Fused-ring compound, preparation method therefor, and use thereof

Abstract

A fused-ring compound, a preparation method therefor, and use thereof. The fused-ring compound has a structure represented by the formula D-E-L-M' and can be used for preparing an antibody-drug conjugate. The prepared antibody-drug conjugate has a relatively good drug-to-antibody conjugation ratio and has excellent targeted killing effects on solid tumors such as gastric cancer, breast cancer, lung cancer, and urothelial carcinoma.

Description

Fused ring compounds, their preparation methods and uses

[0001] This application is based on and claims priority to Chinese patent applications No. 202410585326.9, filed on May 10, 2024, and No. 202411554683.5, filed on November 1, 2024, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0002] This application relates to the pharmaceutical field, specifically to a fused-ring compound and its preparation method and uses. Background Technology

[0003] Antibody-drug conjugates (ADCs) are a new class of drugs used to treat cancer. They consist of three main parts: a monoclonal antibody, a biologically active small molecule (primarily a cytotoxin capable of killing tumor cells), and a linker connecting the two parts. The antibody is responsible for recognizing specific antigens on the surface of tumor cells, guiding the ADC to precisely target the tumor region. Once the ADC is internalized by tumor cells, the cytotoxin is released inside the cell, effectively killing cancer cells by interfering with tumor cell microtubules or directly damaging DNA, while minimizing damage to normal cells.

[0004] Pyrrolobenzodiazepine PBD (protonyldioxanone) is a class of molecules that specifically bind to the minor groove of DNA and belongs to the anthraxmycin antibiotic family. Studies have shown that linking two PBD units through an alkylene chain can form a PBD dimer with extremely high DNA cross-linking ability, and this structure exhibits extremely high activity in various tumor cells.

[0005] In antibody-drug conjugates (ADCs), the linker is a crucial component connecting the antibody and the drug, and its structure plays a decisive role in the drug's efficacy and safety. Therefore, designing and developing novel PBD linker compounds is of great significance for improving the efficacy of ADC drugs and reducing side effects. This not only enhances drug targeting but also reduces adverse effects on normal tissues, thus providing safer and more effective treatment options for cancer patients. Summary of the Invention

[0006] This application relates to a fused-ring compound having the structure shown in the general formula DEL-M'. The fused-ring compound can be used to prepare antibody-drug conjugates, and the resulting antibody-drug conjugates exhibit excellent targeted killing effects against tumors.

[0007] compound

[0008] In one aspect, this application provides a compound or a pharmaceutically acceptable salt thereof having the structure shown in formula DEL-M', wherein:

[0009] 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;

[0010] L is the structural segment connecting M and E;

[0011] E is a structural segment connecting L and D;

[0012] D is a cytotoxic drug fragment.

[0013] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0014] In some embodiments, M is selected from the following structural segments, whether substituted or unsubstituted:

[0015] In some embodiments, M is selected from the following structural segments, whether substituted or unsubstituted:

[0016] In some embodiments, M is selected from the following structural segments, whether substituted or unsubstituted:

[0017] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1- 6-alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, alkenyl, ynyl and ynyl-containing structural segments may optionally be replaced by one or more suitable substituents.

[0018] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1- 6-alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments.

[0019] In some implementations, Lg is selected from halogenated, substituted, or unsubstituted C. 1-6 Alkyl sulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH), and amino (-NH2).

[0020] In some embodiments, Lg is selected from halogens, substituted or unsubstituted methanesulfonyl groups, halophenoxy groups, hydroxyl (-OH), mercapto (-SH), and amino (-NH2).

[0021] In some implementations, Lg is a methanesulfonyl group.

[0022] In some implementations, L is selected from one or more of the following structural segments or stereoisomers thereof: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic groups, -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), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Gly-Lys, 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-A la, 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)), The structural segment is optionally further substituted; wherein R' consists of one or more groups including, but not limited to, hydrogen, C 1-6 Alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups SO3H, -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-heterocyclic, -CH2NH-SO3H, -CH2N(C 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N + (C 1-6Alkylene (-SO3H)3, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl group 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- 6alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylenes -CO2H, -CH2N + (C 1- (6-alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, or polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). 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), or NOA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), where r is selected from an integer from 1 to 20; s is selected from an integer from 1 to 20.

[0023] In some implementations, "-NOTA" refers to

[0024] In some implementations, "-DOTA" refers to

[0025] In some implementations, "-DOTAGA" refers to

[0026] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: natural amino acids 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))). r OCH3), Lys(R'), and short peptides composed of amino acids (such as Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala). R' is composed of one or more of the following groups, including but not limited to hydrogen, 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), or NOA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), wherein s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, or 10.

[0027] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: natural amino acids 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))). r OCH3), and short peptides composed of amino acids (such as Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala). Where r is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, 9, or 10.

[0028] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: Val, Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala, Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10.

[0029] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: Val, Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala, Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10.

[0030] In some embodiments, L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,

[0031] Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

[0032] In some embodiments, L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,

[0033] Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

[0034] In some implementations, L is selected from the following substituted or unsubstituted structures:

[0035] Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

[0036] In some implementations, E is a single bond, substituted or unsubstituted -NH-CH2- or

[0037] In some implementations, E is a single bond, substituted or unsubstituted -NH-CH2-.

[0038] In some implementations, E is a single bond or -NH-CH2-.

[0039] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0040] Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

[0041] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0042] Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

[0043] In some implementation schemes, Selected from the following structures, whether substituted or unsubstituted:

[0044] In some implementation schemes, Selected from:

[0045] In some embodiments, the cytotoxic drug is selected from anti-microtubule agents, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors. In some embodiments, the anti-microtubule agent is an olistatin, maytansine, or eribulin. In some embodiments, the DNA intercalating agent is pyrrolobenzodiazepine. (PBD) compounds, trabectedin, or rubotecin. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecone, or rubotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogues, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin. In some embodiments, the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogues.

[0046] In some embodiments, the cytotoxic drug is selected from pyrrolobenzodiazepines. (PBD) class of compounds.

[0047] The cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), and secondary amino (-NR). a H), tertiary amino group (-NR) b R c ), where R a R b R c In this context, it refers only to non-hydrogen substituents on N, or mercapto groups (-SH), which can react with suitable functional groups in the rest of the conjugate to achieve linkage.

[0048] In some embodiments, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary amino, or tertiary amino group, or a -SH group. In some embodiments, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amino group on the cytotoxic drug. In some embodiments, D is a monovalent structure obtained by losing an H from the -OH or -NH2 group on the cytotoxic drug.

[0049] In some embodiments, the cytotoxic drug is selected from compounds with the structure shown in formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs thereof:

[0050] Among them, R 1 and R 2 Each is independently selected from hydrogen, -CN, halogen, and -OR. a -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C1- 6-alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1- 6-alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0051] R 4 and R 4’ Each is independently selected from hydrogen, halogen, -OH, -NH2, C 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 1-6 Alkoxy, R 5 and R 5’ All are hydrogen;

[0052] Or, R 4 and R 4’ Together with R 4 and R 4’ The carbon atoms form carbon-carbon double bonds or 3-6 membered carbon rings, R 5 and R 5’ All are hydrogen;

[0053] Or, R 4 and R 5 Connect to the key or together with the R connection 4 and R 5 The carbon atoms form 3-6 membered carbon rings, R 5’ and R 4’ Each is independently selected from hydrogen, halogen, -NH2, C 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 1-6 Alkoxy;

[0054] R 6 For hydrogen, C 1-6 alkyl or

[0055] R 6’ R 7 and R 7’ All are hydrogen; or R 6’ With R 7 Linking and bonding, R 7’ It is hydrogen;

[0056] R a Selected from H, C 1-6 Alkyl or C 3-6 cycloalkyl;

[0057] Ring A is selected from 3-6 membered cycloalkyl groups, C 6-10 aryl, 5-10 membered heteroaryl, 3-6 membered heterocyclic; the 3-6 membered cycloalkyl, C 6-10 The aryl, 5-10 membered heteroaryl, and 3-6 membered heterocyclic groups may be optionally substituted by one or more of the following substituents: hydrogen, -CN, halogen, -OR. a -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0058] R 3 Selected from hydrogen, -CN, halogen, -OR a -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0059] X and Y are independently -CH2- and -CD2-, respectively. and -C(O)-;

[0060] t is selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0061] In some implementations, when n in formula (I) is 5 and ring A is phenyl, R 3 It is not methoxylated.

[0062] In some implementation schemes, R 1 Selected from hydrogen, -CN, halogen, -OR a and C 1-6 Alkyl, wherein R a Selected from C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0063] In some implementation schemes, R 1 Selected from -OR a , where R a Selected from C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0064] In some implementation schemes, R 1 It is selected from methoxy, trideuteroxy and -O-cyclopropyl.

[0065] In some implementation schemes, R 1 Selected from hydrogen, -CN, halogen, -OR a and C 1-6 Alkyl, wherein R a C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0066] In some implementation schemes, R 1 For -OR a , where R a C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0067] In some implementation schemes, R 1 It is methoxy or -O-cyclopropyl.

[0068] In some implementation schemes, R 2 Selected from hydrogen, -CN, halogen, -OR a and C 1-6 Alkyl, wherein R a C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0069] In some implementation schemes, R 2 For -OR a , where R a C 1-6 alkyl.

[0070] In some implementation schemes, R 2 It is a methoxy group.

[0071] In some implementation schemes, R 4 R is hydrogen or halogen. 4’ Selected from hydrogen, halogens, -OH, -NH2, C 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 1-6 Alkoxy, R 5 and R 5’ Both are hydrogen; or, R 4 and R 4’ Together with R 4 and R 4’ The carbon atoms form carbon-carbon double bonds or 3-6 membered carbon rings, R 5 and R 5’ Both are hydrogen; or, R 4 With R 5 Connect to the key or together with the R connection 4 and R 5 The carbon atoms form 3-6 membered carbon rings, R 5’ and R 4’ Both are hydrogen.

[0072] In some implementation schemes, R 4 For hydrogen or fluorine, R 4’ Selected from hydrogen, fluorine, -OH, -NH2, methyl, trifluoromethyl, and methoxy, R 5 and R 5’ Both are hydrogen; or, R 4 and R 4’ Together with R 4 and R 4’ The carbon atoms form carbon-carbon double bonds or 3-6 membered carbon rings, R 5 and R 5’ Both are hydrogen; or, R 4 With R 5 Connect to the key or together with the R connection 4 and R 5 The carbon atoms form a 3-membered carbon ring, R 5’ and R 4’ Both are hydrogen.

[0073] In some implementation schemes, R 4 For hydrogen or fluorine, R 4’ Selected from hydrogen, fluorine, -OH, -NH2, methyl, trifluoromethyl, and methoxy, R 5 and R 5’ Both are hydrogen; or, R 4 and R 4’ Together with R 4 and R4’ The carbon atoms form carbon-carbon double bonds, R 5 and R 5’ Both are hydrogen; or, R 4 With R 5 Connect to the key or together with the R connection 4 and R 5 The carbon atoms form a 3-membered carbon ring, R 5’ and R 4’ Both are hydrogen.

[0074] In some implementation schemes, R 4 and R 4’ With connection R 4 and R 4’ The carbon atoms form a 3-membered carbon ring, R 5 and R 5’ Both are hydrogen.

[0075] In some implementation schemes, R 6 for

[0076] Ring A is selected from 3-6 membered cycloalkyl groups, C 6-10 Aryl or 5-10 heteroaryl groups;

[0077] R 3 Selected from hydrogen, -CN, halogen, -OR a -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0078] R a Selected from H and C 1-6 alkyl.

[0079] In some implementation schemes, R 6 for

[0080] Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;

[0081] R 3Selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.

[0082] In some implementation schemes, R 6 for

[0083] Ring A is phenyl;

[0084] R 3 Selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.

[0085] In some implementations, ring A is C 6-10 Aryl or 5-10 heteroaryl compounds.

[0086] In some embodiments, ring A is selected from phenyl, pyrimidinyl, pyridinyl, furanyl, and thiophenyl.

[0087] In some implementation schemes, R 3 Selected from -OH, methoxy, -NH2 and hydroxymethyl.

[0088] In some implementation schemes, R 6 Selected from phenyl, pyridyl,

[0089] In some implementation schemes, R 6 Selected from phenyl, pyridyl,

[0090] R 6’ With R 7 Linking and bonding, R 7’ It is hydrogen.

[0091] In some implementation schemes, R 6 for

[0092] R 6’ R 7 and R 7’ Both are hydrogen.

[0093] In some implementations, t is 3-8, such as 3, 4, 5, 6, 7 or 8.

[0094] In some implementations, X is -CH2- or -CD2-; optionally, Y is selected from -CH2-, -CD2-, And -C(O)-.

[0095] In some implementations, X is -CH2- and Y is -CH2-; X is -CH2- and Y is -CD2- or -C(O)-; or X is -CD2- and Y is -CH2-.

[0096] In some implementations, X is -CH2- and Y is -CH2-.

[0097] In some implementation schemes,

[0098] R 1 Selected from methoxy, trideuteroxy, and -O-cyclopropyl;

[0099] R 2 It is a methoxy group;

[0100] R 4 For hydrogen or fluorine, R 4’ Selected from hydrogen, fluorine, -OH, -NH2, trifluoromethyl, and methoxy, R 5 and R 5’ Both are hydrogen; or, R 4 and R 4’ With connection R 4 and R 4’ The carbon atoms form carbon-carbon double bonds or three-membered carbon rings, R 5 and R 5’ Both are hydrogen; or, R 4 With R 5 Connect to the key or together with the R connection 4 and R 5 The carbon atoms form a 3-membered carbon ring, R 5’ and R 4’ All are hydrogen;

[0101] Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;

[0102] R 3 Selected from hydrogen, fluorine, -OH, methoxy, -NH2, and hydroxymethyl;

[0103] t is 5.

[0104] In some implementation schemes,

[0105] R 1 and R 2 All are methoxyl groups;

[0106] R 4 For hydrogen or fluorine, R 4’ Selected from hydrogen, fluorine, -OH, -NH2, trifluoromethyl, and methoxy, R 5 and R 5’ Both are hydrogen; or, R 4 and R 4’ With connection R 4 and R 4’ The carbon atoms form carbon-carbon double bonds or three-membered carbon rings, R 5 and R 5’Both are hydrogen; or, R 4 With R 5 Connect to the key or together with the R connection 4 and R 5 The carbon atoms form a 3-membered carbon ring, R 5’ and R 4’ All are hydrogen;

[0107] Selected from

[0108] t is 5.

[0109] In some embodiments, the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:

[0110] In some embodiments, the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:

[0111] In some embodiments, the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:

[0112] In some embodiments, the cytotoxic agent is a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof:

[0113] In some embodiments, the cytotoxic drug fragment (D) has the structure shown in formula (I'):

[0114] Among them: rings A and R 1 R 2 R 4 R 4’ R 5 R 5’ R 6’ R 7 R 7’ X, Y, and t are as described in any of the preceding items;

[0115] R 3A Selected from chemical bonds, -NH-, -N(C 1-6 Alkyl group -, -O-, -C1-6 alkylene-O- and -C 1-6 Alkylene-O-.

[0116] In some embodiments, the cytotoxic drug fragment (D) is selected from the structures shown below:

[0117] In some embodiments, the cytotoxic drug fragment (D) is selected from the structures shown below:

[0118] In some embodiments, the cytotoxic drug fragment (D) has the structure shown below:

[0119] In some embodiments, the compound has the following structure:

[0120] Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

[0121] Antibody-drug conjugates

[0122] A second aspect of the invention provides a coupling as shown in formula (II), wherein: A-[MLED] x Equation (II)

[0123] M, L, E, and D are as described in any of the preceding items; x is 1-10;

[0124] A represents the target group, with targets selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, O772P, MPF, Napi3b, Sema 5b, and PSCA. hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2. BMPR1B, Tyro7, c-Met, ApoE, CD1lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10 RA1, IL-6, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL 5. 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, CDllb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and TENB2. .

[0125] In some preferred embodiments, A is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linking two aliphatic indoles, an anthocyanin dye, or IR-783 or a derivative thereof.

[0126] In some preferred embodiments, A is an antibody, such as a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment 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.

[0127] In some preferred embodiments, A is an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase family.

[0128] Those skilled in the art will understand that the antibody-drug conjugates described in this application can be prepared in a modular manner. For example, the compound M'-LED (i.e., the free form of the "drug-linker") described in any of the above-mentioned claims can be obtained first, wherein M' is the structural form of M before covalently linking it with an antibody or its antigen-binding fragment, and then it can be covalently linked with the antibody or its antigen-binding fragment to obtain the antibody-drug conjugates described in this application. Accordingly, in the free form of the "drug-linker", M' is linked to one or more thiol (-SH) or amino (-NH2) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of the -SO2Me or pentafluorophenol structure) or an addition reaction. In some embodiments, -MLED can be formed from the following structure, for example, through a substitution reaction (e.g., removal of the methanesulfonyl group structure).

[0129] In some embodiments, the antibody-drug conjugates of this application are formed by conjugating the compounds A-1 to A-9, B-1 to B-19 or G-1 to G-16 described above with an antibody or antigen-binding fragment.

[0130] In some embodiments, the antibody-drug conjugate of this application is formed by desaturating the methylsulfonyl group of the compounds A-1 to A-9, B-1 to B-19 or G-1 to G-16 described above and then conjugating them with an antibody or antigen-binding fragment.

[0131] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0132] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0133] (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,

[0134] (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;

[0135] 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;

[0136] or,

[0137] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0138] (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,

[0139] (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;

[0140] 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;

[0141] or,

[0142] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0143] (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,

[0144] (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;

[0145] Wherein, the variant described in any one of (3a) and (3b) 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;

[0146] or,

[0147] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0148] (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,

[0149] (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;

[0150] 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.

[0151] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0152] (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:

[0153] (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,

[0154] (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;

[0155] 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;

[0156] or,

[0157] (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:

[0158] (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,

[0159] (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;

[0160] 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;

[0161] or,

[0162] (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:

[0163] (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,

[0164] (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;

[0165] Wherein, the variant described in any one of (3a) and (3b) 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;

[0166] or,

[0167] (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:

[0168] (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,

[0169] (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;

[0170] 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.

[0171] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0172] (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:

[0173] (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 / or, 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,

[0174] (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 / or 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;

[0175] or,

[0176] (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:

[0177] (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 / or, 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,

[0178] (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 / or 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;

[0179] or,

[0180] (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:

[0181] (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 / or, 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,

[0182] (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 / or 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;

[0183] or,

[0184] (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:

[0185] (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 / or, 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,

[0186] (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 / or 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.

[0187] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0188] (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:

[0189] (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,

[0190] (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;

[0191] or,

[0192] (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:

[0193] (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,

[0194] (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;

[0195] or,

[0196] (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:

[0197] (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,

[0198] (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;

[0199] or,

[0200] (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:

[0201] (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,

[0202] (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.

[0203] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0204] (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

[0205] (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;

[0206] 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 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, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0207] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0208] (a) VH as shown in SEQ ID NO: 1, and / or VL as shown in SEQ ID NO: 2; or

[0209] (b) VH as shown in SEQ ID NO: 3, and / or VL as shown in SEQ ID NO: 4.

[0210] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0211] (a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

[0212] (b) VH or a variant thereof shown in SEQ ID NO: 3, and VL or a variant thereof shown in SEQ ID NO: 4.

[0213] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0214] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0215] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0216] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0217] (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

[0218] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has 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).

[0219] In some embodiments, 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.

[0220] In some embodiments, 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, 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: 35.

[0221] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.

[0222] In some embodiments, 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.

[0223] In some embodiments, the antibody or its antigen-binding fragment includes a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0224] In some embodiments, 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.

[0225] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0226] (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

[0227] (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.

[0228] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 80.

[0229] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a 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.

[0230] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0231] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0232] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0233] In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0234] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine residue.

[0235] In some embodiments, the antibody-drug conjugate is selected from:

[0236] Among them, HA-(S- or) in each antibody-drug conjugate It is an antibody or its antigen-binding fragment;

[0237] Wherein, -(S- or represents the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the M fragment;

[0238] n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

[0239] In some implementations, the HA in each antibody-drug conjugate includes:

[0240] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0241] (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,

[0242] (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;

[0243] 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;

[0244] or,

[0245] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0246] (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,

[0247] (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;

[0248] 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;

[0249] or,

[0250] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0251] (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,

[0252] (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;

[0253] Wherein, the variant described in any one of (3a) and (3b) 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;

[0254] or,

[0255] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0256] (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,

[0257] (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;

[0258] 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.

[0259] In some implementations, the HA in each antibody-drug conjugate includes:

[0260] (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:

[0261] (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,

[0262] (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;

[0263] 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;

[0264] or,

[0265] (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:

[0266] (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,

[0267] (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;

[0268] 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;

[0269] or,

[0270] (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:

[0271] (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,

[0272] (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;

[0273] Wherein, the variant described in any one of (3a) and (3b) 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;

[0274] or,

[0275] (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:

[0276] (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,

[0277] (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;

[0278] 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.

[0279] In some implementations, the HA in each antibody-drug conjugate includes:

[0280] (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:

[0281] (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 / or, 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,

[0282] (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 / or 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;

[0283] or,

[0284] (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:

[0285] (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 / or, 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,

[0286] (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 / or 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;

[0287] or,

[0288] (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:

[0289] (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 / or, 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,

[0290] (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 / or 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;

[0291] or,

[0292] (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:

[0293] (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 / or, 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,

[0294] (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 / or 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.

[0295] In some implementations, the HA in each antibody-drug conjugate includes:

[0296] (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:

[0297] (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,

[0298] (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;

[0299] or,

[0300] (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:

[0301] (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,

[0302] (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;

[0303] or,

[0304] (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:

[0305] (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,

[0306] (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;

[0307] or,

[0308] (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:

[0309] (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,

[0310] (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.

[0311] In some implementations, the HA in each antibody-drug conjugate includes:

[0312] (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

[0313] (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;

[0314] 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 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, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0315] In some implementations, the HA in each antibody-drug conjugate includes:

[0316] (a) VH as shown in SEQ ID NO: 1, and / or VL as shown in SEQ ID NO: 2; or

[0317] (b) VH as shown in SEQ ID NO: 3, and / or VL as shown in SEQ ID NO: 4.

[0318] In some implementations, the HA in each antibody-drug conjugate includes:

[0319] (a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

[0320] (b) VH or a variant thereof shown in SEQ ID NO: 3, and VL or a variant thereof shown in SEQ ID NO: 4.

[0321] In some implementations, the HA in each antibody-drug conjugate includes:

[0322] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0323] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0324] In some implementations, the HA in each antibody-drug conjugate further comprises:

[0325] (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

[0326] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has 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).

[0327] In some embodiments, 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.

[0328] In some embodiments, the HA in each antibody-drug conjugate contains a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 35 (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).

[0329] In some implementations, the HA in each antibody-drug conjugate includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.

[0330] In some embodiments, the HA in each antibody-drug conjugate contains a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 36 (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).

[0331] In some implementations, the HA in each antibody-drug conjugate includes a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0332] In some embodiments, the HA in each antibody-drug conjugate includes 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.

[0333] In some implementations, the HA in each antibody-drug conjugate includes:

[0334] (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

[0335] (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.

[0336] In some embodiments, the HA in each antibody-drug conjugate is selected from Trastuzumab or Pertuzumab, wherein the amino acid sequence of Trastuzumab has a lookup accession number (IMGT / mAb-DB ID) of 97 in the IMGT database, and the amino acid sequence of Pertuzumab has a lookup accession number (IMGT / mAb-DB ID) of 80 in the IMGT database.

[0337] In some embodiments, the HA in each antibody-drug conjugate may independently contain C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine and / or contain N-terminal glutamine or glutamate, cyclize the N-terminal amino acid to pyroglutamic acid or cyclize the N-terminal amino acid to pyroglutamate salt.

[0338] In some embodiments, the HA in each antibody-drug conjugate may include its post-translational modifications (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate in the heavy or light chain being converted to pyroglutamate or pyroglutamate salt), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0339] In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0340] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine residue.

[0341] In some implementations, the HA in each antibody-drug conjugate represents trastuzumab, pertuzumab, or their antigen-binding fragments.

[0342] In some implementations, the HA in each antibody-drug conjugate represents the following antibody or antigen-binding fragment:

[0343] (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

[0344] (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;

[0345] in, This indicates the specific way in which the thiol group in the antibody or its antigen-binding fragment is linked to the linker.

[0346] In some implementations, x 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.

[0347] In some implementations, x is 5-8, such as 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8.

[0348] In some implementations, x is 1-6, such as 2-6, 3-6, 4-6.

[0349] In some implementations, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0350] The antibody-drug conjugates of the present invention may optionally be substituted with one or more suitable substituents.

[0351] Composition

[0352] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC comprises x drug-linkers as described herein, where x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining the DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0353] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: 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 to 10, 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 to 10, 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 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.

[0354] In some embodiments, the DAR of the ADC compositions described herein is about 3 to 9, such as 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, 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, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 5.0 to 8.0, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 5.5 to 8.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.5, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 7.5, approximately 6.5 to 8.5, approximately 7.0 to 7.5.

[0355] In some embodiments, the DAR of the ADC composition described herein is about 3 to 8, for example, 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 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0, or about 7.5 to 8.0.

[0356] In some embodiments, the DAR of the ADC composition described herein is 7.5 to 8.5, for example, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5.

[0357] In some embodiments, the DAR of the ADC compositions described herein is about 6.0 to 9.0, preferably about 6.0-8.0, for example about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.3, about 6.31, about 6.32, about 6.33, about 6. .34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.4, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6.53, about 6.54, about 6. 55, approximately 6.56, approximately 6.57, approximately 6.58, approximately 6.59, approximately 6.6, approximately 6.61, approximately 6.62, approximately 6.63, approximately 6.64, approximately 6.65, approximately 6.66, approximately 6.67, approximately 6.68, approximately 6.69, approximately 6.7, approximately 6.71, approximately 6.72, approximately 6.73, approximately 6.74, approximately 6.75, approximately 6.7 6, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97 Approximately 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, 7.1, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18 Approximately 7.19, 7.2, 7.21, 7.22, 7.23, 7.24, 7.25, 7.26, 7.27, 7.28, 7.29, 7.3, 7.31, 7.32, 7.33, 7.34, 7.35, 7.36, 7.37, 7.38, 7.39, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.54, approximately 7.55, approximately 7.56, approximately 7.57, approximately 7.58, approximately 7.59, approximately 7.6, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.7, approximately 7.71, approximately 7.72, approximately 7.73, approximately 7.74, approximately 7.75, approximately 7.76, approximately 7.77, approximately 7.78, approximately 7.79, approximately 7.8, approximately 7.81, approximately 7.82, approximately 7.83, approximately 7.84, approximately 7.85, approximately 7.86, approximately 7.87, approximately 7.88, approximately 7.89, approximately 7.9, approximately 7.91, approximately 7.92, approximately 7.93, approximately 7.94, approximately 7.95, approximately 7.96, approximately 7.97, approximately 7.98, approximately 7.99, approximately 8.0, approximately 8.01, approximately 8.02, approximately 8.03, approximately 8.04, approximately 8.05, approximately 8.06, approximately 8.07, approximately 8.08, approximately 8.09, approximately 8.1, approximately 8.11, approximately 8.12, approximately 8.13, approximately 8.14, approximately 8.15, approximately 8.16, approximately 8.17, approximately 8.18, approximately 8.19, approximately 8.2, approximately 8.21, approximately 8.22, approximately 8.23, approximately 8.24, approximately 8.25, approximately 8.26, approximately 8.27, approximately 8.28, approximately 8.29, approximately 8.3, approximately 8.31, approximately 8.32, approximately 8.33, approximately 8.34, approximately 8.35, approximately 8.36, approximately 8.37, approximately 8.38, approximately 8.39, approximately 8.4, approximately 8.41, approximately 8.42, approximately 8.43, approximately 8.44, approximately 8.45, approximately 8.46, approximately 8.47, approximately 8.48, approximately 8.49, approximately 8.5, approximately 8.51, approximately 8.52, approximately 8.53, approximately 8.54, approximately 8.55, approximately 8.56, approximately 8.57, approximately 8.58, approximately 8.59, approximately 8.6, approximately 8.61, approximately 8.62, approximately 8.63, approximately 8.64, approximately 8.65, Approximately 8.66, 8.67, 8.68, 8.69, 8.7, 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77, 8.78, 8.79, 8.8, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8.87, 8.88, 8.89, 8.9, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.0.

[0358] In some embodiments, the DAR of the ADC composition described herein is about 6.88.

[0359] In some embodiments, the DAR of the ADC composition described herein is about 4.07.

[0360] In some embodiments, the DAR of the ADC composition described herein is about 4.51.

[0361] Pharmaceutical Composition

[0362] In another aspect, this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates or compositions described in any of the preceding claims, and one or more pharmaceutical excipients.

[0363] The antibody-drug conjugates or compositions described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, or intratumoral injection. Optionally, antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16). th (ed., Osol, A.Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates may be administered via any route appropriate for the individual to be treated.

[0364] application

[0365] The antibody-drug conjugates, compositions, or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0366] Therefore, this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the antibody-drug conjugates described in any of the preceding claims in the preparation of a medicament for treating Her2-expressing cancers.

[0367] Additionally, this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the conjugates described above in a medicament for treating Her2-expressing cancer.

[0368] Additionally, this application provides a method for treating Her2-expressing cancer, comprising the step of administering a therapeutically effective amount of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the conjugates described in any of the preceding claims to an individual in need.

[0369] In some embodiments, the antibody-drug conjugate, compound, drug-linker, or pharmaceutical composition containing it is sufficient (e.g., in a subject):

[0370] (1) Inhibits cell (such as tumor cells) proliferation;

[0371] (2) Inhibits tumor growth;

[0372] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0373] (4) Inhibit HER2-mediated signal transduction;

[0374] (5) Prevention and / or treatment of HER2-mediated diseases / disorders; or

[0375] (6) Any combination of (1)-(5) above.

[0376] In some implementations, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

[0377] definition

[0378] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques obvious to those skilled in the art. Furthermore, laboratory procedures used herein, such as those related to genomics, nucleic acid chemistry, and molecular biology, are standard procedures widely used in their respective fields. While it is believed that the following terms will be readily understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0379] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid distribution in each region or domain can follow various numbering systems known in the art.

[0380] The term "antibody" also includes embodiments in which the heavy chain constant region contains 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 into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.

[0381] 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. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0382] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0383] The following general rules (published at www.bioinf.org.uk: Professor Andrew C. Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids that form the antigenic epitope that the antibody binds to. In rare cases, these generally constant features may not appear; however, Cys residues are the most conserved feature.

[0384] V H The complete amino acid sequence is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In some embodiments, V H The amino acid sites in the sequence can be numbered sequentially starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the V mentioned herein... H and V L The amino acid sites in the sequence are defined according to their sequential numbering.

[0385] The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can 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 numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

[0386] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0387] The term "antigen-binding fragment" in the antibody terminology refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology 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 (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0388] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0389] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0390] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0391] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:46) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:47) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0392] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0393] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0394] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0395] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0396] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position 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). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage 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)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0397] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0398] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0399] The term "linker" refers to a structural segment that connects a cytotoxic drug to an antibody or antigen-binding fragment. For example, the formula Ab-[MLED] x The -MLE- structure fragment in the text.

[0400] The term "drug-linker" refers to the structure of the cytotoxic drug and linker described in this invention before they are linked to an antibody or its antigen-binding fragment. For example, "drug-linker" refers to M'-LED, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment. The "drug-linker" is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.

[0401] The "drug-linker" also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the "drug-linker" compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0402] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0403] The term "isotopically labeled compound" means that the compound is structurally identical to the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0404] The term "substitution" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a specified compound or structural segment by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound. For example, each substituent may independently consist of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 (alkylene) group, C1-C6 haloalkyl (alkylene) group, C1-C6 alkoxy group, C2-C6 (alkenyl) group, C2-C6 (alkynyl) group, C3-C8 (cycloalkyl) group, 3-8 membered (heterocyclic) group, C6-C 10 (sub-)aryl and 5-10 nucleotide (sub-)heteroaryl, etc.

[0405] If a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted. Attached Figure Description

[0406] Figure 1. Efficacy of antibody-drug conjugate (ADC) in a mouse model of NCI-N87 subcutaneous tumor-bearing cells.

[0407] Figure 2. Changes in body weight of mice in different groups in the NCI-N87 CDX model of human gastric cancer cells.

[0408] Figure 3. Efficacy results of antibody-drug conjugates (ADCs) on the JIMT-1 model.

[0409] Figure 4. Changes in body weight of mice in each group in the JIMT-1 model.

[0410] Figure 5. Efficacy results of antibody-drug conjugates (ADCs) in a breast cancer xenograft model.

[0411] Figure 6. Changes in body weight of mice in different groups in the breast cancer xenograft model. Detailed Implementation

[0412] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0413] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention.

[0414] Information about the sequences involved in this invention is described in the table below:

[0415] The abbreviations used in this article have the following meanings:

[0416] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0417] Nuclear magnetic resonance (¹H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0418] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0419] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0420] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0421] I. Preparation of Compounds

[0422] Preparation Example 1: (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-5)

[0423] Step 1: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-5-2)

[0424] (S)-8-(benzyloxy)-7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (2 g, 5.29 mmol) was dissolved in tetrahydrofuran (100 mL), sodium borohydride (599.85 mg, 15.86 mmol) was added, and then trifluoroacetic acid (1.21 g, 10.57 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then refluxed at 75 °C for 5 hours. The reaction solution was concentrated, and water (50 mL) was added. The mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.82 g, 4.99 mmol), which was used directly in the next step without purification.

[0425] Its structural characterization data are as follows:

[0426] ESI-MS (m / z): 365.2 [M+H] +

[0427] Step 2: Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-5-3)

[0428] (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (100 mg, 0.274 mmol) was dissolved in dichloromethane (0.8 mL) and cooled to 0 °C. Then, methanesulfonic acid (0.4 mL) was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 1 hour. Water (2 mL) was added to the reaction mixture, and the pH was adjusted to 8 with a saturated sodium bicarbonate solution. The mixture was extracted five times with dichloromethane (3 mL x 5), and the combined organic phases were washed with a saturated sodium chloride solution (3 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (75 mg, 0.273 mmol), which was used directly in the next step without purification.

[0429] Its structural characterization data are as follows:

[0430] ESI-MS (m / z): 275.2 [M+H] +

[0431] Step 3: Preparation of (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-5-5)

[0432] (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl-trifluoromethanesulfonate (550 mg, 0.987 mmol) and 4-(tert-butoxycarbonyl)aminophenylboronic acid (468 mg, 1.97 mmol), sodium carbonate (418 mg, 3.95 mmol) were dissolved in a mixture of toluene (10 mL), ethanol (1 mL), and water (1 mL). Tetra(triphenylphosphine)palladium (114 mg, 0.099 mmol) was added, and the mixture was heated at 80 °C for 3 hours under nitrogen purging twice and protection. The reaction solution was then added to ethyl acetate (20 mL) and water (10 mL), stirred, filtered, and the filtrate was allowed to stand and separated. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (397 mg, 0.661 mmol).

[0433] Its structural characterization data are as follows:

[0434] ESI-MS (m / z): 600.7 [M+H] +

[0435] Step 4: Preparation of tert-butyl carbamate (7-5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (7-5-6)

[0436] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (100 mg, 0.167 mmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (46 mg, 0.167 mmol) were dissolved in DMF (1 mL), and potassium carbonate (69 mg, 0.500 mmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH / DCM = 0%–5%) yielded the title compound (130 mg, 0.165 mmol).

[0437] Its structural characterization data are as follows:

[0438] ESI-MS (m / z): 794.4 [M+H] +

[0439] Step 5: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-5-7)

[0440] 200 mg (0.122 mmol) of tert-butyl carbamate (4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (2 mL) was dissolved in dichloromethane. Trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (31 mg, 0.045 mmol).

[0441] Its structural characterization data are as follows:

[0442] ESI-MS (m / z): 694.4 [M+H]+

[0443] Step Six: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-5)

[0444] (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (31 mg, 0.045 mmol) was dissolved in dry tetrahydrofuran (5 mL), sodium borohydride (8.5 mg, 0.224 mmol) was added, and trifluoroacetic acid (20.4 mg, 0.179 mmol) was slowly added dropwise, producing a large amount of gas. After the gas dissipated, the temperature was slowly raised to 70 °C and refluxed for 16 hours. The reaction solution was cooled to room temperature, and the reaction was quenched dropwise with methanol. The solution was then concentrated under reduced pressure. After purification by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–50%), the solution was freeze-dried to give the title compound (4.65 mg, 0.007 mmol).

[0445] Its structural characterization data are as follows:

[0446] ESI-MS (m / z): 680.4 [M+H] +

[0447] 1 H NMR (400MHz, DMSO): δ7.31(d,J=13.4Hz,2H),7.26(s,1H),7.11(d,J=8.4Hz,2H),6.53(d,J=8.5Hz,3H),6. 29(d,J=8.1Hz,3H),5.19(s,2H),4.13(d,J=4.4Hz,1H),3.93(dd,J=12.0,6.1Hz,4H),3.83(d,J=7.6Hz,1H ),3.65(d,J=7.5Hz,6H),3.56-3.46(m,3H),3.31-3.11(m,4H),2.69(dd,J=16.7,3.9Hz,1H),1.95(dd,J=1 2.5,7.3Hz,1H),1.79(dd,J=12.5,7.4Hz,5H),1.56(d,J=6.8Hz,2H),0.67-0.62(m,1H),0.60-0.50(m,3H).

[0448] Its preparation method is as follows:

[0449] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0450] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0451] Preparation Example 2: Preparation of (S)-8-((5-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-3)

[0452] Step 1: Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-1)

[0453] (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (500 mg, 897.13 μmol), (4-(benzyloxy)phenyl)boronic acid (409.18 mg, 1.79 mmol), tetraphenylphosphine palladium (103.67 mg, 89.71 μmol), and sodium carbonate (380.34 mg, 3.59 mmol) were dissolved in toluene (8 mL), water (2 mL), and ethanol (2 mL). The reaction system was reacted at 80 °C for 3 hours. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The organic phases were combined, dried, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the title compound (303 mg, 461.04 μmol).

[0454] Its structural characterization data are as follows:

[0455] ESI-MS (m / z): 591.4 [M+H] +

[0456] Step 2: Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-10-(2-(trimethylsilyl)ethoxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-2)

[0457] (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (186 mg, 314.46 μmol) was dissolved in tetrahydrofuran (5 mL), and the solution was cooled to -78 °C. Then, n-butyllithium (294.81 μL, 471.69 μmol) was added dropwise. The reaction mixture was kept at this temperature for 0.5 h. Then, 2-(trimethylsilyl)ethoxymethyl chloride (78.64 mg, 471.69 μmol) was added dropwise to the reaction mixture. After the addition was complete, the reaction was continued at this temperature for 1 h. After the reaction was complete, saturated ammonium chloride was added to quench the reaction, followed by extraction with ethyl acetate. The organic phase was dried and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the title compound (200 mg, 263.25 μmol).

[0458] Its structural characterization data are as follows:

[0459] ESI-MS (m / z): 721.3 [M+H] +

[0460] Step 3: Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-3-3)

[0461] Sodium borohydride (52.42 mg, 1.39 mmol) and calcium chloride (153.77 mg, 1.39 mmol) were dissolved in a mixture of tetrahydrofuran (2 mL) and ethanol (2 mL). A solution of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-10-(2-(trimethylsilyl)ethoxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (100 mg, 138.55 μmol) in tetrahydrofuran (2 mL) was slowly added dropwise to the above system. The reaction was carried out at room temperature for 16 hours. After the reaction was complete, formic acid (0.5 mL) was added to quench the reaction. Stirring was continued for 10 minutes. The intermediate state conversion was confirmed. The reaction solution was prepared and purified to obtain the title compound (14 mg, 23.03 μmol).

[0462] Its structural characterization data are as follows:

[0463] ESI-MS (m / z): 577.3 [M+H] +

[0464] Its preparation method is as follows:

[0465] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0466] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0467] Step 4: Preparation of (S)-8-((5-(((S)-2-(4-(benzyloxy)phenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-3-4)

[0468] (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (12 mg, 20.78 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (5.70 mg, 20.78 μmol) were dissolved in DMF (2 mL), and potassium carbonate (8.62 mg, 62.34 μmol) was added. The reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography to obtain the title compound (10 mg, 12.32 μmol).

[0469] Its structural characterization data are as follows:

[0470] ESI-MS (m / z): 771.4 [M+H] +

[0471] Step 5: Preparation of (S)-8-((5-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-3)

[0472] (S)-8-((5-(((S)-2-(4-(benzyloxy)phenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (10.00 mg, 12.97 μmol) was dissolved in dichloromethane (2 mL), and methanesulfonic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 16 hours. After the reaction was completed, the reaction solution was prepared, purified, and lyophilized to obtain the title compound (0.47 mg, 0.69 μmol).

[0473] Its structural characterization data are as follows:

[0474] ESI-MS (m / z): 681.3 [M+H] +

[0475] 1 H NMR (400MHz, DMSO): δ¹H NMR(400MHz,DMSO)δ9.49(s,1H),7.37(s,1H),7.32(s,1H),7.29(s,1H),7.2 6(d,J=8.8Hz,2H),6.73(d,J=8.4Hz,2H),6.54(d,J=6.4Hz,1H),6.31-6.28( m,3H),4.20-4.12(m,1H),3.95-3.89(m,3H),3.86-3.81(m,1H),3.66(s,3H) ,3.64(s,3H),3.57-3.46(m,3H),3.30-3.28(m,1H),3.26-3.20(m,1H),3.17 -3.11(m,1H),2.76-2.67(m,2H),1.98-1.93(m,1H),1.83-1.77(m,4H),1. 59-1.52(m,2H),0.67-0.62(m,1H),0.60-0.56(m,1H),0.56-0.50(m,2H).

[0476] Its preparation method is as follows:

[0477] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0478] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0479] Preparation Example 3: Preparation of (11aS,11a”S)-8,8”-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo][e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one)(7-9)

[0480] Step 1: Preparation of 8,8”-(pentane-1,5-diylbis(oxy))(11aS,11a”S)-bis(7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-carboxylic acid diallyl ester)(7-9-2)

[0481] (S)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-carboxylic acid allyl ester (55 mg, 153.47 μmol) and 1,5-dibromopentane (17.64 mg, 76.73 μmol) were added to DMF (2 mL), followed by potassium carbonate (63.63 mg, 460.40 μmol). The mixture was stirred at room temperature for 20 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (5 mL x 3). The combined organic phases were washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by thin-layer chromatography (petroleum ether / ethyl acetate = 1:2) and concentrated again under reduced pressure to obtain the title compound (100 mg, 127.41 μmol).

[0482] Its structural characterization data are as follows:

[0483] ESI-MS (m / z): 785.4 [M+H] +

[0484] Step 2: Preparation of (11aS,11a”S)-8,8”-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo][e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one)(7-9)

[0485] 8,8”-(pentane-1,5-diylbis(oxy))(11aS,11a”S)-bis(7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-carboxylic acid diallyl ester (80 mg, 101.92 μmol) and tetrahydropyrrole (14.50 mg, 203.85 μmol) were dissolved in DMF (1.5 mL). Pd(PPh3)4 (11.78 mg, 10.19 μmol) was added under nitrogen protection, followed by three nitrogen purgings. The reaction mixture was then stirred at room temperature for 2 hours under nitrogen protection. After the reaction was complete, 5 mL of water was added to the reaction solution, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was first purified by preparative thin-layer chromatography (ethyl acetate:methanol = 20:1) to remove triphenylphosphine oxide, and then concentrated under reduced pressure. After further purification by preparative high-performance liquid chromatography, it was freeze-dried to obtain the title compound (12.83 mg, 20.18 μmol).

[0486] Its structural characterization data are as follows:

[0487] ESI-MS (m / z): 617.4 [M+H] + 309.3 [M / 2+1] +

[0488] 1 H NMR (400MHz, CDCl3) δ7.54(s,2H),6.10(s,2H),3.99(dd,J=16.0,10.4Hz,3H),3.88(d,J=30.0Hz,3H),3.74-3.38(m,4H),2 .04(dd,J=17.2,7.6Hz,1H),1.96-1.83(m,2H),1.71(ddd,J=21.6,12.0,5.6Hz,2H),0.77-0.66(m,1H),0.66-0.50(m,3H).

[0489] Its preparation method is as follows:

[0490] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0491] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0492] Preparation Example 4: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-(methoxy-d3)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-10)

[0493] Step 1: Preparation of methyl 4-bromo-5-fluoro-2-nitrobenzene (7-10-2)

[0494] 4-Bromo-5-fluoro-2-nitrobenzoic acid (5.9 g, 22.35 mmol) was dissolved in methanol (30 mL), thionyl chloride (30 mL) was added, and the mixture was reacted at room temperature for 16 hours. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (3.05 g, 10.97 mmol).

[0495] Step 2: Preparation of methyl 4-bromo-5-(methoxy-d3)-2-nitrobenzene (7-10-3)

[0496] Methyl 4-bromo-5-fluoro-2-nitrobenzene (1.5 g, 5.4 mmol) was dissolved in DMF (6 mL), and deuterated methanol (388.5 mg, 10.8 mmol) and cesium carbonate (2.64 mg, 8.09 mmol) were added. After purging with nitrogen three times, the reaction was carried out at room temperature for 16 hours. The reaction solution was then added to ethyl acetate (20 mL) and water (30 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.08 g, 3.68 mmol).

[0497] Its structural characterization data are as follows:

[0498] 1 H NMR (400MHz, DMSO): δ8.35(s,1H),7.51(s,1H).

[0499] Step 3: Preparation of methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-10-4)

[0500] Methyl 4-bromo-5-(methoxy-d3)-2-nitrobenzene (1.08 g, 3.68 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxoboronane) (2.34 g, 9.21 mmol), bis(triphenylphosphine)-palladium dichloride (775.94 mg, 1.11 mmol), and potassium acetate (1.08 g, 11.05 mmol) were added to a single-necked flask, followed by 1,4-dioxane (15 mL). The mixture was purged with nitrogen three times, heated to 101 °C, and reacted for 16 hours. After cooling to room temperature, the mixture was filtered through diatomaceous earth and concentrated to obtain the crude product (1.1 g, 3.23 mmol), which was used directly in the next step without purification.

[0501] Step 4: Preparation of methyl 4-hydroxy-5-(methoxy-d3)-2-nitrobenzene (7-10-5)

[0502] Methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.1 g, 3.23 mmol) was dissolved in tetrahydrofuran (30 mL), and glacial acetic acid (3.75 mL) was added at 0 °C. Hydrogen peroxide (7 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (30 mL) was added to the reaction solution, and the mixture was stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found. 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and concentrated to obtain the title compound (680 mg, 2.95 mmol).

[0503] Its structural characterization data are as follows:

[0504] ESI-MS (m / z): 230.2 [MH] -

[0505] Step 5: Preparation of methyl 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzene (7-10-6)

[0506] Methyl 4-hydroxy-5-(methoxy-d3)-2-nitrobenzoate (680 mg, 2.95 mmol) was dissolved in DMF (5 mL), potassium carbonate (815.3 mg, 5.91 mmol) and benzyl bromide (1.01 g, 5.91 mmol) were added, and the mixture was reacted at room temperature for 15 hours. After adding 30 mL of water, 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (688 mg, 2.15 mmol).

[0507] Step Six: Preparation of 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoic acid (7-10-7)

[0508] Methyl 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoate (688 mg, 2.15 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL). Lithium hydroxide (103.1 mg, 4.3 mmol) was added, and the mixture was reacted at room temperature for 3 hours. The pH of the system was adjusted to 5 with dilute hydrochloric acid, and a solid precipitated out. The solid was extracted with 30 mL of ethyl acetate, and after separation, the layers were washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered to obtain the title compound (400 mg, 1.31 mmol).

[0509] Step 7: Preparation of (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester (7-10-8)

[0510] 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoic acid (400 mg, 1.31 mmol) and methyl (S)-5-azaspiro[2.4]heptane-6-carboxylate (203 mg, 1.31 mmol) were dissolved in DMF (5 mL), and HATU (597.3 mg, 1.57 mmol) and DIPEA (507 mg, 3.93 mmol) were added. The mixture was reacted at room temperature for 15 hours. After adding 30 mL of water, 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (570 mg, 1.29 mmol).

[0511] Its structural characterization data are as follows:

[0512] ESI-MS (m / z): 444.1 [M+H] +

[0513] Step 8: Preparation of (S)-8-(benzyloxy)-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (7-10-9)

[0514] Methyl (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate (570 mg, 1.29 mmol) was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (4 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (306 mg, 0.8 mmol).

[0515] Its structural characterization data are as follows:

[0516] ESI-MS (m / z): 382.2 [M+H] +

[0517] Step Nine: Preparation of (S)-8-hydroxy-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (7-10-10)

[0518] (S)-8-(benzyloxy)-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (132 mg, 0.29 mmol) was dissolved in dichloromethane (1 mL) and cooled to 0 °C. Then, methanesulfonic acid (0.5 mL) was added dropwise. The reaction mixture was kept at this temperature for 1 hour. Water (3 mL) was added to the reaction mixture, and the pH was adjusted to 8 with a saturated sodium bicarbonate solution. The mixture was extracted three times with dichloromethane (5 mL x 3), and the combined organic phases were washed with a saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (100 mg, 0.27 mmol), which was used directly in the next step without purification.

[0519] Its structural characterization data are as follows:

[0520] ESI-MS (m / z): 292.1 [M+H] +

[0521] Step 10: Preparation of tert-butyl carbamate (7-10-11)

[0522] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (167 mg, 0.27 mmol) and (S)-8-hydroxy-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (100 mg, 0.27 mmol) were dissolved in DMF (2 mL), potassium carbonate (60 mg, 0.54 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH / DCM = 0%–5%) yielded the title compound (120 mg, 0.136 mmol).

[0523] Its structural characterization data are as follows:

[0524] ESI-MS (m / z): 797.3 [M+H] +

[0525] Step 11: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-(methoxy-d3)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-10-12)

[0526] 120 mg (0.136 mmol) of tert-butyl carbamate (4-((S)-7-methoxy-8-((5-((((S)-7-(methoxy-d3))-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (3 mL) was dissolved in dichloromethane. Trifluoroacetic acid (0.6 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (35 mg, 0.05 mmol).

[0527] Its structural characterization data are as follows:

[0528] ESI-MS (m / z): 697.3 [M+H] +

[0529] Step Twelve: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-(methoxy-d3)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-10)

[0530] (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-(methoxy-d3)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (35 mg, 0.05 mmol) was dissolved in dry tetrahydrofuran (5 mL), sodium borohydride (8.8 mg, 0.225 mmol) was added, and trifluoroacetic acid (20.9 mg, 0.18 mmol) was slowly added dropwise, producing a large amount of gas. After the gas dissipated, the temperature was slowly raised to 70 °C and refluxed for 16 hours. The reaction solution was cooled to room temperature, and the reaction was quenched dropwise with methanol. The solution was then concentrated under reduced pressure. After purification by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–50%), the solution was freeze-dried to give the title compound (2.9 mg, 0.004 mmol).

[0531] Its structural characterization data are as follows:

[0532] ESI-MS (m / z): 683.3 [M+H] +

[0533] 1H NMR (400MHz, DMSO): δ7.33(s,1H),7.30(s,1H),7.27(s,1H),7.13(d,J=8.4Hz,1H), 6.58-6.53(m,2H),6.34-6.26(m,2H),4.20-4.10(m,1H),4.0-3.92(m,4H),3.86-3.8 0(m,2H),3.67(s,3H),3.56(s,3H),3.3-3.22(m,3H),3.20-3.12(m,2H),2.76-2.66 (m,2H),2.02-1.94(m,1H),1.85-1.77(m,4H),1.63-1.52(m,2H),0.62-0.52(m,3H).

[0534] Its preparation method is as follows:

[0535] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0536] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0537] Preparation Example 5: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-11)

[0538] Step 1: Preparation of methyl 4-bromo-5-cyclopropoxy-2-nitrobenzene (7-11-1)

[0539] Methyl 4-bromo-5-fluoro-2-nitrobenzoate (1.5 g, 5.4 mmol) was dissolved in DMF (6 mL), and cyclopropanol (635 mg, 10.97 mmol) and cesium carbonate (2.68 mg, 8.3 mmol) were added. After purging with nitrogen three times, the reaction was carried out at room temperature for 16 hours. The reaction solution was then added to ethyl acetate (30 mL) and water (30 mL) and stirred. The mixture was allowed to stand and separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (750 mg, 2.37 mmol).

[0540] Its structural characterization data are as follows:

[0541] 1 H NMR (400MHz, DMSO): δ8.41(s,1H),7.70(s,1H),4.21(tt,J=5.8,2.8Hz,1H),3.88(s,3H),0.92(m,2H),0.80(m,2H).

[0542] Step 2: Preparation of methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-11-2)

[0543] Methyl 4-bromo-5-cyclopropoxy-2-nitrobenzene (750 mg, 2.37 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxoboronane) (1.5 g, 5.93 mmol), bis(triphenylphosphine)-palladium dichloride (499.6 mg, 0.71 mmol), and potassium acetate (700 mg, 7.12 mmol) were added to a single-necked flask, followed by 1,4-dioxane (15 mL). The mixture was purged with nitrogen three times, heated to 101 °C, and reacted for 16 hours. After cooling to room temperature, the mixture was filtered through diatomaceous earth and concentrated to obtain the crude product (1.6 g, 4.41 mmol), which was used directly in the next step without purification.

[0544] Step 3: Preparation of methyl 5-cyclopropoxy-4-hydroxy-2-nitrobenzene (7-11-3)

[0545] Methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.6 g, 4.41 mmol) was dissolved in tetrahydrofuran (40 mL), and glacial acetic acid (5 mL) was added at 0 °C. Hydrogen peroxide (10 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (50 mL) was added to the reaction solution and stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found. 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and concentrated to obtain the title compound (1.05 g, 4.15 mmol).

[0546] Its structural characterization data are as follows:

[0547] ESI-MS (m / z): 253.2 [MH] -

[0548] Step 4: Preparation of methyl 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzene (7-11-4)

[0549] Methyl 5-cyclopropoxy-4-hydroxy-2-nitrobenzoate (1.05 g, 4.15 mmol) was dissolved in DMF (5 mL), potassium carbonate (1.2 g, 8.6 mmol) and benzyl bromide (1.49 g, 8.6 mmol) were added, and the mixture was reacted at room temperature for 15 hours. After adding 30 mL of water, 50 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.09 g, 3.17 mmol).

[0550] Step 5: Preparation of 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoic acid (7-11-5)

[0551] Methyl 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoate (1.09 g, 3.17 mmol) was dissolved in tetrahydrofuran (8 mL) and water (2 mL). Lithium hydroxide (152.1 mg, 6.35 mmol) was added, and the mixture was reacted at room temperature for 3 hours. The pH of the system was adjusted to 5 with dilute hydrochloric acid, and a solid precipitated out. The solid was extracted with 60 mL of ethyl acetate, and after separation, the layers were washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The concentrate yielded the title compound (743 mg, 2.26 mmol), which was used directly in the next step without purification.

[0552] Step Six: Preparation of (S)-5-(4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester (7-11-6)

[0553] 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoic acid (743 mg, 2.26 mmol) and methyl (S)-5-azaspiro[2.4]heptane-6-carboxylate (700 mg, 4.51 mmol) were dissolved in DMF (5 mL), and HATU (1.71 g, 4.51 mmol) and DIPEA (875 mg, 6.77 mmol) were added. The mixture was reacted at room temperature for 2 hours. After adding 30 mL of water, 50 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (910 mg, 1.95 mmol).

[0554] Its structural characterization data are as follows:

[0555] ESI-MS (m / z): 467.1 [M+H] +

[0556] Step 7: Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (7-11-7)

[0557] Methyl (S)-5-(4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate (910 mg, 1.95 mmol) was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (6 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (467 mg, 1.15 mmol).

[0558] Its structural characterization data are as follows:

[0559] ESI-MS (m / z): 405.2 [M+H] +

[0560] Step 8: Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-11-8)

[0561] (S)-8-(benzyloxy)-7-cyclopropoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (467 mg, 1.15 mmol) was dissolved in dry tetrahydrofuran (20 mL), sodium borohydride (128 mg, 3.46 mmol) was added, and trifluoroacetic acid (264 mg, 2.31 mmol) was slowly added dropwise, producing a large amount of gas. After the gas dissipated, the mixture was slowly heated to 70 °C and refluxed for 16 hours. The reaction solution was cooled to room temperature, and the reaction was quenched dropwise with methanol and concentrated under reduced pressure. After purification by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–80%), the solution was freeze-dried to give the title compound (380 mg, 0.97 mmol).

[0562] Its structural characterization data are as follows:

[0563] ESI-MS (m / z): 363.3 [M+H] +

[0564] Step Nine: Preparation of (S)-7-cyclopropoxy-8-hydroxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-11-9)

[0565] (S)-8-(benzyloxy)-7-cyclopropoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (380 mg, 0.97 mmol) was dissolved in dichloromethane (1.5 mL) and cooled to 0 °C. Then, methanesulfonic acid (0.6 mL) was added dropwise. The reaction mixture was kept at this temperature for 1 hour. Water (10 mL) was added to the reaction mixture, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The mixture was extracted three times with dichloromethane (8 mL x 3), and the combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (118 mg, 0.31 mmol), which was used directly in the next step without purification.

[0566] Its structural characterization data are as follows:

[0567] ESI-MS (m / z): 301.1 [M+H] +

[0568] Step 10: Preparation of tert-butyl carbamate (7-11-10)

[0569] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (141 mg, 0.23 mmol) and (S)-7-cyclopropoxy-8-hydroxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (118 mg, 0.31 mmol) were dissolved in DMF (2 mL), potassium carbonate (49 mg, 0.47 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by silica gel column chromatography (MeOH / DCM = 0%–5%) to give the title compound (81 mg, 0.09 mmol).

[0570] Its structural characterization data are as follows:

[0571] ESI-MS (m / z): 806.3 [M+H] +

[0572] Step 11: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-11)

[0573] 81 mg (0.09 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.6 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (3.5 mg, 0.005 mmol).

[0574] Its structural characterization data are as follows:

[0575] ESI-MS (m / z): 706.3 [M+H]+

[0576] 1 H NMR (400MHz, DMSO): δ7.58(s,1H),7.27(d,J=11.8Hz,2H),7.11(d,J=8.4Hz,2H),6.57-6.51( m,2H),6.29(d,J=9.0Hz,2H),5.19(s,2H),4.18-4.07(m,1H),4.01-3.88(m,4H),3.86-3.80( m,1H),3.67(s,3H),3.57-3.51(m,4H),3.27-3.10(m,4H),2.73-2.65(m,1H),2.02-1.93(m,1 H),1.88-1.71(m,5H),1.61-1.48(m,2H),1.24(s,2H),0.88-0.75(m,1H),0.69-0.48(m,6H).

[0577] Its preparation method is as follows:

[0578] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0579] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0580] Preparation Example 6: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one-11,11-d2(7-21)

[0581] Step 1: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one-11,11-d2(7-21-1)

[0582] (S)-8-(benzyloxy)-7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (1.00 g, 2.64 mmol) was dissolved in tetrahydrofuran (3.00 mL). After cooling to 0 °C, a tetrahydrofuran solution of deuterated borane (0.75 M, 7.05 mL) was added dropwise to the reaction system under nitrogen protection. The temperature was restored to 25 °C and stirring was continued for 2 hours. After adding methanol (100 mL) to the reaction system and stirring for another half hour, the mixture was directly concentrated to obtain the crude title compound (1.00 g), which was used directly in the next step without purification.

[0583] Its structural characterization data are as follows:

[0584] ESI-MS (m / z): 367.2 [M+H] +

[0585] Step 2: Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one-11,11-d2(7-21-2)

[0586] (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one-11,11-d2 (1.00 g, 2.73 mmol) was dissolved in methanol (10.0 mL), purged three times with nitrogen, and then Pd / C (0.20 g, 187 μmol, 10% purity) was added. Hydrogen gas was then introduced, and the reaction was continued for 2 hours. The reaction solution was filtered through a silica gel pad and directly concentrated to obtain the crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 97 / 3), it was concentrated again to obtain the title compound (500 mg, 1.77 mmol).

[0587] Its structural characterization data are as follows:

[0588] ESI-MS (m / z): 277.1 [M+H] +

[0589] 1H NMR: (400MHz, CD3OD) δ7.36(s,1H),6.52(s,1H),3.95-4.04(m,1H),3.86(s,3H),3.63(d,J=11.6Hz,1H),3.49(d ,J=12.0Hz,1H),2.26(dd,J=12.8,8.0Hz,1H),1.68(dd,J=12.8,4.4Hz,1H),0.69-0.79(m,2H),0.62-0.69(m,2H)

[0590] Step 3: Preparation of tert-butyl carbamate (7-21-3)

[0591] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (21.23 mg, 36.19 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one-11,11-d2 (10 mg, 36.19 μmol) were dissolved in DMF (1 mL), potassium carbonate (14.98 mg, 108.57 μmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by silica gel column chromatography (MeOH / DCM = 0%–5%) to give the title compound (20 mg, 25.58 μmol).

[0592] Its structural characterization data are as follows:

[0593] ESI-MS (m / z): 794.4 [M+H] +

[0594] Step 4: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one-11,11-d2(7-21)

[0595] 20 mg (25.58 μmol) of tert-butyl carbamate was dissolved in 1 mL of dichloromethane. Trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (4.7 mg, 6.82 μmol).

[0596] Its structural characterization data are as follows:

[0597] ESI-MS (m / z): 682.4 [M+H] +

[0598] 1 H NMR (400MHz, DMSO) δ7.28(d,J=13.4Hz,2H),7.27(s,1H),7.15(d,J=8.5Hz,2H),6. 59(d,J=8.6Hz,2H),6.30(d,J=10.2Hz,2H),3.95(dd,J=12.0,6.1Hz,4H),3.66(d,J =7.5Hz,6H),3.59-3.51(m,2H),3.37-3.15(m,4H),2.76-2.67(m,2H),2.03-1.92( m,2H),1.85-1.75(m,4H),1.62-1.52(m,2H),0.68-0.58(m,2H),0.56-0.48(m,2H).

[0599] Its preparation method is as follows:

[0600] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0601] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0602] Preparation Example 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22)

[0603] Step 1: Preparation of (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-L-proline methyl ester (7-22-2)

[0604] 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (3.00 g, 9.89 mmol) and L-proline methyl ester hydrochloride (1.97 g, 11.87 mmol) were dissolved in DMF (30 mL), and HATU (5.64 g, 14.84 mmol) and DIPEA (3.84 g, 29.68 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. The reaction solution was poured into water (100 mL), and the product was extracted with EA / PE = 1 / 1 (200 mL). The product was washed with brine (30 mL) and concentrated to obtain the title compound (4.10 g, 9.89 mmol), which was used directly in the next step without purification.

[0605] Its structural characterization data are as follows:

[0606] MS m / z (ESI): 415.1 [M+H] +

[0607] Step 2: Preparation of (S)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-22-3)

[0608] The compound (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-L-proline methyl ester (3.80 g, 8.98 mmol) was dissolved in methanol (40 mL) and tetrahydrofuran (40 mL), and 10% palladium on carbon (380 mg) and HOAc (1.08 g, 17.95 mmol) were added. The air was removed under vacuum, hydrogen was introduced, and the reaction was carried out at 25 °C for 16 hours. The palladium on carbon was filtered off, and the filtrate was concentrated to obtain the title compound (2.35 g, 8.98 mmol), which was used directly in the next step without purification.

[0609] Its structural characterization data are as follows:

[0610] MS m / z (ESI): 263.1 [M+H] +

[0611] Step 3: Preparation of (S)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22-4)

[0612] The compound (S)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (100.0 mg, 381.30 μmol) was dissolved in THF (3 mL), and a boranetetrahydrofuran solution (4 M, 476.62 μL) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was then poured into methanol (3 mL) to destroy the compound. The pH was adjusted to 5-6 with 20% formic acid aqueous solution, and the crude product was concentrated. The crude product was purified by C18 reverse column chromatography (H2O:ACN = 0-30%, 0.05% FA) and then lyophilized to obtain the title compound (95.0 mg, 306.11 μmol).

[0613] Its structural characterization data are as follows:

[0614] MS m / z (ESI): 249.1 [M+H] +

[0615] Step 4: Preparation of tert-butyl carbamate (7-22-5)

[0616] Allyl (20 mg, 34.10 μmol) of (S)-8-((5-bromopentyl)oxy)-2-(4-((tert-butoxycarbonyl)amino)phenyl)-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid (1,4.10 μmol) and (S)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid were prepared. 4] Diaza-5-one (12.70 mg, 51.15 μmol) was dissolved in DMF (1 mL), and Cs2CO3 (33.33 mg, 102.30 μmol) was added. The mixture was heated to 40 °C and reacted for 16 hours. EA (20 mL) and H2O (6 mL) were added to the system and stirred for 5 minutes. The EA phase was separated, washed with brine, and concentrated to obtain the crude product. The crude product was purified by thin-layer chromatography (MeOH / DCM = 10%) to obtain the title compound (15.0 mg, 19.90 μmol).

[0617] Its structural characterization data are as follows:

[0618] MS m / z (ESI): 754.4 [M+H] +

[0619] Step 5: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22)

[0620] The compound (4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl) tert-butyl carbamate (15.0 mg, 19.90 μmol) was dissolved in DCM (0.5 mL), and trifluoroacetic acid (226.87 mg, 1.99 mmol) was added. The mixture was reacted at 25 °C for 1 hour, concentrated to obtain the crude product, purified by preparative high performance liquid chromatography, and freeze-dried to obtain the title compound (7.10 mg, 10.53 μmol).

[0621] Its preparation method is as follows:

[0622] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0623] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0624] Its structural characterization data are as follows:

[0625] MS m / z (ESI): 654.4 [M+H] +

[0626] 1 H NMR (400MHz, MeOD): δ7.38 (dd, J=8.2, 2.3Hz, 3H), 6.91 (d, J=8.9Hz, 2H), 6.27 (d, J=9.3Hz, 2H), 4.24 (s, 1H),4.00(dd,J=9.9,6.3Hz,4H),3.80(s,3H),3.77(s,3H),3.76(s,3H),3.64(s,1H),3.61(s,1H),3.56 (d,J=11.1Hz,2H),3.50-3.46(m,2H),3.41(s,1H),3.40-3.33(m,3H),2.80(d,J=13.5Hz,1H),2.08(d,J =5.0Hz,1H),1.95-1.86(m,5H),1.69(d,J=6.7Hz,2H),0.71-0.68(m,1H),0.61(dd,J=12.4,6.0Hz,3H).

[0627] Preparation Example 8: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23)

[0628] Step 1: Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-23-2)

[0629] 4-Benzyloxy-5-methoxy-2-nitrobenzoic acid (10 g, 32.97 mmol), (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride (5.27 g, 36.27 mmol), and HATU (18.81 g, 49.46 mmol) were dissolved in DMF (50 mL), and DIPEA (12.78 g, 98.92 mmol) was added dropwise. The mixture was stirred for 2 hours. Water and EA were added and stirred. The mixture was separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed twice with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (eluent 30-65% ethyl acetate / petroleum ether), the mixture was concentrated again to give the title compound (14 g, 32.53 mmol).

[0630] Step 2: Preparation of (2R,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-3)

[0631] Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylate (14 g, 32.53 mmol) was dissolved in methanol (700 mL), and saturated ammonium chloride aqueous solution (70 mL) was added. Zinc powder (21.27 g, 325.27 mmol) was then added in portions, and the mixture was refluxed at 80 °C for 16 hours. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate) to give the title compound (5.3 g, 14.39 mmol).

[0632] Its structural characterization data are as follows:

[0633] MS m / z (ESI): 368.5 [M+H] +

[0634] Step 3: Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-4)

[0635] (2R,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1 g, 2.71 mmol) was dissolved in DMF (25 mL), and imidazole (1.85 g, 27.15 mmol) and TBSCl (1.12 g, 13.57 mmol) were added. The reaction was allowed to proceed for 16 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed three times with saturated brine and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: 50% ethyl acetate / petroleum ether) and then concentrated again to give the title compound (1.02 g, 2.11 mmol).

[0636] Step 4: Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23-5)

[0637] (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (500 mg, 1.04 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and then concentrated to obtain the title compound (120 mg, 0.26 mmol).

[0638] Its structural characterization data are as follows:

[0639] ESI-MS (m / z): 453.3 [M+H] +

[0640] Step 5: Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23-6)

[0641] (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (120 mg, 0.26 mmol) was dissolved in tetrahydrofuran (3 mL), then 20% palladium on carbon (12 mg) was added. After three hydrogen purgings, the mixture was heated to 45 °C and stirred for 3 hours. The solution was filtered through diatomaceous earth and concentrated to obtain the crude title compound (90 mg, 0.23 mmol), which was used directly in the next step without purification.

[0642] Its structural characterization data are as follows:

[0643] ESI-MS (m / z): 379.2 [M+H] +

[0644] Step Six: Preparation of (4-((S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-23-7)

[0645] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (135 mg, 0.23 mmol) and (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (90 mg, 0.23 mmol) were dissolved in DMF (2 mL), potassium carbonate (64 mg, 0.47 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (20 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH / DCM = 0%–5%), the mixture was concentrated again to give the title compound (82 mg, 0.092 mmol).

[0646] Its structural characterization data are as follows:

[0647] ESI-MS (m / z): 884.3 [M+H] +

[0648] Step 7: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23)

[0649] 82 mg (0.092 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl carbamate was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (3 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 5 hours. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (8.4 mg, 0.012 mmol).

[0650] Its structural characterization data are as follows:

[0651] ESI-MS (m / z): 670.3 [M+H] +

[0652] 1 H NMR (400MHz, DMSO): δ7.37(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J=8.4Hz,2H),6.53(d,J=8.4Hz, 2H),6.31(s,1H),6.26(s,1H),5.28-5.10(m,2H),5.08-4.79(m,1H),4.21-4.11(m,2H),3.97-3.88(m ,4H),3.89-3.76(m,1H),3.66(s,3H),3.64(s,3H),3.55-3.47(m,4H),3.25-3.20(m,2H),3.01-2.93( m,1H),2.68-2.65(m,1H),2.12-2.04(m,1H),1.85-1.76(m,4H),1.74-1.66(m,1H),1.61-1.52(m,2H).

[0653] Its preparation method is as follows:

[0654] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0655] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0656] Preparation Example 9: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24)

[0657] Step 1: Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-24-2)

[0658] 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.0 g, 3.3 mmol) and methyl (2S,4S)-4-hydroxypyrrolidine-2-carboxylate (503 mg, 3.46 mmol) were dissolved in DMF (10 mL), and HATU (2.51 g, 6.51 mmol) and DIPEA (1.28 g, 9.9 mmol) were added. The mixture was reacted at room temperature for 2 hours. After adding 50 mL of water, 70 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.36 g, 3.16 mmol).

[0659] Its structural characterization data are as follows:

[0660] ESI-MS (m / z): 431.1 [M+H] +

[0661] Step 2: Preparation of (2S,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-3)

[0662] Methyl (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylate (1.36 g, 3.16 mmol) was dissolved in methanol (52 mL), and 10% ammonium chloride aqueous solution (10 mL) and zinc powder (2.43 g, 37.2 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 50 mL of ethyl acetate. The mixture was washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (1.3 g, 3.53 mmol).

[0663] Its structural characterization data are as follows:

[0664] ESI-MS (m / z): 369.2 [M+H] +

[0665] Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-4)

[0666] (2S,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.3 g, 3.53 mmol) was dissolved in DMF (15 mL), and tert-butyldimethylchlorosilane (2.66 g, 17.64 mmol) and imidazole (2.4 g, 35.3 mmol) were added. After reacting at room temperature for 16 hours, 60 mL of water was added, followed by extraction with 80 mL of ethyl acetate. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.38 g, 2.86 mmol).

[0667] Its structural characterization data are as follows:

[0668] ESI-MS (m / z): 483.2 [M+H] +

[0669] Step 4: Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24-5)

[0670] (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (500 mg, 1.04 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and concentrated to obtain the title compound (60 mg, 0.13 mmol).

[0671] Its structural characterization data are as follows:

[0672] ESI-MS (m / z): 469.2 [M+H] +

[0673] Step 5: Preparation of (2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24-6)

[0674] (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (60 mg, 0.13 mmol) was dissolved in tetrahydrofuran (3 mL), and then 20% palladium on carbon (6 mg) was added. After three hydrogen purgings, the mixture was kept at 25 °C and stirred for 2 hours. After filtration through diatomaceous earth, the crude product of the title compound (46 mg, 0.12 mmol) was obtained and used directly in the next step without purification.

[0675] Its structural characterization data are as follows:

[0676] ESI-MS (m / z): 379.2 [M+H] +

[0677] Step Six: Preparation of (4-((S)-8-((5-(((2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-24-6)

[0678] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (71 mg, 0.12 mmol) and (2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (46 mg, 0.12 mmol) were dissolved in DMF (2 mL), potassium carbonate (33 mg, 0.24 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (20 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH / DCM = 0%–5%) yielded the title compound (39 mg, 0.04 mmol).

[0679] Its structural characterization data are as follows:

[0680] ESI-MS (m / z): 884.3 [M+H] +

[0681] Step 7: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24)

[0682] 39 mg (0.04 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl carbamate was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (2 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 5 hours. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (7 mg, 0.01 mmol).

[0683] Its structural characterization data are as follows:

[0684] ESI-MS (m / z): 670.3 [M+H]+

[0685] 1 H NMR (400MHz, DMSO): δ7.28(d,J=4.0Hz,2H),7.26(s,1H),7.12(d,J=8.4Hz,2H),6.54(d,J=8.4Hz,2H ),6.30(s,1H),6.26(s,1H),5.37-5.14(m,1H),5.10-5.03(m,1H),4.24-4.06(m,2H),3.96-3.84(m,2 H),3.66(s,3H),3.63(s,3H),3.61-3.56(m,1H),3.55-3.48(m,1H),3.47-3.40(m,3H),3.26-3.21(m ,3H),2.68-2.64(m,1H),2.34-2.30(m,1H),1.85-1.76(m,4H),1.71-1.63(m,1H),1.62-1.51(m,2H).

[0686] Its preparation method is as follows:

[0687] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0688] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0689] Preparation Example 10: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25)

[0690] Step 1: Preparation of (2S,4R)-4-methoxypyrrolidine-2-carboxylic acid methyl ester (7-25-2)

[0691] 1-(tert-butyl)-2-methyl(2S,4R)-4-methoxypyrrolidine-1,2-dicarboxylic acid (1.0 g, 3.53 mmol) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (800.6 mg, 5.01 mmol).

[0692] Its structural characterization data are as follows:

[0693] ESI-MS (m / z): 160.1 [M+H] +

[0694] Step 2: Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methoxypyrrolidine-2-carboxylic acid methyl ester (7-25-3)

[0695] Methyl (2S,4R)-4-methoxypyrrolidine-2-carboxylate (800.6 mg, 5.01 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.56 g, 3.51 mmol).

[0696] Its structural characterization data are as follows:

[0697] ESI-MS (m / z): 445.2 [M+H] +

[0698] Step 3: (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -5,11(10H)-dione (7-25-4)

[0699] Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (1.56 g, 3.1 mmol) was dissolved in methanol (30 mL), zinc powder (2.4 g, 31.1 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.13 g, 2.95 mmol), which was used directly in the next step without purification.

[0700] Its structural characterization data are as follows:

[0701] ESI-MS (m / z): 383.2 [M+H] +

[0702] Step 4: Preparation of (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-5)

[0703] (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (0.5 g, 1.3 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and concentrated to obtain the title compound (80 mg, 0.22 mmol).

[0704] Its structural characterization data are as follows:

[0705] ESI-MS (m / z): 369.1 [M+H] +

[0706] Step 5: Preparation of (2R,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-6)

[0707] (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (80 mg, 0.22 mmol) was dissolved in tetrahydrofuran (3 mL), palladium on carbon (8 mg) was added, and the reaction was carried out under a hydrogen atmosphere for 1 hour. After the reaction was completed, the mixture was filtered, dried over anhydrous sodium sulfate, and then concentrated to give the crude title compound (49 mg, 0.17 mmol), which was used directly in the next step without purification.

[0708] Its structural characterization data are as follows:

[0709] ESI-MS (m / z): 279.2 [M+H] +

[0710] Step Six: Preparation of (4-((S)-8-((5-(((2R,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H)-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)

[0711] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (30.8 mg, 0.053 mmol) and (2R,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (15 mg, 0.05 mmol) were dissolved in DMF (1.5 mL), potassium carbonate (13.8 mg, 0.1 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH / DCM = 0%–5%), the mixture was concentrated again to give the title compound (19.6 mg, 0.25 mmol).

[0712] Its structural characterization data are as follows:

[0713] ESI-MS (m / z): 784.4 [M+H] +

[0714] Step 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25)

[0715] 19.6 mg (0.25 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H)-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (9.2 mg, 13.16 μmol).

[0716] Its structural characterization data are as follows:

[0717] ESI-MS (m / z): 684.4 [M+H] +

[0718] 1H NMR (400MHz, DMSO): δ7.37(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J=8.4Hz,2H),6.53(d,J =8.4Hz,2H),6.30(s,1H),6.26(s,1H),5.19(s,2H),4.19-4.08(m,1H),3.96-3.84(m,5H),3.7 4-3.68(m,2H),3.66(s,3H),3.64(s,3H),3.56-3.46(m,3H),3.29-3.22(m,2H),3.22(s,3H),3 .02-2.93(m,1H),2.73-2.66(m,1H),1.85-1.76(m,4H),1.75-1.66(m,1H),1.61-1.50(m,2H).

[0719] Its preparation method is as follows:

[0720] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0721] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0722] Preparation Example 11: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26)

[0723] Step 1: Preparation of (2S,4S)-4-(methoxy)pyrrolidine-2-carboxylic acid methyl ester (7-26-2)

[0724] 1-(tert-butyl)-2-methyl(2S,4S)-4-(methoxy)pyrrolidine-1,2-dicarboxylic acid (1.0 g, 4.08 mmol) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (797.66 mg, 4.08 mmol).

[0725] Its structural characterization data are as follows:

[0726] ESI-MS (m / z): 196.2 [M+H]+

[0727] Step 2: Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(methoxy)pyrrolidine-2-carboxylic acid methyl ester (7-26-3)

[0728] Methyl (2S,4S)-4-(methoxy)pyrrolidine-2-carboxylate (797.66 mg, 4.08 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.24 g, 4.08 mmol) were dissolved in DMF (10 mL), and HATU (1.86 g, 4.89 mmol) and DIPEA (1.58 g, 12.23 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.8 g, 4.05 mmol).

[0729] Its structural characterization data are as follows:

[0730] ESI-MS (m / z): 445.2 [M+H] +

[0731] Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(methoxy)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-26-4)

[0732] Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(methoxy)pyrrolidine-2-carboxylate (1.8 g, 4.05 mmol) was dissolved in methanol (30 mL), zinc powder (2.63 g, 40.50 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.5 g, 3.92 mmol), which was used directly in the next step without purification.

[0733] Its structural characterization data are as follows:

[0734] ESI-MS (m / z): 383.2 [M+H] +

[0735] Step 4: Preparation of (2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-5)

[0736] (2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (0.8 g, 2.09 mmol) was dissolved in tetrahydrofuran (10 mL), sodium borohydride (158.13 mg, 41.8 mmol) was added, and then trifluoroacetic acid (596 mg, 5.23 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then heated to 75 °C and refluxed for 5 hours. The reaction solution was concentrated, and water (50 mL) was added. The solution was then diluted with ethyl acetate (50 mL). 2) The extract was performed twice, and the combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%). The resulting lyophilized compound yielded the title compound (640 mg, 1.73 mmol). Its structural characterization data are as follows:

[0737] ESI-MS (m / z): 369.1 [M+H] +

[0738] Step 5: Preparation of (2S,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-6)

[0739] (2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (640 mg, 1.73 mmol) was dissolved in methanol (10 mL), palladium on carbon (30 mg) was added, and the reaction was carried out under a hydrogen atmosphere for 1 hour. After the reaction was completed, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (434 mg, 1.56 mmol), which was used directly in the next step without purification.

[0740] Its structural characterization data are as follows:

[0741] ESI-MS (m / z): 279.2 [M+H] +

[0742] Step Six: Preparation of (4-((S)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)

[0743] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (21.07 mg, 35.93 μmol) and (2S,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10 mg, 35.93 μmol) were dissolved in DMF (1 mL), potassium carbonate (14.88 mg, 107.80 μmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH / DCM = 0%–5%), the mixture was concentrated again to give the title compound (20 mg, 25.51 μmol).

[0744] Its structural characterization data are as follows:

[0745] ESI-MS (m / z): 784.4 [M+H] +

[0746] Step 7: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26)

[0747] 20 mg (25.51 μmol) of tert-butyl carbamate (4-((S)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (13.51 mg, 19.56 μmol).

[0748] Its structural characterization data are as follows:

[0749] ESI-MS (m / z): 684.4 [M+H] +

[0750] 1 H NMR (400MHz, DMSO): δ7.39 (s, 1H), 7.28 (d, J = 9.3Hz, 2H), 6.85-6.75 (m, 2H), 6.48- 6.63(m,2H),6.31(s,1H),6.29(s,1H)4.22-4.15(m,1H),3.97-3.86(m,4H),3.75-3 .65(m,2H),3.66(s,3H),3.64(s,3H),3.62-3.50(m,6H),3.24(s,3H),3.32-3.16( m,2H),2.76-2.67(m,1H),2.43-2.36(m,1H),1.87-1.73(m,4H),1.60-1.49(m,4H).

[0751] Its preparation method is as follows:

[0752] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0753] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0754] Preparation Example 12: Preparation of (S)-8-((5-(((2R,11aS)-2-amino-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-aminophenyl)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-27)

[0755] Step 1: Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylic acid methyl ester (7-27-2)

[0756] Methyl (2S,4R)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylate (1 g, 4.09 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.37 g, 4.50 mmol) were dissolved in DMF (10 mL), and HATU (1.71 g, 4.50 mmol) and DIPEA (1.59 g, 12.28 mmol) were added. The reaction mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (50 mL) and saturated brine (50 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20%–60%) and concentrated again to obtain the title compound (2.1 g, 3.97 mmol).

[0757] Its structural characterization data are as follows:

[0758] ESI-MS (m / z): 530.2 [M+H] +

[0759] Step 2: Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-3)

[0760] Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylate (2.1 g, 3.97 mmol) was dissolved in methanol (50 mL), zinc powder (2.58 g, 15.86 mmol) was added, followed by saturated aqueous solution of ammonium chloride (20 mL). The mixture was refluxed at 85 °C for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.8 g, 3.85 mmol), which was used directly in the next step without purification.

[0761] Its structural characterization data are as follows:

[0762] ESI-MS (m / z): 468.3 [M+H]+

[0763] Step 3: Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-4)

[0764] Dissolve tert-butyl carbamate ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl) in tetrahydrofuran (100 mL), add sodium borohydride (843.15 mg, 11.55 mmol), and then add trifluoroacetic acid (877.8 mg, 7.7 mmol) dropwise at room temperature until no large amount of gas is released. Then, reflux the mixture at 75 °C for 5 hours. The reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of the title compound (1.4 g, 3.09 mmol), which was used directly in the next step without purification.

[0765] Its structural characterization data are as follows:

[0766] ESI-MS (m / z): 454.2 [M+H] +

[0767] Step 4: Preparation of ((2R,11aS)-8-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-5)

[0768] ((2R,11aS)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (100 mg, 220.50 μmol) was dissolved in methanol (5 mL), and palladium on carbon (5 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (80.13 mg, 220.50 μmol), which was used directly in the next step without purification.

[0769] Its structural characterization data are as follows:

[0770] ESI-MS (m / z): 364.2 [M+H] +

[0771] Step 5: Preparation of tert-butyl carbamate (7-27-6)

[0772] ((2R,11aS)-8-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (10 mg, 27.52 μmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (16.14 mg, 27.52 μmol) were dissolved in DMF (1 mL), and potassium carbonate (11.39 mg, 82.55 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH / DCM = 0%–5%) yielded the title compound (20 mg, 23.01 μmol).

[0773] Its structural characterization data are as follows:

[0774] ESI-MS (m / z): 869.8 [M+H]+

[0775] Step Six: Preparation of (S)-8-((5-(((2R,11aS)-2-amino-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-aminophenyl)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-27)

[0776] 20 mg (23.01 μmol) of tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butoxycarbonyl)amino)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (5.41 mg, 0.045 mmol).

[0777] Its structural characterization data are as follows:

[0778] ESI-MS (m / z): 670.4 [M+H] +

[0779] 1 H NMR (400MHz, DMSO): δ7.36 (s, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 7.11 (d, J = 8.5Hz, 2H), 6 .53(d,J=8.6Hz,2H),6.29(d,J=9.3Hz,2H),4.17-4.09(m,1H),3.96-3.90(m,4H),3.6 5(d,J=7.1Hz,6H),3.51(d,J=4.9Hz,2H),3.30-3.20(m,4H),2.99(dd,J=12.9,8.8Hz ,2H),2.23-2.14(m,2H),1.96-1.87(m,2H),1.84-1.76(m,4H),1.56(d,J=6.4Hz,2H).

[0780] Its preparation method is as follows:

[0781] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0782] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0783] Preparation Example 13: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29)

[0784] Step 1: Preparation of (S)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester (7-29-2)

[0785] (S)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid (0.6 g, 2.39 mmol) was dissolved in MeOH (3 mL), and 4M HCl / dioxane solution (4 mL) was added dropwise. The mixture was stirred at room temperature for 20 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to obtain the crude hydrochloride of the title compound (460 mg, 2.28 mmol), which was used directly in the next reaction without purification.

[0786] Its structural characterization data are as follows:

[0787] ESI-MS (m / z): 166.1 [M+H] +

[0788] Step 2: Preparation of (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester (7-29-3)

[0789] 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (743.76 mg, 2.45 mmol) was dissolved in DMF (5 mL), and HATU (1.03 g, 2.70 mmol) and DIPEA (316.96 mg, 2.45 mmol) were added. The mixture was stirred at room temperature for 10 minutes. In a separate reaction flask, (S)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester hydrochloride (450 mg, 2.45 mmol) was dissolved in DMF (5 mL), and DIPEA (316.96 mg, 2.45 mmol) was added to make the solution alkaline. The two reaction solutions were then combined and stirred at room temperature for 2 hours. After the reaction was complete, 30 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL x 3). The organic phase was washed with 15 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (1.4 g, 2.18 mmol), which was used directly in the next reaction without purification.

[0790] Its structural characterization data are as follows:

[0791] ESI-MS (m / z): 468.2 [M+H] +

[0792] Step 3: Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-29-4)

[0793] Methanol (12 mL) and saturated ammonium chloride aqueous solution (3 mL) were added to methyl (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxylate (700 mg, 1.09 mmol), and the mixture remained insoluble. Zinc powder (711.41 mg, 10.88 mmol) was added, and the mixture was directly placed in an oil bath preheated to 75 °C and heated for 16 hours. After the reaction was complete, the reaction solution was cooled to room temperature, filtered to remove insoluble matter, and the filter cake was washed with an appropriate amount of methanol. The filtrate was concentrated under reduced pressure. The residue was extracted with 10 mL of water and ethyl acetate (10 mL x 3). The combined organic phases were washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (580 mg, 1.05 mmol), which was used directly in the next reaction without purification.

[0794] Its structural characterization data are as follows:

[0795] ESI-MS (m / z): 389.2 [M+H] +

[0796] Step 4: Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29-5)

[0797] (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,11-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (100 mg, 257.49 μmol) was dissolved in 2 mL of THF. Under nitrogen protection, the mixture was cooled to 0 °C and borane (1 M in THF, 2.57 mL) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for 1.5 hours. After the reaction was complete, the reaction solution was cooled to 0 °C, and the reaction was quenched dropwise with an appropriate amount of methanol. The mixture was stirred at room temperature for 1 hour. 10 mL of water was added to the reaction solution, and the mixture was extracted with 10 mL of ethyl acetate (3 times). The extract was washed with 10 mL of organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate-petroleum ether = 10–70%) and concentrated again under reduced pressure to obtain the title compound (23 mg, 61.43 μmol).

[0798] Its structural characterization data are as follows:

[0799] ESI-MS (m / z): 385.1 [M+H] +

[0800] Step 5: (S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (7-29-6)

[0801] (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (23 mg, 61.43 μmol) was suspended in methanol (2 mL), and ethyl acetate (1 mL) was added to completely dissolve the starting material. Pd / C (7.46 mg, 6.14 μmol, 10% purity) was added, followed by purging with hydrogen three times. The reaction was stirred at room temperature for 3 hours under a hydrogen balloon atmosphere. After the reaction was complete, the reaction solution was filtered, the filter cake was washed with an appropriate amount of ethyl acetate, and the filtrate was directly concentrated under reduced pressure to obtain the crude title compound (23 mg, 59.88 μmol), which was used directly in the next step without purification.

[0802] Its structural characterization data are as follows:

[0803] ESI-MS (m / z): 285.2 [M+H]+

[0804] Step 6: (4-((S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-29-7)

[0805] (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (20.63 mg, 35.18 μmol) and (S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10 mg, 35.18 μmol) were dissolved in DMF (0.5 mL), and K2CO3 (14.59 mg, 105.54 μmol) was added. The mixture was stirred at room temperature for 18 hours. After the reaction was complete, 4 mL of water was added to the reaction solution, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (28 mg, 26.59 μmol), which was used directly in the next step without purification.

[0806] Its structural characterization data are as follows:

[0807] ESI-MS (m / z): 790.4 [M+H] +

[0808] Step 7: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29)

[0809] At room temperature, tert-butyl(4-((S)-8-((5-((((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) Dichloromethane (1 mL) was added to 28 mg (35.45 μmol) of benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate, followed by 0.2 mL of TFA. The mixture was stirred at room temperature for 2 hours after the addition was complete. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The residue was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (4.5 mg, 6.29 μmol).

[0810] Its structural characterization data are as follows:

[0811] ESI-MS (m / z): 690.4 [M+H] + 345.9 [M / 2+1] +

[0812] 1 H NMR (400MHz, DMSO-d6) δ7.33(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J=8.4Hz,1 H),6.53(d,J=8.4Hz,2H),6.49(brs,1H),6.30(s,2H),5.19(brs,1H),4.14-4.10(m ,1H),3.98-3.84(m,7H),3.67(s,3H),3.64(s,3H),3.57-3.50(m,2H),3.25-3.14( m,3H),2.75-2.72(m,2H),1.82-1.78(m,3H),1.82-1.78(m,4H),1.59-1.53(m,2H).

[0813] Its preparation method is as follows:

[0814] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0815] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0816] Preparation Example 14: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30)

[0817] Step 1: Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylic acid methyl ester (7-30-2)

[0818] Methyl (2S,4S)-4-fluoropyrrolidine-2-carboxylate (1.07 g, 7.25 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (2 g, 6.59 mmol) were dissolved in DMF (10 mL), and HATU (2.76 g, 7.25 mmol) and DIPEA (2.56 g, 19.78 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20%–60%) and concentrated again to obtain the title compound (2.3 g, 5.32 mmol).

[0819] Its structural characterization data are as follows:

[0820] ESI-MS (m / z): 433.2 [M+H] +

[0821] Step 2: (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -5,11(10H)-dione (7-30-3)

[0822] Methyl (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylate (2.3 g, 5.32 mmol) was dissolved in methanol (50 mL), zinc powder (3.46 g, 53.19 mmol) was added, followed by saturated aqueous solution of ammonium chloride (10 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.8 g, 4.86 mmol), which was used directly in the next step without purification.

[0823] Its structural characterization data are as follows:

[0824] ESI-MS (m / z): 371.2 [M+H] +

[0825] Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-4)

[0826] (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.8 g, 4.86 mmol) was dissolved in tetrahydrofuran (100 mL), sodium borohydride (1064.34 mg, 14.58 mmol) was added, and then trifluoroacetic acid (1108.1 mg, 9.72 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then refluxed at 75 °C for 5 hours. The reaction solution was concentrated, and water (50 mL) was added. The mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.4 g, 3.93 mmol), which was used directly in the next step without purification.

[0827] Its structural characterization data are as follows:

[0828] ESI-MS (m / z): 357.3 [M+H] +

[0829] Step 4: Preparation of (2S,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-5)

[0830] (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (200 mg, 561.18 μmol) was dissolved in methanol (10 mL), and palladium on carbon (5 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (0.14 g, 525.79 μmol), which was used directly in the next step without purification.

[0831] Its structural characterization data are as follows:

[0832] ESI-MS (m / z): 267.1 [M+H] +

[0833] Step 5: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-6)

[0834] (2S,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10 mg, 37.56 μmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (22.03 mg, 37.56 μmol) were dissolved in DMF (1 mL), and potassium carbonate (15.55 mg, 112.67 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH / DCM = 0%–5%), the mixture was concentrated again to give the title compound (20 mg, 25.91 μmol).

[0835] Its structural characterization data are as follows:

[0836] ESI-MS (m / z): 772.3 [M+H] +

[0837] Step Six: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30)

[0838] (S)-2-(4-aminophenyl)-8-((5-((((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (20 mg, 25.91 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (8.14 mg, 12.00 μmol).

[0839] Its structural characterization data are as follows:

[0840] ESI-MS (m / z): 673.3 [M+H] +

[0841] 1 ¹H NMR (400MHz, DMSO) δ 7.40 (s, ¹H), 7.29 (s, ¹H), 7.24 (s, ¹H), 6.87–6.70 (m, 2H), 6.60–6.45 (m, 2H), 6.31 (s, 2H), 4.17 (s, ¹H), 4.03–3.87 (m, 4H), 3.87–3.76 (m, 2H), 3.67 (s, 3H), 3.65 (s, 3H), 3.58–3.45 (m, 4H), 3.29–3.20 (m, 2H), 2.82–2.61 (m, 2H), 2.20–2.06 (m, 2H), 1.86–1.73 (m, 4H), 1.60–1.53 (m, 2H). The preparation method is as follows:

[0842] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0843] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0844] Preparation Example 15: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31)

[0845] Step 1: Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylic acid methyl ester (7-31-2)

[0846] Methyl (2R,4S)-4-fluoropyrrolidine-2-carboxylate (1.1 g, 7.25 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (2.05 g, 6.59 mmol) were dissolved in DMF (10 mL), and HATU (2.76 g, 7.25 mmol) and DIPEA (2.56 g, 19.78 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20%–60%) and concentrated again to obtain the title compound (2.1 g, 4.85 mmol).

[0847] Its structural characterization data are as follows:

[0848] ESI-MS (m / z): 433.2 [M+H] +

[0849] Step 2: Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-31-3)

[0850] Methyl (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylate (2.1 g, 4.85 mmol) was dissolved in methanol (50 mL), zinc powder (3.4 g, 53.1 mmol) was added, followed by saturated aqueous solution of ammonium chloride (10 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.7 g, 4.7 mmol), which was used directly in the next step without purification.

[0851] Its structural characterization data are as follows:

[0852] ESI-MS (m / z): 371.2 [M+H] +

[0853] Step 3: Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-4)

[0854] (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (400 mg, 1.04 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and concentrated to obtain the title compound (110 mg, 0.31 mmol).

[0855] Its structural characterization data are as follows:

[0856] ESI-MS (m / z): 357.3 [M+H] +

[0857] Step 4: Preparation of (2R,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-5)

[0858] (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (110 mg, 0.31 mmol) was dissolved in tetrahydrofuran (5 mL), and palladium on carbon (10 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (50 mg, 190.1 μmol), which was used directly in the next step without purification.

[0859] Its structural characterization data are as follows:

[0860] ESI-MS (m / z): 267.1 [M+H] +

[0861] Step 5: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-6)

[0862] (2R,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (50 mg, 190.1 μmol) and tert-butyl(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (117.3 mg, 199.5 μmol) were dissolved in DMF (3 mL), and potassium carbonate (52.4 mg, 380 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH / DCM = 0%–5%), the mixture was concentrated again to give the title compound (88 mg, 114.1 μmol).

[0863] Its structural characterization data are as follows:

[0864] ESI-MS (m / z): 772.3 [M+H] +

[0865] Step Six: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31)

[0866] (S)-2-(4-aminophenyl)-8-((5-((((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (88 mg, 114.1 μmol)) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (8.0 mg, 11.7 μmol).

[0867] Its structural characterization data are as follows:

[0868] ESI-MS (m / z): 673.3 [M+H] +

[0869] 1 H NMR (400MHz, DMSO): δ7.39 (s, 1H), 7.29 (s, 2H), 7.14 (d, J = 7.8Hz, 2H), 6.58 (d, J = 7. 4Hz,2H),6.29(d,J=10.0Hz,2H),5.26(d,J=53.6Hz,1H),4.21-4.08(m,1H),4.01-3 .89(m,4H),3.85-3.76(m,2H),3.65(d,J=5.8Hz,6H),3.60-3.49(m,4H),3.29-3.11 (m,4H),3.04-2.95(m,1H),2.74-2.63(m,1H),1.88-1.73(m,4H),1.62-1.50(m,2H).

[0870] Its preparation method is as follows:

[0871] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0872] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0873] Preparation Example 16: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32)

[0874] Step 1: Preparation of (2S,4S)-4-(trifluoromethyl)pyrrolidine-2-carboxylate methyl ester (7-32-2)

[0875] 1-(tert-butyl)-2-methyl(2S,4S)-4-(trifluoromethyl)pyrrolidine-1,2-dicarboxylic acid (1.0 g, 3.53 mmol) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (820.79 mg, 3.51 mmol).

[0876] Its structural characterization data are as follows:

[0877] ESI-MS (m / z): 234.2 [M+H] +

[0878] Step 2: Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-32-3)

[0879] Methyl (2S,4S)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (820.79 mg, 3.51 mmol, CL) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.5 g, 3.1 mmol).

[0880] Its structural characterization data are as follows:

[0881] ESI-MS (m / z): 483.2 [M+H] +

[0882] Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-32-4)

[0883] Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (1.5 g, 3.1 mmol) was dissolved in methanol (30 mL), zinc powder (2.2 g, 35.1 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.4 g, 3.3 mmol), which was used directly in the next step without purification.

[0884] Its structural characterization data are as follows:

[0885] ESI-MS (m / z): 421.2 [M+H] +

[0886] Step 4: Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-5)

[0887] (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (400 mg, 0.95 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and concentrated to obtain the title compound (100 mg, 0.24 mmol).

[0888] Its structural characterization data are as follows:

[0889] ESI-MS (m / z): 408.1 [M+H] +

[0890] Step 5: Preparation of (2S,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-6)

[0891] (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (100 mg, 0.24 mmol) was dissolved in tetrahydrofuran (3 mL), and palladium on carbon (10 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (70 mg, 0.22 mmol), which was used directly in the next step without purification.

[0892] Its structural characterization data are as follows:

[0893] ESI-MS (m / z): 317.1 [M+H] +

[0894] Step Six: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-7)

[0895] (2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (70 mg, 0.22 mmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (136 mg, 0.23 mmol) were dissolved in DMF (2 mL), and potassium carbonate (61 mg, 0.44 mmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The mixture was purified by silica gel column chromatography (MeOH / DCM = 0%–5%) and then concentrated again to give the title compound (72 mg, 88.3 μmol).

[0896] Its structural characterization data are as follows:

[0897] ESI-MS (m / z): 822.5 [M+H] +

[0898] Step 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32)

[0899] (S)-2-(4-aminophenyl)-7-methoxy-8-((5-((((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (72 mg, 88.3 μmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (9.8 mg, 13.6 μmol).

[0900] Its structural characterization data are as follows:

[0901] ESI-MS (m / z): 723.3 [M+H] +

[0902] 1 H NMR (400MHz, DMSO): δ7.41(s,1H),7.27(d,J=12.2Hz,2H),7.11(d,J=8.2Hz,2H),6.5 3(d,J=8.4Hz,2H),6.29(d,J=12.4Hz,2H),5.20(s,2H),4.19-4.08(m,1H),3.97-3.8 8(m,4H),3.87-3.76(m,2H),3.66(s,3H),3.64(s,3H),3.59-3.46(m,4H),3.28-3.16 (m,4H),3.04-2.94(m,1H),2.73-2.64(m,1H),1.84-1.76(m,4H),1.61-1.50(m,2H).

[0903] Its preparation method is as follows:

[0904] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0905] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0906] Preparation Example 17: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33)

[0907] Step 1: Preparation of (2S,4R)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (7-33-2)

[0908] 1-(tert-butyl)-2-methyl(2S,4R)-4-(trifluoromethyl)pyrrolidine-1,2-dicarboxylic acid (1.0 g, 3.53 mmol) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (824.79 mg, 3.53 mmol).

[0909] Its structural characterization data are as follows:

[0910] ESI-MS (m / z): 234.2 [M+H] +

[0911] Step 2: Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-33-3)

[0912] Methyl (2S,4R)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (824.79 mg, 3.53 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.7 g, 3.52 mmol).

[0913] Its structural characterization data are as follows:

[0914] ESI-MS (m / z): 483.2 [M+H] +

[0915] Step 3: Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-33-4)

[0916] Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (1.7 g, 3.52 mmol) was dissolved in methanol (30 mL), zinc powder (2.29 g, 35.24 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.48 g, 3.52 mmol), which was used directly in the next step without purification.

[0917] Its structural characterization data are as follows:

[0918] ESI-MS (m / z): 421.2 [M+H] +

[0919] Step 4: Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-5)

[0920] (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.48 g, 3.52 mmol) was dissolved in tetrahydrofuran (30 mL), sodium borohydride (770.88 mg, 10.56 mmol) was added, and then trifluoroacetic acid (802.56 mg, 7.04 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then refluxed at 75 °C for 5 hours. The reaction solution was concentrated, and water (50 mL) was added. The mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.1 g, 2.71 mmol), which was used directly in the next step without purification.

[0921] Its structural characterization data are as follows:

[0922] ESI-MS (m / z): 408.1 [M+H] +

[0923] Step 5: Preparation of (2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-6)

[0924] (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (100 mg, 246.06 μmol) was dissolved in methanol (10 mL), and palladium on carbon (5 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (77.82 mg, 246.05 μmol), which was used directly in the next step without purification.

[0925] Its structural characterization data are as follows:

[0926] ESI-MS (m / z): 317.1 [M+H] +

[0927] Step Six: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-7)

[0928] (2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10.78 mg, 34.10 μmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (20.00 mg, 34.10 μmol) were dissolved in DMF (1 mL), and potassium carbonate (14.12 mg, 102.30 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH / DCM = 0%–5%), the mixture was concentrated again to give the title compound (20 mg, 24.33 μmol).

[0929] Its structural characterization data are as follows:

[0930] ESI-MS (m / z): 822.5 [M+H] +

[0931] Step 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33)

[0932] (S)-2-(4-aminophenyl)-7-methoxy-8-((5-((((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (20 mg, 24.33 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (9.33 mg, 12.80 μmol).

[0933] Its structural characterization data are as follows:

[0934] ESI-MS (m / z): 723.3 [M+H] +

[0935] 1 H NMR(400MHz,DMSO)δ7.39(s,1H),7.28(s,1H),7.25(s,1H),6.82(s,2H),6. 59-6.43(m,2H),6.31(s,2H),4.18-4.08(m,1H),3.97-3.85(m,4H),3.82-3. 74(m,2H),3.68(s,3H),3.66(s,3H),3.56-3.44(m,4H),3.28-3.19(m,2H), 2.81-2.65(m,2H),2.21-2.04(m,2H),1.87-1.70(m,4H),1.59-1.52(m,2H).

[0936] Its preparation method is as follows:

[0937] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0938] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0939] Preparation Examples 18 and 19: (S)-8-((5-(((2S,11aS)-2-(4-aminophenyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7- 34) and the preparation of (S)-8-((5-(((2R,11aS)-2-(4-aminophenyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-35)

[0940] (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11-tetrahydro-3H,5H-spiro[e]pyrrolo[1,2-a][1,4]aza-2,1'-cyclopropane]-5-one (compound 7-5) (20 mg, 29.42 μmol) was dissolved in a mixed solvent of methanol (5 mL) and tetrahydrofuran (5 mL), and 10% palladium on carbon (2 mg) was added. The mixture was stirred for 2 hours under hydrogen purging and protection. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and purified by preparative high performance liquid chromatography. After freeze drying, compounds 7-34 (2.77 mg, 3.13 μmol) and 7-35 (5.66 mg, 6.69 μmol) were obtained.

[0941] Its structural characterization data are as follows:

[0942] MS m / z (ESI): Bimodal 682.5 [M+H] +

[0943] Compound 7-34: 1H NMR (400MHz, DMSO) δ7.40-7.24(m,4H),7.10(d,J=8.3Hz,2H),6.31(t,J=13.0Hz,2H),3.97(s ,1H),3.92(t,J=6.2Hz,4H),3.83(d,J=7.6Hz,2H),3.65(d,J=5.1Hz,6H),3.55-3.47(m,4H), 3.30(dd,J=19.7,10.5Hz,2H),3.26-3.11(m,2H),2.31(dd,J=12.5,7.5Hz,1H),2.17-2.09(m ,1H),1.96(dd,J=12.4,7.2Hz,1H),1.86-1.72(m,5H),1.60-1.51(m,2H),0.68-0.48(m,4H).

[0944] Compound 7-35: 1 H NMR (400MHz, DMSO) δ7.35(dd,J=18.7,10.3Hz,4H),7.11(d,J=8.2Hz,2H),6.29(d,J=15.3Hz,2H ),4.05(d,J=3.6Hz,1H),3.92(t,J=6.4Hz,4H),3.83(s,2H),3.65(t,J=2.7Hz,6H),3.53(d,J=11 .8Hz,4H),3.33(d,J=11.7Hz,2H),3.12(ddd,J=34.2,13.2,8.2Hz,2H),2.46(d,J=5.1Hz,1H),2 .00-1.93(m,1H),1.87-1.70(m,6H),1.56(d,J=6.6Hz,2H),0.58(ddt,J=15.7,12.7,6.9Hz,4H).

[0945] Its preparation method is as follows:

[0946] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0947] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0948] Preparation Example 20: Preparation of (S)-8-((5-(((S)-2-(furan-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-36)

[0949] Step 1: Preparation of (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-1)

[0950] Furan-3-ylboronic acid (40.15 mg, 358.85 μmol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (100 mg, 179.43 μmol), tetra(triphenylphosphine)palladium (20.73 mg, 17.94 μmol), and sodium carbonate (76.07 mg, 717.70 μmol) were mixed together, and water (0.5 mL), toluene (2 mL), and ethanol (0.5 mL) were added. The mixture was then purged with nitrogen three times and heated to 80 °C for 3 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and then concentrated again under reduced pressure to give the title compound (60 mg, 126.23 μmol).

[0951] Its structural characterization data are as follows:

[0952] ESI-MS (m / z): 475.1 [M+H] +

[0953] Step 2: Preparation of (S)-2-(furan-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-2)

[0954] (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (60 mg, 126.23 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (34.63 mg, 126.23 μmol) were dissolved in DMF (1 mL), and potassium carbonate (52.34 mg, 378.68 μmol) was added. The mixture was stirred at room temperature for 20 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and a large amount of solid precipitated. The solid was extracted with 10 mL of a mixed solvent of ethyl acetate:methanol (10:1) three times, washed with 10 mL of organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by thin-layer chromatography (DCM:MeOH = 8:1) and concentrated again under reduced pressure to obtain the title compound (43 mg, 50.80 μmol).

[0955] Its structural characterization data are as follows:

[0956] ESI-MS (m / z): 669.3 [M+H] +

[0957] Step 3: Preparation of (S)-8-((5-(((S)-2-(furan-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-36)

[0958] (S)-2-(furan-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (24 mg, 35.89 μmol) was dissolved in THF (2 mL), NaBH4 (12.22 mg, 323.00 μmol) was added, and then TFA (24.55 mg, 215.33 μmol) was added dropwise. A large number of bubbles were generated in the system. After the bubbles disappeared, the mixture was transferred to an oil bath and heated to 75°C for 22 hours. After the reaction was complete, 5 mL of water was added to the reaction solution, and the mixture was extracted three times with 5 mL of ethyl acetate (3 times). The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (6.56 mg, 9.77 μmol).

[0959] Its structural characterization data are as follows:

[0960] ESI-MS (m / z): 655.4 [M+H] +

[0961] 1 H NMR (400MHz, DMSO-d6) δ7.66(d,J=10.0Hz,2H),7.32(s,2H),7.28(s,1H),6.85(s,1H),6.53(d,J=6 .4Hz,1H),6.31-6.26(m,3H),4.20-4.13(m,1H),3.98-3.88(m,4H),3.88-3.70(m,1H),3.66(s,3H) ,3.64(s,3H),3.58-3.43(m,4H),3.25-3.11(m,4H),2.62(dd,J=16.8,4.4Hz,1H),1.95(dd,J=12.4 ,7.2Hz,1H),1.79(dd,J=12.4,7.2Hz,5H),1.61-1.49(m,2H),0.68-0.61(m,1H),0.60-0.48(m,3H).

[0962] Its preparation method is as follows:

[0963] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0964] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0965] Preparation Example 21: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2-(thiophen-3-yl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-37)

[0966] Step 1: Preparation of (S)-8-((5-bromopentyl)oxy)-2-(thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-1)

[0967] Thiophene-3-ylboronic acid (91.84 mg, 717.70 μmol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (200 mg, 358.85 μmol), tetra(triphenylphosphine)palladium (41.47 mg, 35.89 μmol), and sodium carbonate (152.14 mg, 1.44 mmol) were mixed together, and water (1 mL), toluene (4 mL), and ethanol (1 mL) were added. The mixture was then purged with nitrogen three times and heated to 80 °C for 2 hours. After the reaction was complete, the reaction solution was extracted three times with 5 mL of ethyl acetate (3 times each), washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) and then concentrated under reduced pressure to obtain the title compound (58 mg, 118.03 μmol).

[0968] Its structural characterization data are as follows:

[0969] ESI-MS (m / z): 491.1 [M+H] +

[0970] Step 2: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrole)[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-(thiophen-3-yl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-2)

[0971] (S)-8-((5-bromopentyl)oxy)-2-(thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (55 mg, 111.93 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (30.70 mg, 111.93 μmol) were dissolved in DMF (2 mL), and potassium carbonate (46.41 mg, 335.78 μmol) was added. The mixture was stirred at room temperature for 40 hours. After the reaction was completed, the reaction solution was filtered to remove insoluble matter, and the filtrate was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (15 mg, 21.90 μmol).

[0972] Its structural characterization data are as follows:

[0973] ESI-MS (m / z): 685.3 [M+H] +

[0974] Its preparation method is as follows:

[0975] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0976] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0977] Step 3: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2-(thiophen-3-yl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-37)

[0978] (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-(thiophen-3-yl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (15 mg, 21.90 μmol) was dissolved in THF (2 mL). Sodium borohydride (8.29 mg, 219.04 μmol) was added, and a few bubbles were produced. TFA (14.99 mg, 131.43 μmol) was added dropwise, and a large number of bubbles were produced. The temperature was raised to 70℃ and the reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, further purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (2.83 mg, 4.01 μmol).

[0979] Its structural characterization data are as follows:

[0980] ESI-MS (m / z): 671.3 [M+H] +

[0981] 1 H NMR (400MHz, DMSO-d6) δ7.53(dd,J=4.8,2.8Hz,1H),7.45(dd,J=3.6,1.2Hz,2H),7.32(s,1H),7.29-7.28(m,2H),6.55(d,J=6. 0Hz,1H),6.31(s,1H),6.30-6.26(m,2H),4.21-4.15(m,1H),3.93(dd,J=13.2,6.6Hz,4H),3.83(dd,J=15.2,7.6Hz,1H),3.66(s ,3H),3.64(s,3H),3.55-3.50(m,2H),3.50-3.44(m,1H),3.24(dd,J=13.2,10.0Hz,2H),3.14(dd,J=12.0,8.8Hz,1H),2.74(dd ,J=16.4,4.0Hz,1H),2.03-1.90(m,2H),1.79(dd,J=12.4,7.6Hz,5H),1.61-1.50(m,2H),0.68-0.61(m,1H),0.61-0.48(m,3H).

[0982] Its preparation method is as follows:

[0983] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0984] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0985] Preparation Example 22: Preparation of (S)-8-((5-(((S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spirocyclic[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-38)

[0986] Step 1: Preparation of (S)-8-((5-bromopentyl)oxy)-2-(5-(hydroxymethyl)thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-1)

[0987] (5-(hydroxymethyl)thiophen-3-yl)boronic acid (113.38 mg, 717.70 μmol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (200 mg, 358.85 μmol), tetra(triphenylphosphine)palladium (41.47 mg, 35.89 μmol), and sodium carbonate (152.14 mg, 1.44 mmol) were mixed together, and water (1 mL), toluene (4 mL), and ethanol (1 mL) were added. The mixture was then purged with nitrogen three times and heated to 80 °C for 2 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted three times with 5 mL of ethyl acetate (3 times). The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–100%) and then concentrated again under reduced pressure to obtain the title compound (30 mg, 50.06 μmol).

[0988] Its structural characterization data are as follows:

[0989] ESI-MS (m / z): 521.1 [M+H] +

[0990] Step 2: Preparation of (S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-2)

[0991] (S)-8-((5-bromopentyl)oxy)-2-(5-(hydroxymethyl)thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (30 mg, 57.53 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (15.78 mg, 57.53 μmol) were dissolved in DMF (1 mL), potassium carbonate (23.85 mg, 172.60 μmol) was added, and the mixture was stirred at room temperature for 40 hours. After the reaction was completed, the reaction solution was filtered to remove insoluble matter, and the filtrate was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (10 mg, 13.99 μmol).

[0992] Its structural characterization data are as follows:

[0993] ESI-MS (m / z): 715.3 [M+H] +

[0994] Its preparation method is as follows:

[0995] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0996] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0997] Step 3: Preparation of (S)-8-((5-(((S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spirocyclic[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-38)

[0998] (S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a)-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (10 mg, 13.99 μmol) was dissolved in THF (2 mL), and sodium borohydride (5.15 mg, 139.89 μmol) was added, producing a few bubbles. TFA (9.57 mg, 83.94 μmol) was added dropwise. The temperature was raised to 70℃ and the reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in methanol, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (2.18 mg, 2.96 μmol).

[0999] Its structural characterization data are as follows:

[1000] ESI-MS (m / z): 701.3 [M+H] +

[1001] 1H NMR (400MHz, DMSO-d6) δ7.35(s,1H),7.32(s,1H),7.28(s,1H),7.24(s,1H),7.15(d,J=1.2Hz,1H),6.55(d,J=6.4Hz,1H ),6.30(d,J=10.8Hz,3H),5.48(s,1H),4.60(d,J=3.6Hz,2H),4.17(dd,J=15.2,10.0Hz,1H),3.93(dd,J=12.8,6.4Hz,4 H),3.83(dd,J=14.8,7.2Hz,1H),3.65(d,J=8.4Hz,6H),3.55-3.46(m,4H),3.29-3.21(m,2H),3.21-3.09(m,2H),2.71( dd,J=16.8,4.0Hz,1H),2.03-1.91(m,2H),1.81-1.75(m,,5H),1.59-1.51(m,2H),0.68-0.61(m,1H),0.60-0.48(m,3H).

[1002] Its preparation method is as follows:

[1003] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1004] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1005] Preparation Example 23: Preparation of (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-39)

[1006] Step 1: Allyl(S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid ester (7-39-1)

[1007] (S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (19 mg, 66.84 μmol) and (S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (39.13 mg, 66.84 μmol) were dissolved in DMF (0.5 mL), and potassium carbonate (27.71 mg, 200.52 μmol) was added. The mixture was stirred at room temperature for 24 hours. Potassium iodide (11.10 mg, 66.84 μmol) was added, and the mixture was stirred at room temperature for 48 hours. After the reaction was complete, 5 mL of water was added to the reaction solution, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (70 mg, 64.51 μmol), which was used directly in the next reaction without purification.

[1008] Its structural characterization data are as follows:

[1009] ESI-MS (m / z): 790.4 [M+H] +

[1010] Step 2: Preparation of (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-39)

[1011] (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (70 mg, 88.74 μmol) was dissolved in DMF (1 mL), and tetrahydropyrrole (12.62 mg, 177.48 μmol) and tetra-triphenylphosphine palladium (10.25 mg, 8.87 μmol) were added. The mixture was purged with nitrogen three times and stirred at room temperature for 3 hours under a nitrogen protective atmosphere. After the reaction was complete, the reaction solution was filtered, and the filtrate was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (9.01 mg, 12.66 μmol).

[1012] Its structural characterization data are as follows:

[1013] ESI-MS (m / z): 705.3 [M+H] +

[1014] 1 H NMR(400MHz,DMSO-d6)δ7.45(s,1H),7.39(s,1H),7.37(s,1H),7.34(s,1H),7.29(s,1H), 6.91(d,J=8.8Hz,2H),6.56(d,J=6.0Hz,1H),6.49(d,J=6.0Hz,1H),6.31(d,J=4.8Hz,1H) ,4.22-4.16(m,1H),3.98-3.86(m,7H),3.75(s,3H),3.66(s,3H),3.64(s,3H),3.57-3.52 (m,2H),3.31-3.14(m,3H),2.78-2.68(m,2H),2.43-2.29(m,1H),1.82-1.77(m,4H),1.59 -1.52(m,2H).

[1015] Its preparation method is as follows:

[1016] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1017] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1018] Preparation Example 24: Preparation of (S)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40)

[1019] Step 1: Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-40-1)

[1020] (S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (45 mg, 76.86 μmol) and (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (34.91 mg, 92.23 μmol) were dissolved in DMF (1 mL), and potassium carbonate (21.24 mg, 153.72 μmol) was added. The mixture was stirred and reacted for 16 hours. Water was added to precipitate the solid, which was then filtered, washed with water, and dried to obtain the crude product (65 mg, 73.60 μmol), which was used directly in the next step without purification.

[1021] Its structural characterization data are as follows:

[1022] MS m / z (ESI): 883.1 [M+H] +

[1023] Step 2: Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40-2)

[1024] The (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,1 1a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (65 mg, 73.60 μmol) was dissolved in DMF (1 mL), and tetra(triphenylphosphine)palladium (8.51 mg, 7.36 μmol) and tetrahydropyrrole (5.23 mg, 73.60 μmol) were added. The mixture was stirred for 12 hours under nitrogen purging and protection. Ethyl acetate and water were added, the mixture was stirred, filtered, allowed to stand, and the organic phase was washed three times with saturated brine, dried, and concentrated under reduced pressure to obtain crude product (58 mg, 73.60 μmol), which was used directly in the next step without purification.

[1025] Its structural characterization data are as follows:

[1026] MS m / z (ESI): 800.5 [M+H] +

[1027] Step 3: Preparation of (S)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40)

[1028] (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (58 mg, 73.60 μmol) was dissolved in tetrahydrofuran (1 mL), and tetrabutylammonium fluoride solution (1 M, 145.17 μL) was added dropwise. The mixture was stirred for 1 hour. The solution was diluted with ethyl acetate, washed twice with saturated ammonium chloride aqueous solution, and twice with saturated brine. The solution was then concentrated under reduced pressure. The title compound (17.11 mg, 24.24 μmol) was obtained by preparative high performance liquid chromatography purification followed by freeze drying.

[1029] Its structural characterization data are as follows:

[1030] MS m / z (ESI): 685.3 [M+H] +

[1031] 1 H NMR (400MHz, DMSO) δ7.45 (s, 1H), 7.42-7.36 (m, 3H), 7.29 (s, 1H), 6.91 (d, J = 8.9Hz, 2H), 6.55 (d, J = 5.7Hz, 1H), 6. 36(d,J=5.9Hz,1H),6.31(s,1H),6.26(s,1H),4.92(d,J=3.2Hz,1H),4.19(s,2H),3.93(dd,J=13.7,6.7Hz,4H),3. 80(d,J=9.1Hz,1H),3.76(s,3H),3.65(d,J=9.5Hz,6H),3.51(dt,J=12.1,9.0Hz,4H),3.28-3.19(m,2H),2.99-2. 93(m,1H),2.79-2.74(m,1H),2.08(dd,J=13.0,6.7Hz,1H),1.80(s,4H),1.73-1.67(m,1H),1.56(d,J=6.4Hz,2H).

[1032] Its preparation method is as follows:

[1033] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1034] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1035] Preparation Example 25: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41)

[1036] Step 1: Preparation of (S)-4-methylenepyrrolidine-2-carboxylate (7-41-2)

[1037] (S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid (941.87 mg, 4.14 mmol) was dissolved in MeOH (7 mL), and HCl / dioxane (4 M, 7 mL) was added dropwise. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to obtain crude hydrochloride of the title compound (794 mg, 4.02 mmol), which was used directly in the next reaction without purification.

[1038] Its structural characterization data are as follows:

[1039] ESI-MS (m / z): 142.2 [M+H] +

[1040] Step 2: Preparation of (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carboxylate (7-41-3)

[1041] 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.35 g, 4.45 mmol) was dissolved in DMF (15 mL), and HATU (2.03 g, 5.34 mmol) and DIPEA (1.72 g, 13.34 mmol, 2.32 mL) were added. The mixture was stirred at room temperature for 10 minutes. Then, a mixture of (S)-4-methylenepyrrolidine-2-carboxylate hydrochloride (790 mg, 4.45 mmol) and DIPEA (574.80 mg, 4.45 mmol) in DMF (15 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was complete, 100 mL of water was added to the reaction mixture, and the mixture was extracted with 50 mL of ethyl acetate three times. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–60%) and concentrated again under reduced pressure to give the title compound (1.53 g, 3.58 mmol).

[1042] Its structural characterization data are as follows:

[1043] ESI-MS (m / z): 427.4 [M+H] +

[1044] Step 3: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-41-4)

[1045] Methyl (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carboxylate (1.53 g, 3.59 mmol) was dissolved in saturated ammonium chloride aqueous solution (8 mL) and methanol (80 mL). Zinc powder (2.35 g, 35.88 mmol) was added, and the mixture was heated to 75 °C and stirred for 4 hours. After the reaction was complete, the reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was extracted with 50 mL of water and 3 times of a mixed solvent of ethyl acetate:methanol (4:1). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (1.3 g, 3.57 mmol), which was used directly in the next reaction without purification.

[1046] Its structural characterization data are as follows:

[1047] ESI-MS (m / z): 365.2 [M+H] +

[1048] Step 4: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-41-5)

[1049] (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza -5,11(10H)-dione (0.2 g, 548.86 μmol) was dissolved in THF (3 mL), and the mixture was cooled to -40 °C under nitrogen protection. Then, n-BuLi (2.5 M, 230.52 μL) was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 30 minutes. Then, 2-(trimethylsilyl)ethoxymethyl chloride (100.66 mg, 603.74 μmol) was added dropwise at -40 °C. After the addition was complete, the mixture was allowed to react naturally overnight. After the reaction was complete, the reaction solution was quenched with 5 mL of ammonium chloride aqueous solution, and then extracted with 10 mL of water and 10 mL of ethyl acetate three times. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solution was then concentrated under reduced pressure to obtain the crude product of the title compound (286 mg, 520.36 μmol), which was used directly in the next reaction without purification.

[1050] Its structural characterization data are as follows:

[1051] ESI-MS (m / z): 496.3 [M+H] +

[1052] Step 5: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41-6)

[1053] (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza 5-one (180 mg, 363.89 μmol) was dissolved in THF (10 mL), and the mixture was cooled to -78 °C under nitrogen protection. Then, triethyllithium borohydride (1 M, 727.78 μL) was added dropwise, and the reaction was maintained at this temperature for 2 hours. Triethyllithium borohydride (1 M, 727.78 μL) was added three times, each time at 1-hour intervals. After the reaction was complete, the reaction mixture was added to 15 mL of water, extracted three times with ethyl acetate (10 mL x 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (200 mg, 344.44 μmol), which was used directly in the next reaction without purification.

[1054] Its structural characterization data are as follows:

[1055] ESI-MS (m / z): 367.3 [M+H+H2O] + 697.4 [2M+H] +

[1056] Step Six: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41-7)

[1057] (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza 5-one (80 mg, 229.62 μmol) was dissolved in dichloromethane (1 mL) and methanol (1 mL), and sodium triacetoxyborohydride (97.33 mg, 459.25 μmol) was added. The mixture was stirred overnight at room temperature for 16 hours. NaBH4 (17.37 mg, 459.25 μmol) was added, and after reacting for 2 hours, NaBH4 (17.37 mg, 459.25 μmol) was added again, and the reaction was continued for another 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, dissolved in methanol, and purified by reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0–50%). The solution was then freeze-dried to obtain the title compound (15 mg, 42.81 μmol).

[1058] Its structural characterization data are as follows:

[1059] ESI-MS (m / z): 351.3 [M+H] +

[1060] Step 7: Preparation of (S)-8-hydroxy-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41-8)

[1061] Dichloromethane (1 mL) was added to (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (15 mg, 42.81 μmol), followed by the addition of methanesulfonic acid (0.1 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 2 mL of water was added to the reaction solution, the pH was adjusted to 8-9 with saturated sodium bicarbonate solution, and the mixture was extracted with 3 mL of dichloromethane three times. The organic phase was washed with 3 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (11 mg, 42.26 μmol), which was used directly in the next reaction without purification.

[1062] Its structural characterization data are as follows:

[1063] ESI-MS (m / z): 261.1 [M+H] +

[1064] Step 8: (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo)[e]pyrrolo[1,2-a][1,4]diazide Preparation of -8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-41-9)

[1065] (S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (24.74 mg, 42.26 μmol) and (S)-8-hydroxy-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (11 mg, 42.26 μmol) were dissolved in DMF (1 mL) and potassium carbonate (17.52 mg, 126.78 μmol) was added. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (5 mg, 6.54 μmol).

[1066] Its structural characterization data are as follows:

[1067] ESI-MS (m / z): 766.3 [M+H] +

[1068] Its preparation method is as follows:

[1069] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1070] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1071] Step Nine: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41)

[1072] The (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo)[e]pyrrolo[1,2-a][1,4]diazide -8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (5 mg, 6.54 μmol) was dissolved in DMF (0.5 mL), and tetrahydropyrrole (464.92 μg, 6.54 μmol) and tetratetraphenylphosphine palladium (755.41 μg, 6.54e-1 μmol) were added. The mixture was purged with nitrogen three times, and then stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (1.89 mg, 2.47 μmol).

[1073] Its structural characterization data are as follows:

[1074] ESI-MS (m / z): 681.4 [M+H] +

[1075] Its preparation method is as follows:

[1076] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1077] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1078] Preparation Example 26: Preparation of (1aR,9aS,10aR)-5-methoxy-6-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1a,8,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3(1H)-one (7-43)

[1079] Step 1: Preparation of (1R,3S,5R)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid methyl ester hydrochloride (7-43-2)

[1080] (1R,3S,5R)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid (2.00 g, 8.80 mmol) was dissolved in methanol (10 mL), and 4 M dioxane hydrochloride solution (10 mL) was added dropwise, followed by stirring for 4 hours. After concentration under reduced pressure, the reaction proceeded directly to the next step.

[1081] Step 2: Preparation of (1R,3S,5R)-2-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid methyl ester (7-43-3)

[1082] 4-Benzyloxy-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.89 mmol), (1R,3S,5R)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid methyl ester hydrochloride (760.26 mg, 4.28 mmol), and HATU (2.22 g, 5.84 mmol) were dissolved in DMF (20 mL), and DIPEA (1.51 g, 11.67 mmol) was added dropwise. The mixture was stirred for 2 hours. Water and EA were added and stirred. The mixture was separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed twice with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (eluent 30-65% ethyl acetate / petroleum ether), the mixture was concentrated again to give the title compound (1.64 g, 3.85 mmol).

[1083] Step 3: Preparation of (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (7-43-4)

[1084] Methyl (1R,3S,5R)-2-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-azabicyclo[3.1.0]hexane-3-carboxylate (870 mg, 2.04 mmol) was dissolved in methanol (10 mL), and saturated ammonium chloride aqueous solution (2 mL) was added. Zinc powder (1.33 g, 20.40 mmol) was then added in portions, and the mixture was refluxed at 80 °C for 16 hours. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 5% methanol / dichloromethane). The filtrate was then concentrated again to give the title compound (682 mg, 1.87 mmol).

[1085] Its structural characterization data are as follows:

[1086] MS m / z (ESI): 365.1 [M+H] +

[1087] Step 4: Preparation of (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (7-43-5)

[1088] (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (180 mg, 493.97 μmol) was dissolved in dry THF (10 mL). Sodium borohydride (56.06 mg, 1.48 mmol) was added with stirring, followed by a 100 μL solution of trifluoroacetic acid (112.65 mg, 987.94 μmol, 75.65 μL) in tetrahydrofuran. A large number of bubbles were generated. The mixture was heated to 75 °C and stirred for 4 hours. The mixture was concentrated under reduced pressure, then water and ethyl acetate were added and stirred. The mixture was separated, and the organic phase was concentrated under reduced pressure. After purification by silica gel column chromatography (eluent: 5% methanol / dichloromethane), the mixture was concentrated again to give the title compound (142 mg, 405.24 μmol).

[1089] Its structural characterization data are as follows:

[1090] MS m / z (ESI): 367.3 [M+H2O] +

[1091] Step 5: Preparation of (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropane[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (7-43-6)

[1092] (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (142 mg, 405.24 μmol) was dissolved in dry dichloromethane (5 mL), and pyridine (48.08 mg, 607.86 μmol) was added dropwise. The mixture was cooled and stirred to 0 °C, and allyl chlorate (195.38 mg, 1.62 mmol, 172.29 μL) was added dropwise. The mixture was then brought back to room temperature and stirred for 1 hour. The solution was diluted with dichloromethane, and 10% citric acid solution was added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with water and then dried and concentrated. After silica gel column purification (eluent: 80% ethyl acetate / petroleum ether), the compound was concentrated again to give the title compound (153 mg, 352.14 μmol).

[1093] Step Six: Preparation of (1aR,9aS,10aR)-6-hydroxy-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropane[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (7-43-7)

[1094] (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (130 mg, 299.21 μmol) was dissolved in dichloromethane (1 mL), and methanesulfonic acid (0.5 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After dilution with dichloromethane, sodium bicarbonate aqueous solution was added and stirred. The mixture was allowed to stand and separated, and the organic phase was dried and concentrated. The solution was purified by preparative high-performance liquid chromatography and freeze-dried to give the title compound (57 mg, 165.52 μmol).

[1095] Its structural characterization data are as follows:

[1096] MS m / z (ESI): 345.1 [M+H] +

[1097] Its preparation method is as follows:

[1098] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1099] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1100] Step 7: Preparation of (1aR,9aS,10aR)-6-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (7-43-8)

[1101] (S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (10.20 mg, 17.42 μmol), (1aR,9aS,10aR)-6-hydroxy-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (5 mg, 14.52 μmol), and potassium carbonate (3.01 mg, 21.78 μmol) were dissolved in DMF (1 mL), and the reaction was stirred for 16 hours. Water was added to precipitate the solid, which was then filtered, washed with water, and dried under vacuum to obtain the title compound (12 mg, 14.14 μmol).

[1102] Its structural characterization data are as follows:

[1103] MS m / z (ESI): 849.4 [M+H] +

[1104] Step 8: Preparation of (1aR,9aS,10aR)-5-methoxy-6-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1a,8,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3(1H)-one (7-43)

[1105] The (1aR,9aS,10aR)-6-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-5-methoxy-3-oxo-1,1a,9,9a,10 10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (12 mg, 14.14 μmol) was dissolved in DMF (1 mL), tetrahydropyrrole (1.01 mg, 14.14 μmol) was added dropwise, followed by tetra(triphenylphosphine)palladium (1.63 mg, 1.41 μmol). The reaction was carried out under nitrogen purging and protection for 16 hours. The product was purified by preparative high-performance liquid chromatography and freeze-dried to give the title compound (6.18 mg, 8.99 μmol).

[1106] Its structural characterization data are as follows:

[1107] MS m / z (ESI): 681.3 [M+H] +

[1108] Its preparation method is as follows:

[1109] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1110] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1111] Preparation Example 27: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-44)

[1112] Step 1: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-44-1)

[1113] (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-L-proline methyl ester (870 mg, 2.04 mmol) was dissolved in methanol (100 mL), and saturated ammonium chloride aqueous solution (10 mL) was added. Zinc powder (2.84 g, 43.44 mmol) was then added in portions, and the mixture was refluxed at 80 °C for 16 hours. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 5% methanol / dichloromethane). The filtrate was then concentrated again to give the title compound (1.15 g, 3.26 mmol).

[1114] Its structural characterization data are as follows:

[1115] MS m / z (ESI): 353.1 [M+H] +

[1116] Step 2: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-44-2)

[1117] (S)-8-(benzyloxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.1 g, 3.12 mmol) was dissolved in dry THF (10 mL), and sodium borohydride (354.29 mg, 9.36 mmol) was added with stirring. Then, a tetrahydrofuran solution (100 μL) of trifluoroacetic acid (711.85 mg, 6.24 mmol, 478.07 μL) was added dropwise, resulting in the generation of numerous bubbles. The mixture was heated to 75 °C and stirred for 4 hours. The solution was concentrated under reduced pressure, and water and ethyl acetate were added with stirring. The mixture was separated, and the organic phase was concentrated under reduced pressure. After purification by silica gel column chromatography (eluent: 5% methanol / dichloromethane), the solution was concentrated again to give the title compound (1.01 g, 2.98 mmol).

[1118] Step 3: Preparation of (S)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-44-3)

[1119] (S)-8-(benzyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (1 g, 2.96 mmol) was dissolved in dry dichloromethane (50 mL), and pyridine (350.62 mg, 4.43 mmol, 358.51 μL) was added dropwise. The mixture was cooled and stirred to 0 °C, and allyl chlorate (1.07 g, 8.87 mmol, 942.30 μL) was added dropwise. The mixture was then brought back to room temperature and stirred for 1 hour. The solution was diluted with dichloromethane, and 10% citric acid solution was added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with water and dried and concentrated. The solution was purified by silica gel column chromatography (eluent: 80% ethyl acetate / petroleum ether) and then concentrated again to give the title compound (530 mg, 1.25 mmol).

[1120] Step 4: Preparation of (S)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-44-4)

[1121] (S)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (530 mg, 1.25 mmol) was dissolved in dichloromethane (4 mL), and methanesulfonic acid (2 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After dilution with dichloromethane, sodium bicarbonate aqueous solution was added and stirred. The mixture was allowed to stand and separated. The organic phase was dried and concentrated to give the title compound (410 mg, 1.23 mmol).

[1122] Its structural characterization data are as follows:

[1123] MS m / z(ESI): 333.2 [M+H] +

[1124] Step 5: Preparation of (S)-8-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-44-5)

[1125] (S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (40 mg, 68.32 μmol), (S)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (22.71 mg, 68.32 μmol), and potassium carbonate (14.16 mg, 102.48 μmol) were dissolved in DMF (1 mL), and the mixture was stirred for 16 hours. Water was added to precipitate the solid, which was then filtered, washed with water, and dried under vacuum to obtain the title compound (55 mg, 65.72 μmol).

[1126] Its structural characterization data are as follows:

[1127] MS m / z (ESI): 838.4 [M+H] +

[1128] Step Six: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-44)

[1129] Allyl (55 mg, 65.72 μmol) of (S)-8-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid (1 mL) was dissolved in DMF, and tetrahydropyrrole (4.67 mg, 65.72 μmol) was added dropwise. Tetra(triphenylphosphine)palladium (7.59 mg, 6.57 μmol) was added, and the reaction was carried out under nitrogen purging and protection for 16 hours. The compound was purified by preparative high-performance liquid chromatography and freeze-dried to obtain the title compound (11.71 mg, 17.33 μmol).

[1130] Its structural characterization data are as follows:

[1131] MS m / z (ESI): 669.3 [M+H] +

[1132] 1 H NMR (400MHz, CDCl3) δ7.49(s,2H),7.31(d,J=8.7Hz,2H),7.26(s,3H),6.89(d,J=8.8Hz,2H),4.29(s,1H),3.99(s,4H),3.87(s,5H),3.81(d,J=11 .6Hz,5H),3.70(s,1H),3.58(d,J=10.2Hz,4H),3.32(s,3H),2.74(dd,J= 16.1,3.6Hz,1H),2.21(dd,J=12.8,6.3Hz,1H),1.90(m,6H),1.65(m,4H).

[1133] Its preparation method is as follows:

[1134] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1135] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1136] Preparation Example 28: Preparation of (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one) (7-45)

[1137] Step 1: Preparation of (S)-8-((5-(((R)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-45-1)

[1138] 1,5-Dibromopentane (12.45 mg, 54.16 μmol), (S)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (40 mg, 120.35 μmol), and potassium carbonate (49.90 mg, 361.06 μmol) were dissolved in DMF (1 mL), and the mixture was stirred for 16 hours. Water was added to precipitate the solid, which was filtered, washed with water, and dried under vacuum to give the title compound (40 mg, 54.58 μmol).

[1139] Its structural characterization data are as follows:

[1140] MS m / z(ESI): 733.4 [M+H] +

[1141] Step 2: Preparation of (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one) (7-45)

[1142] (S)-8-((5-(((R)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (40 mg, 54.58 μmol) was dissolved in DMF (1 mL), tetrahydropyrrole (3.88 mg, 54.58 μmol) was added dropwise, and tetra(triphenylphosphine)palladium (6.31 mg, 5.46 μmol) was added. The reaction was carried out under nitrogen purging and protection for 16 hours. The compound was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (4.96 mg, 8.70 μmol).

[1143] Its structural characterization data are as follows:

[1144] MS m / z (ESI): 565.3 [M+H] +

[1145] 1 H NMR (400MHz, CDCl3) δ7.52(s,2H),6.13(s,2H),3.99(s,4H),3.95-3.73(m,9H),3.70(s,2H),3.57(d,J=11 .7Hz,2H),3.29(s,3H),2.22(dd,J=12.6,6.1Hz,2H),1.90(dt,J=13.4,6.5Hz,8H),1.68(d,J=20.9Hz,6H).

[1146] Its preparation method is as follows:

[1147] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1148] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1149] Example 1 of intermediate preparation: Preparation of 1-(3,5-bis(2-(methanesulfonyl)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-decaazatrione-31-carboxylic acid (INT-1):

[1150] Step 1: 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-decaazatrione-31-carboxylic acid (INT-1-2) using standard solid-phase synthesis methods:

[1151] 1) Resin preparation: 2-CTC resin (3.00 mmol, 3.70 g, 0.81 mmol / g), N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (3.00 mol, 933 mg, 3.00 equivalents), and DIPEA (4.00 equivalents) were added to dichloromethane (10.0 mL) and reacted under a nitrogen atmosphere for 2 hours. Then, MeOH (1.0 mL) was added to the resin over a nitrogen bubbling atmosphere over 30 minutes, and the resin was obtained by filtration.

[1152] 2) Coupling: Under nitrogen bubbling, a DMF solution (10.0 mL) of N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (5....

Claims

1. A compound or a pharmaceutically acceptable salt thereof having the structure shown in formula DEL-M', wherein: M' is -M-Lg, where M is the structural fragment that binds to the target site, and Lg is the leaving group for the nucleophilic substitution reaction; L is the structural segment connecting M and E; E is a structural segment connecting L and D; D is a cytotoxic drug fragment.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: The cytotoxic drug is selected from compounds with the structure shown in formula (I) or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs: Among them, R 1 and R 2 Each is independently selected from hydrogen, -CN, halogen, and -OR. a -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1- 6-alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1- 6-alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; R 4 and R 4’ Each is independently selected from hydrogen, halogen, -OH, -NH2, C 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 1-6 Alkoxy, R 5 and R 5’ All are hydrogen; Or, R 4 and R 4’ Together with R 4 and R 4’ The carbon atoms form carbon-carbon double bonds or 3-6 membered carbon rings, R 5 and R 5’ All are hydrogen; Or, R 4 and R 5 Connect to the key or together with the R connection 4 and R 5 The carbon atoms form 3-6 membered carbon rings, R 5’ and R 4’ Each is independently selected from hydrogen, halogen, -NH2, C 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 1-6 Alkoxy; R 6 For hydrogen, C 1-6 alkyl or R 6’ R 7 and R 7’ Both are hydrogen; or, R 6’ With R 7 Linking and bonding, R 7’ It is hydrogen; R a Selected from H, C 1-6 Alkyl or C 3-6 cycloalkyl; Ring A is selected from 3-6 membered cycloalkyl groups, C 6-10 aryl, 5-10 membered heteroaryl, 3-6 membered heterocyclic; the 3-6 membered cycloalkyl, C 6-10 The aryl, 5-10 membered heteroaryl, and 3-6 membered heterocyclic groups may be optionally substituted by one or more of the following substituents: hydrogen, -CN, halogen, -OR. a -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; R 3 Selected from hydrogen, -CN, halogen, -OR a -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; X and Y are independently -CH2- and -CD2-, respectively. and -C(O)-; t is selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the cytotoxic drug is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs: More preferably, the cytotoxic drug is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs: More preferably, the cytotoxic drug is a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof:

3. The compound of claim 1 or 2, 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 structural segments, whether substituted or unsubstituted:

4. The compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein the Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, alkenyl, ynyl and ynyl-containing structural segments may optionally be substituted by one or more suitable substituents; Preferably, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments; Preferably, Lg is selected from halogens, substituted or unsubstituted C1-6 alkylsulfonyl groups, halophenoxy groups, hydroxyl groups (-OH), mercapto groups (-SH), and amino groups (-NH2); Preferably, Lg is selected from halogens, substituted or unsubstituted methanesulfonyl groups, halophenoxy groups, hydroxyl groups (-OH), mercapto groups (-SH), and amino groups (-NH2); Preferably, Lg is a methanesulfonyl group.

5. The compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein, The L is selected from one or more of the following structural segments or stereoisomers: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic groups, -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)) r OCH3), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Gly-Lys, 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-L ys-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), The structural segment is optionally further substituted; wherein R' consists of one or more groups including, but not limited to, hydrogen, C 1-6 Alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups SO3H, -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-heterocyclic, -CH2NH-SO3H, -CH2N(C 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N + (C 1-6 Alkylene (-SO3H)3, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl group 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- 6alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylenes -CO2H, -CH2N + (C 1- (6-alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, or polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl groups), 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), and NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues). Where r is selected from integers from 1 to 20; s is selected from integers from 1 to 20; Preferably, L is selected from one or more of the following substituted or unsubstituted structural fragments: natural amino acids 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)) r OCH3), Lys(R'), and short peptides composed of amino acids (such as Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala). Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, 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). Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, or 10. Preferably, L is selected from one or more of the following substituted or unsubstituted structural fragments: natural amino acids 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)) r OCH3), and short peptides composed of amino acids (such as Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala). Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10.

6. The compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein: The E is a single bond, substituted or unsubstituted -NH-CH2- or Preferably, E is a single bond, substituted or unsubstituted -NH-CH2-; More preferably, E is a single bond or -NH-CH2-.

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein: Selected from the following substituted or unsubstituted structures: Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10; Preferably, Selected from the following substituted or unsubstituted structures:

8. The compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein: The structure of the compound is as follows: Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

9. An antibody-drug conjugate as shown in formula (II), wherein: A-[M-L-E-D] x Formula (II) M, L, E, and D are as described in any one of claims 1-8; x is 1-10; A represents the target group, with targets selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, O772P, MPF, Napi3b, Sema 5b, and PSCA. hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2, BMPR1B, Tyro7, c-Met, ApoE, CD1lc, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDllb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, and TENB2.

10. The antibody-drug conjugate of claim 9, wherein A 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 its antigen-binding fragment 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, A 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.

11. A composition comprising one or more antibody-drug conjugates as described in claim 9 or 10; Preferably, 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.

12. A pharmaceutical composition comprising the antibody-drug conjugate of claim 9 or 10, the composition of claim 11, and one or more pharmaceutical excipients.

13. Use of the antibody-drug conjugate of claim 9 or 10, the composition of claim 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating Her2-expressing cancer.

14. 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.

15. The antibody-drug conjugate of claim 9 or 10, the composition of claim 11, or the pharmaceutical composition of claim 12, for the treatment of Her2-expressing cancers.

16. The antibody-drug conjugate of claim 9 or 10, the composition of claim 11, or the pharmaceutical composition of claim 12, for the treatment of solid tumors or hematologic malignancies, such as gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) or urothelial carcinoma.

17. A treatment method for Her2-expressing cancer, the method comprising administering to an individual in need a therapeutically effective amount of the antibody-drug conjugate of claim 9 or 10, the composition of claim 11, or the pharmaceutical composition of claim 12.

18. The treatment 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.

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