A (100 mg,0.19 mmol) and 2- (tert-butyl) 3, 3-diethyl-1, 2-oxazolidine-2, 3-tricarboxylic acid ester (54.9 mg,0.19mmol, prepared as described in literature "OrganicLetters,2005, vol.7, #4, p.713-716") were dissolved in 10mL dichloromethane and triethylamine (38.4 mg,0.38 mmol) was added and stirred at 25℃for 12 hours. 20mL of water was added, the diluted reaction solution was separated, the organic phase was washed with saturated sodium chloride solution (10 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by preparative thin layer chromatography to give the title product 14b (55 mg, yield: 53%) as a white solid.

MS m/z(ESI):551.2[M+1]。

Second step

2- (2- (Benzyloxy) acetyl) -2- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) hydrazine-1-carboxylic acid tert-butyl ester 14c

14B (55 mg,0.1 mmol) was dissolved in 10mL of tetrahydrofuran, sodium bicarbonate (840 mg,10 mmol) was added, and a tetrahydrofuran solution of 2-benzyloxyacetyl chloride (184 mg,1.0mmol,5 mL) was added dropwise to the solution at 25℃under nitrogen, and stirring was carried out at 25℃for 1 hour. The reaction solution was poured into 10mL of water, extracted with ethyl acetate (10 mL. Times.2), the organic phase was washed with saturated sodium chloride solution (10 mL. Times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by preparative thin layer chromatography to give the title product 14c (58 mg, yield: 83%) as a white solid.

MS m/z(ESI):699.3[M+1]。

Third step

2- (Benzyloxy) -N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) acethydrazide 14d

14C (58 mg,0.09 mmol) was dissolved in 10mL of methylene chloride, 1mL of trifluoroacetic acid was added, and the mixture was stirred at 25℃for 5 hours. The reaction solution was concentrated by distillation under reduced pressure to give the crude title product 14d (70 mg) as a light brown solid, which was used in the next reaction without purification.

MS m/z(ESI):599.2[M+1]。

Fourth step

N ' -acetyl-2- (benzyloxy) -N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) acethydrazide 14e

14D (70 mg,0.09 mmol) was dissolved in 10mL of tetrahydrofuran, sodium hydrogencarbonate (7516 mg,9 mmol) was added thereto, and a tetrahydrofuran solution of acetyl chloride (71 mg,0.9mmol,3 mL) was added dropwise thereto at 25℃under nitrogen and stirred at 25℃for 1 hour. The reaction solution was poured into 10mL of water, extracted with ethyl acetate (10 ml×2), the organic phase was washed with saturated sodium chloride solution (10 ml×2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by preparative thin layer chromatography to give the title product 14e (45 mg, yield: 78%) as an off-white solid.

MS m/z(ESI):641.7[M+1]。

Fifth step

14E (45 mg,0.07 mmol) was dissolved in 10mL of tetrahydrofuran, the temperature was reduced to 10℃under nitrogen with the ice-salt bath, palladium dichloride (50 mg,0.282 mmol) was added, hydrogen was replaced three times, and the reaction solution was stirred at 0℃for 2 hours. The reaction solution was filtered through celite, and the filtrate was concentrated by distillation under reduced pressure, and the resulting residue was purified by preparative HPLC (separation condition: chromatography column: sunfire Prep C18 OBD 10 μm, 19X 250mm, mobile phase: A-acetonitrile/methanol=1/1, B-water (0.2% FA), gradient elution, flow rate: 20mL/min, instrument: GILSON GX-281), to give the title product 14 (4.5 mg, yield: 12%) as an off-white solid.

MS m/z(ESI):551.1[M+1]。

¹H NMR(400MHz,DMSO-d₆)δ10.22-9.65(m,1H),7.79(d,J＝10.8Hz,1H),7.36-7.28(m,1H),6.58-6.52(m,1H),6.22-5.99(m,1H),5.43(s,2H),5.33(s,1H),5.15-5.00(m,2H),4.26-4.13(m,1H),3.98-3.71(m,1H),3.21-3.04(m,2H),2.42-2.34(m,3H),2.25-2.08(m,1H),1.92-1.82(m,2H),1.67-1.24(m,3H),1.25-1.20(m,1H),0.91-0.84(m,3H).

Examples 2-15 to 2-23

With reference to the methods of examples 2-14, the corresponding substrates were selected to prepare the following compounds 15 to 23:

examples 2 to 24: preparation of Compound 24

(S) -N- ((8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) methyl) -2-hydroxyacetamide 24

First step

4-Bromo-7-fluoro-5-nitro-2, 3-dihydro-1H-indene 24b

24A (17 g,79.05mmol, prepared as disclosed in example 9 on page 31 of patent application "WO 2021093820A 1") was dissolved in 200mL trifluoroacetic acid, the ice-water bath was cooled to 0℃under nitrogen protection, nitric acid (20.45 g,292.08mmol, 90% content) was slowly added dropwise to the reaction mixture, stirred at 0℃for 10 min, removed from the ice-water bath and stirred at 25℃for 30 min. 100mL of water was added, the diluted reaction solution was extracted with ethyl acetate (500 mL. Times.3), the organic phase was washed with water (500 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography to give the title product 24B (8.96 g, yield: 44%) as a colorless oil.

¹H NMR(400MHz,CDCl₃)δ7.45(d,J＝8.0Hz,1H),3.05-3.16(m,4H),2.18-2.27(m,2H)。

Second step

4-Bromo-7-fluoro-2, 3-dihydro-1H-inden-5-amine 24c

24B (8.96 g,34.61 mmol) was dissolved in a mixed solution of 100mL of ethanol and 20mL of water, iron powder (5.80 g,103.82 mmol) and ammonium chloride (5.55 g,103.82 mmol) were added in this order, and the reaction solution was stirred at 80℃for 1 hour under nitrogen protection. The reaction solution was filtered, and the filtrate was concentrated by distillation under the reduced pressure to give the crude title product 24c (7.2 g) as a white solid. The product was used in the next reaction without purification.

MS m/z(ESI):229.9[M+1]。

Third step

Tert-butyl (4-bromo-7-fluoro-2, 3-dihydro-1H-inden-5-yl) (tert-butoxycarbonyl) carbamate 24d

24C (7.2 g,31.29 mmol) was dissolved in 50mL of tetrahydrofuran, and di-tert-butyl dicarbonate (27.32 g,125.18 mmol), 4-dimethylaminopyridine (38.23 mg, 312.94. Mu. Mol) and triethylamine (12.67 g,125.18 mmol) were added thereto, and the reaction mixture was stirred at 25℃for 16 hours. The reaction solution was concentrated by distillation under the reduced pressure, and the obtained residue was purified by silica gel column chromatography in the form of a white solid to give the title product 24d (9.2 g, yield: 68%).

MS m/z(ESI):317.9[M+1-56-56]。

Fourth step

Tert-butyl (tert-butoxycarbonyl) (7-fluoro-4-vinyl-2, 3-dihydro-1H-inden-5-yl) carbamate 24e

24D (1.5 g,4.38 mmol) and pinacol vinylborate (810.20 mg,5.26 mmol) were dissolved in a mixed solution of 6mL of 1, 4-dioxane and 2mL of water, and tripotassium phosphate (2.79 g,13.15 mmol) and methanesulfonic acid (2-dicyclohexylphosphino-2 ',4',6 '-tri-isopropyl-1, 1' -biphenyl) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (371.07 mg, 438.38. Mu. Mol) were added in this order under nitrogen, and the reaction mixture was stirred at 95℃for 10 hours under nitrogen. 20mL of water was added, the diluted reaction solution was extracted with ethyl acetate (10 mL. Times.3), the organic phase was washed with saturated sodium chloride solution (5 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography as a developing system B to give the title product 24e (350 mg, yield: 26%) as a colorless oil.

MS m/z(ESI):222.1[M+1-100-56]。

Fifth step

Tert-butyl (tert-butoxycarbonyl) (7-fluoro-4-formyl-2, 3-dihydro-1H-inden-5-yl) carbamate 24f

24E (755 mg,2.00 mmol) was dissolved in a mixed solution of 1.5mL dioxane and 1.5mL water, sodium periodate (1.29 g,6.01 mmol) and potassium osmium dihydrate (73.8 mg, 200. Mu. Mol) were added and stirred at 25℃for 5 hours. The reaction solution was filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography to give the title product 24f (538 mg, yield: 71%) as a yellow solid.

MS m/z(ESI):180.2[M+1-100-100]。

Sixth step

(S) -8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9 h,12 h-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-9, 12-dione 24h

24F (538 mg,1.42 mmol) and 24g (375 mg,1.42mmol, prepared as disclosed in example 30 on page 28 of patent application "WO 2019238046A 1") were dissolved in 10mL of ethanol, concentrated hydrochloric acid (12M, 1.58 mL) was added and stirred at 80℃for 2 hours. The reaction solution was concentrated by distillation under reduced pressure to remove ethanol, 10mL of water was added, and the resultant was filtered to obtain a cake, and the crude title product was obtained as a yellow solid for 24 hours (500 mg). The product was used in the next reaction without purification.

MS m/z(ESI):407.2[M+1]。

Seventh step

(S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',

4':6,7] Indolizino [1,2-b ] quinoline 6-oxide 24i

24H (500 mg,1.21 mmol) was dissolved in 70mL of acetic acid, and hydrogen peroxide (16.9 g,149mmol, 30%) was added under nitrogen protection, followed by stirring at 75℃for 3 hours. LCMS showed 24h of starting material remaining, hydrogen peroxide (16.5 g,145mmol, 30% content) was added again and stirred at 75 ℃ for 4 hours. The reaction solution was poured into 100mL of water and filtered to give a cake, giving the crude title product 24i (400 mg) as a yellow solid. The product was used in the next reaction without purification.

MS m/z(ESI):423.2[M+1]。

Eighth step

(S) -15-chloro-8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9 h,12 h-cyclopenta [ f ] pyrano [3',4':

6,7] indolizino [1,2-b ] quinoline-9, 12-dione 24j

24I (100 mg, 142. Mu. Mol) was dissolved in 2mL of N, N-dimethylformamide, the temperature was lowered to 0℃in an ice-water bath under nitrogen, oxalyl chloride (45.0 mg, 355. Mu. Mol) was added thereto, and the mixture was stirred at 25℃for 1 hour. The reaction solution was poured into 10mL of water and filtered to give a cake, giving the crude title product 24j (70 mg) as a brown solid. The product was used in the next reaction without purification.

MS m/z(ESI):441.2[M+1]。

Ninth step

Tert-butyl (S) - ((8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ]

Pyrano [3',4':6,7] Indolazino [1,2-b ] quinolin-15-yl) methyl) carbamate 24k

24J (20.0 mg, 26.3. Mu. Mol) and ((t-butoxycarbonylamino) methyl) potassium trifluoroborate (14.0 mg, 59.2. Mu. Mol) were dissolved in a mixed solution of 1mL of dioxane and 0.2mL of water, and 1, 1-bis (diphenylphosphine) ferrocene palladium dichloride (2.89 mg, 3.95. Mu. Mol) and potassium carbonate (25.1 mg, 118. Mu. Mol) were added under nitrogen and stirred at 95℃for 1 hour. The reaction solution was poured into aqueous hydrochloric acid (1 m,2 ml), and the filtrate was concentrated by distillation under the reduced pressure to give the crude title product 24k (20 mg) as a brown oil. The product was used in the next reaction without purification.

MS m/z(ESI):436.2[M+1-100]。

Tenth step

(S) -15- (aminomethyl) -8-ethyl-4-fluoro-8-hydroxy-1,2,3,8,11,14-hexahydro-9H, 12H-cyclopenta [ f ] pyrano [3',

4':6,7] Indolizino [1,2-b ] quinoline-9, 12-dione 24l

24K (1.00 mg, 1.87. Mu. Mol) was dissolved in 0.6mL of methylene chloride, and a dioxane solution (4M, 0.2 mL) of hydrochloric acid was added thereto and stirred at 25℃for 1 hour. 1mL of water was added, the diluted reaction solution was extracted with ethyl acetate (1 mL. Times.3), the aqueous phase was filtered, and the filtrate was concentrated by distillation under reduced pressure to give 24l (0.5 mg) of the crude title product as a yellow solid. The product was used in the next reaction without purification.

MS m/z(ESI):436.1[M+1]。

Eleventh step

24L (13.3 mg, 22.9. Mu. Mol) and 2-hydroxyacetic acid (2.10 mg, 27.5. Mu. Mol) were dissolved in 1mL of methylene chloride, N-diisopropylethylamine (8.90 mg, 68.9. Mu. Mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (6.60 mg, 34.4. Mu. Mol) and 1-hydroxybenzotriazole (4.65 mg, 34.4. Mu. Mol) were added, and stirred at 25℃for 30 minutes. The reaction solution was concentrated by distillation under reduced pressure, 2mL of water was added, extracted with ethyl acetate (1 mL. Times.3), the organic phase was washed with saturated sodium chloride solution (1 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the resulting residue was purified by preparative HPLC (separation conditions: chromatography column: welch Ultate C18150X 25 mm. Times.7 um, mobile phase: A-water (0.225% FA), B-acetonitrile, gradient elution, flow rate: 25mL/min, instrument: GILSON GX-281) to give the title product 24 (0.98 mg, yield: 8%) as a yellow solid.

MS m/z(ESI):494.1[M+1]。

¹H NMR(400MHz,CD₃OD)δ7.71–7.66(m,1H),7.63(s,1H),5.61–5.56(m,1H),5.50–5.48(m,1H),5.43–5.35(m,3H),5.18–5.09(m,3H),3.73–3.65(m,2H),3.20–3.12(m,2H),2.39–2.29(m,2H),2.02–1.93(m,2H),1.05–0.94(m,3H).

Examples 2-25 to 2-33: preparation of Compounds 25 to 33

Referring to examples 2-24, the corresponding substrates were selected to prepare the following compounds 25 to 33:

(S) -N- (((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) methyl) -2-hydroxypropionamide 25

MS m/z(ESI):508.1[M+1]。

¹H NMR(400MHz,CD₃OD)δ7.70(d,J＝9.6Hz,1H),7.63(s,1H),5.63–5.56(m,1H),5.43–5.36(m,3H),5.15–5.10(m,2H),4.25–4.18(m,1H),3.71–3.66(m,2H),3.19–3.13(m,2H),2.39–2.31(m,2H),2.01–1.93(m,2H),1.43–1.38(m,3H),1.04–0.98(m,3H).

Examples 2 to 2626

(R) -N- (((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) methyl) -2-hydroxypropionamide 26

MS m/z(ESI):508.1[M+1]。

¹H NMR(400MHz,CD₃OD)δ7.72(d,J＝9.4Hz,1H),7.66(s,1H),5.63–5.59(m,1H),5.45–5.39(m,3H),5.17–5.14(m,2H),4.26–4.22(m,1H),3.74–3.68(m,2H),3.21–3.16(m,2H),2.40–2.35(m,2H),2.01–1.95(m,2H),1.44–1.39(m,3H),1.06–1.00(m,3H).

Examples 2 to 2727

(S) -2-cyclopropyl-N- (((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) methyl) -2-hydroxyacetamide 27

MS m/z(ESI):534.3[M+1]。

¹H NMR(400MHz,CD₃OD)δ7.72-7.66(m,1H),7.63(s,1H),5.61-5.55(m,1H),5.45-5.34(m,3H),5.12(s,2H),3.75-3.64(m,3H),3.19-3.10(m,2H),2.40-2.30(m,2H),2.02-1.91(m,2H),1.21-1.14(m,1H),1.06-0.93(m,3H),0.57-0.40(m,4H).

Examples 2 to 2828

(R) -2-cyclopropyl-N- (((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) methyl) -2-hydroxyacetamide 28

MS m/z(ESI):534.4[M+1]。

¹H NMR(400MHz,CD₃OD)δ7.75-7.71(m,1H),7.66(s,1H),5.63-5.59(m,1H),5.47-5.43(m,2H),5.40-5.37(m,1H),5.16-5.14(m,2H),3.75-3.66(m,3H),3.21-3.15(m,2H),2.41-2.32(m,2H),2.04-1.95(m,2H),1.21-1.15(m,1H),1.07-1.00(m,3H),0.56-0.42(m,4H).

Examples 2 to 34: preparation of Compound 34

(S) -N- ((8- (cyclopropylmethyl) -4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) methyl) -2-hydroxyacetamide 34

(S) -4- (cyclopropylmethyl) -4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f ] indolizine-3, 6,10 (4H) -trione 34a

1F was isolated by SFC (separation conditions: chromatography: DAICEL CHIRALPAK AS mm. Times.50 mm,10 μm; mobile phase: A-carbon dioxide: B-methanol (0.1% NH ₃·H₂ O), isocratic elution: B:20%, flow rate: 120mL/min, instrument: shimadzu LC-30 ADsf) as a yellow solid to give the title product 34a.

The second to seventh steps are carried out by 34a and 24f in the same manner as in the sixth to eleventh steps of examples 2 to 24 to obtain 34.

Examples 2 to 35: preparation of Compound 35

(S) -N- ((4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) methyl) -2-hydroxyacetamide 35

35 Was prepared by the same method as in the sixth to eleventh steps of examples 2-24 by 34a with 2-amino-4-fluoro-5-methylbenzaldehyde (prepared by the method disclosed in example 1 on page 73 of patent application "WO 2022140504A 1").

Examples 2 to 36: preparation of Compound 36

(S) -N- (((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) methyl) -2-hydroxypropionamide 36

By the same procedure as in the sixth to eleventh steps of examples 2 to 24, 36 was prepared by 34a with 2-amino-4-fluoro-5-methylbenzaldehyde (prepared by the method disclosed in example 1 on page 73 of patent application "WO 2022140504A 1").

Examples 2 to 37: preparation of Compound 37

(R) -N- (((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) methyl) -2-hydroxypropionamide 37

By using 34a and 2-amino-4-fluoro-5-methylbenzaldehyde (prepared by the method disclosed in example 1 on page 73 of patent application "WO 2022140504A 1"), 37 was prepared by the same method as in the sixth to eleventh steps of examples 2 to 24.

Examples 2 to 38: preparation of Compound 38

(S) -2-cyclopropyl-N- (((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) methyl) -2-hydroxyacetamide 38

By using 34a and 2-amino-4-fluoro-5-methylbenzaldehyde (prepared by the method disclosed in example 1 on page 73 of patent application "WO 2022140504A 1"), 38 was prepared by the same method as in the sixth to eleventh steps of examples 2 to 24.

Examples 2 to 39: preparation of Compound 39

(R) -2-cyclopropyl-N- (((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) methyl) -2-hydroxyacetamide 39

39 Was prepared by the same method as in the sixth to eleventh steps of examples 2-24 by 34a and 2-amino-4-fluoro-5-methylbenzaldehyde (prepared by the method disclosed in example 1 on page 73 of patent application "WO 2022140504A 1").

Preparation examples of conjugate intermediate linker-drug

Example 3-1: preparation of LD-1

2- (((S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-pentan-25-yl) m-benzodimethylformamide LD-1

2- (4- (Tert-butoxy) -4-oxobutoxy) -5-nitroisophthalic acid dimethyl ester LD-1b

LD-1a (5 g,19.55mmol, prepared by the method disclosed in literature "Journal ofPhysical Organic Chemistry,2010, vol.16, #10, p.682-690"), tert-butyl 4-hydroxybutyrate (3.76 g,23.47 mmol) and triphenylphosphine (7.69 g,29.33 mmol) were dissolved in 40mL of a mixed solvent of N, N-dimethylformamide and 120mL of tetrahydrofuran, the ice-water bath was cooled to 0℃under nitrogen protection, diisopropyl azodicarboxylate (5.93 g,29.33 mmol) was slowly added dropwise to the reaction solution, and stirred at 25℃for 20 hours. 150mL of water was added, the diluted reaction solution was extracted with ethyl acetate (200 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by reverse phase high performance liquid chromatography (separation conditions: column: I.D.100 mm. Times.H2-mm Welch Ultimate XB-C1820-40 μm, mobile phase: A-water, B-acetonitrile, gradient elution, flow rate: 200mL/min, instrument: AGELA ASTRA), to give the title product LD-1B (4.8 g, yield: 61%) as a yellow solid.

MS m/z(ESI):420.2[M+23]。

Second step

2- (4- (Tert-butoxy) -4-oxobutoxy) -5-nitroisophthalic acid LD-1c

LD-1b (1.5 g,3.71 mmol) was dissolved in a mixed solvent of 15mL of water and 15mL of tetrahydrofuran, and lithium hydroxide monohydrate (311.10 mg,7.41 mmol) was added thereto and stirred at 25℃for 2 hours. To the reaction solution was added 1N hydrochloric acid solution to adjust pH to 2-3, extracted with ethyl acetate (40 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure to give the crude title product LD-1c (1.49 g) as a yellow solid, which was used in the next reaction without purification. MS m/z (ESI): 392.1[ M+23].

Third step

4- (2, 6-Bis ((2,5,8,11,14,17,20,23-octaoxapentac-25-yl) carbamoyl) -4-nitrophenoxy)

Butyl butyrate LD-1d

LD-1c (400 mg, 933.60. Mu. Mol) was dissolved in 5mL of N, N-dimethylformamide, 1-hydroxybenzotriazole (378.45 mg,2.80 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (536.92 mg,2.80 mmol), N, N-diisopropylethylamine (723.97 mg,5.60 mmol) and 3,6,9,12,15,18,21,24-octaoxapentadecan-1-amine (895.03 mg,2.33 mmol) were added, and stirred at 25℃for 2 hours. 10mL of water was added, extracted with methylene chloride (10 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give the title product LD-1d (515 mg, yield: 50%) as a yellow oil.

MS m/z(ESI):1117.7[M+18]。

Fourth step

4- (2, 6-Bis ((2,5,8,11,14,17,20,23-octaoxapentacan-25-yl) carbamoyl) -4-aminophenoxy)

Butyl butyrate LD-1e

LD-1d (515 mg, 466.74. Mu. Mol) was dissolved in a mixed solvent of 20mL of ethanol and 10mL of water, and iron powder (130.32 mg,2.33 mmol) and ammonium chloride (249.66 mg,4.67 mmol) were added thereto and stirred at 80℃for 16 hours. The reaction solution was filtered through celite, 10mL of water was added to the filtrate, the diluted filtrate was extracted with methylene chloride (10 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography as an expanded system A to give the title product LD-1e (435 mg, yield: 87%) as a yellow oil.

MS m/z(ESI):1070.8[M+1]。

Fifth step

4- (2, 6-Bis ((2,5,8,11,14,17,20,23-octaoxapentacan-25-yl) carbamoyl) -4- (2, 5-dioxo-2, 5-)

Dihydro-1H-pyrrol-1-yl) phenoxy) butanoic acid tert-butyl ester LD-1f

LD-1e (380 mg, 352.47. Mu. Mol) and 2, 5-furandione (38.02 mg, 387.71. Mu. Mol) were dissolved in 6mL dioxane, stirred at 25℃for 1 hour, and after TLC monitoring the completion of the reaction of raw material LD-1e, ammonium persulfate (160.87 mg, 704.93. Mu. Mol) and dimethyl sulfoxide (55.08 mg, 704.93. Mu. Mol) were added to the reaction solution, and stirred at 100℃for 1 hour. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by preparative HPLC (separation condition: chromatographic column: welch Xtimate C18150X 25mm X5 μm; mobile phase: A-water (0.1% TFA), B-acetonitrile, gradient elution, flow rate 25 mL/min) to give the title product LD-1f (91 mg, yield: 21%) as a yellow oil.

MS m/z(ESI):1167.8[M+18]。

Sixth step

Dihydro-1H-pyrrol-1-yl) phenoxy) butanoic acid LD-1g

LD-1f (2 mg, 1.74. Mu. Mol) was dissolved in 0.5mL of methylene chloride, and trifluoroacetic acid (0.1 mL) was added thereto and stirred at 25℃for 1 hour. The reaction solution was concentrated by distillation under the reduced pressure to give crude title product LD-1g (1.5 mg) as a colorless oil, which was used in the next reaction without purification. MS m/z (ESI): 1111.8[ M+18].

Subsequent synthetic procedures were prepared by the method disclosed in "WO2020063676A1" to give LD-1.

MS m/z(ESI):972.5[1/2(M+2)]。

Example 3-2: preparation of LD-2

2- (((2 S,10 s) -10-benzyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzobenzamide LD-2

MS m/z(ESI):979.5[1/2(M+2)]。

Examples 3-3: preparation of LD-3

2- (((2 R,10 s) -10-benzyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ di (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzobenzamide LD-3

MS m/z(ESI):979.6[1/2(M+2)]。

Examples 3-4: preparation of LD-4

2- (((2 S,10 s) -10-benzyl-2-cyclopropyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzobenzamide LD-4

MS m/z(ESI):992.5[1/2(M+2)]。

Examples 3 to 5: preparation of LD-5

2- (((2 R,10 s) -10-benzyl-2-cyclopropyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ di (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzobenzamide LD-5

MS m/z(ESI):992.5[1/2(M+2)]。

Examples 3 to 6: preparation of LD-6

2- (((S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-6

MS m/z(ESI):776.8[1/2(M+2)]。

Examples 3 to 7: preparation of LD-7

2- (((2 S,10 s) -10-benzyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-7

Examples 3 to 8: preparation of LD-8

2- (((2 R,10 s) -10-benzyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-8

Examples 3 to 9: preparation of LD-9

2- (((2 S,10 s) -10-benzyl-2-cyclopropyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-9

Examples 3 to 10LD-10

2- (((2 R,10 s) -10-benzyl-2-cyclopropyl-1- (((1 s,9 s) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15,18-hexa-oxo-3-oxa-5, 8,11,14, 17-pentaazaeicosan-21-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-10

Examples 3 to 11LD-11

N- ((S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynamide LD-11

MS m/z(ESI):559.8[1/2(M+2)]。

Examples 3 to 12LD-12

(S) -N ⁵ - ((S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) glutaramide LD-12

MS m/z(ESI):806.9[1/2(M+2)]。

Examples 3 to 13LD-13

(S) -N ⁵ - ((2S, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15-pent-oxo-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) glutaramide LD-13

MS m/z(ESI):813.9[1/2(M+2)]。

Examples 3 to 14LD-14

(S) -N ⁵ - ((2R, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15-pent-oxo-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) glutaramide LD-14

MS m/z(ESI):813.9[1/2(M+2)]。

Examples 3 to 15LD-15

(S) -N ⁵ - ((2S, 10S) -10-benzyl-2-cyclopropyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentacan-25-yl) glutaramide LD-15

Examples 3 to 16LD-16

(S) -N ⁵ - ((2R, 10S) -10-benzyl-2-cyclopropyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentacan-25-yl) glutaramide LD-16

Examples 3 to 17LD-17

(S) -N ⁵ - ((S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-in-25-yl) glutaramide LD-17

Examples 3 to 18LD-18

(S) -N ⁵ - ((2S, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) glutaramide LD-18

Examples 3 to 19LD-19

(S) -N ⁵ - ((2R, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) glutaramide LD-19

Examples 3 to 20LD-20

(S) -N ⁵ - ((2S, 10S) -10-benzyl-2-cyclopropyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentan-25-yl) glutaramide LD-20

Examples 3 to 21LD-21

(S) -N ⁵ - ((2R, 10S) -10-benzyl-2-cyclopropyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1 yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) glutaramide LD-21

Examples 3 to 22LD-22

(S) -N ⁵ - ((S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pentaoxo-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) gluta-namide LD-22

Examples 3 to 23LD-23

(S) -N ⁵ - ((2S, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15-pentaoxo-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ 2S,3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) pentanamide LD-23

Examples 3 to 24LD-24

(S) -N ⁵ - ((2R, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15-pent-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ 2S,3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) glutaramide LD-24

Examples 3 to 25LD-25

(S) -N ⁵ - ((2S, 10S) -10-benzyl-2-cyclopropyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) glutaramide LD-25

Examples 3 to 26LD-26

(S) -N ⁵ - ((2R, 10S) -10-benzyl-2-cyclopropyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) glutaramide LD-26

Examples 3 to 27LD-27

2- (((S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,1,11,14,17,20,23-octaoxapentacosan-25-yl) isophthalamide LD-27

MS m/z(ESI):959.5[1/2(M+2)]。

Examples 3 to 28LD-28

2- (((4S, 12S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4-methyl-3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatric-N-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzodicarboxamide LD-28

MS M/z (ESI): 966.4[1/2 (M+2) ]. Examples 3 to 29LD-29

2- (((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazatric-an-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzodicarboxamide LD-29

Examples 3 to 30LD-30

2- (((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazatric-an-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzodicarboxamide LD-30

Examples 3 to 31LD-31

2- (((S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl) benzamide LD-31

Examples 3 to 32LD-32

2- (((4S, 12S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4-methyl-3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatric-N-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxa-pentac-N-25-yl) benzamide LD-32

Examples 3 to 33LD-33

2- (((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatric-an-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxa-pentac-25-yl) benzamide LD-33

Examples 3 to 34LD-34

2- (((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatric-an-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxa-pentac-25-yl) benzamide LD-34

Examples 3 to 35LD-35

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentazaoctadeca-N-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) glutaramide LD-35

MS m/z(ESI):793.9[1/2(M+2)]。

Examples 3 to 36LD-36

(S) -N ⁵ - ((4S, 12S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4-methyl-3,8,11,14,17-pent-oxy-5-oxa-2,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxa-eicosan-25-yl) glutaramide LD-36

MS m/z(ESI):800.8[1/2(M+2)]。

Examples 3 to 37LD-37

(S) -N ⁵ - ((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxa-eicosan-25-yl) glutaramide LD-37

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Examples 3 to 38LD-38

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) glutaramide LD-38

Examples 3 to 39LD-39

(S) -N ⁵ - ((4S, 12S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4-methyl-3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) glutaramide LD-39

Examples 3 to 40LD-40

(S) -N ⁵ - ((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,1417,20,23-octaoxaeicosan-25-yl) glutaramide LD-40

Examples 3 to 41LD-41

(S) -N ⁵ - ((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) glutaramide LD-41

Examples 3 to 42LD-42

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentazaoctadecan-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-42

Examples 3 to 43LD-43

(S) -N ⁵ - ((4S, 12S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4-methyl-3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-43

Examples 3 to 44LD-44

(S) -N ⁵ - ((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-44

Examples 3 to 45LD-45

2- (((2S, 13S) -13-benzyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18,21-hexa-oxo-6-oxa-3,8,11,14,17,20-hexaazatetrac-N-24-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) isophthalamide LD-45

Examples 3 to 46LD-46

2- (((2S, 5S, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17,20-hexaazatetrac-N-24-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) isophthalamide LD-46

Examples 3 to 47LD-47

2- (((2S, 5r, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17,20-hexaazatetrac-N-24-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) isophthalamide LD-47

Examples 3 to 48LD-48

2- (((2S, 13S) -13-benzyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18,21-hexa-oxo-6-oxa-3,8,11,14,17,20-hexaazatetrac-N-24-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-48

Examples 3 to 49LD-49

2- (((2S, 5S, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17,20-hexaazatetrac-N-24-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-49

Examples 3 to 50LD-50

2- (((2S, 5r, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17,20-hexaazatetrac-N-24-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-N-25-yl) benzamide LD-50

Examples 3 to 51LD-51

(S) -N ⁵ - ((2S, 13S) -13-benzyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pentoxy-6-oxa-3,8,11,14,17-pentaazanonadec-19-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentadec-25-yl) glutaramide LD-51

Examples 3 to 52LD-52

(S) -N ⁵ - ((2S, 5S, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-6-oxa-3,8,11,14,17-pentaazanonadec-19-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-25-yl) glutaramide LD-52

Examples 3 to 53LD-53

(S) -N ⁵ - ((2S, 5R, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-6-oxa-3,8,11,14,17-pentaazanonadec-19-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-25-yl) glutaramide LD-53

Examples 3 to 54LD-54

(S) -N ⁵ - ((2S, 13S) -13-benzyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-oxy-6-oxa-3,8,11,14,17-pentaaza-nonadec-19-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxa-eicosan-25-yl) pentanamide LD-54

Examples 3 to 55LD-55

(S) -N ⁵ - ((2S, 5S, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-oxy-6-oxa-3,8,11,14,17-pentaazanonadec-N-19-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-N-25-yl) glutaramide LD-55

Examples 3 to 56LD-56

(S) -N ⁵ - ((2S, 5R, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-oxy-6-oxa-3,8,11,14,17-pentaazanonadec-N-19-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-N-25-yl) glutaramide LD-56

Examples 3 to 57LD-57

(S) -N ⁵ - ((2S, 13S) -13-benzyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-oxy-6-oxa-3,8,11,14,17-pentaaza-nonadecan-19-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-57

Examples 3 to 58LD-58

(S) -N ⁵ - ((2S, 5S, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-oxy-6-oxa-3,8,11,14,17-pentaazanonadecan-19-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-58

Examples 3 to 59LD-59

(S) -N ⁵ - ((2S, 5R, 13S) -13-benzyl-5-cyclopropyl-2- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4,9,12,15,18-pent-oxy-6-oxa-3,8,11,14,17-pentaazanonadecan-19-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-59

Examples 3 to 60LD-60

2- (((S) -12-benzyl-1- ((S) -8- (cyclopropylmethyl) -4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricin-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) isophthalamide LD-60

Examples 3 to 61LD-61

2- (((S) -12-benzyl-1- ((S) -8- (cyclopropylmethyl) -4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatric-N-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-61

Examples 3 to 62LD-62

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -8- (cyclopropylmethyl) -4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) glutaramide LD-62

Examples 3 to 63LD-63

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -8- (cyclopropylmethyl) -4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxa-pentac-25-yl) pentanediamide LD-63

Examples 3 to 64LD-64

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -8- (cyclopropylmethyl) -4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) gluta-mide LD-64

Examples 3 to 65LD-65

2- (((S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17,20-hexahydro-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) isophthalamide LD-65

Examples 3 to 66LD-66

2- (((4S, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzodicarboxamide LD-66

Examples 3 to 67LD-67

2- (((4R, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzodicarboxamide LD-67

Examples 3 to 68LD-68

2- (((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzodicarboxamide LD-68

Examples 3 to 69LD-69

2- (((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) m-benzodicarboxamide LD-69

Examples 3 to 70LD-70

2- (((S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17,20-hexahydro-5-oxa-2,7,10,13,16,19-hexaazatricin-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-70

Examples 3 to 71LD-71

2- (((4S, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-71

Examples 3 to 72LD-72

2- (((4R, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatricosan-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxapentac-25-yl) benzamide LD-72

Examples 3 to 73LD-73

2- (((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatric-an-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxa-eicosan-25-yl) benzamide LD-73

Examples 3 to 74LD-74

2- (((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17,20-hexa-oxo-5-oxa-2,7,10,13,16,19-hexaazatric-an-23-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-fluoro-N- (2,5,8,11,14,17,20,23-octaoxa-eicosan-25-yl) benzamide LD-74

Examples 3 to 75LD-75

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pent-oxo-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-penta-N-25-yl) glutaramide LD-75

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Examples 3 to 76LD-76

(S) -N ⁵ - ((4S, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylacylamino) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) gluta-mide LD-76

Examples 3 to 77LD-77

(S) -N ⁵ - ((4R, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylacylamino) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) glutaramide LD-77

Examples 3 to 78LD-78

(S) -N ⁵ - ((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentadec-25-yl) glutaramide LD-78

Examples 3 to 79LD-79

(S) -N ⁵ - ((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) glutaramide LD-79

Examples 3 to 80LD-80

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pent-oxo-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-en-25-yl) penta-diamide LD-80

Examples 3 to 81LD-81

(S) -N ⁵ - ((4S, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) pentanamide LD-81

Examples 3 to 82LD-82

(S) -N ⁵ - ((4R, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) pentanamide LD-82

Examples 3 to 83LD-83

(S) -N ⁵ - ((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) pentanamide LD-83

Examples 3 to 84LD-84

(S) -N ⁵ - ((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) pentanamide LD-84

Examples 3 to 85LD-85

(S) -N ⁵ - ((S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pent-oxo-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) glutaramide LD-85

Examples 3 to 86LD-86

(S) -N ⁵ - ((4S, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-86

Examples 3 to 87LD-87

(S) -N ⁵ - ((4R, 12S) -12-benzyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -4-methyl-3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-N-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-87

Examples 3 to 88LD-88

(S) -N ⁵ - ((4S, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-88

Examples 3 to 89LD-89

(S) -N ⁵ - ((4R, 12S) -12-benzyl-4-cyclopropyl-1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -3,8,11,14,17-pent-oxy-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ -methyl-N ¹ - ((2S, 3R,4R, 5R) -2,3,4,5, 6-pentahydroxyhexyl) penta-diamide LD-89

Examples 3 to 90LD-90

5- (2, 5-Dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -2- (((9S, 12S, 15S) -1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -9,12, 15-trimethyl-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaeicosan-20-yl) oxy) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) isophthalamide LD-90

MS m/z(ESI):907.0[1/2(M+2)]。

Examples 3 to 91LD-91

(S) -N ⁵ - ((9S, 12S, 15S) -1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -9, 12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadec-15-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxa-eicosan-25-yl) glutaramide LD-91

Examples 3 to 92LD-92

(S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ⁵ - ((9S, 12S, 15S) -1- ((S) -8-ethyl-4-fluoro-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -9, 12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadecan-15-yl) -N ¹ - (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) glutaramide LD-92

Examples 3 to 93LD-93

N- ((9S, 12S, 15S) -1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -9, 12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadecan-15-yl) -6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide LD-93

Examples 3 to 94LD-94

2- (((9S, 12S, 15S) -1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -9,12, 15-trimethyl-3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaeicosan-20-yl) oxy) -5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N ¹,N³ -bis (2,5,8,11,14,17,20,23-octaoxaeicosan-25-yl) m-benzodicarboxamide LD-94

Examples 3 to 95LD-95

(S) -N ⁵ - ((9S, 12S, 15S) -1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -9, 12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadecan-15-yl) -2- (6- (2- (methylsulfonyl) pyrimidin-5-yl) hex-5-ynylamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapenta-25-yl) glutaramide LD-95

Examples 3 to 96LD-96

(S) -N ⁵ - ((9S, 12S, 15S) -1- ((S) -4- (cyclopropylmethyl) -8-fluoro-4-hydroxy-9-methyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) -9, 12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadecan-15-yl) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -N ¹ - (2,5,8,11,14,17,20,23-octaoxapentan-25-yl) pentanamide LD-96

Examples 3 to 97LD-97

N- ((S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -1- (4- (2- (methylsulfonyl) pyrimidin-5-yl) but-3-yn-1-yl) cyclopropane-1-carboxamide LD-97

Examples 3 to 98LD-98

N- ((2S, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,12-pentoxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -1- (4- (2- (methylsulfonyl) pyrimidin-5-yl) but-3-yn-1-yl) cyclopropane-1-carboxamide LD-98

Examples 3 to 99LD to 99

N- ((2R, 10S) -10-benzyl-1- (((1S, 9S) -9- (cyclopropylmethyl) -5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-methyl-1,6,9,12,15-pentoxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -1- (4- (2- (methylsulfonyl) pyrimidin-5-yl) but-3-yn-1-yl) cyclopropane-1-carboxamide LD-99

Examples 3 to 100LD-100

N- ((S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -3,8,11,14,17-pent-oxo-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -1- (4- (2- (methylsulfonyl) pyrimidin-5-yl) but-3-yn-1-yl) cyclopropane-1-carboxamide LD-100

Examples 3 to 101LD-101

N- ((4S, 12S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4-methyl-3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -1- (4- (2- (methylsulfonyl) pyrimidin-5-yl) but-3-yn-1-yl) cyclopropane-1-carboxamide LD-101

Examples 3 to 102LD-102

N- ((4R, 12S) -12-benzyl-1- ((S) -8-ethyl-4-fluoro-8-hydroxy-9, 12-dioxo-2,3,8,9,12,14-hexahydro-1H, 11H-cyclopenta [ f ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-15-yl) -4-methyl-3,8,11,14,17-pentoxy-5-oxa-2,7,10,13,16-pentaazaoctadeca-18-yl) -1- (4- (2- (methylsulfonyl) pyrimidin-5-yl) but-3-yn-1-yl) cyclopropane-1-carboxamide LD-102

4. ADC conjugate examples

Preparation procedure of antibody conjugate

A, the antibody was replaced with an ultrafiltration tube having a molecular weight cut-off of 50kD to 50mM PBS/1.0mM EDTA buffer (pH 6.0), 5 to 10 equivalents of 10mM TCEP aqueous solution was added, and the mixture was shaken at 37℃for 3 hours. Dissolving the linker-drug conjugate with DMSO, taking 10-20 equivalents of the linker-drug conjugate from the solution, dropwise adding the solution into the reduced antibody solution, stirring the solution by vortex to mix the solution uniformly, oscillating the solution at 25 ℃ for 2 hours, adding 200 equivalents of 100mM NAC aqueous solution, oscillating the solution at 25 ℃ for 20 minutes to terminate the linker reaction, removing excessive small molecules by using an ultrafiltration tube with a molecular weight cut-off of 50kD, replacing the antibody drug conjugate into 10mM PBS buffer (pH 6.0), ultrafiltering and purifying the solution, filtering the sample by a 0.22 mu m filter membrane to obtain the unhydrolyzed antibody drug conjugate, and storing the unhydrolyzed antibody drug conjugate in a refrigerator at 4 ℃. Taking part of unhydrolyzed antibody drug conjugate, replacing the unhydrolyzed antibody drug conjugate into 50mM PBS/1.0mM EDTA buffer (pH 7.8) by using an ultrafiltration tube with a molecular weight cutoff of 50kD, oscillating at constant temperature for 2 hours at 35 ℃, replacing the antibody drug conjugate into 10mM PBS buffer (pH 6.0) by using an ultrafiltration tube with a molecular weight cutoff of 50kD, filtering a sample by using a 0.22 mu m filter membrane after ultrafiltration purification, and obtaining the hydrolyzed antibody drug conjugate, and storing in a refrigerator at 4 ℃. DAR values of the conjugates before and after hydrolysis were determined using RP-HPLC or LCMS, respectively, and the proportion of the conjugates was determined using SEC-HPLC.

Example 4-1ADC-1

The antibody Trastuzumab and LD-1 are used as raw materials, and the antibody Trastuzumab and LD-1 are prepared by adopting the step A. An exemplary product of the coupling mixture (FADC-1A and/or FADC-1B and/or FADC-1C mix) was obtained as a PBS buffer for ADC-1 and stored at 4 ℃.

Examples 4-2ADC-2 to 4-10ADC-10

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Similar preparation methods were employed using LD-2, LD-3, LD-4, LD-5, LD-6, LD-7, LD-8, LD-9 and LD-10, respectively, instead of LD-1, to obtain corresponding ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7, ADC-8, ADC-9 and ADC-10, examples 4-2 to 4-10. EXAMPLES 4-11ADC-11

The antibody Trastuzumab and LD-11 are used as raw materials, and are prepared by a method disclosed in patent application WO2019114666A 1. An exemplary product of the formula FADC-11, ADC-11, in PBS buffer, was obtained and stored at 4 ℃.

Examples 4-12ADC-12 to examples 4-16ADC-16

Similar preparation methods were employed using LD-12, LD-13, LD-14, LD-15 and LD-16, respectively, instead of LD-11, to obtain corresponding ADC-12, ADC-13, ADC-14, LD-15 and ADC-16, examples 4-12 to 4-16.

EXAMPLES 4-17ADC-17

The antibody Trastuzumab and LD-17 are used as raw materials, and are prepared by a method disclosed in patent application WO2022068898A 1. An exemplary product of the coupling mixture (FADC-17A and/or FADC-17B and/or FADC-17C mix) was obtained as a PBS buffer for ADC-17, which was stored at 4 ℃.

Examples 4 to 18ADC-18 to examples 4 to 21ADC-21

Similar preparation methods were employed using LD-18, LD-19, LD-20 and LD-21, respectively, instead of LD-17, to obtain corresponding ADC-18, ADC-19, ADC-20 and ADC-21, examples 4-18 to 4-21.

EXAMPLES 4-22ADC-22

The antibody Trastuzumab and LD-22 are used as raw materials, and are prepared by a method disclosed in patent application WO2022068898A 1. An exemplary product of the coupling mixture (FADC-22A and/or FADC-22B and/or FADC-22C mix) was obtained as a PBS buffer for ADC-22, which was stored at 4 ℃.

Examples 4 to 23ADC-23 to examples 4 to 26ADC-26

Similar preparation methods were employed using LD-23, LD-24, LD-25 and LD-26, respectively, instead of LD-22, to obtain corresponding ADC-23, ADC-24, ADC-25 and ADC-26, examples 4-23 to 4-26.

EXAMPLES 4 to 27ADC-27

The antibody Pertuzumab and LD-1 are used as raw materials, and the method of the step A is adopted for preparation. An exemplary product of the coupling mixture (FADC-27A and/or FADC-27B and/or FADC-27C mix) was obtained as a PBS buffer for ADC-27, which was stored at 4 ℃.

EXAMPLES 4-28ADC-28

The antibody Trastuzumab and LD-27 are used as raw materials, and the method of the step A is adopted for preparation. An exemplary product of the coupling mixture (FADC-28A and/or FADC-28B and/or FADC-28C mix) was obtained as a PBS buffer for ADC-28, which was stored at 4 ℃.

EXAMPLES 4 to 29ADC-29

The antibody Trastuzumab and LD-28 are used as raw materials, and the method of the step A is adopted for preparation. PBS buffer of exemplary product ADC-29 of the coupling mixture (FADC-29A and/or FADC-29B and/or FADC-29C mix) was obtained and stored at 4 ℃.

EXAMPLES 4-30ADC-30

The antibody Trastuzumab and LD-29 are used as raw materials, and the method of the step A is adopted for preparation. An exemplary product of the coupling mixture (FADC-30A and/or FADC-30B and/or FADC-30C mix) was obtained as a PBS buffer for ADC-30, which was stored at 4 ℃.

EXAMPLES 4-31ADC-31

The antibody Trastuzumab and LD-30 are used as raw materials, and the method of the step A is adopted for preparation. An exemplary product of the coupling mixture (FADC-31A and/or FADC-31B and/or FADC-31C mix) was obtained as a PBS buffer for ADC-31 and stored at 4 ℃.

Examples 4-32ADC-32 to examples 4-35ADC-35

The preparation of step A was carried out using LD-31-LD-34, respectively, with Trastuzumab antibody, to give PBS buffers of the exemplary products ADC-32-ADC-35 of the corresponding conjugate mixtures (FADC-A and/or FADC-B and/or FADC-C mixtures), respectively, stored at 4℃as examples 4-32-4-35.

Examples 4-36ADC-36 to examples 4-38ADC-38

The exemplary products of the general formulas ADC-36 to ADC-38, respectively, were prepared using LD-35 to LD-37, respectively, and antibody Trastuzumab using the method disclosed in patent application "WO2019114666A1", respectively, to obtain PBS buffers of the general formulas FADC-36 to FADC-38, and stored at 4℃for examples 4-36 to 4-38.

Examples 4-39ADC-39 to examples 4-42ADC-42

LD-38-LD-41, respectively, were prepared with the antibody Trastuzumab using the method disclosed in patent application "WO2022068898A 1". PBS buffer solutions of the exemplary products ADC-39-ADC-42 of the corresponding coupling mixtures (FADC-A and/or FADC-B and/or FADC-C mixtures), respectively, were obtained and stored at 4℃as examples 4-39-4-42.

Examples 4 to 43ADC-43 to examples 4 to 45ADC-45

LD-42-LD-44, respectively, was prepared with the antibody Trastuzumab using the method disclosed in patent application "WO2022068898A 1". PBS buffer solutions of the exemplary products ADC-43-ADC-45 of the corresponding coupling mixtures (FADC-A and/or FADC-B and/or FADC-C mixtures), respectively, were obtained and stored at 4℃as examples 4-43-4-45.

Examples 4 to 46ADC-46 to examples 4 to 48ADC-48

The preparation of step A was carried out using LD-45-LD-47, respectively, with Trastuzumab antibody, to give PBS buffers of the exemplary products ADC-46-ADC-48 of the corresponding conjugate mixtures (FADC-A and/or FADC-B and/or FADC-C blends), respectively, stored at 4℃as examples 4-46-4-48.

Examples 4 to 49ADC-49 to examples 4 to 51ADC-51

The preparation of step A was carried out using LD-48-LD-50, respectively, with Trastuzumab antibody, to give PBS buffers of exemplary products ADC-49-ADC-51 of the corresponding conjugate mixtures (FADC-A and/or FADC-B and/or FADC-C mixtures), respectively, stored at 4℃as examples 4-49-4-51.

Examples 4-52ADC-52 to examples 4-54ADC-54

The exemplary products of the general formulas ADC-52-ADC-54, respectively, were prepared using LD-51-LD-53, respectively, and antibody Trastuzumab using the method disclosed in patent application "WO2019114666A1", respectively, to obtain PBS buffers of the general formulas FADC-52-FADC-54, stored at 4℃for examples 4-52-4-54.

Examples 4 to 55ADC-55 to examples 4 to 57ADC-57

LD-54-LD-56 were used separately and were prepared with the antibody Trastuzumab using the method disclosed in patent application "WO2022068898A 1". PBS buffer solutions of the exemplary products ADC-55-ADC-57 of the corresponding coupling mixtures (FADC-A and/or FADC-B and/or FADC-C mixtures), respectively, were obtained and stored at 4℃as examples 4-55-4-57.

Examples 4 to 58ADC-58 to examples 4 to 60ADC-60

LD-57 to LD-59 were used separately and prepared with the antibody Trastuzumab using the method disclosed in patent application "WO2022068898A 1". PBS buffers of the exemplary products ADC-58-ADC-60 of the corresponding coupling mixtures (FADC-A and/or FADC-B and/or FADC-C mixtures), respectively, were obtained and stored at 4℃as examples 4-58-4-60.

EXAMPLES 4-61ADC-61

The antibody Trastuzumab and LD-60 are used as raw materials, and the method of the step A is adopted for preparation. An exemplary product of the coupling mixture (FADC-61A and/or FADC-61B and/or FADC-61C mix) was obtained as a PBS buffer for ADC-61, which was stored at 4 ℃.

EXAMPLES 4-62ADC-62

The antibody Trastuzumab and LD-61 are used as raw materials, and the method of the step A is adopted for preparation. PBS buffer of exemplary product ADC-62 of the coupling mixture (FADC-62A and/or FADC-62B and/or FADC-62C mix) was obtained and stored at 4 ℃.

EXAMPLES 4-63ADC-63

The antibody Trastuzumab and LD-62 are used as raw materials, and are prepared by a method disclosed in patent application WO2019114666A 1. An exemplary product of the formula FADC-63, ADC-63, in PBS buffer, was obtained and stored at 4 ℃.

Examples 4-64ADC-64

The antibody Trastuzumab and LD-63 are used as raw materials, and are prepared by a method disclosed in patent application WO2022068898A 1. PBS buffer of exemplary product ADC-64 of the coupling mixture (FADC-64A and/or FADC-64B and/or FADC-64C mix) was obtained and stored at 4 ℃.

EXAMPLES 4 to 65ADC-65

The antibody Trastuzumab and LD-64 are used as raw materials, and are prepared by a method disclosed in patent application WO2022068898A 1. An exemplary product of the coupling mixture (FADC-65A and/or FADC-65B and/or FADC-65C mix) was obtained as a PBS buffer for ADC-65, which was stored at 4 ℃.

EXAMPLES 4 to 66ADC 66

The antibody Trastuzumab and LD-65 are used as raw materials, and the method of the step A is adopted for preparation. PBS buffer of exemplary product ADC-66 of the coupling mixture (FADC-66A and/or FADC-66B and/or FADC-66C mix) was obtained and stored at 4 ℃.

Examples 4-67ADC-67 to examples 4-75ADC-75

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Similar preparation methods were employed using LD-66-LD-74 instead of LD-65, respectively, to give corresponding ADC-67-ADC-75 as in examples 4-67-4-75. Examples 4-76ADC-76 to examples 4-80ADC-80

The antibody Trastuzumab and LD-75-LD-79 are used as raw materials, and are prepared by a method disclosed in patent application WO2019114666A 1. PBS buffers of exemplary products ADC-76-ADC-80 of the general formulas FADC-76-FADC-80 were obtained, respectively, and stored at 4 ℃.

EXAMPLES 4-81ADC-81

The antibody Trastuzumab and LD-80 are used as raw materials, and are prepared by a method disclosed in patent application WO2022068898A 1. PBS buffer of exemplary product ADC-81 of the coupling mixture (FADC-81A and/or FADC-81B and/or FADC-81C mix) was obtained and stored at 4 ℃.

Examples 4-82ADC-82 to examples 4-85ADC-85

Similar preparation methods were employed using LD-81 to LD-84 instead of LD-80, respectively, to obtain corresponding ADC-82 to ADC-85 as examples 4-82 to 4-85.

EXAMPLES 4 to 86ADC 86

The antibody Trastuzumab and LD-85 are used as raw materials, and are prepared by a method disclosed in patent application WO2022068898A 1. PBS buffer of exemplary product ADC-86 of the coupling mixture (FADC-86A and/or FADC-86B and/or FADC-86C mix) was obtained and stored at 4 ℃.

Examples 4-87 ADC-87-examples 4-90ADC-90

Similar preparation methods were employed using LD-86 to LD-89 instead of LD-85, respectively, to obtain corresponding ADC-87 to ADC-90, examples 4-87 to 4-90.

Examples 4 to 91 ADC to 91 to examples 4 to 97 ADC to 97

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Similar preparation methods are adopted by respectively using LD-90-LD-96 and antibody Trastuzumab as raw materials to respectively obtain corresponding ADC-91-ADC-97, which are examples 4-91-4-97.

Examples 4-98ADC-98 to examples 4-100ADC-100

The method disclosed in patent application WO2019114666A1 is adopted to prepare the antibody Trastuzumab by using LD-97-LD-99 and the antibody Trastuzumab as raw materials respectively. Corresponding ADC-98 to ADC-100 were obtained as examples 4-98 to 4-100.

Examples 4 to 101ADC-101 to examples 4 to 103ADC-103

LD-100-LD-102 and antibody Trastuzumab are used as raw materials, respectively, and are prepared by adopting a method disclosed in patent application WO2019114666A 1. Corresponding ADC-101 to ADC-103 were obtained as examples 4-101 to 4-103.

ADC stock drug load analysis

Purpose and principle of experiment

The ADC stock solution is an antibody crosslinking substance drug, and the mechanism of treating diseases is to rely on targeting of antibodies to transport toxin molecules into cells so as to kill the cells. The loading of the drug plays a decisive role in the efficacy. The drug loading of the ADC stock was determined using mass spectrometry, reversed phase high performance liquid chromatography and size exclusion chromatography.

Reversed phase high performance liquid chromatography:

(1) The experimental method comprises the following steps: the sample is diluted to 0.5mg/mL by ultrapure water, reduced by dithiothreitol with the final concentration of 25mM at 37 ℃ for 30min, and then directly injected with the injection amount of 5 mu L. The chromatographic column is reverse phase chromatographic column (AGILENT PLRP-S) (5 Μm) 50X 2.1 mm) was eluted with a gradient of mobile phases A and B (A: 0.1% fa-0.025% tfa-water, B:0.1% FA-0.025% TFA-acetonitrile). The flow rate was 0.25mL/min, the detection wavelength was 280nm, and the column temperature was 70 ℃. (2) data analysis: and (3) distinguishing the positions of the light chains and the heavy chains through the comparison of the spectrograms of the sample and the naked antibody, and then integrating the spectrograms of the detection sample to calculate the DAR value. The calculation formula is as follows: LC (liquid crystal): 0 (number of linked drugs), lc+1:2 (number of linked drugs), HC:0 (number of linked drugs), hc+1:2 (number of linked drugs), hc+2:4 (number of linked drugs), hc+3:6 (number of linked drugs). LC peak area sum=lc peak area+lc+1 peak area; total HC peak area = HC peak area + HC +1 peak area + HC +2 peak area + HC +3 peak area; LC DAR = Σ (number of linked drugs x percentage of peak area)/LC peak area sum; HC DAR = Σ (number of linked drugs x percentage of peak area)/HC peak area sum; dar=lc dar+hc DAR

Mass spectrometry: (1) experimental method: 10. Mu.L of the test sample with the concentration of 1mg/mL is taken into a 1.5mL centrifuge tube, 0.5. Mu.L of fast cutting enzyme (RAPID PNGASE F, adamas) is added, and the mixture is uniformly mixed by vortex, and incubated for 60min at 37 ℃. After completion of N-cut, 6. Mu.L of ultrapure water and 4. Mu.L of a 0.5M aqueous dithiothreitol solution were added to the centrifuge tube, and after vortexing and mixing, incubation was carried out at 37℃for 30 minutes. After the reaction was completed, the supernatant was centrifuged and fed into a sample bottle, and 2. Mu.L of the sample was introduced. The chromatographic column is reverse phase chromatographic column (AGILENT PLRP-S)(5 Μm) 50X 2.1 mm) was eluted with a gradient of mobile phases A and B (A: 0.1% fa-water, B:0.1% of FA-acetonitrile), the flow rate is 0.5mL/min, the column temperature is 60 ℃, the detection wavelength is 280nm, the ESI-Tof (LC-MS) is used for collecting m/z, the mass spectrum collection mode is a positive ion mode, the collection m/z range is 200-3200, and then deconvolution treatment is carried out on the original mass spectrum by software. (2) data analysis: the DAR value is calculated by using the peak height value by using a method similar to the reversed-phase high-performance liquid chromatography.

Size exclusion chromatography:

The experimental method comprises the following steps: the sample loading amount of the test sample is about 40 mug, the chromatographic column is a hydrophilic silica gel size exclusion chromatographic column (TSKgel G3000SWXL (5 mu m) 7.8X300 mM), the mobile phase is (100 mM phosphate+100 mM sodium sulfate+5% isopropyl alcohol, pH 7.0, flow rate 0.5mL/min, detection wavelength 280nm, and column temperature 30 ℃.

III test case

Biological evaluation

Test example 1: determination of the in vitro proliferation inhibitory Activity of Compounds of general formula (GH) on SK-BR-3 tumor cells

1. Test purpose:

The purpose of this experiment was to examine the in vitro killing activity of the disclosed compounds (GH) against SK-BR-3 tumor cells (human breast cancer cells). Cells were treated with different concentrations of the compounds in vitro, and after 3 days of incubation, the number of viable cells was measured using CCK8 (Cell Counting Kit-8, cat# TS 547) reagent and the in vitro activity of the test compounds was evaluated based on IC ₅₀ values.

2. The experimental method comprises the following steps:

taking the test method for the in vitro killing activity of SK-BR-3 cells as an example, the following test is carried out. It should be understood that the present method is equally applicable to, but not limited to, in vitro proliferation inhibition activity assays for other tumor cells (HCC 1569, JIMT-1, DIFI, etc.).

(1) Cell culture: SK-BR-3 cells were cultured in RPMI 1640 medium (Shanghai Yuanzhi Biotech Co., ltd., cat# G211018) with 10% FBS.

(2) Cell preparation: the SK-BR-3 cells in the logarithmic growth phase were taken, washed 1 time with PBS (phosphate buffer, shanghai Yuanzhi Biotechnology Co., ltd., cat# E211004), then digested 2-3 minutes with 2-3mL trypsin (0.25% Trypsin-EDTA (1X), shanghai Yuanzhi Biotechnology Co., ltd., cat# A121002), after the cell digestion was completed, 10mL of cell culture solution containing 2% FBS was added, the digested cells were eluted, 300g was centrifuged for 5 minutes, the supernatant was discarded, and then 10-20mL of cell culture solution containing 2% FBS was added to resuspend the cells to prepare a single cell suspension.

(3) Cell plating: the SK-BR-3 single cell suspension was homogenized, the viable cell density was adjusted to 6X 10 ⁴ cells/mL with cell culture medium containing 2% FBS, and the cell suspension after the density adjustment was homogenized and added to 96-well cell culture plates at 100. Mu.L/well. 200. Mu.L of PBS was added to the wells outside Zhou Kongzhi of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ℃,5% co ₂).

(4) Compound preparation: the compound was dissolved in DMSO (dimethyl sulfoxide, SIGMA) to prepare a 2mM stock solution.

The initial concentration of both small molecule compound and Dxd was 600nM and the dosing method was as follows.

Dxd (MedChemExpress, cat# HY-13631D, CAS: 159940-33-1) and 2mM small molecule compound sample stock solution are diluted to 1200nM by using a cell culture medium containing 2% FBS, filtered and sterilized by using a 0.22 mu M filter head, and then 315 mu L of different samples to be tested are respectively added into the first column of the 96-well U-shaped bottom dispensing plate, wherein the sample concentration is 1200nM; 210. Mu.L of cell culture medium containing 2% FBS was added to each well of columns 2 to 10. Taking 105 mu L of the first row of samples to 210 mu L of the second row of cell culture media, uniformly mixing, taking 105 mu L of samples to the third row, and the like to the 10 th row.

(5) Sample adding operation: to the plates were added 100 μl of each sample with two wells configured to test samples of different concentrations. The plates were incubated in an incubator for 3 days (37 ℃,5% co ₂).

(6) Color development operation: the 96-well cell culture plates were removed, the cell culture supernatant was scraped off, 100. Mu.L of RPMI 1640 basal medium containing 10% CCK8 (no FBS) was added, and then incubated in an incubator for about 1.5 hours (37 ℃,5% CO ₂).

(7) Plate reading operation: the 96-well cell culture plate was removed, placed in an enzyme-labeled instrument (MD SpectraMax i 3X), and A ₄₅₀ was measured by the enzyme-labeled instrument.

(8) Data analysis: data were analyzed by Microsoft Excel, GRAPHPAD PRISM.

The results of the above tests are summarized in table 1:

Table 1: IC ₅₀ values for the in vitro proliferation inhibition of SK-BR-3 cells by the test compounds.

Conclusion: the small molecule fragments in the present disclosure have significant proliferation inhibitory activity on SK-BR-3 cells.

Test example 2: in vitro proliferation inhibition assay of HER2 target tumor cells with the presently disclosed antibody drug conjugates

Determination of SK-BR-3 tumor cell in vitro proliferation inhibition Activity

1. Test purpose:

The purpose of this experiment was to detect the inhibitory activity of the antibody drug conjugates of the present disclosure against HER2 targets on SK-BR-3 cell (human breast cancer cell, shanghai-s-Xin) proliferation in vitro. Cells were treated with different concentrations of the compound in vitro, cultured for 7 days, and proliferation of the cells was detected using CCK8 (Cell Counting Kit-8, cat# TS 547) reagent, and their in vitro activity was evaluated based on IC ₅₀ values.

2. The experimental method comprises the following steps:

Taking the test method for the in vitro proliferation activity of SK-BR-3 cells as an example, the following test was performed. It should be understood that the present method is equally applicable to, but not limited to, in vitro proliferation inhibition activity assays for other tumor cells (HCC 1569, JIMT-1, DIFI, etc.).

(1) Cell culture: SK-BR-3 cells were cultured in RPMI 1640 medium (Shanghai Yuanzhi Biotech Co., ltd., cat# L210 KJ) with 2% FBS.

(2) Cell preparation: taking SK-BR-3 cells in logarithmic growth phase, washing 1 time with PBS (phosphate buffer, shanghai Yuanzhi Biotechnology Co., ltd., cat# E211004), adding 1mL trypsin (0.25% Trypsin-EDTA (1X), shanghai Yuanzhi Biotechnology Co., ltd., cat# A121002), digesting for 2-3min, adding 10mL cell culture solution after cell digestion is complete, eluting digested cells, centrifuging 300g for 5min, discarding supernatant, and then adding 10mL cell culture solution to resuspend cells to prepare single cell suspension.

(3) Cell plating: the SK-BR-3 single cell suspension is uniformly mixed, the density of the living cells is regulated to 3X 10 ⁴ cells/mL by using a cell culture solution, the cell suspension with the regulated density is uniformly mixed, and 100 mu L/hole is added into a 96-hole cell culture plate. 200. Mu.L of PBS was added to the wells outside Zhou Kongzhi of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ℃,5% co ₂).

(4) Preparing a sample to be tested:

diluting a sample to be tested to 10nM by using a cell culture medium containing 2% FBS, and adding 300 mu L of sample with the concentration of 10nM into a first hole of a 96-hole U-shaped bottom dispensing plate; 210. Mu.L of cell culture medium containing 2% FBS was added to each well of columns 2 to 10. Taking 70 mu L of the first column sample to 210 mu L of the second column cell culture medium, uniformly mixing, taking 70 mu L of the first column sample to the third column sample, and the like to the 10 th column sample.

(5) Sample adding operation: to the plates were added 100 μl of configured samples to be tested at different concentrations, two wells per sample. The plates were incubated in an incubator for 7 days (37 ℃,5% co ₂).

(6) Color development operation: the 96-well cell culture plates were removed, the culture supernatant was removed by beating, 100. Mu.L of 10% CCK8 (prepared with RPMI 1640 basal medium without FBS) solution was added to each well, and incubated at room temperature for 2 hours.

(7) Plate reading operation: the 96-well cell culture plate was removed, placed in an enzyme-labeled instrument (SpectraMax i 3X), and a ₄₅₀ was measured with the enzyme-labeled instrument.

(8) Data analysis: data were analyzed by Microsoft Excel, GRAPHPAD PRISM.

Test example 3: antibody drug conjugate bystander killing test of the disclosure

Her2 positive cells SKBR3 cells and Her3 negative cells MDA-MB-468 were adjusted to a cell density of 1.2X10- ⁵ cells and 4X 10 ⁴ cells/mL with RPMI-1640+10% FBS, respectively, and 1mL of each of SK-BR-3 and MDA-MB-468 cells was added to a 6-well plate, and mixed and cultured overnight at 37℃with 5% CO ₂. ADC samples were formulated to a concentration of 4.5 μg/mL (30 nM) and 1mL was added to the cells to give a total volume of 3mL and a final concentration of 10nM. A solvent control group was set and incubated at 37℃for 5 days with 5% CO ₂. Cell pancreatin (0.25% Trypsin-EDTA (1X), shanghai source culture Biotech Co., ltd., cat# A121002) was added to the 6-well plate for digestion, 1mL FACS buffer (PBS+4% FBS) was added for resuspension, and cells were stained with trypan blue and counted. Residual cells were centrifuged, the Trastuzumab was diluted to 10. Mu.g/mL with FACS buffer, the cells were resuspended with 200. Mu.L of antibody solution, incubated on ice for 30min, the supernatant was discarded after centrifugation, washed once with 1mL of FACS buffer, 200. Mu.L of AF 647-labeled goat anti-human Fc antibody (Jackson, cat. No. 109-606-170) solution was added, incubated on ice for 15min, PI (Sigma, cat. No. 31845) solution was added at a concentration of 5. Mu.g/mL, incubated on ice for 5min, the supernatant was discarded after centrifugation, 1mL of FACS buffer was washed once, 400. Mu.L of PBS resuspended cells were assayed by flow cytometry, and the ratio and number of cells were determined from flow and count results.

The embodiments of the technical solutions of the present disclosure have been described above by way of example. It should be understood that the protection scope of the present disclosure is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. which are within the spirit and principles of the present disclosure, by those skilled in the art, should be included in the scope of the present claims.

Claims

1. A conjugate, stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide or pharmaceutically acceptable salt thereof, represented by the following formula (C):

Tp—L—G(C)

wherein Tp is a targeting moiety;

l is selected from a bond or a linker;

g is a group of the formula (G):

wherein A is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

T ^L is selected from the group consisting of bond 、#O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(＝O)*、#O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n*、#N(R¹³)-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)*、#S-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)*、#O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(NR¹³)*、#O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-P(＝O)(OR¹⁴)*、*O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(＝O)#、*O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n#、*N(R¹³)-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)#、*S-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(O)#、*O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-C(NR¹³)#、*O-(CR⁹R¹⁰)_m-(CR¹¹R¹²)_n-P(＝O)(OR¹⁴)#, wherein # represents the site of attachment to L and x represents the site of attachment to B;

Wavy lines indicate the site of attachment to L;

Or B is-N (NR ⁷R⁸) -;

2. The conjugate of claim 1, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein the group in formula (G) is independently selected from the following definitions:

a is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

3. The conjugate of claim 1, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein the group in formula (G) is independently selected from the following definitions:

a is selected from N or C-R ^A;

z is selected from 0 or 1;

R ⁴ is selected from alkyl, alkyloxy, halogen, hydroxy, amino, cyano;

r ⁵ is selected from hydroxy;

b is selected from the absence, -NR ⁷ -or-N (NR ⁷R⁸) -;

4. The conjugate of claim 1, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein the group in formula (G) is independently selected from the following definitions:

a is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

5. The conjugate of claim 1, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein the group in formula (G) is independently selected from the following definitions:

a is selected from N or C-R ^A;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

6. The conjugate of claim 1, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein the group in formula (G) is independently selected from the following definitions:

A is selected from N;

z is selected from 0 or 1;

r ⁵ is selected from hydroxy;

B is selected from-NR ⁷ -or-N (NR ⁷R⁸) -;

7. The conjugate of any one of claims 1-6, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein:

R ¹、R² together with the quinolinyl to which it is attached form one of the following substructures, wherein the substructures may be unsubstituted or substituted with one, two or more R ^E:

Or R ²、R³ together with the quinolinyl to which it is attached form one of the following substructures, wherein the substructures may be unsubstituted or substituted with one, two or more R ^F:

Or preferably B is selected from the group consisting of absent, -NH-or-N (NR ⁷R⁸) -, wherein R ⁷、R⁸ independently of each other has the definition of any one of claims 1 to 6.

8. The conjugate of any one of claims 1-7, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein G is selected from the group represented by the following formula (G-1):

Wherein A, B, T ^L、R¹、R²、R³、R⁶ independently has the definition as defined in any one of claims 1 to 7.

9. The conjugate of any one of claims 1-7, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein G is selected from the group consisting of groups represented by formula (G-2) or (G-3):

wherein R ^A、B、T^L、R¹、R²、R³、R⁶ has the definition as defined in any one of claims 1 to 7.

10. The conjugate of any one of claims 1-7, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein G is selected from the following groups or groups following-T ^L -linkage at the wavy line:

Wherein A, R ¹、R²、R³、R⁴、R⁶、R⁷, z have the definitions as defined in any one of claims 1 to 7.

11. The conjugate of any one of claims 1-7, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein G is selected from the following groups or groups following-T ^L -linkage at the wavy line:

Wherein A, R ¹、R²、R³、R⁴、R⁷ has the definition as defined in any one of claims 1 to 7.

12. The conjugate of any one of claims 1-7, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein G is selected from the following groups or groups following-T ^L -linkage at the wavy line:

Wherein A, R ¹、R⁴、R⁶、R⁷, z have the definitions as defined in any one of claims 1 to 7.

13. The conjugate of any one of claims 1-7, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein G is selected from the group consisting of the following or a group following-T ^L -linkage at the wavy line edge of the group:

/>

14. the conjugate according to any one of claim 1 to 7, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

Wherein L is selected from a bond or a linker of formula (L) as defined below:

#L¹—L²—L³—L⁴—L⁵*(L)

Wherein L ¹ is a linking moiety to the targeting moiety Tp, formed by the reactive group L ¹' and the targeting moiety Tp, # represents a linking site to the targeting moiety Tp;

For example, L ¹' is a maleimide group, then L ¹ is of the structure:

or an open-loop version thereof: /(I)

L ² is absent or a spacer of L ¹ and L ³;

l ³ is a peptide moiety;

L ⁴ is absent or is a spacer to L ⁵ for the peptide moiety;

Optionally:

hydrophilic moiety:

Sterically hindered moiety:

The hydrophilic or sterically hindered moiety as defined above is present or absent.

15. The conjugate according to claim 14, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

L ¹ is formed by any group L ¹ 'which reacts with the targeting moiety Tp, L ¹' is preferably a thiol-reactive group, an amino-reactive group, a carboxyl-reactive group, a proline residue-reactive group, a tyrosine residue-reactive group, a disulfide bridge group, etc.; antibodies directed against the introduction of unnatural amino acids can also be selected from click chemistry reactive groups such as ketones, azides, alkynes, cyclopropenes, or dienes;

L ¹' is preferably a thiol-reactive group;

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

/>

Or L ¹' is preferably a thiol-reactive group of the structure:

Hal-Het-

In a preferred embodiment Hal-Het-is:

The corresponding L ¹ structure is as follows:

and L ¹'-L² is preferably of the structure:

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

Further preferred is:

16. The conjugate according to claim 14, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

Preferably, L ² is linked to the N-terminus of the peptide fragment by-C (R ^z4R^z5) -CO-, wherein

L ² is further preferably -(CH₂)_q-、-(CH₂)_q-C(＝O)-、、-(C≡C)-(CH₂)_q-C(R^z4R^z5)-C(＝O)-, wherein q is an integer selected from 0 to 10, preferably 1,2, 3, 4, 5, 6, 7 or 8;

17. The conjugate according to claim 14, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

Further preferred, L ³ is selected from a divalent peptide group comprising 2,3, 4,5 or 6 optionally substituted natural amino acid residues or a combination of non-natural amino acid residues, each of which is the same or different, independently of the other selected from the residues of amino acids: 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 (gin), 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, norpyrrolysine; for example -ValCit-;-CitVal-;-AlaAla-;-AlaCit-;-CitAla-;-AsnCit-;-CitAsn-;-CitCit-;-ValGlu-;-GluVal-;-SerCit-;-CitSer-;-LysCit-;-CitLys-;-AspCit-;-CitAsp-;-AlaVal-;-ValAla-;-PheAla-;-AlaPhe-;-PheLys-;-LysPhe-;-ValLys-;-LysVal-;-AlaLys-;-LysAla-;-PheCit-;-CitPhe-;-LeuCit-;-CitLeu-;-IleCit-;-CitIle-;-PheArg-;-ArgPhe-;-CitTrp-;-TrpCit-;-PhePheLys-;-LysPhePhe-;-DPhePheLys-;-DLysPhePhe-;-GlyPheLys-;-LysPheGly-;-GlyPheLeuGly-;-GlyLeuPheGly-;-AlaLeuAlaLeu-,-GlyGlyGly-;-GlyGlyGlyGly-;-GlyPheValGly-;-GlyValPheGly-;-GlyGlyPheGly-;-AlaAlaAla-;, most preferably, L ³ is-GLYGLYPHEGLY-.

18. The conjugate according to claim 14, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

L ⁴ is preferably a group having self-cleaving properties, a self-cleaving group or a self-sacrificing group (self-immolative group) being a group that initiates drug release by intramolecular reactions such as 1, 4-elimination, 1, 6-elimination or cyclization elimination without depending on the enzyme;

L ⁴-L⁵ is preferably:

PABC spacer arm, the structure is:

GABA interval arm, the structure is:

the alpha, alpha-dimethyl GABA spacer has the structure:

Or a beta, beta-dimethyl GABA spacer arm.

Most preferably, L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

r ^z1 is H or C1-4 alkyl;

R ^z2 or R ^z3 are independently H or C1-4 alkyl;

T ^L is as defined in claims 1 to 7, and one of T ^L in L ⁴-L⁵ or G is a bond.

19. The conjugate according to any one of claim 14 to 18, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

Hydrophilic moiety: :

20. The conjugate according to any one of claim 14 to 18, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

Sterically hindered moiety:

21. the conjugate according to any one of claim 14 to 18, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

R ^z1 is H or C _1-4 alkyl;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

T ^L is as defined in claims 1 to 13, and one of T ^L in L ⁴-L⁵ or G is a bond;

Further preferred is:

When T ^L in G is a bond, L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-O-(CH₂)_m-C(R^z4R^z5) -CO-;

Wherein:

r ^z1 is H or C1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C1-4 alkyl;

R ^z4 or R ^z5 are independently H, C1-4 alkyl, C3-6 cycloalkyl-C1-4 alkyl or R ^z4 or R ^z5 together form C _3-6 cycloalkyl.

22. The conjugate according to any one of claim 14 to 18, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

L ¹-L² is generated from coupling Tp with L ¹'-L² of the following structure:

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

Further preferably R ^z4 or R ^z5 together form C _3-6 cycloalkyl;

l ¹-L² is most preferably composed of Coupling Tp.

23. The conjugate according to any one of claim 1 to 22, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof,

Tp is a targeting moiety selected from small molecule ligands, proteins, polypeptides, non-protein agents (e.g., sugars, RNA or DNA);

Preferably, the target of Tp is selected from the group consisting of 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、Sodiumphosphate cotransporter 2B、GPNMB、Trophoblast glycoprotein、AGS-16、EGFR、CD33、CD66e、CD74、CD56、PD-L1、TACSTD2、DR5、E16、0772P、MPF、Napi3b、Sema5b、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、LY64、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、CD1 lc、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、SERPINEl、STAT1、TGFBR1、CTSS、PGF、VEGFA、C1QA、C1QB、ANGPTL4、EGLN、EGLN3、BNIP3、AIF1、CCL5、CXCL10、CXCL11、IFI6、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、CDl lb、ADGRE1、EMR2、TNFRSF21、UPK1B、TNFSF9、MMP16、MFI2、IGF-1R、RNF43、NaPi2b and BCMA;

Preferably, tp is a small molecule ligand such as folic acid derivatives, glutamic acid urea derivatives, somatostatin derivatives, arylsulfonamide derivatives (e.g., carbonic anhydrase IX inhibitors), ICG dyes, cyanine dyes, or derivatives thereof;

preferably, tp is selected from an antibody or antigen binding fragment thereof, said antibody being selected from a chimeric, humanized or fully human antibody; preferably monoclonal antibodies;

Preferably, the antibody or antigen binding fragment thereof is selected from at least one of the following: an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-HER 2 (ErbB 2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3 (ErbB 3) antibody, an anti-HER 4 (ErbB 4) antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, or an anti-Mesothelin antibody, which may be bispecific or multispecific antibodies;

further preferred, 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 Glematumamab thereof.

24. The conjugate, stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof of any one of claims 1-23, wherein the conjugate, linker, or linker-drug is selected from one of the following:

Wherein u, v, w are independently from each other integers from 0 to 10, G has the definition of any one of claims 1 to 23, LG has the definition of Tp of any one of claims 1 to 23; r ²⁰、R^20', which are identical or different, are independently of one another selected from H, C _1-4 alkyl, C _3-6 cycloalkyl, C _3-6 cycloalkyl-C _1-4 alkyl, or R ²⁰、R^20' together with the carbon atom to which they are attached form C _3-6 cycloalkyl:

/>

25. the conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate has a structure according to the formula:

wherein ,A、R¹、R²、R³、R⁴、R⁶、R⁷、R¹¹、R¹²、L¹、L²、Tp has the definition as defined in any one of claims 1 to 23.

26. The conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate has a structure according to the formula:

Wherein ,A、R¹、R²、R³、R⁴、R⁷、R¹¹、R¹²、L¹、L²、Tp has the definition as defined in any one of claims 1 to 13.

27. The conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate has a structure according to the formula:

Wherein R ¹¹、R¹²、L¹、L², tp have the definition as defined in any one of claims 1 to 13.

28. The conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from one of the following:

/>

Wherein R ¹¹、R¹² independently of each other have the definition as defined in any one of claims 1 to 23;

R ¹⁶、R^16' independently of one another have the definition as defined in claim 14;

mAb represents a monoclonal antibody;

29. The conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate has a structure according to the formula:

wherein R ¹¹、R¹²、L¹、L², tp have the definition as defined in any one of claims 1 to 23.

30. The conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from one of the following:

/>

mAb represents a monoclonal antibody;

31. The conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate has a structure according to the formula:

32. The conjugate of any one of claims 1-23, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from one of the following:

/>

mAb represents a monoclonal antibody;

33. The conjugate of any one of claims 1-32, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide, or a 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 mab (DAR), which may be selected from integers or fractions, such as integers or fractions selected from 1-50, 1-20, or 1-10;

/>

Wherein R is selected from-CH ₃, and R' is selected from-H;

R is selected from-H, R' is selected from-CH ₃;

R is selected from R' is selected from-H; or R is selected from-H, R' is selected from/>/>

Wherein R is selected from-H, and R' is selected from-H;

r is selected from-CH ₃, R' is selected from-H;

R is selected from-H, R' is selected from-CH ₃; r is selected from R' is selected from-H; or R is selected from-H, R' is selected from/>

/>

Wherein R is selected from-H, and R' is selected from-H;

r is selected from-CH ₃, R' is selected from-H;

/>

Wherein R is selected from-CH ₃, and R' is selected from-H;

R is selected from-H, R' is selected from-CH ₃;

/>

Wherein R is selected from-CH ₃, and R' is selected from-H;

R is selected from-H, R' is selected from-CH ₃;

/>

Wherein R is selected from-H, and R' is selected from-H;

r is selected from-CH ₃, R' is selected from-H; r is selected from R' is selected from-H; or R is selected from-H, R' is selected from/>

Wherein R is selected from-H, and R' is selected from-H; r is selected from-CH ₃, R' is selected from-H; or R is selected fromR' is selected from-H; /(I)

Wherein R is selected from-H, and R' is selected from-H;

r is selected from-CH ₃, R' is selected from-H; r is selected from R' is selected from-H; or R is selected from-H, R' is selected from/>/>

Wherein R is selected from-H, and R' is selected from-H; r is selected fromR' is selected from-H; or R is selected from-H, R' is selected from/>/>

Wherein R is selected from-H, and R' is selected from-H; r is selected fromR' is selected from-H; or R is selected from-H, R' is selected from/>

/>

Wherein R is selected from-CH ₃, and R' is selected from-H;

R is selected from-H, R' is selected from-CH ₃;

Wherein R is selected from-H, and R' is selected from-H;

r is selected from-CH ₃, R' is selected from-H;

Wherein R is selected from-H, and R' is selected from-H;

R is selected from-CH ₃, R' is selected from-H; r is selected from-H, R' is selected from-CH ₃; r is selected from R' is selected from-H; or R is selected from-H, R' is selected from/>

/>

Wherein R is selected from-CH ₃, and R' is selected from-H;

R is selected from-H, R' is selected from-CH ₃;

/>

Wherein R is selected from-CH ₃, and R' is selected from-H;

R is selected from-H, R' is selected from-CH ₃;

/>

Wherein r= -H, R' = -H;

R＝-CH₃，R’＝-H；

R＝-H，R’＝-CH₃；

wherein r= -H, R' = -H;

R＝-CH₃，R’＝-H；

R＝-H，R’＝-CH₃。

34. The method for producing a conjugate, a stereoisomer, a racemate, a tautomer, an isotopologue marker, a nitroxide or a pharmaceutically acceptable salt thereof according to any one of claim 1 to 33,

preferably, in the above-mentioned linker,

Wherein:

R ^z1 is H or C _1-4 alkyl;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

Further preferred is:

Wherein:

R ^z1 is H or C _1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

Wherein the intermediate structure of formula (G') is:

Wherein ,A、B、T^L、R¹、R²、R³、R⁴、R⁵、R⁶、L¹'、L¹、L²、L³、L⁴、L⁵、L⁵' independently has the definition as defined in any one of claims 1 to 33;

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

optionally, if desired, the functional groups of the reaction substrate may also be protected with protecting groups known in the art to allow the reaction to proceed smoothly and to deprotect after the reaction has ended.

35. A compound of formula (GH), a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide or pharmaceutically acceptable salt thereof:

wherein T is selected from H-T ^L;

T ^L、A、B、R¹、R²、R³、R⁴、R⁵、R⁶, z have the definition as defined in any one of claims 1 to 32 independently of one another.

36. The compound of claim 35, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein the compound of formula (GH) can be selected from the compounds of formula (GH-1) as follows:

Wherein R ^A、B、T、R¹、R²、R³、R⁶ independently has the definition as defined in claim 35.

37. The compound of claim 35, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein the compound of formula (GH) can be selected from the compounds of formula (GH-2) or (GH-3) as follows:

wherein R ^A、B、T、R¹、R²、R³、R⁶ independently has the definition as set forth in claim D1.

38. The compound of claim 35, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide, or pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of:

/>

39. a process for the preparation of a compound according to any one of claims 35 to 38, a stereoisomer, racemate, tautomer, isotopologue, isotopic label, nitroxide or pharmaceutically acceptable salt thereof, said process comprising the step of reacting a compound of formula (i) with a compound of formula (ii):

Wherein A, B, T, R ¹、R²、R³、R⁴、R⁵、R⁶, z independently have the definition as defined in any one of claims 35 to 38;

LE represents a group that is removed or left after the reaction.

40. A process for the preparation of a compound of formula (ii) as claimed in claim 35, which comprises the step of reacting a compound of formula (iii) with a compound of formula (iv) to give a compound of formula (v):

wherein A, B, R ¹、R²、R³、R⁴、R⁵、R⁶, z independently have the definition as defined in any one of claims 35 to 38;

R ²¹ is selected from H or a protecting group, preferably an amino protecting group.

41. A compound represented by formula (iii):

Wherein R ⁵、R⁶, z independently have the definition as defined in any one of claims 35 to 38.

42. A compound represented by formula (v):

wherein A, B, R ¹、R²、R³、R⁴、R⁵、R⁶、R²¹, z independently have the definition as defined in any one of claims 35 to 38.

43. A conjugate of the structure:

Tp—L—D(D)

Wherein Tp is a targeting moiety;

#L¹—L²—L³—L⁴—L⁵*(L)

Wherein L ¹ is a linking moiety to the targeting moiety Tp, formed by the reactive group L ¹' and the targeting moiety Tp, # represents a linking site to the Tp moiety;

l ² is absent or a spacer of L ¹ and L ³;

l ³ is a peptide moiety;

L ⁴ is absent or is a spacer to L ⁵ for the peptide moiety;

L ⁵ is the linking moiety of L ⁴ with the bioactive molecule D, generated by the reaction of the reactive group L ⁵' with the bioactive molecule;

Preferably, L ¹'、L¹、L²、L³、L⁴、L⁵、L⁵' is as defined in any one of claims 14 to 22;

The conditions were as follows:

Condition I:

hydrophilic moiety:

Or condition II:

Sterically hindered moiety:

or condition III:

L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

r ^z1 is H or C1-4 alkyl;

R ^z2 or R ^z3 are independently H or C1-4 alkyl;

T ^L is as defined in any one of claims 1 to 13;

In a further embodiment of the present invention,

hydrophilic moiety:

In a further embodiment:

l ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-O-(CH₂)_m-C(R^z4R^z5) -CO-;

Wherein:

r ^z1 is H or C1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C1-4 alkyl;

R ^z4 or R ^z5 are independently H, C1-4 alkyl, C3-6 cycloalkyl-C1-4 alkyl or R ^z4 or R ^z5 together form C3-6 cycloalkyl.

Or condition IV:

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

R ^z4 or R ^z5 are independently H, C1-4 alkyl, C3-6 cycloalkyl-C1-4 alkyl or R ^z4 or R ^z5 together form C3-6 cycloalkyl, wherein at least one of R ^z4 or R ^z5 is other than H;

It is further preferred that R ^z4 or R ^z5 together form a C3-6 cycloalkyl group;

l ¹-L² is most preferably composed of Coupling Tp.

44. The conjugate according to claim 43, wherein the biologically active molecule D is a biologically active or potentially biologically active compound described in the Chinese, U.S. or European pharmacopoeia or disclosed in other publications;

The drug may 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, docamicin, etoposide, maytansinoids, and maytansinoids, taxanes, benzodiazepines Class or benzene-containing dinitrogen/>Class i drugs and vinca alkaloids.

45. A pharmaceutical composition comprising at least one selected from the group consisting of:

A conjugate of formula (C), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof according to any of claims 1 to 33; and/or

A compound of formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof according to any of claims 35-38; and/or

The conjugate of any one of claims 43 to 44, which is represented by formula (D), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof

Preferably, the compound of formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof in the pharmaceutical composition, and the conjugate of formula (C) or (D), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof is present in a therapeutically effective amount.

46. Use of a compound of formula (GH), a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof, or a conjugate of formula (C) according to any of claims 1 to 33 or a conjugate of formula (D) according to any of claims 43 to 44, a stereoisomer, a racemate, a tautomer, an isotopologue, an isotopic label, a nitroxide or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of a disease or disorder and/or for the preparation of a medicament;

Preferably, the disease or disorder may be selected from a tumour, for example a solid tumour or a hematological cancer.

47. The use according to claim 46, wherein the medicament is for the prevention and/or treatment of a disease or condition;

preferably, the disease or disorder may be selected from a tumor, such as a solid tumor or hematological cancer;

The solid tumor is selected from the group consisting of malignant tumors of various organ systems, e.g., sarcomas, adenocarcinomas, blastomas, and carcinomas, such as those affecting the liver, lung, breast, lymph, biliary intestine (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon, rectum, renal cell carcinoma, liver, small cell lung, non-small cell lung, small intestine and esophagus;

The hematologic cancer is selected from leukemia, lymphoma, and malignant lymphoproliferative disorders affecting the blood, bone marrow, and lymphatic system.

48. The conjugate of the linker and the drug has the following structure:

L¹'—L²—L³—L⁴—L⁵-G(C’)

Wherein: g is as defined in any one of claims 1 to 13;

L ¹'、L²、L³、L⁴、L⁵ is as defined in any one of claims 14 to 22.

49. The linker and drug conjugate of claim 4 wherein the conjugate is selected from one of the following:

/>

50. the conjugate of the linker and the drug has the following structure:

L¹'—L²—L³—L⁴—L⁵-D

Wherein L ¹'、L²、L³、L⁴、L⁵ is as defined in claims 14 to 22, D is as defined in claims 43 to 44,

The conditions are as follows:

Condition I:

Between the fragments as defined above, preferably between L ¹' and L ², or between L ² and L ³, or in the form of substitution L ², the following hydrophilic moieties are inserted:

hydrophilic moiety:

Or condition II:

L ¹' is a thiol-reactive group selected from a maleimide group or a substituted maleimide group;

Between L ¹' and L ², or between L ² and L ³, or in the form of substitution for L ², contains a sterically hindered moiety as follows:

Sterically hindered moiety:

or condition III:

L ¹' is a thiol-reactive group selected from Hal-Het-;

L ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-T^L -;

Wherein:

R ^z1 is H or C _1-4 alkyl;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

t ^L is as defined above;

Or condition IV:

L ¹'-L² is the following structure:

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

Further preferably R ^z4 or R ^z5 together form C _3-6 cycloalkyl;

most preferably, L ¹'-L² is:

51. the conjugate of a linker and a drug as claimed in claim 50, wherein

L ¹' is a thiol-reactive group selected from maleimide groups, substituted maleimide groups or Hal-Het-;

hydrophilic moiety:

52. The conjugate of a linker and a drug as claimed in claim 50, wherein

Sterically hindered moiety:

53. The conjugate of a linker and a drug as claimed in claim 50, wherein

L ¹' is a thiol-reactive group selected from Hal-Het-;

l ⁴-L⁵ is: -NR ^z1-C(R^z2R^z3)-O-(CH₂)_m-C(R^z4R^z5) -CO-;

Wherein:

R ^z1 is H or C _1-4 alkyl;

m is an integer of 0 to 4;

R ^z2 or R ^z3 are independently H or C _1-4 alkyl;

54. The conjugate of a linker and a drug as claimed in claim 50, wherein

L ¹'-L² is the following structure:

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

Further preferably R ^z4 or R ^z5 together form C _3-6 cycloalkyl;

most preferably, L ¹'-L² is: />