CN121001750A

CN121001750A - Heterocyclic compounds, their preparation methods and uses

Info

Publication number: CN121001750A
Application number: CN202480022202.8A
Authority: CN
Inventors: 田强; 张毅涛; 叶健; 龙冬; 周琴; 龙虎; 杨禹; 袁晓曦; 宋宏梅; 葛均友
Original assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date: 2023-05-12
Filing date: 2024-05-10
Publication date: 2025-11-21
Also published as: WO2024235136A1; WO2024235136A9

Abstract

A heterocyclic compound, its preparation method, and its uses are disclosed. The compound has the structure shown in the formula M′-L-E-D. The compound can be used to prepare antibody-drug conjugates. The conjugates have a superior drug-antibody conjugate ratio and exhibit excellent targeted killing effects on solid tumors such as gastric cancer, breast cancer, lung cancer, and urothelial carcinoma.

Description

Heterocyclic compound, preparation method and application thereof

The present application is based on the application CN application No. 202310538792.7, application No. 2023, application No. 5, application No. 202311262663.6, application No. 2023, application No. 9, application No. 27, application No. 202410173822.3, application No. 2024, application No. 2, application No. 6, and claims priority of the foregoing applications, all of which are incorporated herein by reference in their entirety.

Technical Field

The application relates to the field of medicines, in particular to a heterocyclic compound, a preparation method and application thereof.

Background

Antibody drug conjugates (antibody drug conjugate, ADC) are typically composed of monoclonal antibodies, bioactive molecules, and linkers (Linker). The antibody can identify specific targets on the surfaces of tumor cells, so that the ADC is guided to reach the tumor microenvironment and the surfaces of the cancer cells, the ADC enters the cancer cells through an endocytic effect, the bioactive molecules are covalently coupled with the antibody through a connector, and then the bioactive molecules are released in the cancer cells, and the purpose of killing the cancer cells without damaging normal tissue cells as much as possible is achieved through the actions of damaging DNA of the cancer cells or inhibiting tubulin of the cancer cells and the like.

With the wide application of the ADC drugs in clinic for treating tumors, safety problems or drug resistance problems such as hepatotoxicity, interstitial pneumonia, neurotoxicity, blood toxicity and the like are gradually developed (Pharmacology&Therapeutics 2019,200,110-125;Breast Cancer Research and Treatment 2020,183,23-39;JAMA Oncol.2021,7,1873-1881;Drug Deliv.2022,29,1335-1344;Cancers 2023,15,1130;Cancers 2023,15,1278.).

As an important component of antibody-conjugated drugs, the structure of the drug-linker moiety is closely related to the effectiveness and safety of the ADC drug as a whole. Therefore, the development of a novel drug connector structure is of great significance for developing an antibody-coupled drug with good drug effect and safety.

Disclosure of Invention

The application relates to a fused ring compound, which has a structure shown in a general formula D-E-L-M'. The fused ring compound can be used for preparing antibody drug conjugates, and the conjugates have good targeted killing effect on tumors.

Compounds of formula (I)

In one aspect, the present application provides a compound, or a pharmaceutically acceptable salt thereof, having a structure according to formula D-E-L-M', wherein:

m' is-M-Lg, wherein Lg is a leaving group for nucleophilic substitution reaction, and M is a structural fragment bound to a targeting moiety;

l is a structural fragment linking M and E;

E is a structural fragment linking L and D;

D is a cytotoxic drug fragment.

In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

In some embodiments, lg is selected from the group consisting of halogen (e.g., F, cl, br, I), halo C _1-6 alkyl, C _1-6 alkylsulfonyl, halo C _1-6 alkylsulfonyl, halosulfonyl, C _1-6 alkylsulfonate, halo C _1-6 alkylsulfonate, C _1-6 alkylsulfinate, C _1-6 alkylsulfoxide, halophenoxy, hydroxyl (-OH), mercapto (-SH), amino (-NH ₂), nitro, azido, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments, the halo C _1-6 alkyl, C _1-6 alkylsulfonyl, halo C _1-6 alkylsulfonyl, halosulfonyl, C _1-6 alkylsulfonate, halo C _1-6 alkylsulfonate, C _1-6 alkylsulfinate, C _1-6 alkylsulfoxide, halophenoxy, alkenyl, alkynyl, and alkynyl-containing structural fragments optionally being substituted with one or more suitable substituents.

In some embodiments, lg is selected from the group consisting of halogen (e.g., F, cl, br, I), halo C _1-6 alkyl, C _1-6 alkylsulfonyl, halo C _1-6 alkylsulfonyl, halosulfonyl, C _1-6 alkylsulfonate, halo C _1-6 alkylsulfonate, C _1-6 alkylsulfinate, C _1-6 alkylsulfoxide, halophenoxy, hydroxyl (-OH), mercapto (-SH), amino (-NH ₂), nitro, azido, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments.

In some embodiments, lg is selected from halogen, substituted or unsubstituted C _1-6 alkylsulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH), or amino (-NH ₂).

In some embodiments, lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH), or amino (-NH ₂).

In some embodiments, lg is selected from methylsulfonyl.

In some embodiments, M' is selected from the following substituted or unsubstituted structural fragments:

In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of C _1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 9-12 membered nitrogen containing heterocyclyl, -N (R '), -NH (R'), -N (R ') ₂, -NHCH (R'), -C (=O) -, carbonyl, -O-, natural or unnatural amino acids and analogs thereof (such as Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH₂CH₂(OCH₂CH₂)rOCH₃))、Glu(R')、Lys(R')、 and amino acid-composed short peptides (such as Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH₂CH₂(OCH₂CH₂)rOCH₃))、Glu(R')、Lys(R')、 Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Cit、Val-Lys、Val-Lys(Ac)、Ala-Ala-Ala、Ala-D-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Gly、D-Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-D-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly(GGFG,SEQ ID NO:41)、Gly-Gly-Val-Ala(GGVA,SEQ ID NO:42)、Gly-Phe-Leu-Gly(GFLG,SEQ ID NO:43)、Glu-Ala-Ala-Ala(EAAA,SEQ ID NO:44)、Gly-Gly-Gly-Gly-Gly(GGGGG,SEQ ID NO:45))、 Wherein R' is composed of one or more groups including, but not limited to, hydrogen, C _1-6 alkyl, C _1-6 alkylene, amine, hydroxyl, carboxyl, acyl, -O-, -C _1-6 alkylene CO ₂H、-C_1-6 alkylene SO ₃H、-SO₃H、-PO₃H₂、-C_1-6 alkylene-NHC _1-6 alkyl, -C _1-6 alkylene-N (C _1-6 alkyl) ₂、-C_1-6 alkylene-heterocycle, -CH ₂N(C_1-6 alkyl) -C (=o) C _1-6 alkylene-heterocycle, -CH ₂NH-SO₃H、-CH₂N(C_1-6 alkyl) -SO ₃H、-CH₂NHC_1-6 alkylene-SO ₃H、-CH₂N(C_1-6 alkyl) C _1-6 alkylene-SO ₃H、-CH₂N(C_1-6 alkylene-SO ₃H)₂、-CH₂N⁺(C_1-6 alkylene-SO ₃H)₃、-CH₂N⁺(C_1-6 alkyl) ₂-C_1-6 alkylene-SO ₃H、-CH₂N(C_1-6 alkyl) -C (=o) C _1-6 alkylene-N ⁺(C_1-6 alkylene-SO ₃H)₃、-CH₂NH-C(＝O)C_1-6 alkylene-N ⁺(C_1-6 alkylene-SO ₃H)₃、-CH₂N(C_1-6 alkyl) -C (=o) C _1-6 alkylene-N ⁺(C_1-6 alkyl) ₃、-CH₂NH-C(＝O)C_1-6 alkylene-N ⁺(C_1-6 alkyl) ₃、-CH₂N(C_1-6 alkyl) -C (=o) OC _2-6 alkylene-N ⁺(C_1-6 alkyl) ₃、-CH₂N(C_1-6 alkyl) -C (=o) OC _2-6 alkylene-N ⁺(C_1-6 alkyl) ₂-CH₂CO₂H、-CH₂N(C_1-6 alkyl) -C _1-6 alkylene-CO ₂H、-CH₂N⁺(C_1-6 alkyl) ₂-C_1-6 alkylene-CO ₂ H, Glucosyl, galactosyl, glucuronyl, galactosyl, -CH ₂N(C_1-6 alkyl) -C (=o) - (CH ₂CH₂O)_r-C_1-6 alkyl, -CH ₂N(C_1-6 alkyl) -C (=o) - (OCH ₂CH₂)_r-OC_1-6 alkyl, - (CH ₂N(Me)-C(＝O))_r-C_1-6 alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e., - (CH ₂CH₂O)_r-C_1-6 alkyl), DOTA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid residue), DOTAGA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, α -propionyl), and, NOTA (1, 4, 7-Triazacyclononane-N, N', N "-triacetic acid residue) -C _1-6 alkylene-N (C _1-6 alkyl) -DOTA, -C _1-6 alkyl-N (C _1-6 alkyl) -DOTAGA, or-C _1-6 alkyl-N (C _1-6 alkyl) -NOTA, wherein r is selected from integers from 1 to 20, and s is selected from integers from 1 to 20.

In some embodiments, r is selected from integers from 1-15, such as integers from 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as r is 1,2,3, 4,5, 6,7,8,9, 10, 11, 12, 13, 14, or 15.

In some embodiments, s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, such as s is 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15.

In some embodiments, s is denoted as n.

In some embodiments, "-NOTA" means

In some embodiments, "-DOTA" means

In some embodiments, "-DOTAGA" means

In some embodiments, L is selected from substituted or unsubstituted structural fragments consisting of one or more of C _1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 9-12 membered nitrogen containing heterocyclyl, -N (R '), -NH (R '), -N (R ') ₂, carbonyl, -O-, natural or unnatural amino acids and analogs thereof (such as Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH₂CH₂(OCH₂CH₂)rOCH₃))、 and amino acid composed of a short peptide (such as Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Cit、Val-Lys、Val-Lys(Ac)、Ala-Ala-Ala、Ala-D-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Gly、D-Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-D-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly)、 Wherein R' represents hydrogen, C _1-6 alkyl, -C _1-6 alkylene CO ₂H、-C_1-6 alkylene SO ₃H、-SO₃H、-PO₃H₂、-C_1-6 alkylene-NHC _1-6 alkyl, -C _1-6 alkylene-N (C _1-6 alkyl) ₂、-CH₂N(C_1-6 alkyl) -C (=o) C _1-6 alkylene-heterocycle, -CH ₂NH-SO₃H、-CH₂N(C_1-6 alkyl) -SO ₃H、-CH₂NHC_1-6 alkylene-SO ₃H、-CH₂N(C_1-6 alkyl) C _1-6 alkylene-SO ₃H、-CH₂N(C_1-6 alkylene-SO ₃H)₂、-CH₂N⁺(C_1-6 alkylene-SO ₃H)₃、-CH₂N⁺(C_1-6 alkyl) ₂-C_1-6 alkylene-SO ₃H、-CH₂N(C_1-6 alkyl) -C (=o) C _1-6 alkylene-N ⁺(C_1-6 alkylene-SO ₃H)₃、-CH₂NH-C(＝O)C_1-6 alkylene-N ⁺(C_1-6 alkylene-SO ₃H)₃、-CH₂N(C_1-6 alkyl) -C (=o) C _1-6 alkylene-N ⁺(C_1-6 alkyl) ₃、-CH₂NH-C(＝O)C_1-6 alkylene-N ⁺(C_1-6 alkyl) ₃、-CH₂N(C_1-6 alkyl) -C (=o) OC _2-6 alkylene-N ⁺(C_1-6 alkyl) ₃、-CH₂N(C_1-6 alkyl) -C (=o) OC _2-6 alkylene-N ⁺(C_1-6 alkyl) ₂-CH₂CO₂H、-CH₂N(C_1-6 alkyl) -C _1-6 alkylene-CO ₂H、-CH₂N⁺(C_1-6 alkyl) ₂-C_1-6 alkylene-CO ₂ H, Glucosyl, galactosyl, glucuronyl, galactosyl, -CH ₂N(C_1-6 alkyl) -C (=o) - (CH ₂CH₂O)_r-C_1-6 alkyl, -CH ₂N(C_1-6 alkyl) -C (=o) - (OCH ₂CH₂)_r-OC_1-6 alkyl, - (CH ₂N(Me)-C(＝O))_r-C_1-6 alkyl, or a polyethylene glycol fragment containing 1-10 EO units (i.e., - (CH ₂CH₂O)_r-C_1-6 alkyl), wherein r is selected from integers from 1-20, and s is selected from integers from 1-20).

In some embodiments, L is selected from the group consisting of substituted or unsubstituted structural fragments consisting of one or more of C _1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N (R') -, carbonyl, -O-, natural amino acids or non-natural amino acids and analogs thereof (e.g., Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH₂CH₂(OCH₂CH₂)rOCH₃))、 and amino acid-composed short peptides (e.g., amino acids Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Cit、Val-Lys、Val-Lys(Ac)、Ala-Ala-Ala、Ala-D-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Gly、D-Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-D-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly)、 Wherein R 'consists of one or more groups including, but not limited to, hydrogen, C _1-6 alkyl, C _1-6 alkylene, amine, hydroxyl, carboxyl, acyl, -O-, glucosyl, galactosyl, glucuronyl, galactosyloxy, -CH ₂N(C_1-6 alkyl) -C (=O) - (CH ₂CH₂O)_r-C_1-6 alkyl, - (CH ₂N(Me)-C(＝O))_r-C_1-6 alkyl), polyethylene glycol fragments containing 1-10 EO units (i.e., - (CH ₂CH₂O)_r-C_1-6 alkyl), DOTA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid residue), DOTAGA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, α -propionyl), or NOTA (1, 4, 7-triazacyclononane-N, N' -triacetic acid residue), wherein R is selected from integers from 1 to 20, preferably R is selected from integers from 1 to 15, for example from 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, 1 to 2, for example R is an integer from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, s is selected from integers from 1 to 20, preferably s is selected from integers from 1 to 15, for example from 1 to 12, 3 to 12, for example s is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In some embodiments, L is selected from the group consisting of substituted or unsubstituted structural fragments consisting of one or more of C _1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N (R') -, carbonyl, -O-, natural amino acids or non-natural amino acids and analogs thereof (e.g., Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH₂CH₂(OCH₂CH₂)rOCH₃))、 and amino acid-composed short peptides (e.g., amino acids Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Cit、Val-Lys、Val-Lys(Ac)、Ala-Ala-Ala、Ala-D-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Gly、D-Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-D-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly)、 Wherein R' represents hydrogen, C _1-6 alkyl, glucosyl, galactosyl, glucuronyl, galactosyl, -CH ₂N(C_1-6 alkyl) -C (=O) - (CH ₂CH₂O)_r-C_1-6 alkyl, - (CH ₂N(Me)-C(＝O))_r-C_1-6 alkyl or a polyethylene glycol fragment containing 1-10 EO units (i.e., - (CH ₂CH₂O)_r-C_1-6 alkyl) wherein R is selected from an integer of 1-20 and s is selected from an integer of 1-20.

In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of C _1-6 alkylene, carbonyl, 9-12 membered nitrogen containing heterocyclyl 、-NH-、Ala-Ala、Ala-Lys、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Gly、D-Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-D-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly、 Wherein s is selected from integers from 1 to 20.

In some embodiments, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following:

s is selected from integers of 1-20.

s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, for example an integer of 1-12, 3-12, for example s is 1,2, 3,4, 5, 6,7,8,9, 10, 11, 12, 13, 14 or 15.

s is selected from integers of 1-20.

In some embodiments, L is formed by linking one or more substituted or unsubstituted structural fragments selected from group I below with one or more substituted or unsubstituted structural fragments selected from group II below:

Group I:

wherein s is selected from integers from 1 to 20;

Group II:

in some embodiments, L is selected from substituted or unsubstituted structural fragments consisting of one or more of:

in some embodiments, L contains the following structural fragments:

In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

Wherein s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, e.g. s is 1,2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15.

Wherein s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, for example an integer of 1-12, 3-12, for example s is 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably s is selected from 5, 8, 10.

In some embodiments, L contains the following structural fragments

In some embodiments, L is formed by linking a structural fragment of group I with one or more substituted or unsubstituted structural fragments selected from group II:

Group I is

Group II consists of The composition, wherein s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, e.g. s is 1,2,3, 4,5, 6,7,8,9, 10, 11, 12, 13, 14 or 15, preferably s is selected from 5, 8, 10.

In some embodiments, group II consists of The composition, wherein s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, e.g. s is 1,2,3, 4,5, 6,7,8,9, 10, 11, 12, 13, 14 or 15, preferably s is selected from 5, 8, 10.

In some embodiments, E is a single bond, a substituted or unsubstituted-NH-CH ₂ -, or a structural fragment selected from the group consisting of:

in some embodiments E is a single bond, substituted or unsubstituted-NH-CH ₂ -or

In some embodiments E is a substituted or unsubstituted-NH-CH ₂ -or

In some embodiments of the present invention, in some embodiments,A substituted or unsubstituted structure selected from the group consisting of:

n is an integer from 1 to 20. In some embodiments, n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, such as 1,2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15.

n is an integer from 1 to 20. In some embodiments, n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, such as n is 1,2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15.

s is selected from integers of 1-20. In some embodiments, s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, such as s is 1,2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15.

n is selected from an integer of 1-20, preferably from an integer of 1-15, such as an integer of 1-12, 3-12, such as 1, 2,3, 4, 5,6, 7,8,9,10, 11,12, 13, 14 or 15, preferably 5,8, 10.

n is selected from integers of 1-20, such as 1-12, 3-12, for example n is 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5,8, 10.

in some embodiments of the present invention, in some embodiments, A substituted or unsubstituted structure selected from the group consisting of:

Wherein n is selected from integers of 1-20, such as 1-12, 3-12, such as n is 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5,8, 10.

In some embodiments, the cytotoxic drug is selected from the group consisting of an anti-tubulin agent, a DNA intercalating agent, a DNA topoisomerase inhibitor, an RNA polymerase inhibitor, and a gene transcription inhibitor. In some embodiments, the tubulin inhibitor is an auristatin-based compound, a maytansinoid compound, or an eribulin-based compound. In some embodiments, the DNA intercalator is a Pyrrolobenzodiazepine (PBD) type compound, trabectedin, or lubidine. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, diflomotecan, lurtotecan, karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or Elomotecan)) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159582, and analogs thereof, docamicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α -amanitine (α -amanitin). In some embodiments, the gene transcription inhibitor is triptolide and pharmaceutically acceptable salts, esters, and analogs thereof.

In some embodiments, the cytotoxic drug is selected from a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, lubitecan, diflomotecan, lurtotecan, karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or Elomotecan).

The cytotoxic drugs disclosed in the present application typically contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH ₂), secondary amino (-NR ₁ H), tertiary amino (-NR ₂R₃), where R ₁、R₂、R₃ here represents only a non-hydrogen substituent on N, or sulfhydryl (-SH), which can react with suitable functional groups in the rest of the conjugate to effect attachment.

In some embodiments, the cytotoxic drug is attached to E in the antibody drug conjugate by-OH, primary amino, secondary amino, or tertiary amino groups thereon, or-SH. In some embodiments, D is a monovalent structure resulting from the loss of one H from the-OH, -NH2 or secondary amine group on the cytotoxic drug.

In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, isotopically labeled compounds, solvates, hydrates, isomers, or any crystalline forms or racemates thereof:

In some embodiments, the cytotoxic drug is selected from the following compounds or isotopically-labeled compounds thereof:

in some embodiments, the cytotoxic drug is selected from the group consisting of formulas III and IV below

Wherein R ₅,R₆ is each independently selected from OH, -NH ₂、-NH(C_1-6 alkyl), C _1-6 alkyl, and halogen, said C _1-6 alkyl optionally being further substituted with one or more substituents selected from halogen, hydroxy, C _1-6 haloalkyl, C _3-6 cycloalkyl;

r ₇ is selected from H, -OH, -NH ₂、-NH(C_1-6 alkyl), and-NH-CO- (C _1-6 alkylene) -OH, said C _1-6 alkyl optionally further substituted with one or more substituents selected from halogen, hydroxy, C _1-6 haloalkyl, C _3-6 cycloalkyl;

q is 1 or 2;

R ₈ and R ₉ are each independently selected from H, halogen and hydroxy, or R ₄ and R ₅ are linked to the linking carbon atom to form a 5-6 membered oxygen containing heterocyclic ring, said 5-6 membered oxygen containing heterocyclic ring optionally substituted with one or more substituents selected from deuterium (D), halogen, hydroxy, C _1-6 haloalkyl, C _3-6 cycloalkyl.

R ₁₀ is selected from hydrogen or-C _1-4 alkylene-NR _aR_b;

R ₁₁ is selected from the group consisting of C _1-6 alkyl and-C _1-4 alkylene-NR _aR_b, and

Wherein R _a、R_b at each occurrence is independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl, -SO ₂-C_1-6 alkyl, and-CO-C _1-6 alkyl, said C _1-6 alkyl and C _1-4 alkylene being optionally further substituted with one or more substituents selected from halogen, hydroxy, C _1-6 haloalkyl, C _3-6 cycloalkyl.

In some embodiments, each R ₅,R₆ is independently selected from OH, -NH ₂、C_1-4 alkyl, and halogen, the C _1-4 alkyl optionally being further substituted with one or more substituents selected from halogen, hydroxy, C _1-4 haloalkyl, C _3-6 cycloalkyl.

In some embodiments, R ₇ is selected from the group consisting of-H, -NH ₂、-NH(C_1-4 alkyl), and-NH-CO- (C _1-4 alkylene) -OH; the C _1-4 alkyl group is optionally further substituted with one or more substituents selected from halogen, hydroxy, C _1-4 haloalkyl, C _3-6 cycloalkyl.

In some embodiments, R _a、R_b is independently at each occurrence selected from H, C _1-4 alkyl, C _3-6 cycloalkyl, -SO ₂-C_1-4 alkyl, and-CO-C _1-4 alkyl, said C _1-4 alkyl optionally being further substituted with one or more substituents selected from halogen, hydroxy, C _1-6 haloalkyl, C _3-6 cycloalkyl.

In some embodiments, each R ₅,R₆ is independently selected from OH, -NH ₂、C_1-6 alkyl, and halogen;

R ₇ is selected from-H, -NH ₂、-NH(C_1-6 alkyl) and-NH-CO- (C _1-6 alkylene) -OH; and is also provided with

Q is 2.

In some embodiments, each R ₅,R₆ is independently selected from OH, -NH ₂、C_1-4 alkyl, and halogen. In some embodiments, each R ₅,R₆ is independently selected from OH, -NH ₂, methyl, ethyl, n-propyl, isopropyl, and halogen. In some embodiments, each R ₅,R₆ is independently selected from OH, -NH ₂, methyl, fluoro, and chloro.

In some embodiments, R ₇ is selected from the group consisting of-H, -NH ₂、-NH(C_1-4 alkyl), and-NH-CO- (C _1-4 alkylene) -OH. In some embodiments, R ₇ is selected from -H、-NH₂、-NH(CH₃)、-NH(CH₂CH₃)、-NH(CH(CH₃)₂)、-NH-CO-CH₂-OH、-NH-CO-CH₂CH₂-OH、-NH-CO-CH₂(CH₃)-OH. in some embodiments, R _7A is selected from-H, -NH ₂ and-NH-CO-CH ₂ -OH.

In some embodiments, the cytotoxic drug fragment D has the structure shown in formula III:

Wherein R _5A is selected from-O-or-N-, R _7A is selected from the group consisting of a bond, -O-, -NH-, -N (C _1-6 alkyl) -, and-NH-CO- (C _1-6 alkylene) -O-, said C _1-6 alkyl optionally being further substituted with one or more substituents selected from halogen, hydroxy, C _1-6 haloalkyl, C _3-6 cycloalkyl;

R ₅～R₇, q are as defined in any one of the preceding claims.

In some embodiments, R _7A is selected from the group consisting of a bond, -O-, -NH-, -N (C _1-4 alkyl) -, and-NH-CO- (C _1-4 alkylene) -O-, said C _1-4 alkyl optionally being further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, C _1-4 haloalkyl, C _3-6 cycloalkyl. In some embodiments, R _7A is selected from the group consisting of a chemical bond 、-O-、-NH-、-N(CH₃)-、-N(CH₂CH₃)-、-N(CH(CH₃)₂)-、-NH-CO-CH₂-O-、-NH-CO-CH₂CH₂-O-、-NH-CO-CH₂(CH₃)-O-. in some embodiments, R _7A is selected from the group consisting of-NH-; and-NH-CO-CH ₂ -O-.

In some embodiments, D is selected from the following structures:

in some embodiments, the cytotoxic drug is selected from the following compounds or isotopically-labeled compounds thereof ：1-1、1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、1-11、1-12、1-13、1-14、1-15、1-16、1-17、1-18、1-19、1-20、1-21、1-22、1-23、1-24、1-25、1-26、1-27、1-28、1-29、1-30、1-31、1-32、1-33、1-34、1-35、1-36、1-37、1-38、1-39,2-1、2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、2-11、2-12、2-13、2-14、2-15、2-16、2-17、2-18、2-19、2-20、2-21、2-22、2-23、2-24、2-25、2-26、2-27、2-28、2-29、2-30、2-31、2-32、2-33、2-34、2-35、2-36、2-37、2-38、2-39、2-40、2-41、2-42、2-43、2-44、2-45、2-46、2-47、2-48、2-49、2-50、2-51、2-52、2-53、2-54、2-55、2-56、2-57、2-58、2-59、2-60、2-61、2-62、2-63、2-64、2-65、2-66、2-67、2-68、2-69、2-70、2-71、2-72、2-73、2-74、2-75、2-76、2-77、2-78、2-79、2-80、2-81、2-82、2-83、2-84、2-85、2-86、2-87、2-88、2-89、2-90、2-91、2-92、2-93、2-94、2-95、2-96、2-97、2-98、2-99、2-100、2-101、2-102、2-103,3-1、3-2、3-3、3-4、3-51-1、1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、1-11、1-12、1-13、1-14、1-15、1-16、1-17、1-18、1-19、1-20、1-21、1-22、1-23、1-24、1-25、1-26、1-27、1-28、1-29、1-30、1-31、1-32、1-33、1-34、1-35、1-36、1-37、1-38、1-39,2-1、2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、2-11、2-12、2-13、2-14、2-15、2-16、2-17、2-18、2-19、2-20、2-21、2-22、2-23、2-24、2-25、2-26、2-27、2-28、2-29、2-30、2-31、2-32、2-33、2-34、2-35、2-36、2-37、2-38、2-39、2-40、2-41、2-42、2-43、2-44、2-45、2-46、2-47、2-48、2-49、2-50、2-51、2-52、2-53、2-54、2-55、2-56、2-57、2-58、2-59、2-60、2-61、2-62、2-63、2-64、2-65、2-66、2-67、2-68、2-69、2-70、2-71、2-72、2-73、2-74、2-75、2-76、2-77、2-78、2-79、2-80、2-81、2-82、2-83、2-84、2-85、2-86、2-87、2-88、2-89、2-90、2-91、2-92、2-93、2-94、2-95、2-96、2-97、2-98、2-99、2-100、2-101、2-102、2-103,3-1、3-2、3-3、3-4、3-5.

In some embodiments, the cytotoxic drug is selected from the following compounds or isotopically-labeled compounds thereof ：1-1、1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、1-11、1-12、1-13、1-14、1-15、1-16、1-17、1-18、1-19、1-20、1-21、1-22、1-23、1-24、1-25、1-26、1-27、1-28、1-29、1-30、1-31、1-32、1-33、1-34、1-35、1-36、1-37、1-38、1-39,2-1、2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、2-11、2-12、2-13、2-14、2-15、2-16、2-17、2-18、2-19、2-20、2-21、2-22、2-23、2-24、2-25、2-26、2-27、2-28、2-29、2-30、2-31、2-32、2-33、2-34、2-35、2-36、2-37、2-38、2-39、2-40、2-41,3-1、3-2、3-3、3-4、3-5.

in some embodiments, the compound or pharmaceutically acceptable salt thereof is selected from the group consisting of E-1~E-50, F-1~F-14, G-1~G-26, I-1~I-50 shown below:

wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, such as n is 1,2, 3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15.

In some embodiments, the application provides a compound as shown below, or a pharmaceutically acceptable salt thereof:

In some embodiments, there is provided a compound selected from E '-1~E' -50, F '-1~F' -3, F '-7~F' -12, G '-1~G' -4, I '-10-I' -49, E-1~E-50, F-1~F-14, G-1~G-26, I-1~I-50, or a pharmaceutically acceptable salt thereof, wherein n is selected from an integer from 1-20, such as an integer from 1-12, 3-12, such as n is 1,2, 3, 4,5, 6,7, 8, 9,10, 11, 12, 13, 14, or 15.

In some embodiments, there is provided a compound selected from E-1~E-50, F-1~F-14, G-1~G-24, or a pharmaceutically acceptable salt thereof, wherein n is selected from an integer from 1-20, such as an integer from 1-12, 3-12, such as n is 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

In some embodiments, a compound selected from E-1~E-50, F-1~F-12, or a pharmaceutically acceptable salt thereof, is provided.

In some embodiments, a compound selected from E-1~E-50, I-1~I-49, or a pharmaceutically acceptable salt thereof is provided.

In some embodiments, the compounds described above, or pharmaceutically acceptable salts thereof, may be optionally substituted with one or more suitable substituents.

Conjugates

In a second aspect, the invention provides a conjugate according to formula (II), wherein:

Ab-[M-L-E-D]x

formula (II)

Ab is a targeting moiety M, L, E and D are as described in any of the preceding claims;

x is 1 to 10.

In some preferred embodiments, the target of Ab is selected from the group consisting of epidermal growth factor 、Trop-2、CD37、HER2、CD70、EGFRvIII、Mesothelin、Folate eceoptor1、Mucin 1、CD138、CD20、CD19、CD30、SLTRK6、Nectin 4、Tissue factor、Mucin16、Endothelinreceoptor、STEAP1、SLC39A6、Guanylylcyclase C、PSMA、CCD79b、CD22、Sodium phosphate cotransporter2B、GPNMB、Trophoblast glycoprotein、AGS-16、EGFR、CD33、CD66e、CD74、CD56、PD-L1、TACSTD2、DR5、E16、STEAP1、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、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2、TENB2、 integrin α5β6,α4β7、FGF2、FGFR2、Her3、CD70、CA6、DLL3、DLL4、P-cadherin、EpCAM、pCAD、CD223、LYPD3、LY6E、EFNA4、ROR1、SLITRK6、5T4、ENPP3、SLC39A6、Claudin18.2、BMPR1B、E16、STEAP1、Tyro7、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2,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、ANGPTL4、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 TENB2.

In some preferred embodiments, ab 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), a polyene linking two aliphatic indoles, a cyanine dye, or IR-783, or a derivative thereof.

In some preferred embodiments, the Ab is an antibody, e.g., a monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof comprises Fab, fab ', F (Ab') ₂, fd, fv, dAb, a complementarity determining region fragment, a single chain antibody (e.g., scFv), a non-human antibody, a humanized antibody, a chimeric antibody, a fully human antibody, a pre-anti (Probody), a bispecific antibody, or a multispecific antibody.

In some preferred embodiments, ab is a monoclonal antibody against Her2, e.g., trastuzumab, pertuzumab (Pertuzumab).

In some embodiments, ab is an antibody or antigen binding fragment thereof. In some embodiments, the Ab is an antibody or antigen binding fragment thereof that specifically binds to ErbB family receptor tyrosine kinase member epidermal growth factor receptor 2 (Her 2).

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(1a) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 5 or a variant thereof, CDR-H2 of SEQ ID NO. 6 or a variant thereof, CDR-H3 of SEQ ID NO. 7 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 8 or a variant thereof, CDR-L2 of SEQ ID NO. 9 or a variant thereof, CDR-L3 of SEQ ID NO. 10 or a variant thereof,

(1B) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 20 or a variant thereof, CDR-H2 of SEQ ID NO. 21 or a variant thereof, CDR-H3 of SEQ ID NO. 22 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 23 or a variant thereof, CDR-L2 of SEQ ID NO. 24 or a variant thereof, CDR-L3 of SEQ ID NO. 25 or a variant thereof.

Wherein the variant of any of (1 a), (1 b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1,2, or3 amino acids) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution;

Or alternatively

(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(2a) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 18 or a variant thereof, CDR-H2 of SEQ ID NO. 19 or a variant thereof, CDR-H3 of SEQ ID NO. 7 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 8 or a variant thereof, CDR-L2 of SEQ ID NO. 9 or a variant thereof, CDR-L3 of SEQ ID NO. 10 or a variant thereof,

(2B) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 33 or a variant thereof, CDR-H2 of SEQ ID NO. 34 or a variant thereof, CDR-H3 of SEQ ID NO. 22 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 23 or a variant thereof, CDR-L2 of SEQ ID NO. 24 or a variant thereof, CDR-L3 of SEQ ID NO. 25 or a variant thereof.

Wherein the variant of any of (2 a), (2 b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1,2, or3 amino acids) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution;

Or alternatively

(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(3a) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 11 or a variant thereof, CDR-H2 of SEQ ID NO. 12 or a variant thereof, CDR-H3 of SEQ ID NO. 7 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 8 or a variant thereof, CDR-L2 of SEQ ID NO. 9 or a variant thereof, CDR-L3 of SEQ ID NO. 10 or a variant thereof,

(3B) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 26 or a variant thereof, CDR-H2 of SEQ ID NO. 27 or a variant thereof, CDR-H3 of SEQ ID NO. 22 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 23 or a variant thereof, CDR-L2 of SEQ ID NO. 24 or a variant thereof, CDR-L3 of SEQ ID NO. 25 or a variant thereof.

Wherein the variant of any of (3 a), (3 b), (3 c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has a substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1,2, or3 amino acids) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution;

Or alternatively

(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(4a) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 13 or a variant thereof, CDR-H2 of SEQ ID NO. 14 or a variant thereof, CDR-H3 of SEQ ID NO. 15 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 16 or a variant thereof, CDR-L2 of SEQ ID NO. 17 or a variant thereof, CDR-L3 of SEQ ID NO. 10 or a variant thereof,

(4B) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 28 or a variant thereof, CDR-H2 of SEQ ID NO. 29 or a variant thereof, CDR-H3 of SEQ ID NO. 30 or a variant thereof, and/or a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 31 or a variant thereof, CDR-L2 of SEQ ID NO. 32 or a variant thereof, CDR-L3 of SEQ ID NO. 25 or a variant thereof.

Wherein the variant of any of (4 a), (4 b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1,2 or3 amino acids) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution.

In some preferred embodiments, the antibody or antigen binding fragment thereof comprises:

(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein CDRs are defined by the Chothia numbering system:

Or alternatively

(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein CDRs are defined by the AbM numbering system:

Or alternatively

(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein CDRs are defined according to the Kabat numbering system:

Or alternatively

(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein CDRs are defined by the IMGT numbering system:

(1) The following heavy chain variable region (VH) and light chain variable region (VL), wherein CDRs are defined by the Chothia numbering system:

(1a) A heavy chain variable region (VH) comprising 3 CDRs of CDR-H1 of SEQ ID NO. 5, CDR-H2 of SEQ ID NO. 6, CDR-H3 of SEQ ID NO. 7, and a light chain variable region (VL) comprising 3 CDRs of CDR-L1 of SEQ ID NO. 8, CDR-L2 of SEQ ID NO. 9, CDR-L3 of SEQ ID NO. 10, or,

(1B) A heavy chain variable region (VH) comprising 3 CDRs from CDR-H1 of SEQ ID NO. 20, CDR-H2 of SEQ ID NO. 21, CDR-H3 of SEQ ID NO. 22, and a light chain variable region (VL) comprising 3 CDRs from CDR-L1 of SEQ ID NO. 23, CDR-L2 of SEQ ID NO. 24, CDR-L3 of SEQ ID NO. 25;

Or alternatively

(2) The following heavy chain variable region (VH) and light chain variable region (VL), wherein CDRs are defined by the AbM numbering system:

(2a) A heavy chain variable region (VH) comprising 3 CDRs from CDR-H1 of SEQ ID NO. 18, CDR-H2 of SEQ ID NO. 19, CDR-H3 of SEQ ID NO. 7, and a light chain variable region (VL) comprising 3 CDRs from CDR-L1 of SEQ ID NO. 8, CDR-L2 of SEQ ID NO. 9, CDR-L3 of SEQ ID NO. 10, or,

(2B) A heavy chain variable region (VH) comprising 3 CDRs from CDR-H1 of SEQ ID NO. 33, CDR-H2 of SEQ ID NO. 34, CDR-H3 of SEQ ID NO. 22, and a light chain variable region (VL) comprising 3 CDRs from CDR-L1 of SEQ ID NO. 23, CDR-L2 of SEQ ID NO. 24, CDR-L3 of SEQ ID NO. 25;

Or alternatively

(3) The following heavy chain variable region (VH) and light chain variable region (VL), wherein CDRs are defined according to the Kabat numbering system:

(3a) A heavy chain variable region (VH) comprising 3 CDRs from CDR-H1 of SEQ ID NO. 11, CDR-H2 of SEQ ID NO. 12, CDR-H3 of SEQ ID NO. 7, and a light chain variable region (VL) comprising 3 CDRs from CDR-L1 of SEQ ID NO. 8, CDR-L2 of SEQ ID NO. 9, CDR-L3 of SEQ ID NO. 10, or,

(3B) A heavy chain variable region (VH) comprising 3 CDRs from CDR-H1 of SEQ ID NO. 26, CDR-H2 of SEQ ID NO. 27, CDR-H3 of SEQ ID NO. 22, and a light chain variable region (VL) comprising 3 CDRs from CDR-L1 of SEQ ID NO. 23, CDR-L2 of SEQ ID NO. 24, CDR-L3 of SEQ ID NO. 25;

Or alternatively

(4) The following heavy chain variable region (VH) and light chain variable region (VL), wherein CDRs are defined by the IMGT numbering system:

(4a) A heavy chain variable region (VH) comprising 3 CDRs from CDR-H1 of SEQ ID NO. 13, CDR-H2 of SEQ ID NO. 14, CDR-H3 of SEQ ID NO. 15, and a light chain variable region (VL) comprising 3 CDRs from CDR-L1 of SEQ ID NO. 16, CDR-L2 of SEQ ID NO. 17, CDR-L3 of SEQ ID NO. 10, or,

(4B) A heavy chain variable region (VH) comprising 3 CDRs from CDR-H1 of SEQ ID NO. 28, CDR-H2 of SEQ ID NO. 29, CDR-H3 of SEQ ID NO. 30, and a light chain variable region (VL) comprising 3 CDRs from CDR-L1 of SEQ ID NO. 31, CDR-L2 of SEQ ID NO. 32, CDR-L3 of SEQ ID NO. 25.

(a) VH shown in SEQ ID NO. 1 or variant thereof, and/or VL shown in SEQ ID NO. 2 or variant thereof, or

(B) A VH shown in SEQ ID No. 3 or a variant thereof, and/or a VL shown in SEQ ID No. 4 or a variant thereof;

wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or the variant has a substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1,2, 3, 4, or 5 amino acids) to the sequence from which it is derived, preferably the substitution is a conservative substitution.

(a) VH shown in SEQ ID NO. 1, and VL shown in SEQ ID NO. 2, or

(B) VH shown in SEQ ID NO. 3, and VL shown in SEQ ID NO. 4.

In some embodiments, the antibody or antigen binding fragment thereof further comprises:

(a) A heavy chain constant region (CH) of a human immunoglobulin or variant thereof having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1,2, 3, 4, or 5 amino acid substitutions, deletions or additions) as compared to the wild-type sequence from which it is derived, and

(B) A light chain constant region (CL) of a human immunoglobulin or a variant thereof having a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., a substitution, deletion or addition of 1,2, 3, 4, or 5 amino acids) as compared to the wild-type sequence from which it is derived.

In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, igG2, igG3, or IgG4 heavy chain constant region, e.g., a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO. 35 or a variant thereof having a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10, or up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, or 5 amino acids) as compared to SEQ ID NO. 35.

In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO:36 or a variant thereof having a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10, or up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, or 5 amino acids) as compared to SEQ ID NO: 36.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO. 35 and a light chain constant region (CL) as set forth in SEQ ID NO. 36.

(1) A heavy chain comprising a VH having the sequence shown in SEQ ID NO. 1 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO. 35, and a light chain comprising a VL having the sequence shown in SEQ ID NO. 2 and a light chain constant region (CL) having the sequence shown in SEQ ID NO. 36, or

(2) A heavy chain comprising a VH of the sequence shown in SEQ ID NO. 3 and a heavy chain constant region (CH) shown in SEQ ID NO. 35, and a light chain comprising a VL of the sequence shown in SEQ ID NO. 4 and a light chain constant region (CL) shown in SEQ ID NO. 36.

(1) A heavy chain comprising the sequence shown in SEQ ID NO. 37, and a light chain comprising the sequence shown in SEQ ID NO. 38, or

(2) A heavy chain comprising the sequence shown in SEQ ID NO. 39, and a light chain comprising the sequence shown in SEQ ID NO. 40.

In some embodiments, the antibody or antigen binding fragment thereof is selected from Trastuzumab whose amino acid sequence is in query accession number (IMGT/mAb-DB ID) 97 in the IMGT database or Pertuzumab whose amino acid sequence is in query accession number (IMGT/mAb-DB ID) 80 in the IMGT database.

In certain embodiments of the antibodies or antigen binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

As known to those skilled in the art, pyroglutamic acid is a conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal glycine-lysine, and/or comprise an N-terminal glutamine or glutamic acid, a cyclisation of an N-terminal amino acid to pyroglutamic acid, or a cyclisation of an N-terminal amino acid to pyroglutamate.

In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen, and may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in a heavy chain, conversion of an N-terminal glutamine or glutamic acid in a heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., CHO cell) or during purification/storage.

In certain embodiments, the VH of the sequence set forth in SEQ ID No. 1 or 3 or variant thereof or the N-terminal glutamine of the heavy chain of the sequence set forth in SEQ ID No. 37 or 39 or variant thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate.

In certain embodiments, the heavy chain constant region (CH) as set forth in SEQ ID NO. 35 or a variant thereof or the heavy chain of the sequence as set forth in SEQ ID NO. 37 or 39 or a variant thereof lacks a C-terminal lysine.

In some embodiments, M is linked to a sulfhydryl (-SH) or amino (-NH ₂) group on the Ab.

In some embodiments, M is linked to a thiol (-SH) group on the Ab.

In some embodiments, in an antibody drug conjugate having the structure shown in Ab- [ M-L-E-D ] _x, M-L-E-D is formed from a compound shown in D-E-L-M ', preferably by removal of Lg from D-E-L-M ', wherein the compound shown in D-E-L-M ' is as previously defined, ab is as previously defined, and x is 1-10.

In some embodiments, in an antibody drug conjugate having the structure shown in Ab- [ M-L-E-D ] _x, M-L-E-D is formed from a compound shown as E ' -1~E ' -50, F ' -1~F ' -3, F ' -7~F ' -12, G ' -1~G ' -4, I ' -10-I ' -49, E-1~E-50, F-1~F-14, G-1~G-26, I-1~I-50, preferably by removal of the compound's-SO ₂ Me or pentafluorophenoxy, ab is as previously defined, and x is 1-10.

In certain embodiments, the antibodies in the antibody drug conjugate are conjugated to 1-4 (e.g., 1,2, 3, or 4) of the following structures:

Wherein is the point of attachment of the thiol group of the cysteine of the conjugated antibody.

In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC E-1-ADC E-50, ADC F-1-ADC F-14, ADC G-1-ADC G-26, ADC I-1-ADC I-50 shown below:

Wherein HA in each antibody drug conjugate is an antibody or antigen-binding fragment thereof, preferably the antibody or antigen-binding fragment thereof is as previously defined, n is selected from integers from 1-20, e.g. 1-12, 3-12, e.g. n is 1,2,3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15;

Wherein, the Representing the specific manner of attachment of the thiol group in the antibody or antigen-binding fragment thereof to the M fragment; Represents the specific manner of linking the amino group in the antibody or antigen binding fragment thereof to the M fragment.

In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC E '-1-ADC E' -50, ADC F '-1-ADC F' -3, ADC F '-7-ADC F' -12, ADC G '-1~G' -4, ADC I '-10-I' -49:

Wherein HA in each antibody drug conjugate represents an antibody or antigen binding fragment thereof that specifically binds to ErbB family receptor tyrosine kinase member epidermal growth factor receptor 2 (Her 2), n is selected from integers from 1-20, such as 1-12, 3-12, such as n is 1,2, 3, 4,5, 6,7,8,9, 10, 11, 12, 13, 14, or 15;

Wherein, the Or represents the specific manner of attachment of the thiol group in the antibody or antigen-binding fragment thereof to the M fragment; Represents the specific manner of linking the amino group in the antibody or antigen binding fragment thereof to the M fragment.

In some embodiments, the antibody or antigen binding fragment thereof is as previously defined.

In some embodiments, x in the conjugate shown in Ab- [ M-L-E-D ] x is 1-10, e.g., 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,2-3,2-4,2-5,2-6,2-7,2-8,2-9,2-10,3-4,3-5,3-6,3-7,3-8,3-9,3-10,4-5,4-6,4-7,4-8,4-9,4-10,5-6,5-7,5-8,5-9,5-10,6-7,6-8,6-9,6-10,7-8,7-9,7-10,8-9,8-10 or 9-10.

In some embodiments, x in the conjugate shown in Ab- [ M-L-E-D ] x is 1,2, 3, 4,5, 6,7,8,9, or 10. In some embodiments, the antibody drug conjugate of Ab- [ M-L-E-D ] x has an x of about 2. In some embodiments, the antibody drug conjugate of Ab- [ M-L-E-D ] x has an x of about 4.

In some embodiments, the conjugates of the invention are Antibody Drug Conjugates (ADCs).

The conjugates of the invention are optionally substituted with one or more suitable substituents.

Intermediate products

In some embodiments, the application provides intermediate compounds having the structure shown below, or salts, stereoisomers, tautomers, or isotopically-labeled compounds thereof:

Wherein the method comprises the steps of

PG ₁ is each independently H or a carboxyl protecting group, e.g. C _1-6 alkyl, allyl, benzyl, 2, 4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzoxybenzyl;

PG ₂ is each independently H or a hydroxy protecting group such as Trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-Tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl;

PG ₃ is each independently H or an amino protecting group such as an alkoxycarbonyl amino protecting group, e.g., carbobenzoxy (Cbz), t-butoxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl) oxycarbonyl, an acyl amino protecting group, e.g., phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o (p) nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl, an alkyl amino protecting group, e.g., trityl (Trt), 2, 4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

lg is a leaving group as defined above;

s is selected from an integer of 1-20, preferably from an integer of 1-15, for example from an integer of 1-12, 3-12, for example 1, 2,3, 4, 5,6, 7,8,9,10, 11,12, 13, 14 or 15, preferably 5,8, 10.

In another aspect, the application provides the use of an intermediate compound as described hereinbefore or a salt, stereoisomer, tautomer or isotopically-labelled compound thereof in the preparation of a compound of the application or a pharmaceutically acceptable salt thereof.

Synthesis method

The invention provides a method for synthesizing the following compounds:

the method comprises a step A of oxidizing the following compounds:

wherein n is any one of the above.

In some embodiments, the oxidation reaction is performed in the presence of sodium periodate and/or ruthenium trichloride hydrate.

In some embodiments, n is 10.

In some embodiments, the method further comprises step B of obtaining compound INT-1-2' by N- (((9H-fluoren-9-yl) methoxy) carbonyl) -N-methylglycine.

In some embodiments, step B is a solid phase synthesis method.

In some embodiments, the step B comprises the steps of resin preparation, coupling, deprotection, polypeptide cleavage, and purification.

In some embodiments, the reaction materials of step B are N- (((9H-fluoren-9-yl) methoxy) carbonyl) -N-methylglycine and 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoic acid.

In some embodiments, step B employs a 2-CTC resin.

In some embodiments, the coupling is performed in the presence of HATU and/or DIPEA reagents.

In some embodiments, the deprotection is performed in the presence of a piperidine reagent.

The invention provides a method for synthesizing the following compound A-26-3':

Wherein R ₅、R₆ is as defined in any one of the preceding claims, said method comprising the step of deprotecting a compound selected from the group consisting of:

In some embodiments, the R ₅ is selected from methyl, and R ₆ is selected from Cl;

In some embodiments, the PG ₃ is selected from Fmoc.

In some embodiments, the method further comprises the step of preparing compound A-26-3' from compound 1-4' and compound A-26-1 ':

in some embodiments, the methods are performed in the presence of HATU and/or DIPEA reagents.

Composition and method for producing the same

In another aspect, the application provides a composition, which may comprise a plurality of ADCs as described herein. Each antibody molecule in the composition may be conjugated to 1,2, 3, 4,5,6,7, 8,9 or 10 compounds of the application. Thus, the compositions are characterized by a "drug-to-antibody ratio" (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to the skilled artisan and include methods using reverse phase chromatography or HPLC-MS.

For example, in any embodiment, the compositions described herein have DAR of about 1 to about 10 or any subrange therebetween, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to10, about 4 to 5, about 4 to 6, about 4 to 8, about 4 to 9, about 4 to10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 6 to10, about 7 to10, about 10 to10, about 8, about 10 to10, about 7 to 9, or about 10 to 10.

In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5 to 6 about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5 about 5.0 to 8.0, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.5, about 7.0 to 7.5.

In certain embodiments, the DAR of the ADC compositions described herein is about 3.5-5.0, e.g., about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0.

In certain embodiments, the DAR of the ADC compositions described herein is about 1.0 to 6.0, e.g., about 1.0 to 5.5, about 1.0 to 5.0, about 1.5 to 6.0, about 1.5 to about 5.5, about 1.5 to 5.0, 2.0 to 5.5, about 2.0 to about 5.0, e.g., 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, About 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, about 1.5, about 1.51, about 1.52, About 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.6, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.7, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.8, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.71, about 1.72, about 1.75, about 1.78, about 1.85, about 1.82, about 1.83, about 1.84, about 1.85, about 1.84, about, About 1.87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2.07, about 2.08, about 2.09, about 2.1, about 2.11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21 About 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2.31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, about 2.54, about 2.55, about 2.4, about, About 2.56, about 2.57, about 2.58, about 2.59, about 2.6, about 2.61, about 2.62, about 2.63, about 2.64, about 2.65, about 2.66, about 2.67, about 2.68, about 2.69, about 2.7, about 2.71, about 2.72, about 2.73, about 2.74, about 2.75, about 2.76, about 2.77, about 2.78, about 2.79, about 2.8, about 2.81, about 2.82, about 2.83, about 2.84, about 2.85, about 2.86, about 2.87, about 2.88, about 2.89, about 2.85, About 2.9, about 2.91, about 2.92, about 2.93, about 2.94, about 2.95, about 2.96, about 2.97, about 2.98, about 2.99, about 3.0, about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, about 3.09, about 3.1, about 3.11, about 3.12, about 3.13, about 3.14, about 3.15, about 3.16, about 3.17, about 3.18, about 3.19, about 3.2, about 3.21, about 3.22, about 3.23, about 3.24, About 3.25, about 3.26, about 3.27, about 3.28, about 3.29, about 3.3, about 3.31, about 3.32, about 3.33, about 3.34, about 3.35, about 3.36, about 3.37, about 3.38, about 3.39, about 3.4, about 3.41, about 3.42, about 3.43, about 3.44, about 3.45, about 3.46, about 3.47, about 3.48, about 3.49, about 3.5, about 3.51, about 3.52, about 3.53, about 3.54, about 3.55, about 3.56, about 3.57, about 3.58, about 3.43, about 3.44, about 3.5, about 3.55, about 3.56, about, About 3.59, about 3.6, about 3.61, about 3.62, about 3.63, about 3.64, about 3.65, about 3.66, about 3.67, about 3.68, about 3.69, about 3.7, about 3.71, about 3.72, about 3.73, about 3.74, about 3.75, about 3.76, about 3.77, about 3.78, about 3.79, about 3.8, about 3.81, about 3.82, about 3.83, about 3.84, about 3.85, about 3.86, about 3.87, about 3.88, about 3.89, about 3.9, about 3.91, about 3.92, about 3.93 About 3.94, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4.0, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.06, about 4.07, about 4.08, about 4.09, about 4.1, about 4.11, about 4.12, about 4.13, about 4.14, about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.2, about 4.21, about 4.22, about 4.23, about 4.24, about 4.25, about 4.26, about 4.27, about 4.12, about 4.1, about 4.2, about 4.19, about 4.2, about 4.22, about 4.23, about 4.24, about 4.25, About 4.28, about 4.29, about 4.3, about 4.31, about 4.32, about 4.33, about 4.34, about 4.35, about 4.36, about 4.37, about 4.38, about 4.39, about 4.4, about 4.41, about 4.42, about 4.43, about 4.44, about 4.45, about 4.46, about 4.47, about 4.48, about 4.49, about 4.5, about 4.51, about 4.52, about 4.53, about 4.54, about 4.55, about 4.56, about 4.57, about 4.58, about 4.59, about 4.6, about 4.61, about 4.62, About 4.63, about 4.64, about 4.65, about 4.66, about 4.67, about 4.68, about 4.69, about 4.7, about 4.71, about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78, about 4.79, about 4.8, about 4.81, about 4.82, about 4.83, about 4.84, about 4.85, about 4.86, about 4.87, about 4.88, about 4.89, about 4.9, about 4.91, about 4.92, about 4.93, about 4.94, about 4.95, about 4.96, about 4.81, about 4.82, about 4.89, about 4.85, about 4.86, about 4.93, about 4.94, about 4.95, about 4.96, About 4.97, about 4.98, about 4.99, about 5.0.

In certain embodiments, the DAR of the ADC compositions described herein is about 4.41, about 4.34, about 4.51, about 4.36, about 4.63, about 4.28, about 4.38, about 3.86, about 4.16, about 3.99, or about 2.45.

In certain embodiments, the DAR for the ADC compositions described herein is 4.41, 4.34, 4.51, 4.36, 4.63, 4.28, 4.38, 3.86, 4.16, 3.99, or 2.45.

Pharmaceutical composition

In another aspect, the application provides a pharmaceutical composition comprising a compound of any one of the preceding claims or a pharmaceutically acceptable salt thereof, a conjugate of any one of the preceding claims or a composition of any one of the preceding claims, and one or more pharmaceutically acceptable excipients.

The compounds or conjugates described herein are typically formulated in unit injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, e.g., bolus injection, intravenous injection, intratumoral injection, and the like. Optionally, the antibody drug conjugate of desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilizate or solution (Remington's Pharmaceutical Sciences (1980) 16 ^th edition, osol, a.ed.). The antibody drug conjugates described herein or pharmaceutical compositions containing the antibody drug conjugates may be administered by any route appropriate to the individual to be treated.

Application of

The present application provides the use of a compound as hereinbefore described, or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of cancer.

The present application provides a compound as described hereinbefore, or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, for use in the treatment of cancer.

The present application provides a compound as described hereinbefore, or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, for use in the treatment of a solid tumor or hematological malignancy, e.g., selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, such as lung adenocarcinoma in particular), and urothelial cancer.

The present application provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a compound as described herein before or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof.

In some embodiments, the cancer is selected from a solid tumor or a hematological malignancy, e.g., selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, such as lung adenocarcinoma in particular), and urothelial cancer.

In some embodiments, the antibody drug conjugate, compound, drug-linker, or pharmaceutical composition containing the same is sufficient (e.g., in a subject):

(1) Inhibit proliferation of cells (e.g., tumor cells);

(2) Inhibiting tumor growth;

(3) Inducing and/or increasing antibody dependent cellular cytotoxicity activity;

(4) Inhibit signal transduction;

(5) Preventing and/or treating cancer, or

(6) Any combination of the above (1) - (5).

In some embodiments, the cancer disease is selected from solid tumors or hematological malignancies, for example, from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, such as lung adenocarcinoma in particular), and urothelial cancer.

All technical features disclosed in this specification, such as the definition of individual groups, etc., except for mutually exclusive technical features, can be combined in any way to obtain different general formula ranges or embodiments. These ranges and schemes are within the scope of the invention.

Definition of the definition

Unless defined otherwise hereinafter, all technical and scientific terms used herein are intended to be identical to what is commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those that are obvious to those skilled in the art or alternatives to equivalent techniques. Moreover, the laboratory procedures of genomics, nucleic acid chemistry, molecular biology, and the like, as used herein, are all routine procedures widely used in the corresponding fields. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

The term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a Light Chain (LC) and a Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs arranged from amino-terminus to carboxyl-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The assignment of amino acids to regions or domains may follow various numbering systems known in the art. The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized to pyroglutamate. Thus, in a composition comprising the antibodies disclosed herein, each of the antibodies may independently comprise a C-terminal lysine, lack a C-terminal glycine-lysine, and/or comprise N-terminal glutamine or glutamate or an N-terminal amino acid cyclize to pyroglutamate.

The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Three CDRs are contained in each of the variable regions of the heavy and light chains, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, such as may be defined in accordance with Kabat numbering system (Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883), IMGT numbering system (LEFRANC ET al, dev. Comparat. Immunol.27:55-77,2003) or AbM numbering system (Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:Acombined algorithm.Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to the person skilled in the art (see, for example, LEFRANC ET al. Dev. Comparat. Immunol.27:55-77,2003).

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

The following general rules (published in www.bioinf.org.uk: andrew C.R.Martin professor) can be used to define the CDRs in an antibody sequence, including those that specifically interact with amino acids that are comprised of an epitope to which the antibody binds. In rare cases, these generally constant features do not occur, but Cys residues are the most conserved features.

The entire amino acid sequence of V _H is generally numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the numbering systems described above. In certain embodiments, the amino acid positions in V _H may be numbered sequentially starting at amino acid position 1 and proceeding to the end of the sequence, and may also be numbered according to Kabat. Unless otherwise indicated, the amino acid positions in V _H and V _L described herein are defined according to sequential numbering.

The numbering of the amino acid sites in the heavy chain constant region may be sequentially numbered from amino acid site 1 to the end of the sequence, or may be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence according to Eu numbering starts at position 118 and ends at position 447. Unless otherwise indicated, the amino acid positions of the heavy and light chains described herein are defined according to sequential numbering.

The term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. 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.

The term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, e.g., a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, which is also referred to as an "antigen-binding portion. Generally, see Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, RAVEN PRESS, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies non-limiting examples of antigen binding fragments include Fab fragments, fab ' fragments, F (ab) ' ₂ fragments, F (ab) ' ₃ fragments, fd, fv, scFv, di-scFv, (scFv) ₂, disulfide stabilized Fv proteins ("dsFv"), single domain antibodies (sdabs, nanobodies) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.

The term "Fd" means an antibody fragment consisting of a VH and CH1 domain, the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al, nature 341:544 546 (1989)), the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CH1 domain, the term "F (ab ') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and the term "Fab ' fragment" means a fragment obtained by reducing the disulfide bond joining the two heavy chain fragments in the F (ab ') ₂ fragment, consisting of one complete light and heavy chain Fd fragment consisting of a VH and CH1 domain.

The term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

The term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody to the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.

The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckaphun, the Pharmacology of Monoclonal Antibodies, vol 113, roseburg and Moore, springer-Verlag, new York, pp 269-315 (1994)). 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 a repeated GGGGS (SEQ ID NO: 46) amino acid sequence or variant thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ (SEQ ID NO: 47) may be used, but variants thereof may also be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56, and Roovers et al (2001), cancer Immunol. In some cases, disulfide bonds may also exist between VH and VL of scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, an scFv comprising NH ₂-VH-VH-COOH、NH_2- VL-VL-COOH.

The term "single-domain antibody" (sdAb) has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which a full-length antibody binds (Holt, l. Et al, trends biotechnology (Trends in Biotechnology), 21 (11): 484-490, 2003). Single domain antibodies are also known as nanobodies (nanobodies).

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

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.

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

The term "murine antibody" refers to an antibody obtained by fusing B cells of an immunized mouse with myeloma cells, screening a mouse hybrid fused cell capable of both immortalizing and secreting the antibody, followed by screening, antibody production and antibody purification, or to an antibody secreted by plasma cells formed by differentiation and proliferation of B cells after an antigen has invaded the body of a mouse.

The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain the desired properties of the donor antibody including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having the desired properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or ability to enhance an immune response).

The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be achieved using, for example, the method of Needleman et al (1970) J.mol.biol.48:443-453, which may be conveniently performed by a computer program such as the Align program (DNAstar, inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which has been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available on www.gcg.com) using the Blossum 62 matrix or PAM250 matrix and the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8,6 or 4 and the length weights of 1,2, 3, 4, 5 or 6.

The term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., ,Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),, incorporated by reference herein. In the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

The term "linker" refers to a structural fragment that links a cytotoxic drug to an antibody or antigen-binding fragment. For example, the structural fragment of-M-L-E-in formula Ab- [ M-L-E-D ] _x.

The term "drug-linker" refers to the structure of the cytotoxic drug and linker of the present application prior to attachment to an antibody or antigen binding fragment thereof. For example, a "drug-linker" refers to M '-L-E-D, wherein M' is the structural form of M prior to covalent attachment to an antibody or antigen-binding fragment thereof. The "drug-linker" is covalently linked to an antibody or antigen-binding fragment thereof to provide an antibody drug conjugate of the application.

The term "drug-linker" also includes all pharmaceutically acceptable isotopically-labeled compounds thereof, which are identical to the "drug-linker" compounds of the present invention, except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevailing in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., ²H、³ H, deuterium D, tritium T), isotopes of carbon (e.g., ¹¹C、¹³ C and ¹⁴ C), isotopes of chlorine (e.g., ³⁷ Cl), isotopes of fluorine (e.g., ¹⁸ F), isotopes of iodine (e.g., ¹²³ I and ¹²⁵ I), isotopes of nitrogen (e.g., ¹³ N and ¹⁵ N), isotopes of oxygen (e.g., ¹⁵O、¹⁷ O and ¹⁸ O), and isotopes of sulfur (e.g., ³⁵ S).

The terms "comprising," "including," "having," "containing," or "involving," and other variations thereof herein, are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps.

The term "alkyl" means a group obtained by removing 1 hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C _1-20 alkyl", "C _1-10 alkyl", "C _1-6 alkyl", "C _1-4 alkyl", "C _1-3 alkyl", and the like, and specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 1, 2-dimethylpropyl, and the like.

The term "alkylene" refers to a straight or branched hydrocarbon group from which 2 hydrogen atoms have been removed, such as "C _1-6 alkylene", "C _1-5 alkylene", "C _1-4 alkylene", "C _1-3 alkylene", and specific examples include, but are not limited to, methylene, ethylene, propylene, butylene, and the like.

The term "aryl" refers to an unsaturated carbocyclic group having a conjugated pi electron system, such as "6-10 membered aryl", specific examples include, but are not limited to, phenyl, naphthyl.

The term "heteroaryl" refers to an unsaturated group having a conjugated pi-electron system consisting of ring atoms with at least one (e.g., 1,2,3, or 4) ring atom being a heteroatom, such as N, O and S, wherein the nitrogen atom is optionally quaternized and the nitrogen and sulfur heteroatoms are optionally oxidized. Such as "5-12 membered heteroaryl", "5-11 membered heteroaryl", "5-10 membered heteroaryl", "5-9 membered heteroaryl", "5-6 membered heteroaryl", specific examples include, but are not limited to, phenyl, naphthylfuryl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indazolyl, indolyl, quinolinyl, isoquinolinyl.

The term "heterocyclyl" refers to a saturated or partially unsaturated cyclic group consisting of ring atoms, at least one (e.g., 1,2,3,4, or 5 heteroatoms) of which is a heteroatom, e.g., N, O and S, wherein the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms are optionally oxidized, and the carbon atom is optionally oxo. The heterocyclic group includes monocyclic, bicyclic or polycyclic rings including spiro, fused or bridged rings. The term "nitrogen-containing heterocyclyl" refers to a nitrogen-containing heterocyclic group having at least one heteroatom such as "5-12 membered nitrogen-containing heterocyclyl", "5-9 membered nitrogen-containing heterocyclyl", "9-12 membered nitrogen-containing heterocyclyl", and specific examples include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, 1,2,3, 4-tetrahydroquinolinyl.

The term "isotopically-labeled compound" means that the compound has the same structure as the compound of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevailing in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., ²H、³ H, deuterium D, tritium T), isotopes of carbon (e.g., ¹¹C、¹³ C and ¹⁴ C), isotopes of chlorine (e.g., ³⁷ Cl), isotopes of fluorine (e.g., ¹⁸ F), isotopes of iodine (e.g., ¹²³ I and ¹²⁵ I), isotopes of nitrogen (e.g., ¹³ N and ¹⁵ N), isotopes of oxygen (e.g., ¹⁵O、¹⁷ O and ¹⁸ O), and isotopes of sulfur (e.g., ³⁵ S).

As used herein, the term "suitable substituent" refers to variations of a compound that can be made by one skilled in the art as desired for the compound substituent. "suitable substituents" include oxo (=o), halogen, cyano, NR ⁸R⁹, carboxyl, mercapto, hydroxy, ester (e.g. -C _1-6 alkyl-C (=o) -OC _1-6 alkyl), C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyl-O-C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _3-6 cycloalkyl, 3-to 10-membered heterocyclyl, 5-to 10-membered heteroaryl, C _6-10 aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C _1-6 haloalkoxy, R ⁸、R⁹ are each independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl, 3-to 10-membered heterocyclyl, 5-to 10-membered heteroaryl, C _6-10 aryl, C _1-6 alkoxy, c _1-6 haloalkyl, C _1-6 haloalkoxy, halogen, hydroxy, carboxy and ester groups (e.g. -C _1-6 alkyl-C (=o) -OC _1-6 alkyl).

The term "substitution" refers to replacement of one or more (e.g., 1,2, 3, 4, or 5) hydrogens on the designated compound or structural fragment with a substituent, provided that the designated atom's normal valency under the present circumstances is not exceeded and the substitution forms a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. In some embodiments, the substituents each independently consist of one or more of the following structures: -O-, -S-, -NR' -, halogen, -CN, -OH, -NH ₂、-NO₂、-CN、＝O、C₁-C₆ (alkylene) alkyl, C ₁-C₆ (alkylene) halogeno, C ₁-C₆ (alkoxy), C ₂-C₆ alkenyl (ene), C ₂-C₆ alkynyl (ene), C ₃-C₈ cycloalkyl (ene), 3-8 membered heterocyclyl (ene), C ₆-C₁₀ aryl (ene), and 5-10 membered heteroaryl (ene), and the like. In some embodiments, the substituents are each independently composed of one or more of NR ⁸R⁹, -O-, -S-, -NR' -, halogen, -CN, -OH, -SH, -NH ₂、-NO₂、-C(O)-、-CN、＝O、C₁-C₆ (alkylene), C ₁-C₆ halo (alkylene) alkyl, C ₁-C₆ alkoxy, C ₂-C₆ (ene) alkenyl, C ₂-C₆ (ene) alkynyl, C ₃-C₈ (ene) cycloalkyl, 3-10 membered heterocyclyl, C ₆-C₁₀ (arylene) and 5-10 membered heteroaryl, and the like, wherein R ⁸、R⁹ and R' are as previously defined. For example, the substituents may be suitable substituents as described above.

If a functional group or structural fragment is described as "substituted or unsubstituted," the functional group or structural fragment may be (1) unsubstituted or (2) substituted.

The numerical values of the present application are modified by the term "about," whether or not explicitly indicated. The term "about" means within + -20%, within + -10%, preferably within + -5%, more preferably within + -2% of the stated value.

Drawings

FIG. 1 results of drug efficacy of anti-human HER2 antibody-conjugated drug ADC on NCI-N87 cell subcutaneous tumor-bearing murine model FIG. 2 changes in weight of mice of each group in human gastric cancer cell NCI-N87 CDX model

FIG. 3 results of drug efficacy of anti-human HER2 antibody conjugated drug ADC on NCI-N87 cell subcutaneous tumor-bearing murine model

FIG. 4 results of drug efficacy of anti-human HER2 antibody conjugated drug ADC on NCI-N87 cell subcutaneous tumor-bearing murine model FIG. 5 changes in weight of mice of each group in human gastric cancer cell NCI-N87 CDX model

Detailed Description

The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting. Various modifications and improvements may be made by those skilled in the art in light of the teachings of this invention without deviating from the basic spirit and scope of the invention.

The information of the sequences to which the invention relates is described in the following table:

abbreviations used herein have the following meanings:

The structures of the compounds described in the examples below were determined by nuclear magnetic resonance (¹ H NMR) or Mass Spectrometry (MS).

Nuclear magnetic resonance (1H NMR) was measured using a Bruker 400MHz NMR apparatus, the deuterating agent was hexadeuterated dimethyl sulfoxide (DMSO-d 6), and the internal standard substance was Tetramethylsilane (TMS).

Abbreviations in Nuclear Magnetic Resonance (NMR) spectra used in the examples are shown below.

S: singlet (singlet), d: doublet (doublet), t: triplet (triplet), q: quartet (quartet), m: multiplet (multiplet), br: broad peak (broad), J: coupling constant, hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. Delta values are expressed in ppm values.

Mass Spectrometry (MS) was determined using an Agilent (ESI) mass spectrometer model Agilent 6120B.

Preparation example preparation of (R) -N- ((1S, 9S) -4-chloro-9-ethyl-5-fluoro-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ d ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) -3-hydroxybutyramide (1-36)

(1S, 9S) -1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H, 13H-benzo [ d ] pyrano [3',4':6,7] indolizine [1,2-b ] quinoline-10, 13-dione (30 mg, 52.64. Mu. Mol), (R) -3-hydroxybutyric acid (10.96 mg, 105.28. Mu. Mol), HATU (30.02 mg, 78.96. Mu. Mol) and DIPEA (20.41 mg, 157.93. Mu. Mol) were dissolved in DMF (2 mL), the reaction system was stirred at 25℃for 2 hours, and the reaction solution was prepared by high performance liquid chromatography and then freeze-dried to give the title compound (17 mg, 31.42. Mu. Mol).

The separation and purification method comprises the following steps:

chromatography column Waters XBridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

The structural characterization data are as follows:

ESI-MS(m/z):542.1[M+H]⁺

¹H NMR(400MHz,DMSOd6)δ8.43(d,J＝8.8Hz,1H),8.06(d,J＝10.4Hz,1H),7.33(s,1H),6.54(s,1H),5.63-5.55(m,1H),5.43(s,2H),5.27(d,J＝18.8Hz,1H),5.21(d,J＝18.8Hz,1H),4.66(d,J＝4.8Hz,1H),4.09-3.98(m,1H),3.32-3.26(m,2H),2.32-2.25(m,1H),2.24-2.12(m,3H),1.93-1.78(m,2H),1.08(d,J＝6.0Hz,3H),0.87(t,J＝7.2Hz,3H).

Preparation of (S) -N- ((1S, 9S) -4-chloro-9-ethyl-5-fluoro-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ d ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) -3-hydroxybutyramide (1-39)

(1S, 9S) -1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H, 13H-benzo [ d ] pyrano [3',4':6,7] indolizine [1,2-b ] quinoline-10, 13-dione (30 mg,0.053mmol, trifluoroacetate), (S) -3-hydroxybutyric acid (16.5 mg,0.158 mmol) was dissolved in DMF (0.5 mL), then HATU (40.0 mg,0.105 mmol) and DIPEA (20.4 mg,0.158 mmol) were added and the reaction was continued at room temperature for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (13.5 mg,0.024 mmol).

The structural characterization data are as follows:

MS m/z(ESI):542.1[M+H]⁺

¹HNMR(400MHz,DMSOd6)δ8.44(d,J＝8.8Hz,1H),8.09(d,J＝10.4Hz,1H),7.34(s,1H),6.56(m,1H),5.64-5.60(m,1H),5.44(s,2H),5.32-5.25(m,2H),4.63(d,J＝4.8Hz,1H),4.06-4.01(m,1H),3.31-3.26(m,2H),2.28-2.23(m,1H),2.20-2.13(m,3H),1.90-1.82(m,2H),1.08(d,J＝6.4Hz,3H),0.87(t,J＝7.2Hz,3H).

The separation and purification method comprises the following steps:

Chromatography column Waters SunFire Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Intermediate example one preparation of 1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decazatriacontane-31-carboxylic acid (INT-1):

Step one preparation of 1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decazatriacontane-31-carboxylic acid (INT-1-2)

Standard solid phase synthesis methods were used:

1) Resin preparation 2-CTC resin (3.00 mmol,3.70g,0.81 mmol/g) and N- (((9H-fluoren-9-yl) methoxy) carbonyl) -N-methylglycine (3.00 mol,933mg,3.00 eq.) and DIPEA (4.00 eq.) were added to dichloromethane (10.0 mL) and reacted under nitrogen for 2 hours. MeOH (1.0 mL) was then added to the resin under nitrogen bubbling over 30 minutes and the resin was obtained by filtration.

2) Coupling A solution of N- (((9H-fluoren-9-yl) methoxy) carbonyl) -N-methylglycine (5.60 g,6.00 eq.) and HATU (6.58 g,5.70 eq.) in DMF (10.0 mL) was added to the resin with nitrogen bubbling. After dropping DIPEA (6.00 eq.) nitrogen was bubbled for 30 minutes at 20 ℃. The resin was washed with DMF (30.0 mL. Times.5) before proceeding to the next step.

3) Deprotection 20% piperidine in DMF (30.0 mL) was added to the resin and sparged with nitrogen for 30 minutes at 20 ℃. The resin was then washed with DMF (30.0 mL. Times.5).

4) Steps 2 and 3 were repeated using materials numbered 2-10 in table 1 and coupling reagents.

5) The resin was then washed with DMF (30.0 mL. Times.5), meOH (30.0 mL. Times.5) and then dried under vacuum.

TABLE 1

Cleavage and purification of the polypeptide:

1) The cleavage solution (TFA/DCM, 1/100, v/v,200.0 mL) was added to the flask containing the dried resin from solid phase synthesis 5 described above and stirred for 3 min, filtered and this procedure repeated twice.

2) After filtration, the filtrates were combined and concentrated.

3) The crude product was purified by high performance liquid chromatography and lyophilized to give the title compound (1117.1 mg, TFA salt).

The structural characterization data are as follows:

ESI-MS(m/z):1010.4(M+H)⁺。

the purification method is as follows:

Step two preparation of 1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decazatriacontane-31-carboxylic acid (INT-1)

1- (3, 5-Bis (2- (methylthio) pyrimidin-5-yl) phenyl) -2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31-carboxylic acid (665 mg, 615.04. Mu. Mol) was added to water (7.5 mL) and acetonitrile (15 mL), sodium periodate (1.32 g,6.15 mmol) and ruthenium trichloride hydrate (51.03 mg, 246.02. Mu. Mol) were added and reacted at 25℃for 1 hour. The reaction was directly purified by reverse phase (acetonitrile/water (0.05% formic acid) =0-25%), and freeze-dried to give the title compound (515 mg, 449.69. Mu. Mol).

The structural characterization data are as follows:

ESI-MS(m/z):1074.4(M+H)⁺。

intermediate example two preparation of 1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid (INT-2)

Step one preparation of methyl 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoate (INT-2-2)

Raw material 3, 5-dibromobenzoate (720 mg,2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg,5.14 mmol), XPhosPd G (207 mg,245 μmol), K ₃PO₄ (1.56 g,7.35 mmol) were added to dioxane (12 mL) and water (4 mL), the reaction system was stirred under nitrogen atmosphere at 90℃for 3 hours, the reaction was monitored by LC-MS, celite was filtered, water and ethyl acetate were added to the filtrate, extraction and concentration were carried out to obtain crude product, and 710mg of methyl 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoate was purified by column chromatography (EA/PE=0 to 25%).

The structural characterization data are as follows:

ESI-MS(m/z):385.1[M+H]⁺。

Step two preparation of 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoic acid (INT-2-3)

The compound methyl 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoate (650 mg,1.69 mol), lithium hydroxide (121 mg,5.07 mmol) was dissolved in THF (2 mL), meOH (2 mL) and H ₂ O (2 mL). The reaction was stirred at 25℃for 2 hours, monitored by LC-MS, the pH of the system was adjusted to about 2 with 1N HCl, a large amount of solids precipitated, the filter cake was collected by filtration, and 560mg of 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoic acid was obtained by drying.

The structural characterization data are as follows:

ESI-MS(m/z):371.1[M+H]⁺。

Step III preparation of 1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosahederane-29-oic acid tert-butyl ester (INT-2-4)

3, 5-Bis (2- (methylthio) pyrimidin-5-yl) benzoic acid (3.00 g,8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-carboxylate (4.03 g,8.10 mmol) were added to DMF (40 mL), HOBt (3.28 g,24.3 mmol), EDCI (4.66 g,24.3 mmol) and DIPEA (4.19 g,32.4mmol,5.64 mL) were added sequentially and the reaction stirred at 60℃for 2 h. Water (100 mL) and ethyl acetate were added to the reaction solution to extract (60 mL. Times.3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give tert-butyl 1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosahederane-29-carboxylate (4.20 g,4.14 mmol) which was used in the next step without purification.

Preparation of 1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosahederan-29-oic acid (INT-2-5)

1- (3, 5-Bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosahederane-29-oic acid tert-butyl ester (3.60 g,4.24 mmol) was dissolved in dichloromethane (30 mL), TFA (15.3 g,134mmol,10 mL) was added and the reaction stirred at 25℃for 6 h. To the reaction solution was added water (60 mL) and ethyl acetate for extraction (40 mL. Times.3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated to give crude product, which was purified by preparative high performance liquid chromatography and freeze-dried to give 1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosahederane-29-acid (2.93 g,3.63 mmol).

The structural characterization data are as follows:

ESI-MS(m/z):794.3[M+H]⁺。

the purification method is as follows:

phenomenex luna C18 chromatographic columns (250 mm. Times.70 mm. Times.10 μm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Step five preparation of 1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosahederane-29-oic acid (INT-2)

1- (3, 5-Bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosahedan-29-oic acid (148 mg,0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), sodium periodate (398.71 mg,1.86 mmol) and ruthenium trichloride hydrate (15.47 mg, 74.56. Mu. Mol) were further added to the reaction system, and the reaction system was stirred at 25℃for 30 minutes, extracted with water and ethyl acetate, and concentrated to give 1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-aza-icosaan-29-oic acid (155 mg).

The structural characterization data are as follows:

ESI-MS(m/z):858.3[M+H]⁺。

Examples of intermediates preparation of allyl (5-amino-2- (((tert-butyldiphenylsilyl) oxy) methyl) benzyl) (meth) carbamate (INT-3)

Step one preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-3-2)

6-Nitroisobenzofuran-1 (3H) -one (50.0 g,279 mmol) was added to a solution of methylamine in tetrahydrofuran (2M, 500 mL), warmed to 75℃and stirred for 5 hours. The reaction was concentrated directly to give crude title compound (58.0 g, crude) which was used in the next step without purification.

The structural characterization data are as follows:

ESI-MS(m/z):211.0[M+H]⁺。

Preparation of step two (2- ((methylamino) methyl) -4-nitrophenyl) methanol (INT-3-3)

2-Hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g,119 mmol) was dissolved in tetrahydrofuran (500 mL), and after cooling to 0 ℃, borane-dimethyl sulfide solution (10M, 89.3 mL) was added dropwise to the reaction system. After the completion of the dropwise addition, the temperature was raised to 70℃and stirred for 5 hours. Cooling to 0 ℃ again, adding hydrogen chloride-methanol solution (2M, 100 mL) dropwise into the reaction solution, heating to 65 ℃ again, and stirring for 12 hours. The reaction was filtered and the filtrate was concentrated directly to give crude title compound (44.3 g, crude) which was used in the next step without purification.

The structural characterization data are as follows:

ESI-MS(m/z):197.0[M+H]⁺。

Step three preparation of 1- (2- (((tert-butyldiphenylsilyl) oxy) methyl) -5-nitrophenyl) -N-methyl methylamine (INT-3-4)

(2- ((Methylamino) methyl) -4-nitrophenyl) methanol (40.3 g,205 mmol) was dissolved in dichloromethane (800 mL), cooled to 0℃and imidazole (55.9 g,82 mmol) and t-butyldiphenylchlorosilane (84.0 g,308mmol,78.8 mL) were added and after resumption of stirring at 25℃for 1 hour. The reaction was quenched by adding water (200 mL), extracted 3 times with dichloromethane (300 mL. Times.3), the organic phases were combined, washed with saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. After purification on a silica gel column (dichloromethane/methanol=20/1 to 5/1), the mixture was concentrated again to give the title compound (59.7 g,137 mmol).

The structural characterization data are as follows:

ESI-MS(m/z):435.1[M+H]⁺。

Preparation of allyl (2- (((tert-butyldiphenylsilyl) oxy) methyl) -5-nitrobenzyl) (methyl) carbamate (INT-3-5)

1- (2- (((Tert-butyldiphenylsilyl) oxy) methyl) -5-nitrophenyl) -N-methylmethylamine (54.9 g,126 mmol) was dissolved in dichloromethane (550 mL), cooled to 0℃and DIPEA (48.9 g,379mmol,66.0 mL) and allyl chloroformate (30.5 g, 255 mmol,26.8 mL) were added and after a return to 25℃was made for additional stirring for 1 hour. The reaction was quenched by adding water (300 mL), extracted 3 times with dichloromethane (200 mL. Times.3), the organic phases were combined, washed with saturated aqueous sodium chloride (300 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. After purification on a silica gel column (petroleum ether/ethyl acetate=20/1 to 3/1), the mixture was concentrated again to give the title compound (65.5 g,126 mmol).

The structural characterization data are as follows:

ESI-MS(m/z):519.1[M+H]⁺。

Step five preparation of allyl (5-amino-2- (((tert-butyldiphenylsilyl) oxy) methyl) benzyl) (methyl) carbamate (INT-3)

Allyl (2- (((tert-butyldiphenylsilyl) oxy) methyl) -5-nitrobenzyl) (methyl) carbamate (62.8 g,121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL), and after adding iron powder (33.8 g,605 mmol) and ammonium chloride (64.8 g,1.21 mol), the temperature was raised to 80℃and stirred for 2 hours. The reaction mixture was filtered, the filtrate was extracted 3 times with dichloromethane (100 mL x 3), the organic phases were combined and washed with saturated aqueous sodium chloride (300 mL), dried over anhydrous sodium sulfate, and concentrated by filtration to give crude title compound (49.3 g).

The structural characterization data are as follows:

ESI-MS(m/z):511.7[M+H]⁺。

Intermediate example tetrakis (S) -2-amino-N- ((S) -1- (((S) -1-) ((2- ((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-oxoethoxy) methyl) amino) -1-oxopropan-2-yl) amine) -1-propionamide (A-26-3)

Step one:

Compound A-26-1 (657 mg,1.22 mmol) and compound 1-4 (500 mg,1.11 mmol) were dissolved in N, N-dimethylformamide (10 mL), followed by addition of HATU (630.67 mg,1.66 mmol) and N, N-diisopropylethylamine (428 mg,3.32 mmol) and stirring at room temperature for 1 hour. After the reaction is finished, the reaction solution is directly purified by a preparative high performance liquid chromatography and then is freeze-dried to obtain 700mg of A-26-2 compound.

The preparation method comprises the following steps:

Chromatography column Waters SunFire Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Step two:

Compound A-26-2 (500 mg,0.513 mmol) was dissolved in N, N-dimethylformamide (2 mL), and diethylamine (75.05 mg,1.03 mmol) was added thereto and reacted at room temperature for 1 hour. After the reaction, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain 307mg of the A-26-3 compound.

The preparation method comprises the following steps:

Chromatography column Waters SunFire Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example one N- ((7S, 10S, 13S) -1- (((1S, 9S) -5-chloro-9-ethyl-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) -7,1,13-trimethyl-1,6,9,12,15-pentoxy-3,18,21,24,27,30,33,36,39-nonyloxy-5, 8,11, 14-tetraazatetraen-41-yl) -3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (E-5)

Step one:

3, 5-bis (2-methylsulfanylmethylsulfadiazine-5-yl) benzoic acid (3.00 g,8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-carboxylate (4.03 g,8.10 mmol) were added to DMF (40 mL), HOBt (3.28 g,24.3 mmol), EDCI (4.66 g,24.3 mmol) and DIEA (4.19 g,32.4mmol,5.64 mL) were added sequentially and stirred at 60℃for 2h. Water (100 mL) was added to the reaction solution, extraction was performed with ethyl acetate (60 mL. Times.3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give the E-5-3 compound (4.20 g,4.14 mmol), which was used in the next step without purification.

Step two:

1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid tert-butyl ester (E-5-3, 3.60g,4.24 mmol) was dissolved in dichloromethane (30 mL) and TFA (15.3 g,134mmol,10 mL) was added and stirred at 25℃for 6 h. Water (60 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (40 mL. Times.3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to give crude product, which was purified by preparative high performance liquid chromatography and lyophilized to give E-5-4 compound (2.93 g,3.63 mmol).

The separation and purification method comprises the following steps:

phenomenex luna C18 chromatographic column (250 mm. Times.70 mm. Times.10m)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Step three:

1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid (E-5-4, 148mg, 186.41. Mu. Mol) was added to acetonitrile (15 mL) and water (7.5 mL), sodium periodate (398.71 mg,1.86 mmol) and ruthenium trichloride hydrate (15.47 mg, 74.56. Mu. Mol) were added to the reaction system, the reaction was stirred at 25℃for 30min, the reaction was monitored by LC-MS, water and EA were added for extraction, and the mixture was concentrated to give 155mg of E-5-5 as a pale yellow solid.

Step four:

(S) -2-amino-N- ((S) -1- (((S) -1-) ((2- (((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) amino) -2-oxoethoxy) methyl) amino) -1-oxopropan-2-yl) amine) -1-propan-amide (A-26-3, 27.91mg, 34.97. Mu. Mol), E-5-5 (30 mg, 34.97. Mu. Mol), HATU (26.59 mg, 69.93. Mu. Mol), DIPEA (22.60 mg, 174.84. Mu. Mol) were added to DMF (3 mL) and the reaction system was reacted at 25℃for 1 hour. The reaction mixture was purified by preparative high performance liquid chromatography and lyophilized to give 15mg of the title compound.

The structural characterization data are as follows:

ESI-MS(m/z):1591.7[M+H]+

The separation and purification method comprises the following steps:

chromatography column Waters XBridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example two 2,2' - (10- (2- ((5- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -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 ] pyran [3',4':6,7] azepino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazadodecane-1, 4, 7-tri-yl) triacetic acid (F-1)

Preparation of 2, 5-Dioxopyrrolidin-1-yl 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoate (F-1-2) 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoic acid (0.6 g,1.62 mmol) was dissolved in THF (15 mL), N-hydroxysuccinimide (278.89 mg,2.43 mmol) was added, and dicyclohexylcarbodiimide (0.4 g,1.94 mmol) was then added and stirred at room temperature for 2 hours. After the reaction was completed, the filtrate was collected by suction filtration, and concentrated under reduced pressure to give a crude product (1.5 g,3.42 mmol) of the title compound, which was used in the next reaction without purification.

Step two preparation of 1- (3, 5-bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5, 8,11,14, 17-pentaoxa-2-azaeicosane-20-carboxylic acid (F-1-3)

2, 5-Dioxopyrrolidin-1-yl 3, 5-bis (2- (methylthio) pyrimidin-5-yl) benzoate (331.5 mg,0.99 mmol) was dissolved in DMF (4 mL), 1-amino-3, 6,9,12, 15-pentaoxaoctadecane-18-carboxylic acid (0.3 g,0.67 mmol), DIPEA (588.24 mg,4.56 mol) was added and stirred at room temperature for 2 hours. After completion of the reaction, flash column chromatography (C18, water/acetonitrile=0.5) afforded the title compound (315.50 mg,0.44 mmol).

The structural characterization data are as follows:

MS m/z(ESI):662.2[M+H]⁺

Step three preparation of 1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -1-oxo-5, 8,11,14, 17-pentaoxa-2-azaeicosane-20-carboxylic acid (F-1-4)

1- (3, 5-Bis (2- (methylthio) pyrimidin-5-yl) phenyl) -1-oxo-5, 8,11,14, 17-pentaoxa-2-azaeicosane-20-carboxylic acid (315.50 mg,0.44 mmol) was dissolved in acetonitrile (3 mL), water (1.5 mL), sodium periodate (470.50 mg,2.20 mmol) was added, ruthenium trichloride monohydrate (9.11 mg,0.04 mmol) and stirred at room temperature for 0.5 h. After completion of the reaction, flash column chromatography (C18, water/acetonitrile=2/1) followed by lyophilization gave the title compound (115.50 mg,0.16 mmol).

The structural characterization data are as follows:

MS m/z(ESI):726.1[M+H]⁺

Preparation of step four (1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -1-oxo-5, 8,11,14, 17-pentaoxa-2-azaeicosan-20-yl) -L-valine (F-1-5)

1- (3, 5-Bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -1-oxo-5, 8,11,14, 17-pentaoxa-2-azaeicosane-20-carboxylic acid (115.50 mg,0.16 mmol) was dissolved in DMF (4 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (122.50 mg,0.32 mmol) and DIPEA (62.0 mg,0.48 mmol) were added, and L-valine (50.0 mg,0.48 mmol) was added after stirring at room temperature for 0.5 hours. Stirring at room temperature for 15 hours, after completion of the reaction, purification by flash column chromatography (C18, water/acetonitrile=2/1) followed by lyophilization afforded the title compound (30.50 mg,0.04 mmol).

The structural characterization data are as follows:

MS m/z(ESI):825.2[M+H]⁺

Preparation of allyl (S) - (5- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -2- (((tert-butyldiphenylsilyl) oxy) methyl) benzyl) (methyl) carbamate (F-1-6) allyl (5-amino-2- (((tert-butyldiphenylsilyl) oxy) methyl) benzyl) (methyl) carbamate (150.0 mg,0.31 mmol), (((9H-fluoren-9-yl) methoxy) carbonyl) -L-alanine (115.1 mg,0.37 mmol) was dissolved in DCM (8 mL), meOH (2 mL), 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (229.7 mg,0.93 mmol) was added, stirred at room temperature for 15 hours, concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0-90%) and concentrated again under reduced pressure to give the title compound (194.2.25 mmol).

The structural characterization data are as follows:

MS m/z(ESI):782.2[M+H]⁺

step six preparation of (S) - (5- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -2- (hydroxymethyl) benzyl) (methyl) carbamate (F-1-7)

Allyl (S) - (5- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -2- (((tert-butyldiphenylsilyl) oxy) methyl) benzyl) (methyl) carbamate (194.2 mg,0.25 mmol) was dissolved in DMF (5 mL), pyridine hydrofluoric acid salt (390.2 mg,3.93 mmol) was added and stirred at room temperature for 15 hours. After the completion of the reaction, 20mL of water was added to the reaction mixture, which was extracted 3 times with ethyl acetate (10 ml×3), washed with brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a crude product of the title compound, which was purified by flash column chromatography (C18, water/acetonitrile=2/1), and freeze-dried to give the title compound (109.3 mg,0.21 mmol).

The structural characterization data are as follows:

MS m/z(ESI):566.1[M+Na]⁺

Step seven preparation of allyl (S) - (5- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -2- ((((4-nitrophenoxy) carbonyl) oxy) methyl) benzyl) (methyl) carbamate (F-1-8)

(S) - (5- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -2- (hydroxymethyl) benzyl) (methyl) carbamate (109.3 mg,0.21 mmol) was dissolved in DMF (5 mL), DIPEA (81.3 mg,0.63 mmol) was added, p-nitrophenyl chloroformate (50.8 mg,0.25 mmol) was added and stirred at room temperature for 2 hours. After completion of the reaction, purification by flash column chromatography (C18, water/acetonitrile=2/1) followed by lyophilization afforded the title compound (134.50 mg,0.19 mmol).

The structural characterization data are as follows:

MS m/z(ESI):731.2[M+Na]⁺

Step eight preparation of allyl (5- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -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 ] pyran [3',4':6,7] indolizinyl [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (F-1-9)

Allyl (S) - (5- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -2- (((((4-nitrophenoxy) carbonyl) oxy) methyl) benzyl) (methyl) carbamate (134.50 mg,0.19 mmol), (1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H, 13H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13-dione (165.50 mg,0.38 mmol) was dissolved in DMF (5 mL), 1-hydroxybenzotriazole (30.80 mg,0.23 mmol), pyridine (16.5 mg,0.21 mmol), DIPEA (73.5 mg,0.57 mmol) was added and stirred at room temperature for 2 hours. After the reaction was completed, the title compound (114.70 mg,0.12 mmol) was obtained by purification by flash column chromatography (C18, water/acetonitrile=0.7) and freeze-drying.

The structural characterization data are as follows:

MS m/z(ESI):1005.2[M+H]⁺

preparation of allyl (5- ((S) -2-aminopropionamido) -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 ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (F-1-10)

Allyl (5- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -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 ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (114.70 mg,0.12 mmol) was dissolved in DMF (3 mL), diethylamine (87.8 mg,1.2 mmol) was added and stirred at room temperature for 1 hour. After the reaction was completed, the title compound (62.6 mg,0.08 mmol) was obtained by flash column chromatography (C18, water/acetonitrile=0.7) followed by lyophilization.

The structural characterization data are as follows:

MS m/z(ESI):783.1[M+H]⁺

Preparation of allyl (5- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -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 ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (F-1-11)

(1- (3, 5-Bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -1-oxo-5, 8,11,14, 17-pentaoxa-2-azaeicosan-20-yl) -L-valine (30.50 mg,0.05 mmol) was dissolved in DMF (4 mL), 2- (7-benzotriazol) -N, N ' -tetramethylurea hexafluorophosphate (22.06 mg,0.06 mmol) was added to (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (39.10 mg,0.05 mmol), and the mixture was dried by flash chromatography (5 mg,0.06 mmol) at room temperature after flash chromatography (1.5 mg, 6.02 g,5 mg) was added.

The structural characterization data are as follows:

MS m/z(ESI):1589.3[M+H]⁺

Preparation of 4- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -2- ((methylamino) methyl) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamate (F-1-12)

Allyl (5- ((22 s,25 s) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -2- (((((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyran [3',4':6,7] naphthyrido [1,2-b ] quinolin-1-yl) oxy) methyl) benzyl) (methyl) carbamate (31.7 mg,0.02 mmol) was dissolved in DMF (2 mL), tetrakis triphenylphosphine palladium (5.2 mg,4.2 mmol) was added, 15 μl formic acid, 30 μ L N-methyl morpholine was added at room temperature, 1.00 g, and the reaction was dried after flash chromatography (1.00 mmol) under stirring to give the title compound, 0.00 g of water after flash chromatography (1 m.8 mmol).

The structural characterization data are as follows:

MS m/z(ESI):1505.2[M+H]⁺

Preparation of 2,2' - (10- (2- ((5- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -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 ] pyran [3',4':6,7] azepino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazadodecane-1, 4, 7-tri-yl) triacetic acid (F-1)

4- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-ne-26-amido) -2- ((methylamino) methyl) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamate (12.1 mg,0.008 mmol) was dissolved in DMF (1 mL), DIPEA (5.2 mg,0.04 mmol) was added, 2',2"- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-ethyl) -1, 7-labelling-1, 5-oxa-2-hydroxy-ethyl) -1, 7,10, 5-trioxaprop-ane (0.04 mmol) was prepared, and the solution was directly purified by HPLC (0.0.0 mg,0.0 mmol) to give the title compound.

The structural characterization data are as follows:

MS m/z(ESI):1892.2[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

EXAMPLE three preparation of N- ((20S, 23S) -24- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -20-isopropyl-23-methyl-18,21,24-trioxo-3, 6,9,12, 15-pentaoxa-19, 22-diazatetracosyl) -3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-1)

F-1-4 (25.3 mg,0.03 mmol), (S) -2-amino-N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro) -1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (20.0 mg,0.03 mmol) was dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (19.9 mg,0.05 mmol) and DIPEA (13.5 mg,0.1 mmol) were added and after completion of the reaction, the reaction mixture was directly purified by preparative high performance liquid chromatography and dried title compound (26.9 mg,0.02 mmol) after completion of the reaction.

The structural characterization data are as follows:

MS m/z(ESI):1281.3[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example IV preparation of N- ((29S, 32S) -33- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28, 31-diazatriacontyl) -3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-2)

E-5-5 (25.7 mg,0.03 mmol) and G-1-1 (20.0 mg,0.03 mmol) were dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (19.9 mg,0.05 mmol) and DIPEA (13.5 mg,0.1 mmol) were added and stirred at room temperature for 1 hour, after completion of the reaction, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to give the title compound (24.5 mg,0.02 mmol).

The structural characterization data are as follows:

MS m/z(ESI):1413.3[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

EXAMPLE five preparation of N- (2- ((2- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] Indolo [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethyl) -3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-5)

Step one preparation of (9H-fluoren-9-yl) methyl (2- ((2- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13) -dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethyl) carbamate (G-5-1)

((9H-fluoren-9-yl) methoxy) carbonyl) glycylglycine (10.6 mg,0.03 mmol), G-1-1 (20.0 mg,0.03 mmol) were dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (19.9 mg,0.05 mmol) and DIPEA (13.5 mg,0.1 mmol) were added and stirred at room temperature for 1 hour, after which time 10mL of water was added, the solid was precipitated, filtered to give a filter cake and the lyophilized water gave the title compound (23.5 mg,0.025 mmol).

The structural characterization data are as follows:

MS m/z(ESI):910.3[M+H]⁺

Preparation of (S) -2- (2- (2-amino-acetamido) -N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (G-5-2)

G-5-1 (23.5 mg,0.025 mmol) was dissolved in DMF (2 mL), diethylamine (18.3 mg,0.25 mmol) was added and stirred at room temperature for 1 hour. After completion of the reaction, purification by flash column chromatography (C18, water/acetonitrile=0.5) followed by lyophilization afforded the title compound (8.2 mg,0.012 mmol).

The structural characterization data are as follows:

MS m/z(ESI):688.2[M+H]⁺

step three preparation of N- (2- ((2- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] Indolo [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethyl) -3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-5)

G-5-2 (8.2 mg,0.012 mmol) was dissolved in DMF (1 mL), DIPEA (7.8 mg,0.06 mmol) was added, 2, 5-dioxopyrrolidin-1-yl 3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoate (19.1 mg,0.036 mmol) was added, and after the reaction was complete, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze dried to give the title compound (1.45 mg, 1.3. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1104.2[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

EXAMPLE six preparation of N- ((31S, 34S) -35- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -31-isopropyl-3,6,9,12,15,18,21,24,27,34-decamethyl-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxo-3,6,9,12,15,18,21,24,27,30,33-undecazapenta-nyl) -N-methyl-3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-3)

INT-1 (20.0 mg, 17.46. Mu. Mol) and G-1-1 (10.0 mg, 17.46. Mu. Mol) were dissolved in DMF (1 mL), and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (8.0 mg, 20.0. Mu. Mol) and DIPEA (6.8 mg, 52.39. Mu. Mol) were added thereto, followed by stirring at room temperature for 1 hour, and after completion of the reaction, the reaction mixture was directly purified by preparative high performance liquid chromatography and freeze-dried to give the title compound (3.2 mg, 1.86. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1700.6[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example seven preparation of 2,2' - (10- (2- ((5- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -2- ((((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] azepino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazadodecane-1, 4, 7-tri-yl) triacetic acid (F-2)

Step one (5- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) -2- ((((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizinyl [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) allyl carbamate (F-2-1)

F-1-8 (200.0 mg,0.28 mmol), (1S, 9S) -1-amino-9-ethyl-5-chloro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H, 13H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13-dione (253.0 mg,0.56 mmol) was dissolved in DMF (6 mL), 1-hydroxybenzotriazole (45.5 mg,0.34 mmol), pyridine (24.5 mg,0.31 mmol), DIPEA (108.5 mg,0.57 mmol) was added and stirred at room temperature for 2 hours. After the reaction was completed, the title compound (183.70 mg,0.18 mmol) was obtained by freeze-drying after purification by flash column chromatography (C18, water/acetonitrile=0.7).

The structural characterization data are as follows:

MS m/z(ESI):1022.2[M+H]⁺

Preparation of allyl (5- ((S) -2-aminopropionamido) -2- (((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (F-2-2)

F-2-1 (183.70 mg,0.18 mmol) was dissolved in DMF (3 mL), diethylamine (131.8 mg,1.8 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the title compound (136.6 mg,0.17 mmol) was obtained by freeze-drying after purification by flash column chromatography (C18, water/acetonitrile=0.6).

The structural characterization data are as follows:

MS m/z(ESI):800.1[M+H]⁺

Preparation of allyl (F-2-3) methyl) carbamate (5- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -2- (((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl)

F-1-5 (140.13 mg,0.17 mmol), F-2-2 (136.6 mg,0.17 mmol) was dissolved in DMF (5 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (97.40 mg,0.26 mmol) and DIPEA (531.9 mg,0.51 mmol) were added and stirred at room temperature for 1 hour, after completion of the reaction, after purification by flash column chromatography (C18, water/acetonitrile=0.6) and lyophilization gave the title compound (81.7 mg,0.05 mmol).

The structural characterization data are as follows:

MS m/z(ESI):1606.3[M+H]⁺

Preparation of 4- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -2- ((methylamino) methyl) benzyl ((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamate (F-2-4)

F-2-3 (81.7 mg,0.05 mmol) was dissolved in DMF (3 mL), tetrakis triphenylphosphine palladium (11.6 mg, 10.0. Mu. Mol) was added, 30. Mu.L of formic acid, 60. Mu. L N-methylmorpholine was added, stirred at room temperature under nitrogen for 1 hour, and after completion of the reaction, purified by flash column chromatography (C18, water/acetonitrile=0.5) and freeze-dried to give the title compound (32.1 mg,0.02 mmol).

The structural characterization data are as follows:

MS m/z(ESI):1522.3[M+H]

step five preparation of 2,2' - (10- (2- ((5- ((22S, 25S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -22-isopropyl-25-methyl-1,20,23-trioxo-5, 8,11,14, 17-pentaoxa-2,21,24-triazahexa-hexa-ne-26-amido) -2- (((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] azepino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-2)

F-2-4 (32.1 mg,0.02 mmol) was dissolved in DMF (2 mL), DIPEA (12.9 mg,0.1 mmol) was added, 2' - (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (30.1 mg,0.06 mmol) was added, and after completion of the reaction, the reaction solution was purified directly by preparative high performance liquid chromatography and freeze-dried to give the title compound (5.91 mg, 3.1. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1908.1[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example eight preparation of 2,2' - (10- (2- ((5- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl)) benzoylamino) -3-methylbutanamide) propanamido) -2- ((((((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ des ] pyran [3',4':6,7] indolizinyl [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-13)

Step one preparation of 3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoic acid (F-13-1)

3, 5-Bis (2- (methylthio) pyrimidin-5-yl) benzoic acid (0.5 g,1.35 mmol) was dissolved in a mixed solvent of ACN (20 mL) and water (10 mL), sodium periodate (470.50 mg,2.20 mol) was added, and ruthenium trichloride monohydrate (9.11 mg,0.04 mmol) was stirred at room temperature for 0.5 hours. After completion of the reaction, purification by flash column chromatography (C18, water/acetonitrile=5/1) followed by lyophilization afforded the title compound (350.50 mg,0.81 mmol).

The structural characterization data are as follows:

MS m/z(ESI):435.0[M+H]⁺

Preparation of step two (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoyl) -L-valine (F-13-2)

F-13-1 (350.50 mg,0.81 mmol) was dissolved in DMF (3 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (349mg, 0.9 mmol) was added, DIPEA (313.5 mg,2.43 mmol) was added after 10min of reaction, L-valine (189.5 mg,1.62 mmol) was added, stirred at room temperature for 1h, after completion of the reaction, the title compound (120.2 mg,0.22 mmol) was obtained by flash column chromatography (C18, water/acetonitrile=0.5) purification followed by lyophilization.

The structural characterization data are as follows:

MS m/z(ESI):534.1[M+H]⁺

Preparation of allyl (F-13-3) carbamate (5- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamido) -3-methylbutanamido) propanamido) -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) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate

F-13-2 (50.0 mg,0.094 mmol), F-1-10 (86.0 mg,0.11 mmol) was dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (41.8 mg,0.11 mmol) and DIPEA (36.8 mg,0.28 mmol) were added and stirred at room temperature for 1 hour, after completion of the reaction, after purification by flash column chromatography (C18, water/acetonitrile=2:1) the title compound (62.3 mg,0.047 mmol) was obtained by freeze drying.

The structural characterization data are as follows:

MS m/z(ESI):1298.4[M+H]⁺

Preparation of 4- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -3-methylbutanamido) propanamido) -2- ((methylamino) methyl) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamate (F-13-4)

F-13-3 (62.3 mg,0.047 mmol) was dissolved in DMF (3 mL), tetrakis triphenylphosphine palladium (11.6 mg, 10.0. Mu. Mol) was added, 30. Mu.L of formic acid, 60. Mu. L N-methylmorpholine was added, stirred at room temperature under nitrogen for 1 hour, after completion of the reaction, purified by flash column chromatography (C18, water/acetonitrile=0.5) and freeze-dried to give the title compound (40.6 mg,0.033 mmol).

The structural characterization data are as follows:

MS m/z(ESI):1214.4[M+H]⁺

Step five preparation of 2,2' - (10- (2- ((5- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl)) benzamido) -3-methylbutanamido) propanamido) -2- ((((((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ des ] pyran [3',4':6,7] indolizinyl [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-13)

F-13-4 (40.6 mg,0.033 mmol) was dissolved in DMF (2 mL), DIPEA (12.9 mg,0.1 mmol) was added, 2' - (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (30.1 mg,0.06 mmol) was added, and after completion of the reaction, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to give the title compound (1.37 mg, 0.85. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1600.6[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

EXAMPLE nine preparation of N- ((35S, 38S) -39- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -35-isopropyl-3,6,9,12,15,18,21,24,27,38-decamethyl-2,5,8,11,14,17,20,23,26,29,33,36,39-trideoxo-3,6,9,12,15,18,21,24,27,30,34,37-dode-azetidinyl) -N-methyl-3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-4)

Step one preparation of (9H-fluoren-9-yl) methyl (3- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -3-oxopropyl) carbamate (G-4-2)

3- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propionic acid (13.03 mg, 41.84. Mu. Mol), (S) -2-amino-N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro) -1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (20.0 mg, 34.87. Mu. Mol) was dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (15.90 mg, 41.84. Mu. Mol) and DIPEA (13.52 mg, 104.60. Mu. Mol) were added, after completion of the reaction, flash column chromatography (C18, water/acetonitrile) was stirred for 1 hour, and the title compound was dried (26.0 mg, 29.26. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):867.4[M+H]⁺

Preparation of (S) -2- (3-aminopropionamido) -N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (G-4-3)

(9H-fluoren-9-yl) methyl (3- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -3-oxopropyl) carbamate (30.0 mg,34.60 μmol) was dissolved in DMF (1 mL), diethylamine (0.5 mL) was added, stirred at room temperature for 1 hour, after the reaction was purified by flash column chromatography (C18, water/acetonitrile=2:1) and lyophilized to give the title compound (22.31 mg,34.60 μmol).

The structural characterization data are as follows:

MS m/z(ESI):645.6[M+H]⁺

Preparation of N- ((35S, 38S) -39- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -35-isopropyl-3,6,9,12,15,18,21,24,27,38-decamethyl-2,5,8,11,14,17,20,23,26,29,33,36,39-trideoxo-3,6,9,12,15,18,21,24,27,30,34,37-dode azetidinyl) -N-methyl-3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-4)

(S) -2- (3-aminopropionamido) -N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (22.31 mg, 34.60. Mu. Mol) and 1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontan-31-carboxylic acid (22.31 mg, 34.60. Mu. Mol) were dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (15.78 mg, 41.53. Mu. Mol) and PEA (13.13.13.90. Mu. Mol) were added to the solution, and the title compound was directly purified by high performance chromatography (90 mg, 1.81. Mu. Mol) after drying.

The structural characterization data are as follows:

MS m/z(ESI):1771.6[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example ten preparation of 2,2' - (10- (2- ((5- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amino) -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) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-4)

Step one preparation of 2, 5-Dioxopyrrolidin-1-yl 3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoate (F-4-1)

3, 5-Bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoic acid (0.1 g, 230.18. Mu. Mol) was dissolved in DMF (3 mL), EDCI (132.58 mg, 690.54. Mu. Mol) was added and stirred at room temperature for 2 hours. After completion of the reaction, ethyl acetate (20 mL) and water (15 mL) were added and the organic phase was evaporated to dryness under reduced pressure to give the title compound (80 mg, 150.51. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):532.3[M+H]⁺

Preparation of (S) -2- (((3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoyl) glycylglycinoyl) oxy) -3-methylbutanoic acid (F-4-2)

2, 5-Dioxopyrrolidin-1-yl 3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoate (30 mg, 56.44. Mu. Mol) was dissolved in DMF (1 mL), 2-hydroxyacetyl) glycyl-L-valine (26.22 mg, 112.88. Mu. Mol), DIPEA (14.59 mg, 112.88. Mu. Mol) was added, stirred at room temperature for 1 hour, and after completion of the reaction, flash column chromatography (C18, water/acetonitrile=0.5) was purified and freeze-dried to give the title compound (25 mg, 38.54. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):649.4[M+H]⁺

Preparation of allyl (F-4-3) carbamate of 5- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amino) -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) carbamoyl) oxy) methyl) benzyl) (methyl)

(S) -2- (((3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoyl) glycinyl) oxy) -3-methylbutanoic acid (25 mg, 38.54. Mu. Mol), (5- ((S) -2-aminopropionamido) -2- ((((((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate allyl (36.20 mg, 46.25. Mu. Mol) in DMF (2 mL) was added to 2- (7-azobenzotriazol) -N, N ' -tetramethylurea hexafluorophosphate (21.09 mg, 55.50. Mu. Mol) and pea (17.17. Mu. Mg, 17. Mu. M) were dried at room temperature, and the column was cooled to dryness after flash chromatography (35 mg ) was performed, and the column was frozen after the reaction was dried.

The structural characterization data are as follows:

MS m/z(ESI):1413.6[M+H]⁺

Preparation of 4- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amido) -2- ((methylamino) methyl) benzyl ((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) carbamate (F-4-4)

Allyl (5- ((9 s,12 s) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetrazatridecan-13-amino) -2- (((((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyrano [3',4':6,7] naphthyridin [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (30 mg,21.23 μmol) was dissolved in DMF (2 mL), triphenylphosphine (2.45 mg,2.12 μmol) was added, 30 μL formic acid, 60 μ L N-methylmorpholine was added, and after stirring at room temperature under nitrogen protection, the title compound was dried by flash chromatography (22.23.22 mg) after flash chromatography (22.23 mg).

The structural characterization data are as follows:

MS m/z(ESI):1329.4[M+H]⁺

Step five preparation of 2,2' - (10- (2- ((5- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amino) -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) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-4)

4- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxatridecan-13-ylamino) -2- ((methylamino) methyl) benzyl ((1S, 9S) -9-ethyl-9-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] azepino [1,2-b ] quinolin-1-yl) carbamate (28.22 mg, 21.23. Mu. Mol) was dissolved in DMF (2 mL), DIPEA (12.9 mg,0.1 mmol) was added, 2"- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxo-ethyl) -1, 7,10, 7-oxa-4, 7. Mu. 5mg was added, and the title compound was purified by high performance chromatography (3.20 mg, 7.3 mg,7. Mu. Mol) followed by direct purification.

The structural characterization data are as follows:

MS m/z(ESI):1716.8[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example eleven preparation of 2,2' - (10- (2- ((5- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -3-methylbutanamide) propanamido) -2- ((((((1S, 9S) -5-chloro-9-ethyl-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) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (F-14)

Step one (preparation of allyl 5- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamido) -3-methylbutanamido) propanamido) -2- ((((((1S, 9S) -5-chloro-9-ethyl-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) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (F-14-1)

(3, 5-Bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoyl) -L-valine (14.80 mg, 27.84. Mu. Mol), (5- ((S) -2-aminopropionamido) -2- ((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (20.84 mg, 23.20. Mu. Mol) was dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (12.70 mg, 33.41. Mu. Mol) and DIPEA (8.99mg, 69.59. Mu. Mol) were added, and after stirring at room temperature, 1 mol, the title compound was dried by flash chromatography (18 mg, 19.77. Mu. Mol) and the title compound was purified by flash chromatography (2.77 mg).

The structural characterization data are as follows:

MS m/z(ESI):1315.6[M+H]⁺

Preparation of 4- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -3-methylbutanamido) propanamido) -2- ((methylamino) methyl) benzyl ((1S, 9S) -5-chloro-9-ethyl-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) carbamate (F-14-2)

Allyl (5- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -3-methylbutanamido) propanamido) -2- ((((((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (26 mg, 19.77. Mu. Mol) was dissolved in DMF (1 mL), tetrakis triphenylphosphine palladium (2.28 mg, 1.98. Mu. Mol) was added, 30. Mu.l formic acid, 60 mu L N-methylmorpholine was stirred at room temperature under nitrogen protection for 1 hour, after the reaction was completed, flash column (C18, water/acetonitrile=2:1) was purified and dried to give the title compound (20 mg, 16.25. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1231.5[M+H]⁺

Step three preparation of 2,2' - (10- (2- ((5- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamido) -3-methylbutanamido) propanamido) -2- ((((((1S, 9S) -5-chloro-9-ethyl-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) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-14)

4- ((S) -2- ((S) -2- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamido) -3-methylbutanamido) propanamido) -2- ((methylamino) methyl) benzyl ((1S, 9S) -5-chloro-9-ethyl-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) carbamate (20 mg, 16.25. Mu. Mol) was dissolved in DMF (1 mL), DIPEA (6.30 mg, 48.75. Mu. Mol) was added, 2' - (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-trioxyacetic acid (24.45 mg,48. Mu. Mol) was prepared, and the title compound was directly purified by high performance liquid chromatography (0.45 mg,48. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1618.1[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example twelve preparation of 2,2' - (10- (2- ((5- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amino) -2- (((((1S, 9S) -9-ethyl-5-chloro-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) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-5)

Preparation of allyl (F-5-1) carbamate (5- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amino) -2- (((((1S, 9S) -9-ethyl-5-chloro-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) carbamoyl) oxy) methyl) benzyl) (methyl)

(S) -2- (((3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoyl) glycinyl) oxy) -3-methylbutanoic acid (30 mg, 46.32. Mu. Mol), (5- ((S) -2-aminopropionamido) -2- (((((1S, 9S) -9-ethyl-5-chloro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate allyl (44.43 mg, 55.58. Mu. Mol) in DMF (2 mL) was added to 2- (7-azobenzotriazol) -N, N ' -tetramethylurea hexafluorophosphate (26.40 mg, 69.48. Mu. Mol) and pea (17.17. Mu. Mg, 96. Mu. M) and the column was dried after stirring to obtain the title compound (1, 96. Mu. Mg, 96. Mu. Mol) after flash chromatography, 3mg, 96. Mu. M, and after stirring to obtain the title compound.

The structural characterization data are as follows:

MS m/z(ESI):1429.3[M+H]⁺

Preparation of 4- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amido) -2- ((methylamino) methyl) benzyl ((1S, 9S) -9-ethyl-5-chloro-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) carbamate (F-5-2)

Allyl (5- ((9 s,12 s) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxatridec-13-amino) -2- (((((1 s,9 s) -9-ethyl-5-chloro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyrano [3',4':6,7] naphthyrido [1,2-b ] quinolin-1-yl) carbamoyl) oxy) methyl) benzyl) (methyl) carbamate (F-5-1) (30 mg, 20.99. Mu. Mol) was dissolved in DMF (2 mL), triphenylphosphine palladium (2.32 mg, 2.01. Mu. Mol) was added, 30. Mu. L formic acid, 60. Mu. L N-methyl was added, morpholine was dried at room temperature under nitrogen, and the title compound was dried by flash chromatography (25 mg, 18.25 mg) and dried after flash chromatography (25 mg).

The structural characterization data are as follows:

MS m/z(ESI):1331.6[M+H]⁺

Preparation of 2,2' - (10- (2- ((5- ((9S, 12S) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridec-13-amino) -2- (((((1S, 9S) -9-ethyl-5-chloro-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) carbamoyl) oxy) methyl) benzyl) (methyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (F-5)

4- ((9 S,12 s) -1- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) phenyl) -9-isopropyl-12-methyl-1, 4,7, 10-tetraoxo-2, 5,8, 11-tetraazatridecan-13-ylamino) -2- ((methylamino) methyl) benzyl ((1 s,9 s) -9-ethyl-5-chloro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyrano [3',4':6,7] Indolo [1,2-b ] quinolin-1-yl) carbamate (F-5-2) (10 mg, 7.44. Mu. Mol) was dissolved in DMF (2 mL), DIPEA (2.88 mg, 22.31. Mu. Mol) was added, 2' - (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (11.19 mg, 22.31. Mu. Mol) was added, and after the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to give the title compound (1.01 mg, 3.29. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1732.2[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example thirteen preparation of 2,2' - (10- (2- (((S) -5- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -6- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] azepino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (G-25)

Preparation of tert-butyl ((S) -5- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -6- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexyl) carbamate (G-25-1)

N ² - (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoyl) -N ⁶ - (tert-butoxycarbonyl) -L-lysine (64.12 mg, 96.75. Mu. Mol) and (S) -2-amino-N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (G-1-1) (37 mg, 64.50. Mu. Mol) were dissolved in DMF (2 mL) and DIPEA (25.01 mg, 193.50. Mu. Mol) HATU (36.79 mg, 96.75. Mu. Mol) was added and reacted for one hour at room temperature. After the reaction, the reaction mixture was directly purified by preparative high performance liquid chromatography and lyophilized to give the title compound (20 mg, 16.42. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1219.3[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Preparation of tert-butyl ((S) -5- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -6- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexyl) carbamate (G-25-2)

Tert-butyl ((S) -5- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -6- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexyl) carbamate (G-25-1) (20 mg, 16.42. Mu. Mol) was dissolved in DCM (1 mL) and then TFA (0.1 mL) was added and reacted at room temperature for two hours, after which the reaction solution was directly purified by preparative high performance liquid chromatography and lyophilized to give the title compound (20 mg, 16.42. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1119.3[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Preparation of 2,2' - (10- (2- (((S) -5- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -6- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] azepino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexyl) amino) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (G-25)

2,2',2"- (10- (2, 5-Dioxopyrrolidin-1-yl) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (2.02 mg, 4.02. Mu. Mol) and ((S) -5- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide) -6- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] Indolo [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl amino) -3-methyl-1-oxobutan-2-yl amino) -6-oxohexyl carbamic acid tert-butyl ester (G-25-2) (3.00 mg, 2.68. Mu. Mol) was dissolved in DMF (1 mL) and DIPEA (346.72. Mu.g, 2.68. Mu. Mol) was added and reacted at room temperature for one hour. After the reaction, the reaction mixture was directly purified by preparative high performance liquid chromatography and lyophilized to give the title compound (1.06 mg,6.55 e-1. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1504.8[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Example fourteen preparation of 2,2' - (10- ((2S, 5S, 14S) -14- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -5-isopropyl-2-methyl-1,4,7,10,13,20-hexaoxo-3,6,9,12,19-pentaazaheneicosan-21-yl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (G-26)

Step one preparation of (9H-fluoren-9-yl) methyl (2- ((2- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13) -dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethyl) carbamate (G-26-1)

((9H-fluoren-9-yl) methoxy) carbonyl) glycylglycine (31.88 mg, 89.95. Mu. Mol) and (S) -2-amino-N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro) -1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (G-1-1) (43 mg, 74.96. Mu. Mol) were dissolved in DMF (1 mL), HATU (34.20 mg, 89.95. Mu. Mol) and DIPEA (29.06 mg, 224.88. Mu. Mol) were added and reacted at room temperature for one hour. The solvent was then removed under reduced pressure to give the crude title compound which was directly carried forward without purification.

The structural characterization data are as follows:

MS m/z(ESI):910.5[M+H]⁺

Preparation of (S) -2- (2- (2-amino-acetamido) -N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (G-26-2)

(9H-fluoren-9-yl) methyl (2- ((2- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13) -dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethyl) carbamate (G-26-1) crude was dissolved in DMF (2.42 mL) and DEA (54.65 mg, 747.27. Mu. Mol, 77.30. Mu.L) was added and reacted at room temperature for one hour. The solvent was then removed under reduced pressure to give the crude product. The crude product was purified by flash column chromatography (C18, water/acetonitrile=0.7) and lyophilized to give the title compound (38 mg,55.25 μmol).

The structural characterization data are as follows:

MS m/z(ESI):689.2[M+H]⁺

Preparation of tert-butyl ((2S, 5S, 14S) -14- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -5-isopropyl-2-methyl-1, 4,7,10, 13-pentoxy-3, 6,9, 12-tetraazaoctadeca-18-yl) carbamate (G-26-3)

N ² - (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoyl) -N ⁶ - (tert-butoxycarbonyl) -L-lysine (54.93 mg, 82.88. Mu. Mol) and (S) -2- (2- (2-aminoacetamido) acetamido) -N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (G-26-2) (38 mg, 55.25. Mu. Mol) were dissolved in DMF (2 mL) and HATU (31.51 mg, 82.88. Mu. Mol), DIPEA (14.28 mg, 110.51. Mu. Mol) was added and reacted at room temperature for four hours. After the reaction, the reaction mixture was directly purified by preparative high performance liquid chromatography and lyophilized to give the title compound (27 mg, 20.26. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1333.4[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

Preparation of N- ((2S, 5S, 14S) -18-amino-1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] Indol [1,2-b ] quinolin-4-yl) amino) -5-isopropyl-2-methyl-1, 4,7,10, 13-pentoxy-3, 6,9, 12-tetraazaoctadeca-14-yl) -3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-26-4)

Tert-butyl ((2S, 5S, 14S) -14- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzoylamino) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -5-isopropyl-2-methyl-1, 4,7,10, 13-pentoxy-3, 6,9, 12-tetraazaoctadeca-18-yl) carbamate (G-26-3) (27 mg,20.26 μmol) was dissolved with DCM (2 mL) and TFA (0.2 mL) was added for two hours at room temperature. The solvent was then removed to give the crude title compound, which was carried forward directly without purification.

The structural characterization data are as follows:

MS m/z(ESI):1233.4[M+H]⁺

Preparation of 2,2' - (10- ((2S, 5S, 14S) -14- (3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamido) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -5-isopropyl-2-methyl-1,4,7,10,13,20-hexaoxo-3,6,9,12,19-pentaazaeicosan-21-yl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid (G-26)

2,2',2"- (10- (2, 5-Dioxopyrrolidin-1-yl) -2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (14.18 mg, 29.21. Mu. Mol) and N- ((2S, 5S, 14S) -18-amino-1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyran [3',4':6,7] azepino [1,2-b ] quinolin-4-yl) amino) -5-isopropyl-2-methyl-1, 4,7,10, 13-pentaoxo-3, 6,9, 12-tetraazaoctadecan-14-yl) -3, 5-bis (2- (methylsulfonyl) pyrimidin-5-yl) benzamide (G-26-4) crude was dissolved in DMF (2 mL) and added to the mixture at room temperature of two times (7.55. Mu. Mol) at room temperature of PEA and at two small reaction times. After the reaction, the reaction mixture was directly purified by preparative high performance liquid chromatography and lyophilized to give the title compound (15.30 mg, 8.79. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1619.9[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

EXAMPLE fifteen Pentafluorophenol ester of 1- (N- ((S) -1- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] Indol [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethoxy carbonyl) sulfamoyl piperidine-4-carboxylate (I-50)

Step one:

Chlorosulfonyl isocyanate (2.14 g,15.13 mmol) was added to methylene chloride (50 mL), the temperature was lowered in an ice-water bath for 10 minutes, and tert-butyl 2-glycolate (2 g,15.13 mmol) was added to the above reaction system, and the reaction was continued with stirring in an ice-water bath for 2 hours. Piperidine-4-carboxylic acid allyl ester hydrochloride (3.74 g,18.16 mmol) and triethylamine (4.59 g,45.40 mmol) were added to the reaction system, the ice-water bath was evacuated, and the temperature was returned to room temperature naturally and stirring was continued for 3 hours. The reaction was quenched by adding water (100 mL), extracted 3 times with ethyl acetate (50 mL. Times.3), the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. Purification by silica gel column (DCM/pe=0-100%) and concentration again gave compound I-50-2 (1.87 g,4.19 mmol).

The structural characterization data are as follows:

¹H NMR(400MHz,DMSO)δ11.64(s,1H),6.06-5.79(m,1H),5.35-5.17(m,2H),4.61-4.48(m,4H),4.03(q,J＝7.1Hz,1H),3.59(dt,J＝12.4,3.3Hz,2H),3.04-2.88(m,2H),1.93(dd,J＝13.5,3.2Hz,2H),1.59(dd,J＝13.4,3.6Hz,2H),1.43(s,9H).

step two:

Allyl 1- (N- ((2- (tert-butoxy) -2-oxoethoxy) carbonyl) sulfamoyl) piperidine-4-carboxylate (I-50-2) (1.75 g,4.31 mmol) was added to a mixed solvent of trifluoroacetic acid (5 mL) and dichloromethane (10 mL) and reacted at 25℃for 2 hours. The reaction was concentrated to dryness, diluted with ethyl acetate, pH was adjusted to about 8 with aqueous sodium bicarbonate, the impurities were removed by extraction, pH was adjusted to about 3 with 3N diluted hydrochloric acid, extraction was performed 3 times with ethyl acetate (30 mL. Times.3), the organic phases were combined and dried over anhydrous sodium sulfate, and then concentrated by filtration to give Compound I-50-3 (1.4 g,4.00 mmol).

The structural characterization data are as follows:

ESI-MS(m/z):351.1[M+H]⁺。

step three:

(I-50-3) (18.32 mg,52.30 μmol), (S) -2-amino-N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (30 mg,52.30 μmol) was dissolved in DMF (1 mL), 2- (7-azobenzotriazole) -N, N ' -tetramethylurea hexafluorophosphate (23.85 mg,62.76 μmol) and DIPEA (20.28 mg,156.89 mol) were added, stirred for 1 hour at room temperature, and after reaction, the mixture was purified by flash chromatography (18/4 mg, 40 mg) with water after flash chromatography (18/40 mg, 4) to give the compound after flash chromatography (4-40 mg).

The structural characterization data are as follows:

MS m/z(ESI):906.4[M+H]⁺

Step four:

Allyl 1- ((2- (((S) -1- ((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethoxy) carbonyl) sulfamoyl) piperidine-4-carboxylate (I-50-4) (40 mg,44.15 μmol) was dissolved in DMF (1 mL), tetrakis triphenylphosphine palladium (5.1 mg,4.41 μmol) was added, 15 μl formic acid, 30 μ L N-methylmorpholine was added, and after reaction was stirred at room temperature for 1 hour under nitrogen protection, and after purification by flash column chromatography (C18, water/acetonitrile=3/1), compound I-50-20 mg, 10.5 μmol was obtained.

The structural characterization data are as follows:

MS m/z(ESI):867.4[M+H]⁺

Step five:

1- (N- ((2- (((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -2-oxoethoxy) carbonyl) sulfamoyl) piperidine-4-carboxylic acid (I-50-5) (20 mg, 23.10. Mu. Mol) was dissolved in DMF (1 mL), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (27.72 mg, 144.36. Mu. Mol), and pentafluorophenol (15.94 mg, 86.61. Mu. Mol) was directly subjected to a preparative high performance liquid chromatography and then lyophilized to give compound I-50 (12.53 mg, 12.02. Mu. Mol).

The structural characterization data are as follows:

MS m/z(ESI):1033.4[M+H]⁺

The preparation method comprises the following steps:

Chromatography column Waters Xbridge Prep C OBD (5 μm 19mm 150 mm)

Mobile phase A acetonitrile, mobile phase B water (0.05% formic acid)

2. Preparation of antibodies and determination of binding Activity

Antibody acquisition and purification

According to Trastuzumab amino acid sequence (IMGT/mAb-DB ID: 97) in IMGT database, pertuzumab amino acid sequence (IMGT/mAb-DB ID: 80), codon optimization is carried out, coding genes are synthesized, the coding genes are constructed into an expression vector, CHO cells are transfected, and a stable expression cell strain is constructed by pressurized screening. Expressing and collecting supernatant, purifying by Protein A affinity filler to obtain corresponding antibodies Trastuzumab and Pertuzumab.

3. Conjugation of compounds comprising a cellular bioactive molecule and a linker to an antibody

The antibodies Trastuzumab and Pertuzumab involved in the antibody drug conjugates prepared in the examples below are those described in the second section above.

The conjugation of antibody drug conjugate samples was prepared as follows:

1. preparation of Trastuzumab-E-5

0.617ML of Trastuzumab antibody (16.2 mg/mL) was diluted with 30.86. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 7.60 with 1M Na ₂HPO₄ solution, mixed with 19.83mM TCEP (tris (2-carboxyethyl) phosphine, 19.1. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then sequentially adding 5.5 times of the amount of the substances of the antibody into E-5 (38.28 mu L,10 mM) solution dissolved in dimethyl sulfoxide, uniformly mixing, standing at room temperature for 18h, and after the completion, replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by using NAP-5 gel column (Cytiva) to obtain the antibody drug conjugate (namely Trastuzumab-E-5). The DAR value was determined by mass spectrometry to be 4.41.

2. Preparation of Trastuzumab-F-1

0.884ML of trastuzumab (14.7 mg/mL) was taken, diluted with 44. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 8.0 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 49.26. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then, the solution of F-1 (47.65. Mu.L, 10 mM) dissolved in dimethyl sulfoxide was mixed with 5 times the amount of the antibody, and left at room temperature for 2 hours, after which the buffer was replaced with 20mM histidine buffer solution at pH 6.0 using NAP gel column (Cytiva) to obtain antibody drug conjugate (Trastuzumab-F-1). The DAR value was determined by mass spectrometry to be 4.34.

3. Preparation of Trastuzumab-F-2

2.333ML of trastuzumab (15 mg/mL) was taken, diluted with 117. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 7.60 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 132.6. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding F-2 (171.3 mu L,10 mM) solution of 6.5 times of the antibody dissolved in dimethyl sulfoxide, mixing, standing at room temperature for 18h, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by NAP gel column (Cytiva) to obtain antibody drug conjugate (Trastuzumab-F-2). The DAR value was determined by mass spectrometry to be 4.51.

4. Preparation of Trastuzumab-F-4

0.238ML of trastuzumab (14.7 mg/mL) was taken, diluted with 12. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 7.6 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 13.26. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding 5 times of the amount of the antibody into F-4 (12.18 mu L,10 mM) solution dissolved in dimethyl sulfoxide, mixing uniformly, standing for 18h at room temperature, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by using NAP gel column (Cytiva) after finishing, thereby obtaining the antibody drug conjugate (namely Trastuzumab-F-4). The DAR value was determined by mass spectrometry to be 4.36.

5. Preparation of Trastuzumab-F-13

0.204ML of trastuzumab (14.7 mg/mL) was taken, diluted with 10. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 7.6 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 11.4. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding F-13 (12.5 mu L,10 mM) solution of 6 times of the antibody dissolved in dimethyl sulfoxide into the mixture, mixing the mixture evenly, standing the mixture at room temperature for 18 hours, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by using NAP gel column (Cytiva) after the completion of the reaction, thus obtaining the antibody drug conjugate (namely Trastuzumab-F-13). The DAR value was determined by mass spectrometry to be 4.63.

6. Preparation of Trastuzumab-G-1

2.041ML of trastuzumab (14.7 mg/mL) was taken, diluted with 102. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 8.0 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 113.7. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding the solution of G-1 (155.4 mu L,10 mM) dissolved in dimethyl sulfoxide with the amount of 7.5 times of the antibody in sequence, uniformly mixing, standing at room temperature for 18h, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by using NAP gel column (Cytiva) after finishing, thereby obtaining the antibody drug conjugate (namely Trastuzumab-G-1). The DAR value was determined by mass spectrometry to be 4.28.

7. Preparation of Trastuzumab-G-2

2.041ML of trastuzumab (14.7 mg/mL) was taken, diluted with 102. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 8.0 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 113.7. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding 6 times of the amount of G-2 (126.2 mu L,10 mM) solution of the antibody dissolved in dimethyl sulfoxide into the mixture, mixing the mixture evenly, standing the mixture at room temperature for 18 hours, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by using NAP gel column (Cytiva) after the completion of the reaction, thus obtaining the antibody drug conjugate (namely Trastuzumab-G-2). The DAR value was determined by mass spectrometry to be 4.38.

8. Preparation of Trastuzumab-G-3

2.041ML of trastuzumab (14.7 mg/mL) was taken, diluted with 102. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 8.0 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 113.7. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding 6 times of the amount of the antibody into G-3 (125.2 mu L,10 mM) solution dissolved in dimethyl sulfoxide, mixing uniformly, standing at room temperature for 18h, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by using NAP gel column (Cytiva) after finishing, thereby obtaining the antibody drug conjugate (namely Trastuzumab-G-3). The DAR value was determined by mass spectrometry to be 3.86.

9. Preparation of Trastuzumab-G-4

2.041ML of trastuzumab (14.7 mg/mL) was taken, diluted with 102. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 7.6 with 1M Na ₂HPO₄ solution, mixed well with 10mM TCEP (tris (2-carboxyethyl) phosphine, 113.7. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding 6 times of the amount of the antibody into G-4 (125.3 mu L,10 mM) solution dissolved in dimethyl sulfoxide, mixing uniformly, standing at room temperature for 18h, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by using NAP gel column (Cytiva) after finishing, thereby obtaining the antibody drug conjugate (namely Trastuzumab-G-4). The DAR value was determined by mass spectrometry to be 4.16.

10. Preparation of Trastuzumab-G-5

0.884ML of trastuzumab (14.7 mg/mL) was diluted with 44.2. Mu.L 20mM PB+0.1M EDTA (pH 7.60), then adjusted to pH 7.6 with 1M Na ₂HPO₄ solution, mixed with 10mM TCEP (tris (2-carboxyethyl) phosphine, 49.3. Mu.L, pH 7.60) solution, and left at room temperature for 1.5h. Then adding 6 times of G-5 (54 mu L,10 mM) solution of the antibody dissolved in dimethyl sulfoxide, mixing, standing at room temperature for 18h, and replacing the buffer solution with 20mM histidine buffer solution with pH of 6.0 by NAP gel column (Cytiva) to obtain antibody drug conjugate (Trastuzumab-G-5). The DAR value was determined by mass spectrometry to be 3.99.

11. Preparation of Trastuzumab-I-50

2.04ML of Trastuzumab (14.7 mg/mL) was taken, the pH was adjusted to 7.40 with 1M Na ₂HPO₄ solution, and I-50 (146. Mu.L, 10mM, equivalent to 7 times the amount of the substance of the antibody) solution dissolved in dimethyl sulfoxide was added and mixed well, and left standing at room temperature for 18 hours, after which the buffer was replaced with 20mM histidine buffer solution at pH 6.0 using NAP-5 gel column (Cytiva), to obtain an antibody drug conjugate (Trastuzumab-I-50). The average load (DAR value) was measured by mass spectrometry to be 2.45.

4. Evaluation of tumor growth inhibition by antibody-drug conjugate on mouse subcutaneous transplantation tumor model

The preparation containing the ADC is respectively injected into a CDX model of a subcutaneously transplanted human gastric cancer cell NCI-N87 mouse through tail vein injection, the tumor volume and the animal weight change are measured 2 times a week, and the tumor inhibiting effect of the ADC on the tumor-bearing mouse is calculated.

Test agent

The medicine name, source and preparation method are that a proper amount of ADC is taken, and 0.9% NaCl injection is used for diluting mother liquor according to the dosage of 1 mg/kg. A Vehicle control (Vehicle) was 0.9% NaCl injection.

Experimental animals and cell lines

Balb/c Nude mice (Chengdu Kangshengzhikang biotechnology Co., ltd., production license number: SCXK (Sichuan) 2020-0034, animal eligibility number: 511214900025102)

Human gastric cancer cell NCI-N87 (ATCC)

Experimental grouping and evaluation method

Tumor bearing murine individuals with an average tumor volume of about 150mm ³ were selected for random grouping (grouping number was determined based on the number of samples). According to the group, 0.9% NaCl injection (hereinafter referred to as Vehicle control or Vehicle) and ADC were administered respectively, the frequency of administration was as shown in the specific example, the administration was by tail vein injection, and the administration volume was 10mL/kg. Tumor diameter was measured 2 times per week after dosing with vernier calipers and tumor volume was calculated as v=0.5a×b ², where a and b represent the long and short diameters of the tumor, respectively. Daily observations record animal death.

The tumor growth inhibition TGI (%) was calculated using the following formula for evaluating the tumor inhibition effect of ADC:

Or (b)

Wherein the method comprises the steps ofTumor volume mean at the end of treatment group experiments

V _T0 mean tumor volume at the start of treatment group dosing

Tumor volume mean at the end of the negative control experiments

V _C0 tumor volume mean at the beginning of administration of negative control group

The tumor relative proliferation rate T/C (%) was calculated using the following formula for evaluating the tumor-inhibiting effect of ADC:

Drug effect detection of anti-human Her2 antibody coupled drug in NCI-N87 model

NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37℃under 5% CO ₂. NCI-N87 cells in an exponential growth phase are collected, PBS is resuspended to a proper concentration, and inoculated into female Balb/c-Nude mice subcutaneously to establish a gastric cancer model. When the average tumor volume is about 150mm ³, the tumors are randomly grouped according to the tumor size, and a solvent control group (namely a negative control group and a Vehicle group) is sequentially selected, wherein Trastuzumab-F-2 1mg/kg, trastuzumab-G-1 1mg/kg group and control DS8201 mg/kg (remark: DS8201 is a first co-developed ADC (analog) for targeting human HER 2), and samples are prepared by using Columbitai in the experiment. The drug was administered 3 times in Day0, day7, day14 by tail vein injection (i.v.).

The ADC has remarkable effect of inhibiting the tumor growth of NCI-N87 gastric cancer transplantation tumor model. Compared with the Vehicle group, the Trastuzumab-F-2 1mg/kg, trastuzumab-G-1 mg/kg group and the control DS82011mg/kg have Tumor Growth Inhibition (TGI) of 72.86%, 167.97% and 69.02% in sequence, and the Day27 treatment group has no animal death and obvious animal weight reduction, no obvious drug toxicity reaction is seen, and the mice have good tolerance to the ADC of the invention during the treatment period. The specific results are shown in Table 1, FIG. 1 and FIG. 2.

TABLE 1 human gastric cancer cell NCI-N87CDX model

Note that TGI is the tumor growth inhibition rate, T/C is the relative tumor proliferation rate, and the same applies below.

5. Evaluation of tumor growth inhibition by antibody-drug conjugate on mouse subcutaneous transplantation tumor model

Test agent

The medicine name, source and preparation method are that a proper amount of ADC is taken, and the mother liquor is diluted by 0.9 percent NaCl injection according to the dosage of 1mg/kg and 2 mg/kg. A Vehicle control (Vehicle) was 0.9% NaCl injection.

Experimental animals and cell lines

Balb/c Nude mice (Chengdu Kangshengzhikang biotechnology Co., ltd., production license number: SCXK (Sichuan) 2020-0034, animal eligibility number: 511214900026661)

Human gastric cancer cell NCI-N87 (ATCC)

Experimental grouping and evaluation method

Or (b)

V _T0 mean tumor volume at the start of treatment group dosing

Tumor volume mean at the end of the negative control experiments

Drug effect detection of anti-human Her2 antibody coupled drug in NCI-N87 model

NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37℃under 5% CO ₂. NCI-N87 cells in an exponential growth phase are collected, PBS is resuspended to a proper concentration, and inoculated into female Balb/c-Nude mice subcutaneously to establish a gastric cancer model. When the average tumor volume is about 150mm ³, the tumor is randomly grouped according to the tumor size, and a solvent control group (namely a negative control group and a Vehicle group) is sequentially selected from the Trastuzumab-F-1 2mg/kg group, the Trastuzumab-G-5 1mg/kg group, the Trastuzumab-G-2 1mg/kg group, the Trastuzumab-G-3 1mg/kg group, the Trastuzumab-G-4 1mg/kg group and the control DS8201 mg/kg group (note: DS8201 is a first three co-developed ADC (analog to digital) for targeting human HER2, and samples are prepared in the Columbtai for the test). The drug was administered 3 times in Day0, day7, day14 by tail vein injection (i.v.).

The ADC has remarkable effect of inhibiting the tumor growth of NCI-N87 gastric cancer transplantation tumor model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-F-1 2mg/kg group, the Trastuzumab-G-5 1mg/kg group, the Trastuzumab-G-21mg/kg group, the Trastuzumab-G-3 1mg/kg group, the Trastuzumab-G-4 1mg/kg group and the control DS82011mg/kg group are 156.31%, 80.06%, 122.21%, 99.55%, 130.19% and 60.21% in sequence, and the Day27 treatment group has no animal death and remarkable animal weight reduction, no obvious drug toxicity reaction is seen, and mice have good tolerance to the ADC of the invention during the treatment period. The specific results are shown in Table 2, FIG. 3, FIG. 4 and FIG. 5.

TABLE 2 human gastric cancer cell NCI-N87CDX model

Note that TGI is the tumor growth inhibition rate and T/C is the relative tumor proliferation rate.

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, M’-L-E-D Formula (I)

Where: M’ is -M-Lg, where Lg is the leaving group of the nucleophilic substitution reaction, and M is the structural fragment that binds to the target site;

L is a structural segment connecting M and E;

E is a structural segment connecting L and D;

D is a cytotoxic drug fragment.

The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein M is selected from the following substituted or unsubstituted structural fragments:

Preferably, M is selected from the following substituted or unsubstituted structural segments:

The compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein L is selected from one or more of the following substituted or unsubstituted structural fragments: _C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic, -N(R')-, -NH(R'), -N(R') ₂ , -NHCH(R')-C(=O)-, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( _COCH2CH2 ₍ _OCH2CH2 ₎ rOCH3 ₎ ), Glu(R'), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, G ly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Ly s-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, _C1-6 alkyl, _C1-6 alkylene, amino, hydroxyl, carboxyl, acyl, _-O- , _-C1-6 alkylene CO2H, _-C1-6 alkylene _SO3H , _-SO3H , -PO3H2, _-C1-6 alkylene _-NHC1-6 _alkyl , _-C1-6 alkylene -N( _C1-6 _alkyl ) ₂ , _-C1-6 alkylene - _heterocyclic , -CH2N( _C1-6 alkyl)-C(=O) _C1-6 alkylene - _heterocyclic , _-CH2NH -SO3H, _-CH2N ₍ _C1-6 _alkyl ) _-SO3H , _-CH2NHC1-6 alkylene _- _SO3H , _-CH2N ( _C1-6 _alkyl )C ... _1-6 alkylene- _SO3H ) ₂ , _-CH2N ⁺ ( _C1-6 alkylene-SO3H) ₃ , -CH2N ⁺ ( _C1-6 alkyl) ₂ _-C1-6 alkylene-SO3H, -CH2N( _C1-6 alkyl)-C ₍ ＝O) _C1-6 alkylene- _N ⁺ ( _C1-6 alkylene-SO3H) ₃ , _{-CH2NH-C(＝O)C1-6} _alkylene -N ⁺ ( _C1-6 alkylene- _SO3H ) ₃ , _-CH2N ( _C1-6 alkyl)-C(＝O) _C1-6 alkylene-N ⁺ ₍ _C1-6 alkyl) ₃ , _-CH2NH -C(＝O) _C1-6 alkylene-N ⁺ ( _C1-6 _alkyl ) ₃ , _-CH2N ( _C1-6 alkyl) _1-6 alkyl)-C(＝O)OC _2-6 alkylene-N ⁺ (C _1-6 alkyl) ₃ , -CH ₂ N(C _1-6 alkyl)-C(＝O)OC _2-6 alkylene-N ⁺ (C _1-6 alkyl) ₂ -CH ₂ CO ₂ H, -CH ₂ N(C _1-6 alkyl)-C _1-6 alkylene-CO ₂ H, -CH ₂ N ⁺ (C _1-6 alkyl) ₂ -C _1-6 alkylene-CO ₂ H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH ₂ N(C _1-6 alkyl)-C(＝O)-(CH ₂ CH ₂ O) _r -C _1-6 alkyl, -CH ₂ N(C _1-6 alkyl)-C(＝O)-(OCH ₂ CH ₂ ) _r -OC _1-6 alkyl, -(CH ₂ N(Me)-C(＝O)) _r -C _1-6 alkyl, polyethylene glycol fragments containing 1-10 EO units (i.e., -(CH ₂ CH ₂ O) _r -C _1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), -C _1-6 alkylene-N(C _1-6 alkyl)-DOTA, -C _1-6 alkyl-N(C _1-6 alkyl)-DOTAGA, or -C _1-6 alkyl-N(C _1-6 alkyl)-NOTA, wherein r is selected from integers from 1 to 20, preferably from integers from 1 to 15, such as integers from 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, 1 to 2, for example r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; s is selected from integers from 1 to 20, preferably from integers from 1 to 15, such as integers from 1 to 12, 3 to 12, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

Preferably, L is selected from substituted or unsubstituted structural fragments composed of one or more of the following groups: s is selected from integers from 1 to 20, preferably from integers from 1 to 15, such as integers from 1 to 12 or 3 to 12, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

Preferably, L is selected from substituted or unsubstituted structural fragments composed of one or more of the following groups: Wherein, s is selected from integers from 1 to 20, preferably from integers from 1 to 15, such as integers from 1 to 12 or 3 to 12, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

Preferably, L is selected from substituted or unsubstituted structural fragments composed of one or more of the following groups: Wherein, s is selected from an integer from 1 to 20, preferably s is selected from an integer from 1 to 15, such as an integer from 1 to 12 or 3 to 12, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably s is selected from 5, 8 or 10;

Preferably, L is selected from substituted or unsubstituted structural fragments composed of one or more of the following groups:

Wherein, s is selected from an integer from 1 to 20, preferably s is selected from an integer from 1 to 15, such as an integer from 1 to 12 or 3 to 12, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably s is selected from 5, 8 or 10;

Preferably, L contains the following structural segments

Preferably, L is formed by linking structural segments from group I with one or more substituted or unsubstituted structural segments selected from group II:

Group I is

Group II consists of Composition, preferably group II consists of Composition, preferably group II consists of The composition, wherein s is selected from integers from 1 to 20, preferably s is selected from integers from 1 to 15, such as integers from 1 to 12 or 3 to 12, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably s is selected from 5, 8, 10;

Alternatively, L can be selected from one or more of the following substituted or unsubstituted structural segments: _C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic group, -N(R')-, -NH(R'), -N(R') ₂ , carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( _COCH2CH2 ₍ _OCH2CH2 ₎ rOCH3 ₎ () and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-A) rg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Al a. Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, _C1-6 alkyl, _-C1-6 alkyleneCO2H, _-C1-6 alkyleneSO3H, _-SO3H , _-PO3H2 , _-C1-6 alkylene- _NHC1-6 alkyl, -C1-6 alkylene _-N(C1-6 _alkyl ) ₂ _, -CH2N( _C1-6 alkyl)-C(=O) _C1-6 alkylene-heterocyclic, _-CH2NH -SO3H, -CH2N( _C1-6 alkyl) _-SO3H _{, -CH2NHC1-6} _alkylene - _SO3H , _-CH2N ( _C1-6 _alkyl ) _C1-6 _alkylene _- _SO3H , _-CH2N ( _C1-6 alkylene _-SO3H)2 _, _-CH2N ₊ ⁽ _C1-6 alkylene _- _SO3H ) ₃ _-CH₂N ⁺ (C₁ _- 6alkyl) _₂- C₁ _-6alkylene - _SO₃H , _-CH₂N (C₁ _- 6alkyl)-C(＝O)C₁ _- 6alkylene-N ⁺ (C₁ _- 6alkylene-SO₃H) _₃ , -CH₂NH-C(＝O)C₁-6alkylene-N ⁺ (C₁-6alkylene-SO₃H) _₃ , _-CH₂N ( _C₁ _- 6alkyl)-C(＝O) _C₁ -6alkylene-N + (C₁ _- 6alkyl) _₃ , _-CH₂NH -C(＝O)C₁ _- 6alkylene-N ⁺ (C₁ _- 6alkyl) _₃ , _-CH₂N (C₁-6alkyl)-C(＝O) _OC₂ -6alkylene-N ⁺ (C₁ _- 6alkyl) _₃ , _-CH₂N (C₁ _- 6alkyl)-C(＝O)OC₂ _- 6alkylene-N ⁺ (C₁-6alkyl) _₃ , _-CH₂N (C₁ _- 6alkyl)-C₁ ... _1-6 alkyl)-C(＝O)OC _2-6 alkylene-N ⁺ (C _1-6 alkyl) ₂ -CH ₂ CO ₂ H, -CH ₂ N(C _1-6 alkyl)-C _1-6 alkylene-CO ₂ H, -CH ₂ N ⁺ (C _1-6 alkyl) ₂ -C _1-6 alkylene-CO ₂ H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH ₂ N(C _1-6 alkyl)-C(＝O)-(CH ₂ CH ₂ O) _r -C _1-6 alkyl, -CH ₂ N(C _1-6 alkyl)-C(＝O)-(OCH ₂ CH ₂ ) _r -OC _1-6 alkyl, -(CH ₂ N(Me)-C(＝O)) _r -C _1-6 alkyl, or polyethylene glycol fragments containing 1-10 EO units (i.e. -(CH ₂ CH ₂ O) _r -C _1-6 alkyl), where r is selected from integers from 1 to 20; s is selected from integers from 1 to 20;

Preferably, L is selected from substituted or unsubstituted structural fragments composed of one or more of the following groups: s is an integer selected from 1 to 20.

The compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein E is a single bond, a substituted or unsubstituted -NH- _CH2- , or a structural fragment selected from the following substituted or unsubstituted segments:

Preferably, E is a single bond, substituted or unsubstituted -NH- _CH2- , or

The compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug is selected from anti-microtubule agents, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors and gene transcription inhibitors;

Preferably, the cytotoxic drug is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, ixotecan, topotecan, belototecan, rubotecan, diflomotecan, lurtotecan, Karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or elomotecan);

Preferably, the cytotoxic drug is selected from the following compounds or compounds labeled with their isotopes:

Alternatively, the cytotoxic drug is selected from the following compounds or compounds labeled with their isotopes: 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33. 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2 -34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, 2-48, 2-49, 2-50, 2-51, 2-52, 2-53, 2-54, 2-55, 2-56, 2-57, 2-58, 2-59, 2-60, 2-61, 2-62, 2-63, 2-64, 2-65, 2-66, 2-67, 2-68, 2-69, 2-70, 2- 71, 2-72, 2-73, 2-74, 2-75, 2-76, 2-77, 2-78, 2-79, 2-80, 2-81, 2-82, 2-83, 2-84, 2-85, 2-86, 2-87, 2-88, 2-89, 2-90, 2-91, 2-92, 2-93, 2-94, 2-95, 2-96, 2-97, 2-98, 2-99, 2-100, 2-101, 2-102, 2-103, 3-1, 3-2, 3-3, 3-4, 3-5

Preferably, the cytotoxic drug is linked to the E in the compound via a -OH, primary amino, secondary amino, or tertiary amino group.

The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown below:

Where n is selected from integers from 1 to 20, such as integers from 1 to 12 or 3 to 12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

Alternatively, the structure of the compound is selected from: E-1 to E-50, F-1 to F-14, G-1 to G-5;

Alternatively, the structure of the compound is selected from: E-1 to E-50, F-1 to F-12;

Alternatively, the structure of the compound is selected from: E-1 to E-50, I-1 to I-49.

A coupling as shown in formula (II), wherein: Ab-[M-L-E-D]x Formula (II)

Ab is the target portion; M, L, E, and D are as described in any one of claims 1-7;

x is between 1 and 10.

The conjugate of claim 8, wherein the Ab is an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof includes Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, bispecific antibody, or multispecific antibody;

Preferably, Ab is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

Preferably, the antibody or its antigen-binding fragment comprises:

(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

or,

(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

or,

(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

or,

(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

Preferably, the antibody or its antigen-binding fragment comprises:

(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

or,

(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

or,

(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

or,

(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

Preferably, the antibody or its antigen-binding fragment comprises:

(1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

or,

(2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:33, CDR-H2 of SEQ ID NO:34, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

or,

(3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:26, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

or,

(4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25;

Preferably, the antibody or its antigen-binding fragment comprises:

(a) VH or a variant thereof shown in SEQ ID NO: 1, and/or VL or a variant thereof shown in SEQ ID NO: 2; or

(b) VH or a variant thereof shown in SEQ ID NO: 3, and/or VL or a variant thereof shown in SEQ ID NO: 4;

The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions;

Preferably, the antibody or its antigen-binding fragment comprises:

(a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

(b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4;

Preferably, the antibody or its antigen-binding fragment further comprises:

(a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

(b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35;

Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36;

Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36;

Preferably, the antibody or its antigen-binding fragment comprises:

(1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

(2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36;

Preferably, Ab is trastuzumab or pertuzumab or its antigen-binding fragment.

The coupling agent according to claim 8 or 9 is selected from:

Wherein, HA in each antibody-drug conjugate is an antibody or its antigen-binding fragment, preferably the antibody or its antigen-binding fragment as defined in claim 9; n is selected from integers from 1 to 20, such as integers from 1 to 12 or 3 to 12, for example n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

in, This indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the M fragment; This indicates the specific way in which the amino group in the antibody or its antigen-binding fragment is linked to the M fragment.

A composition comprising one or more conjugates as described in any one of claims 8-10, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7- 10, 8-9, 8-10, or 9-10, preferably 3-8, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 7.0-7.5, 7.0-8.0 or 7.5-8.0; preferably 3.5-5.0 (e.g., 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0);

Alternatively, the composition has a DAR value of about 1.0 to 6.0, such as about 1.0 to 5.5, about 1.0 to 5.0, about 1.5 to 6.0, about 1.5 to about 5.5, about 1.5 to 5.0, 2.0 to 5.5, about 2.0 to about 5.0, such as about 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, etc. Approximately 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1 .45, approximately 1.46, approximately 1.47, approximately 1.48, approximately 1.49, approximately 1.5, approximately 1.51, approximately 1.52, approximately 1.53, approximately 1.54, approximately 1.55, approximately 1.56, approximately 1.57, approximately 1.58, approximately 1.59, approximately 1.6, approximately 1.61, approximately 1.62, approximately 1.63, approximately 1.64, approximately 1.65, approximately 1.66, approximately 1.67, approximately 1.68, approximately 1.69, approximately 1.7, approximately 1.71, approximately 1.72, approximately 1.73, approximately 1.74, approximately 1.7 5. Approximately 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.0, 2.01, 2.02, 2.03, 2.04, 2.05. Approximately 2.06, 2.07, 2.08, 2.09, 2.1, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.2, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.3, 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.4, 2.41. Approximately 2.42, 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.49, 2.5, 2.51, 2.52, 2.53, 2.54, 2.55, 2.56, 2.57, 2.58, 2.59, 2.6, 2.61, 2.62, 2.63, 2.64, 2.65, 2.66, 2.67, 2.68, 2.69, 2.7, 2.71, 2.72, 2.73, 2.74, 2.75, 2.76, 2.77, 2.78 Approximately 2.79, approximately 2.8, approximately 2.81, approximately 2.82, approximately 2.83, approximately 2.84, approximately 2.85, approximately 2.86, approximately 2.87, approximately 2.88, approximately 2.89, approximately 2.9, approximately 2.91, approximately 2.92, approximately 2.93, approximately 2.94, approximately 2.95, approximately 2.96, approximately 2.97, approximately 2.98, approximately 2.99, approximately 3.0, approximately 3.01, approximately 3.02, approximately 3.03, approximately 3.04, approximately 3.05, approximately 3.06, approximately 3.07, approximately 3.08, approximately 3.09, approximately 3.1, approximately 3.11, approximately 3.12, approximately 3.13, approximately 3.14, approximately 3.15 Approximately 3.16, 3.17, 3.18, 3.19, 3.2, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.3, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36, 3.37, 3.38, 3.39, 3.4, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.5, 3.51, 3.52. Approximately 3.53, 3.54, 3.55, 3.56, 3.57, 3.58, 3.59, 3.6, 3.61, 3.62, 3.63, 3.64, 3.65, 3.66, 3.67, 3.68, 3.69, 3.7, 3.71, 3.72, 3.73, 3.74, 3.75, 3.76, 3.77, 3.78, 3.79, 3.8, 3.81, 3.82, 3.83, 3.84, 3.85, 3.86, 3.87, 3.88, 3.89 Approximately 3.9, approximately 3.91, approximately 3.92, approximately 3.93, approximately 3.94, approximately 3.95, approximately 3.96, approximately 3.97, approximately 3.98, approximately 3.99, approximately 4.0, approximately 4.01, approximately 4.02, approximately 4.03, approximately 4.04, approximately 4.05, approximately 4.06, approximately 4.07, approximately 4.08, approximately 4.09, approximately 4.1, approximately 4.11, approximately 4.12, approximately 4.13, approximately 4.14, approximately 4.15, approximately 4.16, approximately 4.17, approximately 4.18, approximately 4.19, approximately 4.2, approximately 4.21, approximately 4.22, approximately 4.23, approximately 4.24, approximately 4.25, approximately 4.26 Approximately 4.27, 4.28, 4.29, 4.3, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.4, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 4.5, 4.51, 4.52, 4.53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, 4.6, 4.61, 4.62, 4.63 Approximately 4.64, 4.65, 4.66, 4.67, 4.68, 4.69, 4.7, 4.71, 4.72, 4.73, 4.74, 4.75, 4.76, 4.77, 4.78, 4.79, 4.8, 4.81, 4.82, 4.83, 4.84, 4.85, 4.86, 4.87, 4.88, 4.89, 4.9, 4.91, 4.92, 4.93, 4.94, 4.95, 4.96, 4.97, 4.98, 4.99, 5.0.

A pharmaceutical composition comprising a compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, a conjugate of any one of claims 8-10, or a composition of claim 11, and one or more pharmaceutical excipients.

Use of the compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 8-10, the composition of claim 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating cancer.

The use according to claim 13, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 8-10, the composition of claim 11, or the pharmaceutical composition of claim 12, for the treatment of cancer.

The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 8-10, the composition of claim 11, or the pharmaceutical composition of claim 12, for the treatment of solid tumors or hematologic malignancies, for example selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), and urothelial carcinoma.

A cancer treatment method comprising, based on an individually therapeutically effective amount of any one of claims 1-7, a pharmaceutically acceptable salt thereof, a conjugate of any one of claims 8-10, a composition of claim 11, or a pharmaceutical composition of claim 12.

The method of claim 17, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

The following are examples of compounds or their salts, stereoisomers, tautomers, or isotopically labeled compounds:

in

Each of PG ₁ is independently protected by an H or a carboxyl group, such as a _C1-6 alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, or 4-p-methylbenzyloxybenzyl.

Each of PG ₂ is independently protected by an H or hydroxyl group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl.

Each of PG ₃ is independently protected by an H or amino group. The amino protecting group may be an alkoxycarbonyl group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), or methoxycarbonyl (or ethoxycarbonyl); or an acyl group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), or o-( p-Nitrobenzenesulfonyl (Ns), p-pentanoyl, benzoyl, tert-butoxycarbonyl, 9-fluorenmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as triphenylmethyl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

Lg is a leaving group; preferably, Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated _C1-6 alkyl, _C1-6 alkylsulfonyl, halogenated _C1-6 alkylsulfonyl, halogenated sulfonyl, _C1-6 alkyl sulfonate, halogenated _C1-6 alkyl sulfinate, _C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxyl (-OH), mercapto ( _-SH ), amino ( _-NH2 ), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments. The halogenated _C1-6 alkyl, C1-6 alkylsulfonyl, halogenated _C1-6 alkylsulfonyl, halogenated sulfonyl, _C1-6 alkyl sulfonate, halogenated _C1-6 alkyl sulfonate, C1-6 alkyl sulfinate, _C1-6 alkyl sulfinate, _C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide ... _{The 1-6} alkyl sulfoxide, halophenoxy, alkenyl, alkynyl and alkynyl-containing structural segments are optionally substituted by one or more suitable substituents; preferably, Lg is selected from halogens, substituted or unsubstituted _C1-6 alkyl sulfonyl, halophenoxy, hydroxyl (-OH), mercapto (-SH) or amino ( _-NH2 );

s is selected from an integer from 1 to 20, preferably from an integer from 1 to 15, such as an integer from 1 to 12 or 3 to 12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, or 10.