Heterocyclic compound, antibody-drug conjugate thereof, method for preparing same, and use thereof

Abstract

The present application provides an antibody-drug conjugate, a method for preparing same, and use thereof. The antibody-drug conjugate has a structure of formula I. The drug is selected from a topoisomerase inhibitor, a DNA-damaging agent, a microtubule inhibitor, an RNA polymerase inhibitor, and an antimetabolite. The prepared antibody-drug conjugate has an optimized drug-to-antibody ratio and targeted killing effects against breast cancer, gastric cancer, lung cancer, and colorectal cancer.

Description

Heterocyclic compounds and their antibody-drug conjugates, preparation methods and uses

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application No. 202411989594.3 filed on December 31, 2024 and CN application No. 202411654443.2 filed on November 19, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application belongs to the pharmaceutical field and relates to heterocyclic compounds and their antibody-drug conjugates, preparation methods and uses. Background Technology

[0004] Significant progress has been made in the development of antitumor drugs and targeted tumor therapy using biopharmaceuticals (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands. However, while biopharmaceuticals are highly targeted, their therapeutic effects on solid tumors are limited; and while bioactive molecules have high killing power against cancer cells, they often lack targeting and frequently damage normal cells, leading to serious toxic side effects.

[0005] Recent studies have found that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). ADCs combine the targeting properties of antibodies with the activity of bioactive molecules. Antibodies guide ADCs to bind to target cells, where they are subsequently internalized, releasing the drug to kill cells and treat the disease. Because antibodies are specific and targeted to tumor cell-related targets, their application value lies not only in treatment but also in serving as ideal carriers for targeted drug delivery, reducing drug side effects.

[0006] Pyrrolobenzodiazepine (PBD) is a sequence-selective DNA minor groove binder and a member of the anthraxmycin family of antibiotics. This type of substance can intercalate into the DNA minor groove via diazotization. The electrophilic imine group at the N-10 / C-11 position within the ring forms a covalent bond with the amino group at the C-2 position of guanine in the minor groove of DNA, fixing the helical structure of DNA, blocking cell division, and thus killing tumor cells. Due to its unique mechanism of action and highly efficient cytotoxicity, PBD toxin-loaded ADCs play an important role in ADC drug development, but there is still a need to develop ADCs with higher efficacy and safety. Summary of the Invention

[0007] This application provides novel drug linker compounds and their drug conjugates, which exhibit good antitumor activity, pharmacokinetic properties, and safety.

[0008] Drug linker

[0009] On the one hand, this application provides a drug linker, wherein the drug linker is a compound with the structure shown in Formula I or a drug-acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug:

[0010] in:

[0011] Q is the structure before it is linked to the antibody or antigen-binding fragment;

[0012] L is the connector sub-part;

[0013] E is the self-eliminating part;

[0014] D represents the bioactive molecule portion;

[0015] n is selected from 1-10.

[0016] In some embodiments, the bioactive molecules are each independently selected from antitumor drugs or compounds with antitumor effects.

[0017] In some embodiments, the bioactive molecules are each independently selected from cytotoxic compounds or antimetabolites.

[0018] In some embodiments, the cytotoxic compound is a DNA damaging agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, or a microtubule inhibitor.

[0019] In some embodiments, the cytotoxic compound is pyrrolobenzodiazepine. Class of compounds (PBD).

[0020] In some implementations, Q is selected individually from the following structures each time it appears:

[0021] Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears.

[0022] In some implementations, Q is selected from the following structures:

[0023] In some implementations, Q has the following structure:

[0024] In some implementations, L is selected from a divalent structure composed of one or more substituted or unsubstituted structural segments: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 5-12 heterocyclic, -N(R')-, carbonyl, -O-, glycosyl, tromethamine, 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)) and Lys(R') r and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Al). a-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Ala-Ala-Glu, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:44), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:45), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:46), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:47), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 48), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 49), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 50), EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, NOPO, Wherein Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R' represents hydrogen, C 1-6 Alkyl groups, polyhydroxy fragments, glycosyl groups, polyethylene glycol-containing fragments, -(CH2CH2O) r -C 1-6 Alkyl group, -C(=O)-(CH2CH2O) r -C 1-6 Alkyl, polysarcosine, -(C(=O)-CH2N(Me)) r -C 1-6 Alkyl groups, carboxylic acid-containing fragments, tetracarboxylic acid residues and their derivatives, EDTA and its derivatives, or DOTA and its derivatives; r is independently selected from integers from 1 to 20 each time it appears.

[0025] In some implementations, L is selected from the following structures:

[0026] Each time r appears, it is independently selected from an integer between 1 and 20.

[0027] In some implementations, L is selected from the following structures:

[0028] Each time r appears, it is independently selected from an integer between 1 and 20.

[0029] In some implementations, L is selected from the following structures:

[0030] In some implementations, L is selected from the following structures:

[0031] In some implementations, L is selected from the following structures:

[0032] In some implementations, E is selected from a single bond, -NH-CH2-.

[0033] In some implementations, E is selected from single bonds or

[0034] In some implementations, E is independently selected from single bonds and

[0035] In some implementations, E is a single bond.

[0036] In some implementations, n in equation (I) is 1, and -ELQ is selected from the following structures:

[0037] The bioactive molecules 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 ), or thiol (-SH), where R A R B R C This refers only to the non-hydrogen substituents on N. These functional groups can be connected to the rest of the part through chemical reactions.

[0038] In some embodiments, the bioactive molecules are each independently linked to the E via -OH, primary amino, secondary amino, tertiary amino, or -SH groups, or by reducing the double bonds in their structure.

[0039] In some embodiments, the bioactive molecule is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, or prodrugs, wherein the compound has the structure shown in formula (III):

[0040] in,

[0041] R 1 and R 2 Each is independently selected from hydrogen and 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; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally and independently selected from H, deuterium (D), 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; or R1 and R 2 It is linked to adjacent atoms to form a ring, wherein the ring is optionally selected by one or more atoms independently selected from H, deuterium (D), 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;

[0042] X is selected from C 2-6 alkenyl, C 2-6 alkynyl, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 2-6 alkynyl, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally selected independently by one or more halogens, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxyalkyl, amino, aminealkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 amine alkyl-amide group, C 1-6 Amine alkoxy-amide group, C 1-6 Aminoalkylamine-amide group, substituted or unsubstituted C 6-10 The aryl group is substituted with substituents of substituted or unsubstituted 5-10 heteroaryl groups.

[0043] In some implementation schemes, R 1 and R 2 Each was independently selected from C 1-6 alkyl.

[0044] In some implementation schemes, R 1 and R 2 It is a methyl group.

[0045] In some implementations, X is selected from C. 2-6 alkenyl, C 6-10 Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally separated by one or more C groups independently selected from amino, substituted or unsubstituted C. 6-10 The aryl group is substituted with substituents of substituted or unsubstituted 5-10 heteroaryl groups.

[0046] In some implementations, X is selected from -C 2-6 alkenyl-C 6-10 Aryl, -C 2-6 alkenyl-5-10 heteroaryl, C 6-10 Aryl, 5-10 heteroaryl, -C 6-10 Aryl-amino and 5-10 heteroaryl-amino.

[0047] In some embodiments, X is selected from -vinyl-phenyl, -vinyl-pyridyl, naphthyl, benzopyridyl, -naphthyl-NH2, and -benzopyridyl-NH2.

[0048] In some embodiments, X is selected from -vinyl-phenyl, -vinyl-pyridyl, naphthyl, benzopyridyl, -naphthyl-NH2, -benzopyridyl-NH2 and -vinyl-phenyl-NH2.

[0049] In some embodiments, X is selected from -naphthyl-NH2, -benzopyridyl-NH2, and -vinyl-phenyl-NH2.

[0050] In some implementations, X is -vinyl-phenyl-NH2.

[0051] In some embodiments, the bioactive molecule is selected from the following compounds:

[0052] In some embodiments, the bioactive molecule is selected from the following compounds:

[0053] In some embodiments, the bioactive molecule is

[0054] In some implementations, D is selected from the following structures:

[0055] Among them, R 1 R 2 X is as described in any of the above; X' is a group of X after removing a hydrogen atom (preferably, X' is a group of X after removing a hydrogen atom from the terminal -NH2).

[0056] In some implementations, X' is selected from the following structures:

[0057] The structure described above is connected to the rest of D via position 1.

[0058] In some implementations, D is selected from the following structures:

[0059] In some implementations, D is selected from the following structures:

[0060] In some implementations, D is

[0061] In some embodiments, the "drug linker" is selected from the following structures:

[0062] Antibody-drug conjugates

[0063] In another aspect, this application provides an antibody-drug conjugate having the structure shown in formula (II):

[0064] Where L' is as described in any of the L items above;

[0065] E' is as described in any of the above E items;

[0066] D' is as described in any of the above D';

[0067] A represents an antibody or its antigen-binding fragment;

[0068] Q' is the structural form of Q covalently linked to an antibody or its antigen-binding fragment as described in any of the above items;

[0069] x is selected from 1 to 10.

[0070] In the antibody-drug conjugate, D' can be conjugated to the antibody or its antigen-binding fragment via a linker.

[0071] In some implementations, Q' is selected from the following structures:

[0072] Each time p appears, it is independently selected from an integer between 1 and 12.

[0073] In some implementations, Q' is selected from the following structures:

[0074] In some implementations, Q' has the following structure:

[0075] In some embodiments, the antibody or its antigen-binding fragment 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.

[0076] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0077] (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:

[0078] (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,

[0079] (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;

[0080] 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;

[0081] or,

[0082] (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:

[0083] (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,

[0084] (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;

[0085] 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;

[0086] or,

[0087] (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:

[0088] (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,

[0089] (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;

[0090] 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;

[0091] or,

[0092] (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:

[0093] (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,

[0094] (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;

[0095] 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.

[0096] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0097] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0098] (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,

[0099] (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;

[0100] or,

[0101] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0102] (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,

[0103] (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;

[0104] or,

[0105] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0106] (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,

[0107] (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;

[0108] or,

[0109] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0110] (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,

[0111] (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.

[0112] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0113] (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

[0114] (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;

[0115] 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.

[0116] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0117] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0118] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0119] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0120] (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

[0121] (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).

[0122] 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.

[0123] 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.

[0124] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 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: 41.

[0125] 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.

[0126] 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.

[0127] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0128] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0129] (1) A heavy chain comprising the VH 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 of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36;

[0130] (2) A heavy chain comprising the VH region 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 region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0131] (3) 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: 41, 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

[0132] (4) 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: 41, 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.

[0133] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0134] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38;

[0135] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40;

[0136] (3) The heavy chain comprising the sequence shown in SEQ ID NO: 42, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0137] (4) The heavy chain comprising the sequence shown in SEQ ID NO: 43, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0138] 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 may 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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, 39, 42 or 43, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0143] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or 41 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37, 39, 42 or 43 or a variant thereof, lacks a C-terminal lysine residue.

[0144] In some embodiments, A is selected from an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase family.

[0145] In some implementations, A is selected from trastuzumab, pertuzumab, trastuzumab mutant, pertuzumab mutant, or a biepisode antibody or its antigen-binding fragment constructed from trastuzumab and pertuzumab.

[0146] 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.

[0147] 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, a free form of the "drug-linker" (QLED, where Q is the structural form of Q' before covalently linking it to an antibody or its antigen-binding fragment) described above can be obtained first, and then covalently linked to 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," Q 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 -Br structure) or an addition reaction.

[0148] In some implementation schemes, Selected from the following structures:

[0149] Among them, the -S) in each antibody-drug conjugate x -Ab or -NH) x -Ab represents an antibody or its antigen-binding fragment as described in any of the above descriptions. This indicates the specific linkage between the thiol and pyrimidine groups in the antibody or its antigen-binding fragment. This indicates the specific connection between the amino and carbonyl groups in the antibody or its antigen-binding fragment, where x is selected from 1 to 10.

[0150] In some implementation schemes, the -S in each antibody-drug conjugate x -Ab represents 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.

[0151] in, This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment.

[0152] In some implementation schemes, This indicates the specific linkage between the thiol group and the pyrimidine group of cysteine ​​in the antibody or its antigen-binding fragment.

[0153] In some implementation schemes, the -NH in each antibody-drug conjugate x -Ab represents 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.

[0154] in, This indicates the specific linkage between the amino and carbonyl groups in the antibody or its antigen-binding fragment.

[0155] In some implementation schemes, This indicates the specific way in which an amino group (such as the side chain amino group of lysine) is linked to a carbonyl group in an antibody or its antigen-binding fragment.

[0156] In some implementations, -S) x -Ab or -NH) x -Ab represents an antibody or antigen-binding fragment thereof containing VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2; wherein x is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0157] This indicates the specific linkage between the thiol and pyrimidine groups in the antibody or its antigen-binding fragment. This indicates the specific linkage between the amino and carbonyl groups in the antibody or its antigen-binding fragment.

[0158] In some embodiments, the antibody or its antigen-binding fragment is trastuzumab or pertuzumab.

[0159] Bioactive molecules

[0160] Another aspect of the present invention provides a bioactive molecule selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, or prodrugs, said compounds having the structure shown in formula (III):

[0161] in,

[0162] R 1 and R 2 Each is independently selected from hydrogen and 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; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally and independently selected from H, deuterium (D), halogen, -OH, -NH2, C. 1-6Alkyl, 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; or R 1 and R 2 It is linked to adjacent atoms to form a ring, wherein the ring is optionally selected by one or more atoms independently selected from H, deuterium (D), 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;

[0163] X is selected from C 2-6 alkenyl, C 2-6 alkynyl, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 2-6 alkynyl, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally selected independently by one or more halogens, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxyalkyl, amino, aminealkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 amine alkyl-amide group, C 1-6 Amine alkoxy-amide group, C 1-6 Aminoalkylamine-amide group, substituted or unsubstituted C 6-10 The aryl group is substituted with substituents of substituted or unsubstituted 5-10 heteroaryl groups.

[0164] In some implementation schemes, R 1 and R 2 Each was independently selected from C 1-6 alkyl.

[0165] In some implementation schemes, R 1 and R 2 It is a methyl group.

[0166] In some implementations, X is selected from C. 2-6 alkenyl, C 6-10Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally separated by one or more C groups independently selected from amino, substituted or unsubstituted C. 6-10 The aryl group is substituted with substituents of substituted or unsubstituted 5-10 heteroaryl groups.

[0167] In some implementations, X is selected from -C 2-6 alkenyl-C 6-10 Aryl, -C 2-6 alkenyl-5-10 heteroaryl, C 6-10 Aryl, 5-10 heteroaryl, -C 6-10 Aryl-amino and 5-10 heteroaryl-amino.

[0168] In some embodiments, X is selected from -vinyl-phenyl, -vinyl-pyridyl, naphthyl, benzopyridyl, -naphthyl-NH2, and -benzopyridyl-NH2.

[0169] In some embodiments, the bioactive molecule is selected from the following compounds:

[0170] In some embodiments, the bioactive molecule is selected from the following compounds:

[0171] Connector

[0172] On the other hand, the present invention provides a connector with the structural formula -E'-L'). x -Q'-; wherein E', L', Q', and x are as described in any of the above.

[0173] In some embodiments, E' is used to link with D' as described in any one of the present applications, and Q' is used to link with an antibody or its antigen-binding fragment as described in any one of the present applications.

[0174] In some embodiments, the connector structure is as follows:

[0175] In some embodiments, the connector structure is as follows:

[0176] In some embodiments, the present invention provides a connector, the connector having the structural formula -EL). n -Q; wherein E, L, Q and n are as described in any of the above.

[0177] In some embodiments, E is used to connect with D as described in any one of the claims of this application.

[0178] In some embodiments, the connector structure is as follows:

[0179] In some embodiments, the connector structure is as follows:

[0180] Synthetic intermediates

[0181] In another aspect, the present invention also provides the following synthetic intermediates and their pharmaceutically acceptable salts:

[0182] In each instance, PG1 is an amino protecting group, PG2 is a hydroxyl protecting group, and LG is a leaving group.

[0183] In some embodiments, the amino protecting group is selected from benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), phosphomethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), (trimethylsilyl)ethoxymethyl (SEM), methoxycarbonyl (or eth)oxycarbonyl, or tert-butylsulfinyl; preferably, the amino protecting group is allyloxycarbonyl (Alloc), phosphomethoxycarbonyl (Fmoc), or (trimethylsilyl)ethoxymethyl (SEM).

[0184] In some embodiments, the hydroxyl protecting group is selected from trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl ether (Me), tert-butyl ether (tBu), benzyl ether (Bn), p-methoxybenzyl ether (PMB), triphenylmethyl ether (Tr), methoxymethyl ether (MOM), and tetrahydropyran (THP). Preferably, the hydroxyl protecting group is tert-butyldimethylsilyl (TBS).

[0185] In some embodiments, the leaving group is selected from halogenated C. 1-6 Alkyl, maleimide, halogenated maleimide, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6Alkyl sulfonate group; preferably, the leaving group is a trifluoromethanesulfonate group (-OTf).

[0186] In some embodiments, the structure of the synthetic intermediate is shown below:

[0187] Composition

[0188] 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 contains a drug-linker as described herein, wherein x independently is 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 DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0189] 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.

[0190] In some embodiments, the DAR of the ADC compositions described herein is about 1 to 8, for example, about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, 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 3.5 to 7.0, about 3.5 to 7.5, about 3.5 to 8.0, 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 7. 5, approximately 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 4.5 to 8.0, approximately 5.0 to 5.5, 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 8.0, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 8.0, approximately 7.0 to 7.5, approximately 7.0 to 8.0, approximately 7.5 to 8.0.

[0191] In some embodiments, the DAR of the ADC composition described herein is about 4.0 to 8.0.

[0192] In some embodiments, the DAR of the ADC composition described herein is about 6.0 to 8.0.

[0193] In some embodiments, the DAR of the ADC composition described herein is about 7.0 to 7.5.

[0194] In some embodiments, the DAR of the ADC composition described herein is about 5.5.

[0195] In some embodiments, the DAR of the ADC composition described herein is about 5.8.

[0196] In some embodiments, the DAR of the ADC composition described herein is about 6.2.

[0197] In some embodiments, the DAR of the ADC composition described herein is about 6.9.

[0198] In some embodiments, the DAR of the ADC composition described herein is about 5.1.

[0199] In some embodiments, the DAR of the ADC composition described herein is about 6.5.

[0200] In some embodiments, the DAR of the ADC composition described herein is about 8.1.

[0201] In some embodiments, the DAR of the ADC composition described herein is about 6.3.

[0202] In some embodiments, the DAR of the ADC composition described herein is about 7.9.

[0203] In some embodiments, the DAR of the ADC composition described herein is about 8.2.

[0204] In some embodiments, the DAR of the ADC composition described herein is about 7.1.

[0205] In some embodiments, the DAR of the ADC composition described herein is about 8.0.

[0206] In some embodiments, the DAR of the ADC composition described herein is about 7.6.

[0207] In some embodiments, the DAR of the ADC composition described herein is about 7.4.

[0208] Pharmaceutical Composition

[0209] In another aspect, this application provides a pharmaceutical composition comprising any of the bioactive molecules, drug linker compounds, antibody-drug conjugates or combinations thereof described in any of the preceding claims, and one or more pharmaceutical excipients.

[0210] The bioactive molecules, drug-conjugate compounds, antibody-drug conjugates, or combinations thereof 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 a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer in the form of a lyophilized or solution form (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.

[0211] application

[0212] The bioactive molecules, antibody-drug conjugates, drug-linkers, ADC 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 colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0213] Therefore, this application provides the use of any of the bioactive molecules, antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the foregoing in the preparation of a medicament for treating Her2-expressing cancers.

[0214] In addition, this application provides any of the bioactive molecules, antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the thereof described above for the treatment of Her2-expressing cancers.

[0215] Additionally, this application provides a method for treating Her2-expressing cancer, comprising administering to a subject in need an effective amount of any of the preceding bioactive molecules, antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the thereof.

[0216] In some embodiments, the bioactive molecule, antibody-drug conjugate, drug-linker, ADC composition, or pharmaceutical composition is sufficient (e.g., in a subject):

[0217] (1) Inhibits cell (such as tumor cells) proliferation;

[0218] (2) Inhibits tumor growth;

[0219] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0220] (4) Inhibit HER2-mediated signal transduction;

[0221] (5) Prevention and / or treatment of HER2-mediated diseases / disorders; or

[0222] (6) Any combination of (1)-(5) above.

[0223] In some implementations, the cancer or tumor is selected from solid tumors or hematologic malignancies, such as colon cancer, stomach cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0224] definition

[0225] 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.

[0226] 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.

[0227] 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).

[0228] 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.

[0229] 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.

[0230] The term "antigen-binding fragment" in antibody 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.

[0231] 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).

[0232] 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.

[0233] 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.

[0234] 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:51) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:52) 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0240] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0241] 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.

[0242] 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).

[0243] 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.

[0244] 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.

[0245] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20Alkyl", C 1- 10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3 Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0246] The term "alkylene" refers to any divalent group obtained by removing one hydrogen atom from any "alkyl" as defined above.

[0247] As used herein, the term “heterocyclic group” or “heterocycle” refers to a monocyclic or polycyclic (e.g., fused, spirocyclic, or bridged) group having two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) carbon atoms and one or more (e.g., 1, 2, 3, or 4) heteroatoms, said heteroatoms including, but not limited to, oxygen, nitrogen, and sulfur atoms, wherein the carbon atoms and heteroatoms on said heterocyclic group are optionally substituted with oxo groups (e.g., forming C=O, S(=O) or S(=O)2).

[0248] As used herein, the term "5-12 membered heterocyclic group" refers to a heterocyclic group containing 5-12 ring atoms. Examples of 5-12 membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, pyrrolylalkyl, and pyrrolidone groups (such as...). ), imidazoalkyl, pyrazolyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl.

[0249] As used herein, the term "heterocyclic group" encompasses fused ring structures, wherein the connection point between the fused ring structure and other groups can be on any ring of the fused ring structure. Therefore, the heterocyclic groups of the present invention also include, but are not limited to, heterocyclic fused heterocyclic groups, heterocyclic fused cycloalkyl groups, monoheterocyclic fused monoheterocyclic groups, and monoheterocyclic fused monocycloalkyl groups, such as 3-7 membered (mono)heterocyclic fused 3-7 membered (mono)heterocyclic groups, 3-7 membered (mono)heterocyclic fused (mono)cycloalkyl groups, and 3-7 membered (mono)heterocyclic fused C 4-6 (Mono)cycloalkyl groups, examples of which include, but are not limited to, pyrrolidinylcyclopropyl, cyclopentylazirylpropyl, pyrrolidinylcyclobutyl, pyrrolidinylpyrrolidinyl, pyrrolidinylpiperidinyl, pyrrolidinylpiperazinyl, and piperidinylmorpholinyl.

[0250] As used in this article, the term "heterocyclic group" encompasses both bridged heterocyclic groups and spirocyclic groups.

[0251] As used herein, the term "bridged heterocycle" refers to a ring structure containing one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, nitrogen, and / or sulfur atoms) formed by two saturated rings sharing two non-directly connected ring atoms. This includes, but is not limited to, 7-10 membered bridged heterocycles, 8-10 membered bridged heterocycles, 7-10 membered nitrogen-containing bridged heterocycles, 7-10 membered oxygen-containing bridged heterocycles, and 7-10 membered sulfur-containing bridged heterocycles, etc. The "nitrogen-bridged heterocycle", "oxygen-bridged heterocycle", and "sulfur-bridged heterocycle" may optionally also contain one or more other heteroatoms selected from oxygen, nitrogen, and sulfur.

[0252] As used herein, the term "spiroheterocycle" refers to a ring structure containing one or more heteroatoms (e.g., oxygen, nitrogen, sulfur) formed by two or more saturated rings sharing a single ring atom. This includes, but is not limited to, 5-10 membered spiroheterocycles, 6-10 membered spiroheterocycles, 6-10 membered nitrogen-containing spiroheterocycles, 6-10 membered oxygen-containing spiroheterocycles, and 6-10 membered sulfur-containing spiroheterocycles. The "nitrogen-containing spiroheterocycle", "oxygen-containing spiroheterocycle", and "sulfur-containing spiroheterocycle" may optionally also contain one or more other heteroatoms selected from oxygen, nitrogen, and sulfur. The term "6-10-membered nitrogen-containing spiroheterocycle group" refers to a spiroheterocycle group containing a total of 6-10 ring atoms, of which at least one ring atom is a nitrogen atom.

[0253] In this invention, heterocyclic groups can fuse with aryl groups to form fused ring structures, examples of which include, but are not limited to:

[0254] As used herein, the term "aryl" or "aromatic ring" refers to a fully carbon monocyclic or fused polycyclic aromatic group having a conjugated π-electron system. As used herein, the term "C" refers to a carbon monocyclic or fused polycyclic aromatic group. 6-12 "Aryl (aromatic ring)" refers to an aryl (aromatic ring) containing 6 to 12 carbon atoms, preferably C64-12 ... 6-10 Aryl (aromatic ring), preferably phenyl or naphthyl. The aryl group is optionally substituted by one or more (such as 1 to 3) identical or different substituents (e.g., halogen, OH, CN, NO2, C1-C6 alkyl, etc.).

[0255] As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to a monocyclic or polycyclic aromatic group containing one or more identical or different heteroatoms, including monocyclic heteroaryl groups and bicyclic or polycyclic ring systems containing at least one heteroaromatic ring (an aromatic ring system containing at least one heteroatom), which may have 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, for example, 5, 6, 7, 8, 9, or 10 ring atoms. The heteroatom may be oxygen, nitrogen, or sulfur. The carbon atom and heteroatom on the heteroaryl group are optionally substituted with oxo groups (e.g., forming C=O, S(=O) or S(=O)2).

[0256] The term "5-6 membered heteroaryl" refers to a heteroaryl group (heteroaryl ring) containing 5 to 6 ring atoms, including 5-6 membered nitrogen-containing heteroaryl, 5-6 membered oxygen-containing heteroaryl, and 5-6 membered sulfur-containing heteroaryl. Each of the "nitrogen-containing heteroaryl," "oxygen-containing heteroaryl," and "sulfur-containing heteroaryl" optionally contains one or more other heteroatoms selected from oxygen, nitrogen, and sulfur. Examples include, but are not limited to, thiopheneyl, furanyl, pyrroleyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl, etc., or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc.

[0257] The term "heteroaryl" encompasses fused ring structures, where the connection point between the fused ring structure and other groups can be on any ring within the fused ring structure. Therefore, the heteroaryls of this invention also include, but are not limited to, (mono)heteroaryl and (mono)heteroaryl, (mono)heteroaryl and (mono)heterocyclic, (mono)heteroaryl and (mono)heterocyclic, such as 5-6 membered (mono)heteroaryl and 5-6 membered (mono)heteroaryl, 5-6 membered (mono)heteroaryl and phenyl, 5-6 membered (mono)heteroaryl and 5-6 membered (mono)heterocyclic, or 5-6 membered (mono)heterocyclic and C 4-6 (Mono)cycloalkyl groups (e.g., 5-6-membered heteroarylcyclobutyl, 5-6-membered heteroarylcyclopentyl, or 5-6-membered heteroarylcyclohexyl), examples of which include, but are not limited to, indole, isoindole, indazole, benzimidazole, quinolinyl, isoquinolinyl, wait.

[0258] 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.

[0259] Information about the sequences involved in this invention is described in the table below:

[0260] The abbreviations used in this article have the following meanings:

[0261] 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).

[0262] 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).

[0263] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0264] 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.

[0265] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0266] I. Preparation of intermediates

[0267] Example 1 of intermediate preparation: N 6 -((allyloxy)carbonyl)-N 2 Preparation of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-1)

[0268] Step 1: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine tert-butyl ester (INT-1-2)

[0269] 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynyl acid (10 g, 37.27 mmol) and L-valine tert-butyl ester (7.75 g, 44.73 mmol) were dissolved in DMF (50 mL), and HATU (21.25 g, 55.91 mmol) and DIPEA (14.45 g, 111.82 mmol, 19.48 mL) were added. The mixture was stirred at room temperature for 5 hours. Water (500 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to give the crude title compound (15.7 g, 37.07 mmol), which was used directly in the next step without purification.

[0270] Its structural characterization data are as follows:

[0271] MS m / z (ESI): 424.2 [M+H] +

[0272] Step 2: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (INT-1-3)

[0273] (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine tert-butyl ester (15.7 g, 37.07 mmol) was dissolved in DCM (70 mL), and TFA (20 mL) was added dropwise. The mixture was then reacted at room temperature for 2 hours.

[0274] Add water (200 mL) to the reaction solution, extract three times with dichloromethane (50 mL), combine the organic phases, wash seven times with purified water (50 mL), dry with anhydrous sodium sulfate, filter and concentrate to obtain crude product of the title compound (13.6 g, 37.01 mmol), which was used directly in the next step without purification.

[0275] Its structural characterization data are as follows:

[0276] MS m / z (ESI): 368.1 [M+H] +

[0277] Step 3: N 6 -((allyloxy)carbonyl)-N 2 Preparation of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-1)

[0278] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (1 g, 2.72 mmol) and N 6-((allyloxy)carbonyl)-L-lysine (689.37 mg, 2.99 mmol) was dissolved in DMF (15 mL), and HATU (1.09 g, 2.86 mmol) was added. The mixture was reacted at room temperature for 1 h. Saturated brine (150 mL) and ethyl acetate (150 x 2) were added to the reaction solution for extraction. The combined organic phases were dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (MeOH / DCM = 0–10%, 50 min) and then concentrated again to obtain the title compound (1.3 g, 2.24 mmol).

[0279] Its structural characterization data are as follows:

[0280] MS m / z (ESI): 580.2 [M+H] +

[0281] Example 2 of intermediate preparation: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (INT-2):

[0282] Step 1: Preparation of (2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-carboxylic acid benzyl ester (INT-2-2):

[0283] (2S,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidine-1-carboxylate (10 g, 39.80 mmol) was dissolved in dichloromethane (100 mL), and triethylamine (6.04 g, 59.69 mmol, 8.30 mL) and DBU (1.21 g, 7.96 mmol, 1.19 mL) were added dropwise with stirring. Tert-butyldimethylchlorosilane (6.60 g, 43.78 mmol) was added in portions, and the reaction was continued for 18 hours. A saturated ammonium chloride aqueous solution was added with stirring, and the mixture was allowed to stand and separated. The aqueous phase was extracted with dichloromethane, the organic phases were combined, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 20-60% ethyl acetate / petroleum ether) to give the title compound (11.3 g, 30.91 mmol).

[0284] Step 2: Preparation of (3R,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-3-ol (INT-2-3):

[0285] (2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-carboxylic acid benzyl ester (11.3 g, 30.91 mmol) was dissolved in ethanol (100 mL), and 10% Pd / C (1.1 g, 3.09 mmol) was added. The mixture was stirred for 16 hours under hydrogen purging and protection. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the crude title compound (7.02 g, 30.34 mmol), which was directly used for the next reaction.

[0286] Step 3: Preparation of ((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-yl)(4,5-dimethoxy-2-nitrophenyl)methyl ketone (INT-2-4):

[0287] 4,5-Dimethoxy-2-nitrobenzoic acid (6.53 g, 28.74 mmol), (3R,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-3-ol (7 g, 30.25 mmol), and HATU (12.64 g, 33.27 mmol) were dissolved in DMF (140 mL). DIPEA (11.73 g, 90.75 mmol, 15.81 mL) was added dropwise with stirring, and the reaction was continued for 1 hour. Water and ethyl acetate were added, the mixture was stirred, allowed to stand, and the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed three times with saturated brine, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-5% methanol / dichloromethane) to give the title compound (13.01 g, 29.53 mmol).

[0288] Its structural characterization data are as follows:

[0289] MS m / z (ESI): 441.3 [M+H] +

[0290] Step 4: Preparation of (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)pyrrolidine-3-one (INT-2-5):

[0291] ((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-yl)(4,5-dimethoxy-2-nitrophenyl)methyl ketone (5.4 g, 12.26 mmol) was dissolved in dichloromethane (50 mL). Tetrapropylammonium perruthenate (1.29 g, 3.68 mmol) and N-methylmorpholine oxide (4.31 g, 36.77 mmol) were added with stirring, and the reaction was continued for 1 hour. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 0-5% methanol / dichloromethane) to give the title compound (3.3 g, 7.52 mmol).

[0292] Step 5: Preparation of (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrole-3-yltrifluoromethanesulfonate (INT-2-6):

[0293] (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)pyrrolidine-3-one (2.8 g, 6.38 mmol) was dissolved in dry dichloromethane (30 mL). The solution was cooled and stirred to -35 °C under nitrogen purging and protection. 2,6-Lutidine (1.37 g, 12.77 mmol, 1.49 mL) was added dropwise, followed by a slow dropwise addition of a dichloromethane (10 mL) solution of trifluoromethanesulfonic anhydride (2.70 g, 9.58 mmol, 1.61 mL). The reaction was maintained at this temperature for 0.5 hours. The reaction was quenched with 40 mL of water, and the mixture was allowed to stand and separated. The aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-5% methanol / dichloromethane) to give the title compound (2.2 g, 3.86 mmol).

[0294] Step Six: Preparation of (S,E)-(4-(4-aminostyryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (INT-2-7):

[0295] (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrole-3-yltrifluoromethanesulfonate (4.57 g, 8.01 mmol), [(E)-2-(4-aminophenyl)vinyl]boronic acid (2.61 g, 16.02 mmol), Pd(dppf)₂Cl₂ (1.31 g, 1.60 mmol), and K₂CO₃ (2.21 g, 16.02 mmol) were dissolved in a mixed solvent of tetrahydrofuran (60 mL) and water (6 mL). The reaction mixture was heated to 75 °C for 2 hours under nitrogen purging and protection. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 10-50% ethyl acetate / petroleum ether) to give the title compound (1.25 g, 2.32 mmol).

[0296] Its structural characterization data are as follows:

[0297] MS m / z (ESI): 540.2 [M+H] +

[0298] Step 7: Preparation of (S,E)-(4-(2-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (INT-2-8):

[0299] (S,E)-(4-(4-aminostyryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (1.2 g, 2.22 mmol) was dissolved in dry dichloromethane (60 mL), cooled and stirred to 0 °C, and DIPEA (862.09 mg, 6.67 mmol, 1.16 mL) was added dropwise, followed by allyl chloroformate (536.01 mg, 4.45 mmol, 472.67 μL). The reaction was stirred for 1 hour. The reaction was quenched with saturated brine, allowed to stand and separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried and concentrated, and purified by silica gel column chromatography (eluent: 0-35% ethyl acetate / petroleum ether) to give the title compound (1.14 g, 1.83 mmol).

[0300] Step 8: Preparation of (S,E)-(4-(2-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (INT-2-9):

[0301] (S,E)-(4-(2-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (1.14 g, 1.83 mmol) was dissolved in methanol (50 mL), and saturated ammonium chloride aqueous solution (10 mL) was added dropwise. Zinc powder (1.20 g, 18.28 mmol) was added with stirring, and the reaction was continued for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and dissolved in dichloromethane and water. After standing, the liquid was separated, and the organic phase was dried, concentrated, and purified by silica gel column chromatography (eluent: 0-35% ethyl acetate / petroleum ether) to give the title compound (927 mg, 1.56 mmol).

[0302] Its structural characterization data are as follows:

[0303] MS m / z (ESI): 594.2 [M+H] +

[0304] Step Nine: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-((E)-4-(((allyloxy)carbonyl)amino)styryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-2-10):

[0305] (S,E)-(4-(2-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (400 mg, 673.65 μmol) was dissolved in dry tetrahydrofuran (8 mL). Triethylamine (95.43 mg, 943.10 μmol, 131.09 μL) was added dropwise under nitrogen purging and protection. The mixture was cooled and stirred to -10 °C, and triphosgene (85.96 mg, 28 μL) was slowly added dropwise. A solution of 9.67 μmol) tetrahydrofuran (2 mL) was stirred for about 10 minutes. Then, a mixed solution of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (228.83 mg, 606.28 μmol) and DMAP (115.22 mg, 943.10 μmol) in tetrahydrofuran (8 mL) and DMF (2.5 mL) was slowly added dropwise. The mixture was heated slowly to 40 °C and reacted for 1 hour. The reaction was quenched by adding 1 mL of water. After concentration under reduced pressure, water and dichloromethane were added and stirred. The mixture was allowed to stand and separated. The organic phase was dried and concentrated. The solution was purified by silica gel column chromatography (eluent: 0-3% methanol / dichloromethane) to give the title compound (486 mg, 487.36 μmol).

[0306] Its structural characterization data are as follows:

[0307] MS m / z (ESI): 998.4 [M+H] +

[0308] Step 10: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-((E)-4-(((allyloxy)carbonyl)amino)styryl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-2-11):

[0309] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-((E)-4-(((allyloxy)carbonyl)amino)styryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (486 mg, 487.36 μmol) was dissolved in a mixed solvent of tetrahydrofuran (10 mL) and water (2 mL). Glacial acetic acid (10 mL) was added dropwise with stirring, and the mixture was heated to 40 °C and reacted for 2 hours. After concentration under reduced pressure, the mixture was purified by reverse-phase column chromatography (eluent: 0-60% acetonitrile / 0.5% formic acid water) and lyophilized to give the title compound (219 mg, 248.03 μmol).

[0310] Its structural characterization data are as follows:

[0311] MS m / z (ESI): 883.3 [M+H] +

[0312] Step 11: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carbonate (INT-2-12):

[0313] Dry DMSO (66.37 mg, 849.42 μmol, 60.33 μL) was dissolved in dry dichloromethane (5 mL). The solution was cooled and stirred to -78 °C under nitrogen purging and protection. Oxaloyl chloride (51.75 mg, 407.72 μmol, 34.50 μL) was added dropwise. After stirring for 0.5 hours, 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl (2... A solution of 300 mg (339.77 μmol) of 4-((S)-4-(((allyloxy)carbonyl)amino)styryl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate in dichloromethane (5 mL) was added, and the reaction was continued for 1 hour. Then, triethylamine (171.91 mg, 1.70 mmol, 236.14 μL) was added dropwise, and the reaction was allowed to return to room temperature for 1 hour. The reaction was quenched with water, allowed to stand, and separated. The aqueous phase was extracted twice with dichloromethane, the organic phases were combined, dried, concentrated, and purified by reverse-phase column chromatography (eluent: 0-55% acetonitrile / 0.5% formic acid in water). The purified compound was lyophilized to give the title compound (110 mg, 124.87 μmol).

[0314] Its structural characterization data are as follows:

[0315] MS m / z (ESI): 881.3 [M+H] +

[0316] Step Twelve: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (INT-2):

[0317] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (100 mg, 113.52 μmol), tetra(triphenylphosphine)palladium (26.23 mg, 22.70 μmol), and 1,3-dimethylbarbituric acid (26.59 mg, 170.27 μmol) were dissolved in DMF (2 mL) and reacted with stirring for 1 hour under nitrogen purging and protection. Reversed-phase column purification (eluent: 0-70% acetonitrile / 0.5% ammonium bicarbonate in water), followed by lyophilization to obtain the title compound (65 mg, 91.19 μmol).

[0318] Its structural characterization data are as follows:

[0319] MS m / z (ESI): 713.2 [M+H] +

[0320] Example 3 of intermediate preparation: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (INT-3):

[0321] Step 1: Preparation of (S)-(4-(6-aminonaphthyl-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (INT-3-1):

[0322] (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrole-3-yltrifluoromethanesulfonate (2.0 g, 3.51 mmol), 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)naphthyl-2-amine (A-6-2) (1.42 g, 5.26 mmol), Pd(dppf)2Cl2 (256.57 mg, 350.50 μmol), K2CO3 (1.21 g, 8.76 mmol) were dissolved in a mixed solvent of dioxane (30 mL) and water (10 mL), and the mixture was heated to 75 °C for 2 hours under nitrogen purging and protection. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent: 10-50% ethyl acetate / petroleum ether) to give the title compound (1.4 g, 2.48 mmol).

[0323] Its structural characterization data are as follows:

[0324] MS m / z (ESI): 564.2 [M+H] +

[0325] Step 2: Preparation of (S)-(6-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (INT-3-2):

[0326] (S)-(4-(6-aminonaphthyl-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (2.2 g, 3.90 mmol) was dissolved in dry dichloromethane (30 mL), cooled and stirred to 0 °C, and DIPEA (1.51 g, 11.71 mmol) was added dropwise, followed by allyl chloroformate (564.49 mg, 4.68 mmol). The reaction was stirred for 1 hour. The reaction was quenched with saturated brine, allowed to stand and separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried and concentrated, and purified by silica gel column chromatography (eluent: 0-35% ethyl acetate / petroleum ether) to give the title compound (2.12 g, 3.27 mmol).

[0327] Its structural characterization data are as follows:

[0328] MS m / z (ESI): 648.2 [M+H] +

[0329] Step 3: Preparation of (S)-(6-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (INT-3-3):

[0330] (S)-(6-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (2.12 g, 3.27 mmol) was dissolved in methanol (70 mL), and saturated ammonium chloride aqueous solution (20 mL) was added dropwise. Zinc powder (2.14 g, 32.73 mmol) was added with stirring, and the reaction was continued for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and dissolved in dichloromethane and water. After standing, the liquid was separated, and the organic phase was dried, concentrated, and purified by silica gel column chromatography (eluent: 0-55% ethyl acetate / petroleum ether) to give the title compound (1.78 g, 2.88 mmol).

[0331] Its structural characterization data are as follows:

[0332] MS m / z (ESI): 618.3 [M+H] +

[0333] Step 4: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-3-4):

[0334] (S)-(6-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (1 g, 1.62 mmol) was dissolved in dry tetrahydrofuran (8 mL). Triethylamine (229.31 mg, 2.27 mmol) was added dropwise under nitrogen purging and protection. The mixture was cooled and stirred to -10 °C, and triphosgene (158.51 mg, 534.15 μmol) was slowly added dropwise. A solution of tetrahydrofuran (2 mL) was stirred for about 10 minutes, and then a mixture of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (610.93 mg, 1.62 mmol) and DMAP (276.85 mg, 2.27 mmol) in tetrahydrofuran (8 mL) and DMF (2.5 mL) was slowly added dropwise. The mixture was heated slowly to 40 °C and reacted for 1 hour. The reaction was quenched by adding 1 mL of water. After concentration under reduced pressure, water and dichloromethane were added and stirred. The mixture was allowed to stand and separated. The organic phase was dried and concentrated, and purified by silica gel column chromatography (eluent: 0-65% ethyl acetate / petroleum ether) to give the title compound (1.58 g, 1.55 mmol).

[0335] Its structural characterization data are as follows:

[0336] MS m / z (ESI): 1022.4 [M+H] +

[0337] Step 5: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-3-5):

[0338] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (1.58 g, 1.55 mmol) was dissolved in tetrahydrofuran (10 mL), and a THF solution of TBAF (1 M, 8.37 mL) was added dropwise with stirring. The mixture was concentrated under reduced pressure and purified by normal-phase column chromatography (0-90% ethyl acetate / petroleum ether). The solution was then lyophilized to give the title compound (998 mg, 1.10 mmol).

[0339] Its structural characterization data are as follows:

[0340] MS m / z (ESI): 907.3 [M+H] +

[0341] Step Six: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carboxylic acid ester (INT-3-6):

[0342] Dry DMSO (116.29 mg, 1.49 mmol, 105.72 μL) was dissolved in dry dichloromethane (5 mL). The solution was cooled and stirred to -78 °C under nitrogen purging and protection. Oxaloyl chloride (94.46 mg, 744.23 μmol, 62.98 μL) was added dropwise. After stirring for 5 minutes, 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl (2... A solution of 450 mg (496.16 μmol) of 4-((S)-4-(6-((((allyloxy)carbonyl)amino)naphth-2-yl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate in dichloromethane (5 mL) was added. After reacting for 1 hour, triethylamine (251.03 mg, 2.48 mmol, 344.82 μL) was added dropwise, and the reaction was allowed to return to room temperature for 1 hour. The reaction was quenched with water, allowed to stand, and separated. The aqueous phase was extracted twice with dichloromethane, the organic phases were combined, dried, concentrated, and purified by reverse-phase column chromatography (eluent: 0-18% 30% methanol / dichloromethane). The purified compound was lyophilized to give the title compound (324 mg, 358.03 μmol).

[0343] Its structural characterization data are as follows:

[0344] MS m / z (ESI): 906.3 [M+H] +

[0345] Step 7: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (INT-3):

[0346] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (324 mg, 358.03 μmol), tetrakis(triphenylphosphine)palladium (76.58 mg, 66.30 μmol), and 1,3-dimethylbarbituric acid (155.15 mg, 994.52 μmol) were dissolved in DMF (2 mL) and reacted with stirring for 1 hour under nitrogen purging and protection. Reversed-phase column purification (eluent: 0-40% acetonitrile / 0.5% ammonium bicarbonate in water), followed by lyophilization to obtain the title compound (228 mg, 309.44 μmol).

[0347] Its structural characterization data are as follows:

[0348] MS m / z (ESI): 737.3 [M+H] +

[0349] Example 4 of intermediate preparation: Preparation of (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid (INT-4)

[0350] Step 1: Preparation of perfluorophenyl 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionate (INT-4-2)

[0351] 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (3 g, 6.36 mmol) and pentafluorophenol (4.69 g, 25.45 mmol) were dissolved in dichloromethane (50 mL), and EDCI (4.88 g, 25.45 mmol) was added. The mixture was stirred at room temperature for one hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether, 30-100%), concentrated, and dried to obtain the title compound (5.0 g, 5.16 mmol).

[0352] Its structural characterization data are as follows:

[0353] MS m / z (ESI): 970.6 [M+H] +

[0354] Step 2: Preparation of ((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)benzyl carbamate (INT-4-3)

[0355] Perfluorophenyl 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionate (5.0 g, 5.16 mmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentanol (4.03 g, 20.63 mmol) were dissolved in DMF (15 mL). DIPEA (2.00 g, 15.47 mmol, 2.69 mL) was added, and the mixture was reacted at room temperature for 2 hours. After removing most of the solvent from the reaction solution under vacuum, the solution was purified by C18 column chromatography (acetonitrile: 0.05% formic acid aqueous solution, 10%–60%) and lyophilized to obtain the title compound (5 g, 4.98 mmol).

[0356] Its structural characterization data are as follows:

[0357] MS m / z (ESI): 1003.4 [M+H] +

[0358] Step 3: Preparation of 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxo))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(INT-4-4)

[0359] 5 g (4.98 mmol) of benzyl carbamate ((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)carbamate was dissolved in a mixed solvent of ethanol (20 mL) and water (20 mL), 10% Pb / C (0.1 g) was added, and the mixture was purged with nitrogen three times. The mixture was then subjected to catalytic hydrogenation for 4 h. The reaction solution was filtered and concentrated to obtain the crude title compound (4.0 g, 4.60 mmol), which was used directly in the next step without purification.

[0360] Its structural characterization data are as follows:

[0361] MS m / z (ESI): 869.9 [M+H] +

[0362] Step 4: Preparation of (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-5-oxopentanoic acid allyl ester (INT-4-5)

[0363] Add 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxo))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(910 mg, 1.05 mm (ol) and (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxovaleric acid (471.67 mg, 1.15 mmol) were dissolved in DMF (2 mL), and DIPEA (541.40 mg, 4.19 mmol) and HATU (597.27 mg, 1.57 mmol) were added. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction solution was directly purified by C18 column (acetonitrile: 0.05% formic acid aqueous solution, 10%–65%), and lyophilized to obtain the crude product. It was then purified again by C18 column (acetonitrile: 0.05% formic acid aqueous solution, 10%–50%), and lyophilized to obtain the title compound (843 mg, 601.98 μmol).

[0364] Its structural characterization data are as follows:

[0365] MS m / z (ESI): 1261.4 [M+H] +

[0366] Step 5: Preparation of (S)-4-amino-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-5-oxopentanoic acid allyl ester (INT-4-6)

[0367] The (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-13-dimethyl ... Hydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-5-oxopentaic acid allyl ester (833 mg, 660.94 μmol, FR) was dissolved in DMF (3 mL), and diethylamine (146.73 mg, 1.98 mmol) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent from the reaction solution under vacuum, the solution was purified by C18 column chromatography (acetonitrile: 0.05% trifluoroacetic acid aqueous solution, 10%–70%), and lyophilized to give the trifluoroacetate of the title compound (699 mg, 606.71 μmol).

[0368] Its structural characterization data are as follows:

[0369] MS m / z(ESI): 1038.1 [M+H] +

[0370] Step Six: Preparation of (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid allyl ester (INT-4-7)

[0371] The (S)-4-amino-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazadi Allyl pentadecane-13-yl)amino)-5-oxopentanoate (620 mg, 538.14 μmol, TF) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (158.81 mg, 591.95 μmol, FR) were dissolved in DMF (6 mL), and DIPEA (278.20 mg, 2.15 mmol) and DMTMM (297.51 mg, 1.08 mmol) were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by C18 column chromatography (acetonitrile: 0.05% trifluoroacetic acid aqueous solution, 10%–70%) and lyophilized to give the title compound (569 mg, 388.65 μmol).

[0372] Its structural characterization data are as follows:

[0373] MS m / z (ESI): 1288.4 [M+H] +

[0374] Step 7: Preparation of (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid (INT-4)

[0375] The (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazatocetane Allyl 1,3-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)-5-oxovalerate (520 mg, 403.61 μmol) was dissolved in a mixed solvent of dichloromethane (5 mL) and DMF (5 mL). Under nitrogen protection, tetrakis(triphenylphosphine)palladium (233.09 mg, 201.81 μmol), formic acid (0.4 mL), and N-methylmorpholine (0.8 mL) were added, and the reaction was stirred at room temperature for 2 hours. The reaction solution was directly purified by C18 column chromatography (acetonitrile: 0.05% formic acid aqueous solution, 10%–50%), and lyophilized to give the title compound (340 mg, 245.13 μmol).

[0376] Its structural characterization data are as follows:

[0377] MS m / z (ESI): 1248.3 [M+H] +

[0378] Example 5 of intermediate preparation: Preparation of (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-5-oxovaleric acid (INT-5)

[0379] Step 1: Preparation of 5-(tert-butyl)-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid allyl ester (INT-5-2)

[0380] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid (6.00 g, 14.10 mmol) and lithium hydroxide monohydrate (1.18 g, 28.20 mmol) were added sequentially to DMF (30 mL). After the addition was complete, the mixture was stirred at 25 °C for 10 min, and then allyl bromide (5.12 g, 42.31 mmol) was added. The reaction mixture was reacted at 25 °C for 1 h. The reaction solution was poured into dilute hydrochloric acid (100 mL of 0.5 N HCl), extracted with EA (40 mL * 2), washed with water (20 mL), dried over anhydrous sodium sulfate (3 g), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (SiO2, EA / PE = 0–45%). The title compound (4.67 g, 10.03 mmol) was obtained.

[0381] Its structural characterization data are as follows:

[0382] ESI-MS (m / z): 410.3 [M+H-56] + .

[0383] Step 2: Preparation of 5-(tert-butyl)-L-glutamic acid allyl ester (INT-5-3)

[0384] 5-(tert-butyl)-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid allyl ester (4.40 g, 9.45 mmol) and DBU (4.32 g, 28.35 mmol) were added sequentially to DMF (22 mL). After the addition was complete, the mixture was stirred at 25 °C for 1 h. The reaction solution was concentrated under reduced pressure to obtain the crude title compound (2.29 g, 9.41 mmol), which was used directly in the next step without purification.

[0385] Its structural characterization data are as follows:

[0386] ESI-MS (m / z): 244.2 [M+H] + .

[0387] Step 3: Preparation of 5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (INT-5-4)

[0388] Crude 5-(tert-butyl)-L-glutamic acid allyl ester (2.29 g, 9.41 mmol), 6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (3.03 g, 11.29 mmol), DIPEA (4.87 g, 37.65 mmol), and HATU (5.37 g, 14.12 mmol) were added sequentially to DMF (22 mL). After addition, the mixture was stirred at 25 °C for 1 h. The reaction was monitored by LC-MS until complete. The crude product was concentrated and purified by column chromatography (ACN / H2O = 0.75%, 0.05% formic acid). The product was then lyophilized to obtain the title compound (1.42 g, 2.87 mmol).

[0389] Its structural characterization data are as follows:

[0390] ESI-MS (m / z): 511.2 [M+H2O] + .

[0391] Step 4: Preparation of (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovalerate (INT-5-5)

[0392] 5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (830.00 mg, 1.48 mmol) was dissolved in DCM (10 mL), and TFA (5 mL) was added. After the addition was complete, the mixture was stirred at 25 °C for 3 h. The reaction solution was subjected to reduced pressure to remove DCM and TFA to obtain the crude product, which was purified by high performance liquid chromatography to obtain the title compound (361.00 mg, 825.21 μmol).

[0393] The purification method is as follows:

[0394] Column: Phenomenex C18 250mm×50mm×10μm

[0395] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0396] Its structural characterization is as follows:

[0397] ESI-MS (m / z): 438.1 [M+H] + .

[0398] Step 5: N 5 -((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazatocetane-13-yl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (INT-5-6):

[0399] (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxovaleric acid (200 mg, 457.18 μmol), 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl) Di(oxy)bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide (436.97 mg, 502.90 μmol) was dissolved in DMF (5 mL), and HATU (208.47 mg, 548.61 μmol) was added with stirring. DIPEA (177.26 mg, 1.37 mmol) was added dropwise, and the reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-35% acetonitrile / 0.5% formic acid water), and lyophilized to give the title compound (1.05 g, 781.51 μmol).

[0400] Its structural characterization data are as follows:

[0401] MS m / z (ESI): 1338.6 [M+H] +

[0402] Step Six: N 5 Preparation of -((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (INT-5):

[0403] Allyl N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19- (diazapentadecane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (311 mg, 241.39 μmol), tetra(triphenylphosphine)palladium (27.89 mg, 24.14 μmol), and 1,3-dimethylbarbituric acid (37.69 mg, 241.39 μmol) were dissolved in DMF (6 mL). The reaction mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-30% acetonitrile / 0.5% trifluoroacetic acid in water) and lyophilized to give the title compound (292 mg, 233.92 μmol).

[0404] Its structural characterization data are as follows:

[0405] MS m / z (ESI): 1249.4 [M+H] +

[0406] Example 6 of intermediate preparation: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6)

[0407] Step 1: Preparation of 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionic acid (INT-6-2)

[0408] 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.94 g, 4.71 mmol) and 2-benzyloxy-1-methylpyridine trifluoromethanesulfonate (1.65 g, 4.71 mmol) were reacted at 80–85 °C for 16 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10–70%) and lyophilized to give the title compound (0.78 g, 1.51 mmol).

[0409] Its structural characterization data are as follows:

[0410] ESI-MS (m / z): 515.2 (M+H) +

[0411] Step 2: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-6-3)

[0412] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.78 g, 1.51 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (0.82 g, 6.80 mmol) were dissolved in DMF (15 mL), DIPEA (1.17 g, 9.07 mmol) was added, and HATU (2.59 g, 6.80 mmol) was added in portions. The reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.74 g, 0.90 mmol).

[0413] Its structural characterization data are as follows:

[0414] ESI-MS (m / z): 824.4 (M+H) +

[0415] Step 3: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6)

[0416] Benzyl 3-(3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.74 g, 0.90 mmol) was dissolved in ethanol (30 mL), 10% palladium on carbon (0.15 g) and acetic acid (0.14 g, 2.42 mmol) were added, the air was removed, hydrogen gas (balloon) was introduced, the temperature was raised to 40 °C and reacted for 4 hours, palladium on carbon was filtered off, the filtrate was concentrated, the residue was dissolved in water (15 mL) and acetonitrile (15 mL) and clarified, and lyophilized to give the title compound (0.59 g, 0.80 mmol).

[0417] Its structural characterization data are as follows:

[0418] ESI-MS (m / z): 734.3 (M+H) +

[0419] Example 7 of intermediate preparation: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-7)

[0420] Step 1: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-7-1)

[0421] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (1.07 g, 2.08 mmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol (1.62 g, 8.32 mmol) were dissolved in DMF (15 mL), DIPEA (1.34 g, 10.40 mmol) was added, and HATU (3.56 g, 9.36 mmol) was added in portions. The reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.66 g, 0.63 mmol).

[0422] Its structural characterization data are as follows:

[0423] ESI-MS (m / z): 1046.5 (M+H) +

[0424] Step 2: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-7)

[0425] Benzyl 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.66 g, 0.63 mmol) was dissolved in ethanol (14 mL) and water (7 mL). 10% palladium on carbon (0.13 g) and acetic acid (0.10 g, 1.70 mmol) were added. The air was removed, and hydrogen gas (in a balloon) was introduced. The mixture was heated to 40 °C and reacted for 4 hours. The palladium on carbon was filtered off, the filtrate was concentrated, and the residue was dissolved and clarified in water (15 mL) and acetonitrile (15 mL). The solution was lyophilized to give the title compound (0.54 g, 0.56 mmol).

[0426] Its structural characterization data are as follows:

[0427] ESI-MS (m / z): 956.4 (M+H) +

[0428] Example 8 of intermediate preparation: Preparation of (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid (INT-8)

[0429] Step 1: Preparation of 1-amino-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azacosanodecane-22-acid (INT-8-2):

[0430] 17,17-bis((2-carboxyethoxy)methyl)-15-oxo-1-triazyl-3,6,9,12,19-pentaoxa-16-azadocoadecan-22-acid (670 mg, 1.10 mmol) was dissolved in tetrahydrofuran (5 mL), and 10% palladium on carbon (50 mg, 109.73 μmol) was added. The reaction mixture was stirred for 18 hours under hydrogen purging and protection. The reaction solution was filtered through a diatomaceous earth filter, and the filtrate was concentrated under reduced pressure to give the crude title compound (640 mg, 1.09 mmol).

[0431] Its structural characterization data are as follows:

[0432] MS m / z (ESI): 585.2 [M+H] +

[0433] Step 2: Preparation of 21,21-bis((2-carboxyethoxy)methyl)-1-(9H-fluorene-9-yl)-3,19-dioxo-2,7,10,13,16,23-hexaoxa-4,20-diazahexacosane-26-acid (INT-8-3):

[0434] 1-Amino-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocoadecan-22-acid (950 mg, 1.63 mmol) was dissolved in a mixture of acetonitrile (10 mL) and water (10 mL). Sodium bicarbonate (682.51 mg, 8.13 mmol) was added with stirring, followed by 9-fluorenemethyl-N-succinimide carbonate (657.74 mg, 1.95 mmol). The reaction mixture was stirred for 5 hours. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-50% acetonitrile / 0.5% formic acid in water) and lyophilized to give the title compound (870 mg, 1.08 mmol).

[0435] Its structural characterization data are as follows:

[0436] MS m / z (ESI): 807.3 [M+H] +

[0437] Step 3: Preparation of (9H-fluorene-9-yl)methyl[(25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis[(3-(methyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino]-3-oxopropoxy]methyl]-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazaeicosyl]carbamate (INT-8-4):

[0438] 21,21-bis((2-carboxyethoxy)methyl)-1-(9H-fluorene-9-yl)-3,19-dioxo-2,7,10,13,16,23-hexaoxa-4,20-diazahexacosane-26-acid (870 mg, 1.08 mmol), meglumine (841.97 mg, 4.31 mmol), and HATU (2.05 g, 5.39 mmol) were dissolved in DMF (10 mL). DIPEA (696.78 mg, 5.39 mmol, 939.05 μL) was added dropwise with stirring, and the reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-45% acetonitrile / 0.5% trifluoroacetic acid in water) and lyophilized to give the title compound (1.05 g, 781.51 μmol).

[0439] Its structural characterization data are as follows:

[0440] MS m / z (ESI): 1338.6 [M+H] +

[0441] Step 4: Preparation of 3,3'-((2-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(INT-8-5):

[0442] (9H-fluorene-9-yl)methyl[(25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis[(3-(methyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino]-3-oxopropoxy]methyl]-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazaeicosyl]carbamate (200 mg, 149.43 μmol) was dissolved in DMF (2 mL), and diethylamine (0.2 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The solvent in the reaction solution was evaporated under reduced pressure, ethyl acetate and water were added and stirred, and the mixture was allowed to stand and separated. The aqueous phase was lyophilized to give the crude product of the title compound (166 mg, 148.72 μmol).

[0443] Its structural characterization data are as follows:

[0444] MS m / z (ESI): 1116.6 [M+H] +

[0445] Step 5: Preparation of (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid allyl ester (INT-8-6):

[0446] (S)-5-(allyloxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid (65.06 mg, 148.72 μmol), 33,3'-((2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl 166 mg (148.72 μmol) of propane-1,3-diyl)bis(oxy)bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide (2 mL) was dissolved in DMF. HATU (84.99 mg, 223.66 μmol) was added with stirring, followed by dropwise addition of DIPEA (57.66 mg, 446.16 μmol, 77.71 μL). The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (145 mg, 94.42 μmol).

[0447] Its structural characterization data are as follows:

[0448] MS m / z (ESI): 1535.5 [M+H] +

[0449] Its preparation method is as follows:

[0450] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0451] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0452] Step Six: Preparation of (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid (INT-8):

[0453] (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28 -trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid allyl ester (145 mg, 94.42 μmol) was dissolved in DMF (3 mL), and tetrakis(triphenylphosphine)palladium (10.91 mg, 9.44 μmol) and 1,3-dimethylbarbituric acid (14.74 mg, 94.42 μmol) were added. The reaction mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (108 mg, 72.21 μmol).

[0454] Its structural characterization data are as follows:

[0455] MS m / z (ESI): 1496.5 [M+H] +

[0456] Its preparation method is as follows:

[0457] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0458] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0459] Example 9 of intermediate preparation: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-9)

[0460] Step 1: Preparation of L-alanyl-L-alanine (INT-9-2)

[0461] (tert-butoxycarbonyl)-L-alanyl-L-alanine (100 mg, 384.19 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, yielding the pale yellow title compound (160 mg, 370.93 μmol).

[0462] Its structural characterization data are as follows:

[0463] MS m / z (ESI): 161.1 [M+H] +

[0464] Step 2: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-9)

[0465] L-alanyl-L-alanine (160 mg, 412.14 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyl ester (150.58 mg, 412.14 μmol) were dissolved in N,N-dimethylacetamide (2 mL), and N,N-diisopropylethylamine (266.33 mg, 2.06 mmol) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (125 mg, 304.55 μmol).

[0466] Its structural characterization data are as follows:

[0467] MS m / z (ESI): 428.1 [M+H2O] +

[0468] II. Compound Preparation Examples

[0469] Preparation Example 1: Preparation of (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-5)

[0470] Step 1: Preparation of (2S,4R)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (A-5-2)

[0471] 4,5-Dimethoxy-2-nitrobenzoic acid (5 g, 22.01 mmol) and (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride (4.4 g, 24.21 mmol) were dissolved in DMF (80 mL), and HATU (8.78 g, 23.11 mmol) and DIPEA (8.53 g, 66.03 mmol) were added. The reaction mixture was reacted for 16 hours. After the reaction was complete, the reaction solution was concentrated, saturated brine (100 mL) was added, and the mixture was extracted twice with ethyl acetate (100 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (methanol / dichloromethane = 0%–30%) and concentrated again to obtain the title compound (3.6 g, 10.16 mmol).

[0472] Its structural characterization data are as follows:

[0473] ESI-MS (m / z): 355.1 [M+H] +

[0474] Step 2: Preparation of (2R,11aS)-2-hydroxy-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-5-3)

[0475] Methyl (2S,4R)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid (3.6 g, 10.16 mmol, FR) was dissolved in methanol (60 mL), zinc powder (6.6 g, 101.6 mmol) and saturated ammonium chloride aqueous solution (20 mL) were added, and the mixture was heated to 80 °C and reacted for 16 hours. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography (methanol / dichloromethane = 0%–30%). The solution was then concentrated again to obtain the title compound (2.1 g, 7.18 mmol).

[0476] Its structural characterization data are as follows:

[0477] ESI-MS (m / z): 293.1 [M+H] +

[0478] Step 3: Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-5-4)

[0479] (2R,11aS)-2-hydroxy-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (2.1 g, 7.18 mmol) was dissolved in DMF (30 mL), and imidazole (2.45 g, 35.92 mmol) and tert-butyldimethylchlorosilane (2.38 g, 15.81 mmol) were added. The mixture was stirred for 16 hours. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by silica gel column chromatography (methanol / dichloromethane = 0%–15%) to give the title compound (1 g, 2.46 mmol).

[0480] Its structural characterization data are as follows:

[0481] ESI-MS (m / z): 407.2 [M+H] +

[0482] Step 4: Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-5-5)

[0483] Under nitrogen protection, NaH (147.58 mg, 3.69 mmol) was dissolved in 30 mL of DMF at 0 °C. (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (1 g, 2.46 mmol) was added and reacted for 30 minutes. Then, 2-(trimethylsilyl)ethoxymethyl chloride (616.17 mg, 2.46 mmol) was added and the reaction was carried out for 1 hour. After the reaction was complete, water was added to quench the reaction, and the system was poured into ice water. A solid precipitated, and the crude product (1.3 g, 2.42 mmol) was obtained by filtration and used directly in the next reaction step.

[0484] Its structural characterization data are as follows:

[0485] ESI-MS (m / z): 537.3 [M+H] +

[0486] Step 5: Preparation of (2R,11aS)-2-hydroxy-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-5-6)

[0487] (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (1.3 g, 2.42 mmol) was dissolved in tetrahydrofuran (20 mL), and tetrabutylammonium fluoride (1.26 g, 4.84 mmol) was added. The reaction was allowed to proceed for 2 hours. Saturated brine was added, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, dried, and concentrated. The solution was purified by silica gel column chromatography (methanol / dichloromethane = 0%–10%) to give the title compound (0.8 g, 1.89 mmol).

[0488] Its structural characterization data are as follows:

[0489] ESI-MS (m / z): 423.1 [M+H] +

[0490] Step Six: Preparation of (S)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2,5,11(3H,10H)-trione (A-5-7)

[0491] (2R,11aS)-2-hydroxy-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (0.8 g, 1.89 mmol) was dissolved in dichloromethane (20 mL), and Dys-Martin oxidant (2.01 g, 4.73 mmol) was added. The reaction was allowed to proceed for 1 hour. The reaction was quenched with saturated sodium bicarbonate solution, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was used directly in the next step of the reaction (0.65 g, 1.55 mmol).

[0492] Its structural characterization data are as follows:

[0493] ESI-MS (m / z): 421.1 [M+H] +

[0494] Step 7: Preparation of (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (A-5-8)

[0495] (S)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2,5,11(3H,10H)-trione (0.65 g, 1.55 mmol) was dissolved in dichloromethane (20 mL) under nitrogen protection. After cooling to -40 °C, 2,6-dimethylpyridine (1.32 g, 12.37 mmol) and trifluoromethanesulfonic anhydride (3.05 g, 10.82 mmol) were added, and the reaction was carried out at -40 °C for 1 hour. After the reaction was completed, the reaction was quenched with water, extracted three times with dichloromethane (20 mL x 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. Silica gel column purification (methanol / dichloromethane = 0%–5%) yielded the title compound (0.6 g, 1.09 mmol).

[0496] Its structural characterization data are as follows:

[0497] ESI-MS (m / z): 553.3 [M+H] +

[0498] Step 8: Preparation of (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-5-9)

[0499] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (0.6 g, 1.09 mmol) and (E)-(4-aminostyryl)boronic acid (210.88 mg, 1.29 mmol) were dissolved in toluene (20 mL), ethanol (5 mL), and water (5 mL). Tetra(triphenylphosphine)palladium (62.95 mg, 54.5 μmol) and sodium carbonate (346.62 mg, 3.27 mmol) were added, and the mixture was then heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (30 mL) was added, and the mixture was extracted twice with ethyl acetate (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (0.35 g, 670.91 μmol).

[0500] Its structural characterization data are as follows:

[0501] ESI-MS (m / z): 521.2 [M+H] +

[0502] Step Nine: Preparation of (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-5)

[0503] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (0.35 g, 670.91 μmol) was dissolved in dry tetrahydrofuran (10 mL). The mixture was reacted under nitrogen protection at -78 °C with triethyllithium borohydride (1 M in tetrahydrofuran) (1.34 mL, 1.34 mmol) for 30 min. 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 reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (7.3 mg, 19.25 μmol).

[0504] Its structural characterization data are as follows:

[0505] ESI-MS (m / z): 376.2 [M+H] +

[0506] 1H NMR (400MHz, CDCl3) δ7.88(d,J=3.8Hz,1H),7.52(s,1H),7.24(s,2H),7.09(s,1H),6.83(s,1H),6.81(d,J=18.2Hz,1H),6.66(d,J =8.2Hz,2H),6.34(d,J=16.0Hz,1H),4.41-4.34(m,1H),3.97(s,3H),3.95(s,3H),3.49-3.41(m,1H),3.27(dd,J=16.0,4.4Hz,1H).

[0507] Its preparation method is as follows:

[0508] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0509] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0510] Preparation Example 2: Preparation of (S,E)-7,8-dimethoxy-2-styryl-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-1)

[0511] Step 1: Preparation of (E)-4,4,5,5-tetramethyl-2-styryl-1,3,2-dioxaborane (A-1-2)

[0512] Phenylacetylene (2 g, 19.58 mmol) was dissolved in pinacolborane (10 mL) and reacted at 110 °C for 16 h under nitrogen protection. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–30%) yielded the title compound (3.77 g, 16.38 mmol).

[0513] Its structural characterization data are as follows:

[0514] ESI-MS (m / z): 231.2 [M+H] +

[0515] Step 2: Preparation of (S,E)-7,8-dimethoxy-2-styryl-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-1-3)

[0516] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (120.07 mg, 217.29 μmol) and (E)-4,4,5,5-tetramethyl-2-styryl-1,3,2-dioxaborane (50 mg, 217.29 μmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL). DPPF palladium dichloride (15.90 mg, 21.73 μmol) and potassium carbonate (90.09 mg, 651.86 μmol) were added, and the mixture was then heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (10 mL) was added, and the mixture was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (60 mg, 118.42 μmol).

[0517] Its structural characterization data are as follows:

[0518] ESI-MS (m / z): 507.2 [M+H] +

[0519] Step 3: Preparation of (S,E)-7,8-dimethoxy-2-styryl-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-1)

[0520] (S,E)-7,8-dimethoxy-2-styryl-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (60 mg, 118.42 μmol) was dissolved in dry tetrahydrofuran (5 mL). Under nitrogen protection, triethyllithium borohydride (1 M in tetrahydrofuran) (236.84 μL, 236.84 μmol) was added at -78 °C and reacted for 30 min. 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 reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (4.15 mg, 11.40 μmol).

[0521] Its structural characterization data are as follows:

[0522] ESI-MS (m / z): 361.2 [M+H] +

[0523] 1 H NMR (400MHz, CDCl3) δ7.90 (d, J = 4.0Hz, 1H), 7.57-7.50 (m, 1H), 7.46-7.36 (m, 3H), 7.37-7.31 (m, 2H), 7.26-7.22 (m, 1H), 7.1 7(s,1H),7.03-6.97(m,1H),6.85(s,1H),4.44-4.37(m,1H),3.97(s,3H),3.95(s,3H),3.52-3.41(m,1H),3.34-3.26(m,1H).

[0524] Its preparation method is as follows:

[0525] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0526] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0527] Preparation Example 3: Preparation of (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-6)

[0528] Step 1: Preparation of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)naphthyl-2-amine (A-6-2)

[0529] 100 mg (270.81 μmol) of tert-butyl (6-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)naphth-2-yl)carbamate was dissolved in 1 mL of dichloromethane, and 1 mL of TFA was added. The reaction was carried out for 1 hour. After the reaction was completed, the crude product of the title compound (72.89 mg, 270.81 μmol) was concentrated.

[0530] Its structural characterization data are as follows:

[0531] ESI-MS (m / z): 270.1 [M+H] +

[0532] Step 2: Preparation of (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-6-3)

[0533] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (149.65 mg, 270.82 μmol) and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)naphthyl-2-amine (72.89 mg, 270.81 μmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL). DPPF palladium dichloride (7.64 mg, 27.08 μmol) and potassium carbonate (112.29 mg, 812.46 μmol) were added, and the mixture was then heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (10 mL) was added, and the mixture was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (110 mg, 201.58 μmol).

[0534] Its structural characterization data are as follows:

[0535] ESI-MS (m / z): 546.3 [M+H] +

[0536] Step 3: Preparation of (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-6)

[0537] (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (110 mg, 201.58 μmol) was dissolved in dry tetrahydrofuran (5 mL). Under nitrogen protection, triethyllithium borohydride (1 M in tetrahydrofuran) (403.15 μL, 403.15 μmol) was added at -78 °C and reacted for 30 min. 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 reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (7.6 mg, 18.84 μmol).

[0538] Its structural characterization data are as follows:

[0539] ESI-MS (m / z): 400.3 [M+H]+

[0540] 1 H NMR (400MHz, CDCl3) δ7.94(d,J=4.0Hz,1H),7.63(d,J=8.6Hz,1H),7.62-7.44(m,5H),7.01-6.93(m, 2H),6.85(s,1H),4.50-4.43(m,1H),3.99(s,3H),3.96(s,3H),3.74-3.67(m,1H),3.55-3.48(m,1H).

[0541] Its preparation method is as follows:

[0542] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0543] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0544] Preparation Example 4: Preparation of (S)-2-(7-aminoquinolin-3-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-7)

[0545] Step 1: Preparation of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)quinoline-7-amine (A-7-2)

[0546] 3-Bromoquinoline-7-amine (100.00 mg, 448.29 μmol) and pinacol diborate (227.68 mg, 896.58 μmol) were dissolved in 1,4-dioxane (5 mL), followed by the addition of DPPF palladium dichloride (32.80 mg, 44.83 μmol) and potassium acetate (131.99 mg, 1.34 mmol). The mixture was then heated to 90 °C for 2 hours under nitrogen protection. After the reaction was complete, the mixture was allowed to return to room temperature before proceeding to the next step.

[0547] Its structural characterization data are as follows:

[0548] ESI-MS (m / z): 271.2 [M+H] +

[0549] Step 2: Preparation of (S)-2-(7-aminoquinoline-3-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (A-7-3)

[0550] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (149.65 mg, 270.82 μmol), DPPF palladium dichloride (7.64 mg, 27.08 μmol), potassium carbonate (112.29 mg, 812.46 μmol), and water (1 mL) were added to the reaction system of the previous step, and then the mixture was heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (10 mL) was added, and the mixture was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (132 mg, 144.87 μmol).

[0551] Its structural characterization data are as follows:

[0552] ESI-MS (m / z): 548.3 [M+H] +

[0553] Step 3: Preparation of (S)-2-(7-aminoquinolin-3-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (A-7)

[0554] (S)-2-(7-aminoquinoline-3-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (132 mg, 144.87 μmol) was dissolved in dry tetrahydrofuran (5 mL). Under nitrogen protection, triethyllithium borohydride (1 M in tetrahydrofuran) (403.15 μL, 403.15 μmol) was added at -78 °C and reacted for 30 min. 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 reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (5.3 mg, 12.97 μmol).

[0555] Its structural characterization data are as follows:

[0556] ESI-MS (m / z): 401.2 [M+H] +

[0557] 1 H NMR (400MHz, CDCl3) δ7.96 (d, J = 4.0Hz, 1H), 7.63 (m, 3H), 7.55-7.52 (m, 2H), 7.00 (s, 2H), 6.8 6(s,1H),4.50-4.43(m,1H),3.99(s,3H),3.96(s,3H),3.74-3.67(m,1H),3.55-3.48(m,1H).

[0558] Its preparation method is as follows:

[0559] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0560] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0561] Example 1: Preparation of N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-9)

[0562] Step 1: Preparation of (9H-fluorene-9-yl)methyl[(S)-1-((4-(((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxo-5-ureidopentane-2-yl)carbamate (L-9-1)

[0563] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (50 mg, 133.18 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (51.64 mg, 399.55 μmol), HATU (126.53 mg, 332.96 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-citrulline (158.79 mg, 399.55 μmol). The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water / acetonitrile = 1 / 1) and then freeze-dried to give the title compound (10 mg, 13.25 μmol). Its structural characterization data are as follows:

[0564] MS m / z (ESI): 755.3 [M+H] +

[0565] Step 2: Preparation of (S)-2-amino-N-(4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)-5-ureidopentanamide (L-9-2)

[0566] (9H-fluorene-9-yl)methyl[(S)-1-((4-(((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (10 mg, 13.25 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (7 mg, 13.14 μmol).

[0567] Its structural characterization data are as follows:

[0568] MS m / z(ESI): 533.1 [M+H] +

[0569] Step 3: Preparation of N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-9)

[0570] (S)-2-amino-N-(4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)-5-ureidopentanamide (7 mg, 13.14 μmol) was dissolved in DMF (1 mL), followed by the addition of HATU (12.49 mg, 32.86 μmol), DIPEA (8.49 mg, 65.72 μmol), and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (INT-1, 14.49 mg, 39.43 μmol). The reaction mixture was then reacted at 25 °C for 2 hours. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (1.1 mg, 1.23 μmol).

[0571] Its structural characterization data are as follows:

[0572] MS m / z (ESI): 882.4 [M+H] +

[0573] Its preparation method is as follows:

[0574] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0575] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0576] Example 2: Preparation of N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-10)

[0577] Step 1: Preparation of (9H-fluorene-9-yl)methyl[(S)-1-((4-(((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)carbamate (L-10-1)

[0578] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (20 mg, 53.27 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (20.66 mg, 159.82 μmol), HATU (24.29 mg, 63.93 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (19.90 mg, 63.93 μmol). The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water / acetonitrile = 1 / 1) and then freeze-dried to give the title compound (11 mg, 16.45 μmol). Its structural characterization data are as follows:

[0579] MS m / z (ESI): 669.3 [M+H] +

[0580] Step 2: Preparation of (S)-2-amino-N-(4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)propionamide (L-10-2)

[0581] (9H-fluorene-9-yl)methyl[(S)-1-((4-(((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)carbamate (11 mg, 16.45 μmol)) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (6.68 mg, 14.96 μmol).

[0582] Its structural characterization data are as follows:

[0583] MS m / z (ESI): 447.1 [M+H] +

[0584] Step 3: Preparation of N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-10)

[0585] (S)-2-amino-N-(4-(((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)propionamide (6.68 mg, 14.96 μmol) was dissolved in DMF (1 mL), followed by the addition of HATU (5.68 mg, 14.96 μmol), DIPEA (5.79 mg, 44.88 μmol), and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (INT-1, 5.50 mg, 14.96 μmol). The reaction mixture was then reacted at 25 °C for 2 hours. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (2.36 mg, 2.92 μmol).

[0586] Its structural characterization data are as follows:

[0587] MS m / z (ESI): 796.3 [M+H] +

[0588] Its preparation method is as follows:

[0589] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0590] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0591] Example 3: Preparation of N-((S)-1-(((S)-6-amino-1-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-12)

[0592] Step 1: Preparation of (9H-fluorene-9-yl)methyl(S,E)-(2-((4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-2-oxoethyl)carbamate (L-12-1)

[0593] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (50 mg, 133.18 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (51.64 mg, 399.55 μmol), HATU (126.53 mg, 332.96 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (118.79 mg, 399.55 μmol). The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water / acetonitrile = 1 / 1) and then freeze-dried to give the title compound (43 mg, 65.68 μmol). Its structural characterization data are as follows:

[0594] MS m / z (ESI): 655.6 [M+H] +

[0595] Step 2: Preparation of (S,E)-2-amino-N-(4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)acetamide (L-12-2)

[0596] ((9H-fluorene-9-yl)methyl(S,E)-(2-((4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-2-oxoethyl)carbamate (43 mg, 65.68 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (28 mg, 64.74 μmol).

[0597] Its structural characterization data are as follows:

[0598] MS m / z (ESI): 433.5 [M+H] +

[0599] Step 3: Preparation of ((S)-6-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazo-2-yl)vinyl)phenyl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-6-oxohexyl)carbamate (L-12-3)

[0600] (S,E)-2-amino-N-(4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)acetamide (28 mg, 64.74 μmol) was dissolved in DMF (1 mL), and HATU (27.06 mg, 71.22 μmol), DIPEA (25.10 mg, 194.23 μmol), and N6-((allyloxy)carbonyl)-N2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-2, 45.04 mg, 77.69 μmol) were added sequentially. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. The reaction solution was purified directly by rapid column chromatography (C18, water / acetonitrile = 1 / 1) and then freeze-dried to give the title compound (11 mg, 11.07 μmol).

[0601] Its structural characterization data are as follows:

[0602] MS m / z (ESI): 995.3 [M+H] +

[0603] Step 4: Preparation of N-((S)-1-(((S)-6-amino-1-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-2-oxoethyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-12)

[0604] ((S)-6-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)vinyl)phenyl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-6-oxohexyl)carbamate (11 mg, 11.07 μmol) was dissolved in DMF (1 mL). 1,3-dimethylbarbituric acid (5.18 mg, 33.21 μmol) and tetrakis(triphenylphosphine)palladium (1.28 mg, 1.11 μmol) were added sequentially under nitrogen atmosphere. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (2.36 mg, 2.92 μmol).

[0605] Its structural characterization data are as follows:

[0606] MS m / z(ESI): 911.2 [M+H]+

[0607] Its preparation method is as follows:

[0608] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0609] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0610] Example 4: Preparation of N-((S)-1-(((S)-6-amino-1-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-4)

[0611] Step 1: Preparation of (9H-fluorene-9-yl)methyl(S)-(2-((6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)-2-oxoethyl)carbamate (L-4-1)

[0612] (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (30 mg, 60.08 μmol) was dissolved in DMF (1 mL). Pyridine (0.5 mL), EDCI (57.68 mg, 300.42 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (35.73 mg, 120.17 μmol) were added sequentially with stirring, and the reaction was continued for 1 hour. After removing the solvent under vacuum, the reaction solution was purified by reverse-phase column chromatography (eluent: 0-70% acetonitrile / 0.05% formic acid water) and then freeze-dried to obtain the title compound (10 mg, 14.73 μmol).

[0613] Its structural characterization data are as follows:

[0614] MS m / z (ESI): 693.3 [M+H] +

[0615] Step 2: Preparation of (S)-2-amino-N-(6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)naphthyl-2-yl)acetamide (L-4-2)

[0616] (9H-fluorene-9-yl)methyl(S)-(2-((6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)-2-oxoethyl)carbamate (10 mg, 14.73 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After removing the solvent from the reaction solution under vacuum, the crude product of the title compound was obtained (theoretical amount: 6.5 mg, 14.24 μmol).

[0617] Its structural characterization data are as follows:

[0618] MS m / z (ESI): 457.4 [M+H] +

[0619] Step 3: Preparation of ((S)-6-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)allyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)butamido)-6-oxohexyl)carbamate (L-4-3)

[0620] (S)-2-amino-N-(6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yl)naphth-2-yl)acetamide (6.5 mg, 14.24 μmol) was dissolved in DMF (1 mL), and DIPEA (5.10 mg, 39.43 μmol), HATU (5.99 mg, 15.77 μmol), and N were added sequentially with stirring. 6 -((allyloxy)carbonyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (11.43 mg, 19.72 μmol), reaction continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-60% acetonitrile / 0.05% formic acid water) and then freeze-dried to give the title compound (11 mg, 10.80 μmol).

[0621] Its structural characterization data are as follows:

[0622] MS m / z (ESI): 1018.4 [M+H] +

[0623] Step 4: Preparation of N-((S)-1-(((S)-6-amino-1-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-4)

[0624] ((S)-6-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)allyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)-6-oxohexyl)carbamate (11 mg, 10.80 μmol) was dissolved in DMF (1 mL), and 1,3-dimethylbarbituric acid (5.06 mg, 32.41 μmol) and tetrakis(triphenylphosphine)palladium (1.25 mg, 1.08 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (2.14 mg, 2.14 μmol).

[0625] Its structural characterization data are as follows:

[0626] MS m / z (ESI): 934.5 [M+H2O+H] +

[0627] Its preparation method is as follows:

[0628] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0629] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0630] Example 5: Preparation of N-((S)-1-(((S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-2)

[0631] Step 1: Preparation of (9H-fluorene-9-yl)methyl[(S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphthyl)amino]-1-oxopropyl-2-yl)carbamate (L-2-1)

[0632] (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (30 mg, 60.08 μmol) was dissolved in DMF (1 mL). Pyridine (0.5 mL), EDCI (57.68 mg, 300.42 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (28.06 mg, 90.13 μmol) were added sequentially with stirring, and the reaction was continued for 1 hour. After removing the solvent under vacuum, the reaction solution was purified by reverse-phase column chromatography (eluent: 0-65% acetonitrile / 0.05% formic acid water) and then freeze-dried to obtain the title compound (19 mg, 27.43 μmol).

[0633] Its structural characterization data are as follows:

[0634] MS m / z (ESI): 693.3 [M+H] +

[0635] Step 2: Preparation of (S)-2-amino-N-(6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)propionamide (L-2-2)

[0636] (9H-fluorene-9-yl)methyl[(S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino]-1-oxopropyl-2-yl)carbamate (19 mg, 27.43 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After removing the solvent from the reaction solution under vacuum, the crude product of the title compound was obtained (theoretical amount: 12 mg, 25.50 μmol).

[0637] Its structural characterization data are as follows:

[0638] MS m / z (ESI): 471.3 [M+H] +

[0639] Step 3: Preparation of N-((S)-1-(((S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (L-2)

[0640] (S)-2-amino-N-(6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)naphth-2-yl)propionamide (6 mg, 12.75 μmol) was dissolved in DMF (1 mL). With stirring, DIPEA (4.94 mg, 38.26 μmol), HATU (7.27 mg, 19.13 μmol), and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (5.62 mg, 15.30 μmol) were added in batches, and the reaction was continued for 2 hours. The reaction solution was directly purified by high-performance liquid chromatography and then freeze-dried to obtain the title compound (1.3 mg, 1.57 μmol).

[0641] Its structural characterization data are as follows:

[0642] MS m / z (ESI): 820.3 [M+H] +

[0643] Its preparation method is as follows:

[0644] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0645] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0646] Example 6: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carbonate (M-7):

[0647] 2,5-Dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (5.13 mg, 14.03 μmol) and 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (10 mg, 14.03 μmol) were dissolved in DMF (1 mL), and DIPEA (5.44 mg, 42.09 μmol) was added dropwise. The mixture was stirred and reacted for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (1.58 mg, 1.56 μmol).

[0648] Its structural characterization data are as follows:

[0649] MS m / z (ESI): 963.4 [M+H] +

[0650] Its preparation method is as follows:

[0651] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0652] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0653] Example 7: Preparation of 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (M-10)

[0654] Step 1: Preparation of (4-((E)-2-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrole-3-yl)vinyl)phenyl)carbamate (M-10-1)

[0655] (S,E)-(4-(2-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (0.5 g, 842.06 μmol) was dissolved in dry tetrahydrofuran (10 mL), cooled and stirred to -10 °C under nitrogen purging and protection, and then triethylamine (119.29 mg, 1.18 mmol) was added dropwise, followed by slow dropwise addition of triphosgene (84.96 mg, 286.30 μmol, dissolved in 5 mL of dry tetrahydrofuran). A solution of 2 mL of dry tetrahydrofuran was reacted, and after 10 minutes, a mixture of allyl ((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (581.75 mg, 842.06 μmol) and DMAP (144.02 mg, 1.18 mmol) was slowly added dropwise (dissolved in 2 mL of dry tetrahydrofuran and 2 mL of dry DMF). The mixture was allowed to return to room temperature for 1 hour. The reaction solution was quenched with water, and ethyl acetate and saturated brine were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-50% ethyl acetate / petroleum ether) to obtain the title compound (1 g, 762.98 μmol).

[0656] Its structural characterization data are as follows:

[0657] MS m / z (ESI): 1311.7 [M+H] +

[0658] Step 2: Preparation of (4-((E)-2-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrole-3-yl)vinyl)phenyl)carbamate (M-10-2)

[0659] Allyl carbamate (1 g, 762.98 μmol) was dissolved in tetrahydrofuran (5 mL), and a tetrabutylammonium fluoride solution in tetrahydrofuran (1 M, 1.52 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent: 20-75% ethyl acetate / petroleum ether) to obtain the title compound (820 mg, 685.4 μmol).

[0660] Its structural characterization data are as follows:

[0661] MS m / z (ESI): 1197.4 [M+H] +

[0662] Step 3: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carbonate (M-10-3)

[0663] Dry DMSO (165.00 mg, 2.11 mmol) was dissolved in dry dichloromethane (10 mL), cooled and stirred to -78 °C under nitrogen purging and protection, and oxaloyl chloride (112.50 mg, 886.32 μmol) was slowly added dropwise. After reacting for 5 minutes, allyl(4-((E)-2-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-(( Diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (820 mg, 685.4 μmol, dissolved in 5 mL of dry dichloromethane) solution was added and the reaction was continued at this temperature for 0.5 hours. Triethylamine (218.40 mg, 2.16 mmol) was added dropwise, and the mixture was allowed to cool naturally to -35 °C for 0.5 hours. The reaction solution was quenched with water, and after returning to room temperature, ethyl acetate and saturated brine were added. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-50% ethyl acetate / petroleum ether) to obtain the title compound (400 mg, 334.9 μmol).

[0664] Its structural characterization data are as follows:

[0665] MS m / z (ESI): 1195.4 [M+H] +

[0666] Step 4: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (M-10-4)

[0667] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (400 mg, 334.9 μmol) was dissolved in DMF (4 mL), and 1,3-dimethylbarbituric acid (78.37 mg, 502.36 μmol) and tetrakis(triphenylphosphine)palladium (19.34 mg, 16.75 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-40% acetonitrile / 0.05% formic acid aqueous solution) and then freeze-dried to give the title compound (310 mg, 302.08 μmol).

[0668] Its structural characterization data are as follows:

[0669] MS m / z (ESI): 1026.8 [M+H] +

[0670] Step 5: Preparation of 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carbonate (M-10-5)

[0671] 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (310 mg, 302.08 μmol) was dissolved in DMF (5 mL), and DIPEA (58.56 mg, 453.12 μmol) and 2,5-dioxopyrrolidine-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (121.4 mg, 166.14 μmol) were added sequentially. The reaction was continued for 1 hour. Ethyl acetate and water were added to the reaction solution and stirred. The mixture was allowed to stand and separated. The organic phase was washed three times with saturated brine and dried. The solution was concentrated under reduced pressure to obtain the crude product of the title compound (380 mg, 297.68 μmol), which was then directly used for the next reaction.

[0672] Its structural characterization data are as follows:

[0673] MS m / z (ESI): 1277.2 [M+H] +

[0674] Step Six: Preparation of 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carbonate (M-10)

[0675] 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (380 mg, 297.68 μmol) was dissolved in dichloromethane (5 mL), and formic acid (4 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure, purified by high performance liquid chromatography, and freeze-dried to give the title compound (120 mg, 117.62 μmol).

[0676] Its structural characterization data are as follows:

[0677] MS m / z(ESI): 1021.3 [M+H] +

[0678] Its preparation method is as follows:

[0679] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0680] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0681] Example 8: N 5 -((S)-1-(((S)-1-((4-((((11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (M-12):

[0682] Step 1: Preparation of 1-allyl-5-(tert-butyl)[((9H-fluorene-9-yl)methoxy)carbonyl]-L-glutamate ester (M-12-2):

[0683] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid (20 g, 47.01 mmol) and 3-bromoprop-1-ene (7.39 g, 61.11 mmol, 5.29 mL) were dissolved in DMF (100 mL). The mixture was cooled and stirred to 0 °C, and DIPEA (8.51 g, 65.81 mmol, 11.46 mL) was added dropwise. The mixture was allowed to return to room temperature naturally for 16 hours. Water and ethyl acetate were added, and the mixture was allowed to stand and separate. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-25% ethyl acetate / petroleum ether) to give the title compound (20.8 g, 44.68 mmol).

[0684] Step 2: Preparation of 1-allyl-5-(tert-butyl)-L-glutamate (M-12-3):

[0685] 1-Allyl-5-(tert-butyl)[((9H-fluorene-9-yl)methoxy)carbonyl]-L-glutamate (20.8 g, 44.68 mmol) was dissolved in DMF (200 mL), and diethylamine (20 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. Water and ethyl acetate were added, and the mixture was allowed to stand and separated. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, and concentrated under reduced pressure. The residue was added to methyl tert-butyl ether with stirring, filtered, and the filtrate was concentrated under reduced pressure to give the crude product of the title compound (10 g, 41.10 mmol), which was directly used for the next reaction.

[0686] Its structural characterization data are as follows:

[0687] MS m / z(ESI): 244.2 [M+H] +

[0688] Step 3: Preparation of 1-allyl-5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamate ester (M-12-4):

[0689] 6-(2-Methylsulfonylmyn-5-yl)hex-5-alkynic acid (11.03 g, 41.10 mmol) and 1-allyl-5-(tert-butyl)-L-glutamate (10 g, 41.10 mmol) were dissolved in DMF (100 mL). HATU (20.30 g, 53.43 mmol) was added with stirring, followed by dropwise addition of DIPEA (15.94 g, 123.31 mmol, 21.48 mL). The reaction was continued for 1 hour. Water and ethyl acetate were added, and the mixture was filtered. The filtrate was allowed to stand and separated. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, and concentrated. The purified compound (20.2 g, 40.93 mmol) was obtained by silica gel column chromatography (eluent: 0-65% ethyl acetate / petroleum ether).

[0690] Step 4: Preparation of (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxovalerate (M-12-5):

[0691] 1-Allyl-5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamate (20 g, 40.52 mmol) was dissolved in dichloromethane (200 mL), and trifluoroacetic acid (50 mL) was added dropwise with stirring. The reaction was continued for 2 hours. The reaction solution was concentrated under reduced pressure, purified by reverse-phase column chromatography (eluent: 0-36% acetonitrile / 0.5% trifluoroacetic acid in water), and lyophilized to give the title compound (13.88 g, 31.73 mmol).

[0692] Its structural characterization data are as follows:

[0693] MS m / z (ESI): 438.1 [M+H] +

[0694] Step 5: Preparation of 4-((S)-2-((S)-2-((S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxopentanamido)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carbonate (M-12-6):

[0695] (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxovaleric acid (14.73 mg, 33.67 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5- Oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (24 mg, 33.67 μmol) was dissolved in DMF (1 mL), and HATU (19.19 mg, 50.51 μmol) was added with stirring, followed by dropwise addition of DIPEA (13.05 mg, 101.01 μmol, 18.03 μL). The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (15 mg, 13.25 μmol).

[0696] Its structural characterization data are as follows:

[0697] MS m / z(ESI): 1133.3 [M+H] +

[0698] Its preparation method is as follows:

[0699] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0700] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0701] Step Six: N 5-((S)-1-(((S)-1-((4-((((11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (M-12):

[0702] 4-((S)-2-((S)-2-((S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxopentanamido)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11 α-Dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (15 mg, 13.25 μmol), tetrakis(triphenylphosphine)palladium (1.53 mg, 1.32 μmol), and 1,3-dimethylbarbituric acid (2.07 mg, 13.25 μmol) were dissolved in DMF (1 mL). The reaction mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (3.15 mg, 2.77 μmol).

[0703] Its structural characterization data are as follows:

[0704] MS m / z(ESI): 1092.3 [M+H] +

[0705] Its preparation method is as follows:

[0706] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0707] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0708] Example 9: 4-((2S,5S,10S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-5-isopropyl-2,19-dimethyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl- Preparation of 5-acetylamido)-4,7,11,18-tetraoxo-15-oxa-3,6,12,19-tetraazapentacarbamate)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-10(5H)-carbonate (M-14):

[0709] (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxovalerate (35.03 mg, 28.0) 6 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (20 mg, 28.06 μmol), HATU (12.79 mg, 33.67 μmol) were dissolved in DMF (1 mL), and DIPEA (7.25 mg, 56.12 μmol) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (13.67 mg, 6.89 μmol).

[0710] Its structural characterization data are as follows:

[0711] MS m / z(ESI): 972.0 [1 / 2M+H] +

[0712] Its preparation method is as follows:

[0713] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0714] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0715] Example 10: 4-((2S,5S,10S,37S,38R,39R,40R)-37,38,39,40,41-pentahydroxy-5-isopropyl-2,35-dimethyl-29,29-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-4 Preparation of 7,11,27,34-pentoxo-15,18,21,24,31-pentoxa-3,6,12,28,35-pentazanonadecanamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (M-15):

[0716] (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid (27.28 mg, 18.24 μmol), 4-((S)-2-(( S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (13 mg, 18.24 μmol) was dissolved in DMF (1 mL), and HATU (8.32 mg, 21.89 μmol) was added with stirring. DIPEA (4.71 mg, 36.48 μmol, 6.35 μL) was added dropwise, and the reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (2.57 mg, 1.16 μmol).

[0717] Its structural characterization data are as follows:

[0718] MS m / z (ESI): 1095.5 [1 / 2M+H] +

[0719] Its preparation method is as follows:

[0720] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

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

[0722] Example 11: 4-((2S,5S,8S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-5-isopropyl-2,19-dimethyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex- Preparation of 5-acetylamido)-4,7,11,18-tetraoxo-15-oxa-3,6,12,19-tetraazapentacarbamate)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-10(5H)-carbonate (M-16):

[0723] N 5 -((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazatocetane-13-yl)-N 2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (15 mg, 12.02 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (8.57 mg, 12.02 μmol) were dissolved in DMF (1 mL), HATU (5.02 mg, 13.22 μmol) was added with stirring, and DIPEA (3.11 mg, 24.03 μmol, 4.19 μL) was added dropwise. The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (2.57 mg, 1.16 μmol).

[0724] Its structural characterization data are as follows:

[0725] MS m / z (ESI): 1095.5 [1 / 2M+H] +

[0726] Its preparation method is as follows:

[0727] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0728] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0729] Example 12: 4-((2S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-19-methyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5 Preparation of (-acetylamido)butamido)-8,18-dioxo-11,15-dioxa-7,19-diazatenecapentanoamide)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazatene-10(5H)-carbonate (M-17)

[0730] 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (50 mg, 49.01 μmol) was dissolved in DMF (1 mL), and the following were added sequentially with stirring: DIPEA (18.99 mg, 147.03 μmol), HATU (22.35 mg, 58.81 μmol), and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (70.28 mg, 73.52 μmol) were added, and the reaction was continued for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to give the title compound formate (24.7 mg, 11.95 μmol).

[0731] Its structural characterization data are as follows:

[0732] MS m / z(ESI): 1958.7 [M+H] +

[0733] Its preparation method is as follows:

[0734] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0735] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0736] Example 13: 4-((S)-9,9-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-1-hydroxy-2,2-bis(hydroxymethyl)-20-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butyramido)-4,14-dioxo- Preparation of 7,11-dioxa-3,15-diazacotetraane-21-amido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (M-18)

[0737] 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (10 mg, 9.80 μmol) dissolved in DMF ( In a 1 mL container, DIPEA (3.80 mg, 29.41 μmol), HATU (4.10 mg, 10.78 μmol), and 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (7.91 mg, 10.78 μmol) were added sequentially with stirring, and the reaction was continued for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound formate (3.52 mg, 2.01 μmol).

[0738] Its structural characterization data are as follows:

[0739] MS m / z (ESI): 1736.6 [M+H] +

[0740] Its preparation method is as follows:

[0741] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0742] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0743] Example 14: 4-((2S,5S,10S,37S,38R,39R,40R)-37,38,39,40,41-pentahydroxy-5-isopropyl-2,35-dimethyl-29,29-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido) Preparation of -4,7,11,27,34-pentoxo-15,18,21,24,31-pentoxa-3,6,12,28,35-pentazatetradecanoamide)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-10(5H)-carboxylic acid ester (M-21)

[0744] 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (12 mg, 16.29 μmol) was dissolved in DMF (1 mL), and HATU (6.81 mg, 17.92 μmol), DIPEA (6.31 mg, 48.86 μmol), and (S)-2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-N1-((25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazanonyl)pentanediamide (26.78 mg, 17.92 μmol, INT-8), reacted for 1 hour. The reaction solution was purified by high performance liquid chromatography to give the title compound (7.84 mg, 3.40 μmol).

[0745] Its structural characterization data are as follows:

[0746] ESI-MS (m / z): 1107.6 [M / 2+H] +

[0747] Its preparation method is as follows:

[0748] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0749] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0750] Example 15: 4-((2S,5S,8R,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-5-isopropyl-2,19-dimethyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl) Preparation of (-5-acetylamido)-4,7,11,18-tetraoxo-15-oxa-3,6,12,19-tetraazapecopentacarbamate)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-10(5H)-carboxylic acid ester (M-22)

[0751] 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (12 mg, 16.29 μmol) was dissolved in DMF (1 mL), and HATU (6.81 mg, 17.92 μmol), DIPEA (6.31 mg, 48.86 μmol), and N5-((2R,3R,4R, 5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-D-glutamine (22.36 mg, 17.92 μmol, INT-4), reacted for 1 hour. The reaction solution was purified by high performance liquid chromatography to give the title compound (4.86 mg, 2.32 μmol).

[0752] Its structural characterization data are as follows:

[0753] ESI-MS (m / z): 1967.6 [M+H] +

[0754] Its preparation method is as follows:

[0755] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0756] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0757] Example 16: 4-((2S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-19-methyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex- Preparation of 5-acetylamido)butamido)-8,18-dioxo-11,15-dioxa-7,19-diazaticopentanoamido)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazatrol-10(5H)-carboxylic acid ester (M-24)

[0758] Step 1: Preparation of allyl (6-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (M-24-1)

[0759] Allyl (S)-(6-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (200 mg, 323.73 μmol) was dissolved in dry tetrahydrofuran (5 mL), cooled and stirred to -10 °C under nitrogen purging and protection, and then triethylamine (45.86 mg, 453.22 μmol) was added dropwise, followed by the slow addition of triphosgene (31.70 mg, 106.83 μmol, dissolved in 5 mL dry tetrahydrofuran). A solution of dry tetrahydrofuran was reacted, and after 10 minutes, a mixed solution of allyl ((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (223.65 mg, 323.73 μmol) and DMAP (55.37 mg, 453.22 μmol) was slowly added dropwise (dissolved in 1 mL of dry tetrahydrofuran and 1 mL of dry DMF). The mixture was allowed to return to room temperature naturally for 1 hour. The reaction solution was quenched with water, and ethyl acetate and saturated brine were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-50% ethyl acetate / petroleum ether) to obtain the title compound (340 mg, 203.80 μmol).

[0760] Its structural characterization data are as follows:

[0761] MS m / z (ESI): 1336.5 [M+H] +

[0762] Step 2: Preparation of allyl (6-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphthalene-2-yl)carbamate (M-24-2)

[0763] Allyl (6-((S)-1-(2-((((4-((S)-2-((S)-2-((((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (340 mg, 203.80 μmol) was dissolved in tetrahydrofuran (5 mL), and a tetrabutylammonium fluoride tetrahydrofuran solution (1 M, 1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent: 20-75% ethyl acetate / petroleum ether) to obtain the title compound (180 mg, 120.94 μmol).

[0764] Its structural characterization data are as follows:

[0765] MS m / z(ESI): 1221.4 [M+H] +

[0766] Step 3: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carboxylic acid ester (M-24-3)

[0767] Dry DMSO (34.57 mg, 442.48 μmol) was dissolved in dry dichloromethane (10 mL). The solution was cooled and stirred to -78 °C under nitrogen purging and protection. Oxaloyl chloride (28.08 mg, 221.24 μmol) was slowly added dropwise. After reacting for 5 minutes, allyl(6-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl( A solution of p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (180 mg, 120.94 μmol, dissolved in 5 mL of dry dichloromethane) was prepared and the reaction was continued at this temperature for 0.5 h. Triethylamine (74.62 mg, 737.46 μmol) was added dropwise, and the mixture was allowed to cool naturally to -35 °C for 0.5 h. The reaction solution was quenched with water, and after returning to room temperature, ethyl acetate and saturated brine were added. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-70% ethyl acetate / petroleum ether) to obtain the title compound (112 mg, 91.92 μmol).

[0768] Its structural characterization data are as follows:

[0769] MS m / z (ESI): 1219.5 [M+H] +

[0770] Step 4: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carboxylic acid ester (M-24-4)

[0771] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (40 mg, 32.83 μmol) was dissolved in DMF (1 mL), and 1,3-dimethylbarbituric acid (15.36 mg, 98.49 μmol) and tetrakis(triphenylphosphine)palladium (3.79 mg, 3.28 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-40% acetonitrile / 0.05% formic acid aqueous solution) and then freeze-dried to give the title compound (26 mg, 22.28 μmol).

[0772] Its structural characterization data are as follows:

[0773] MS m / z (ESI): 1050.5 [M+H] +

[0774] Step 5: Preparation of 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carboxylic acid ester (M-24-5)

[0775] 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (26 mg, 22.28 μmol) was dissolved in DMF (1 mL), and HATU (11.29 mg, 29.71 μmol), DIPEA (9.60 mg, 74.27 μmol), and 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-alkynic acid (7.97 mg, 29.71 μmol) were added sequentially. The reaction was carried out for 1 hour. Ethyl acetate and water were added to the reaction solution and stirred. The mixture was allowed to stand and separated. The organic phase was washed three times with saturated brine and dried. The solution was concentrated under reduced pressure to obtain the crude product of the title compound (34 mg, 23.84 μmol), which was then directly used for the next reaction.

[0776] Its structural characterization data are as follows:

[0777] MS m / z (ESI): 1301.4 [M+H] +

[0778] Step Six: Preparation of 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (M-24-6)

[0779] 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (34 mg, 23.84 μmol) was dissolved in dichloromethane (1 mL), and formic acid (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated and purified by reverse-phase column chromatography (eluent: 0-40% acetonitrile / 0.05% trifluoroacetic acid in water), followed by freeze-drying to obtain the title compound (14 mg, 13.41 μmol).

[0780] Its structural characterization data are as follows:

[0781] MS m / z (ESI): 1044.4 [M+H] +

[0782] Step 7: 4-((2S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-19-methyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5 Preparation of (-acetylamido)butamido)-8,18-dioxo-11,15-dioxa-7,19-diazatenecapentanoamide)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazatene-10(5H)-carboxylic acid ester (M-24)

[0783] 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (14 mg, 13.41 μmol) was dissolved in DMF (1 mL), and then added sequentially... HATU (5.60 mg, 14.75 μmol), DIPEA (5.20 mg, 40.22 μmol), and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (15.38 mg, 16.09 μmol) were reacted for 1 hour. The reaction solution was purified by high performance liquid chromatography to obtain the title compound (7.56 mg, 3.74 μmol).

[0784] Its structural characterization data are as follows:

[0785] ESI-MS (m / z): 1982.8 [M+H] +

[0786] Its preparation method is as follows:

[0787] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0788] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0789] Other compounds of the present invention can be prepared by referring to the methods described in the above embodiments.

[0790] III. Examples of Antibody-Drug Conjugate Preparation

[0791] 1. Preparation of Trastuzumab-L-10

[0792] Take 0.65 mL of trastuzumab (24 mg / mL), add 47.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 59.1 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 10 times the amount of the antibody dissolved in L-10 (109.2 μL, 10 mM) in dimethyl sulfoxide solution and mix well. Incubate at room temperature for 3 h. After that, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-L-10). The DAR value was determined by mass spectrometry to be 5.47.

[0793] 2. Preparation of Trastuzumab-L-9

[0794] Take 0.476 mL of trastuzumab (22.5 mg / mL), add 23.8 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 40.6 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 15 times the amount of the antibody dissolved in L-9 (112.2 μL, 10 mM) in dimethyl sulfoxide solution and mix well. Incubate at room temperature for 3 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-L-9). The DAR value was determined by mass spectrometry to be 5.8.

[0795] 3. Preparation of Trastuzumab-L-12

[0796] Take 0.667 mL of trastuzumab (22.5 mg / mL), add 33.3 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 15 times the amount of the antibody dissolved in L-12 (156.6 μL, 10 mM) in dimethyl sulfoxide solution and mix well. Incubate at room temperature for 3 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-L-12). The DAR value was determined by mass spectrometry to be 6.2.

[0797] 4. Preparation of the antibody-drug conjugate Trastuzumab-L-4

[0798] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 166.0 μL of L-4 solution (10 mM, 15 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-L-4). The DAR value was determined by mass spectrometry to be 6.90.

[0799] 5. Preparation of the antibody-drug conjugate Trastuzumab-L-2

[0800] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, L-2 solution (156.6 μL, 10 mM, 15 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-L-2). The DAR value was 5.11 as determined by mass spectrometry.

[0801] 6. Preparation of the antibody-drug conjugate Trastuzumab-M-7

[0802] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 163.2 μL of M-7 solution (10 mM, 15 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-7). The DAR value was 6.45 as determined by mass spectrometry.

[0803] 7. Preparation of the antibody-drug conjugate Trastuzumab-M-10

[0804] 0.985 mL of Trastuzumab antibody (20.3 mg / mL) was diluted with 49.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 208.8 μL of M-10 solution (10 mM, 15 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-10). The DAR value was determined by mass spectrometry to be 8.08.

[0805] 8. Preparation of the antibody-drug conjugate Trastuzumab-M-12

[0806] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 161.5 μL of M-12 solution (10 mM, 15 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-12). The DAR value was determined by mass spectrometry to be 6.25.

[0807] 9. Preparation of the antibody-drug conjugate Trastuzumab-M-14

[0808] 0.847 mL of Trastuzumab antibody (17.7 mg / mL) was diluted with 42.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 55.7 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 134.4 μL of M-14 solution (10 mM, 13 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-14). The DAR value was determined by mass spectrometry to be 7.85.

[0809] 10. Preparation of the antibody-drug conjugate Trastuzumab-M-15

[0810] 0.36 mL of Trastuzumab antibody (20.6 mg / mL) was diluted with 18.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 28.1 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 75.3 μL of M-15 solution (10 mM, 14 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-15). The DAR value was determined by mass spectrometry to be 8.15.

[0811] 11. Preparation of the antibody-drug conjugate Trastuzumab-M-16

[0812] 0.847 mL of Trastuzumab antibody (17.7 mg / mL) was diluted with 42.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 55.7 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 133.0 μL of M-16 solution (10 mM, 13 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-16). The DAR value was 7.08 as determined by mass spectrometry.

[0813] 12. Preparation of the antibody-drug conjugate Trastuzumab-M-17

[0814] 0.728 mL of Trastuzumab antibody (20.6 mg / mL) was diluted with 36.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 146.1 μL of M-17 solution (10 mM, 14 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-17). The DAR value was determined by mass spectrometry to be 8.0.

[0815] 13. Preparation of the antibody-drug conjugate Trastuzumab-M-18

[0816] 0.728 mL of Trastuzumab antibody (20.6 mg / mL) was diluted with 36.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 146.1 μL of M-18 solution (10 mM, 14 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-18). The DAR value was determined to be 8.0 by mass spectrometry.

[0817] 14. Preparation of the antibody-drug conjugate Trastuzumab-M-21

[0818] 0.769 mL of Trastuzumab antibody (19.09 mg / mL) was diluted with 38.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 158 μL of M-21 solution (10 mM, 15 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-21). The DAR value was determined by mass spectrometry to be 7.61.

[0819] 15. Preparation of the antibody-drug conjugate Trastuzumab-M-22

[0820] 0.769 mL of Trastuzumab antibody (19.09 mg / mL) was diluted with 38.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 161.5 μL of M-22 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-22). The DAR value was 7.36 as determined by mass spectrometry.

[0821] 16. Preparation of the antibody-drug conjugate Trastuzumab-M-24

[0822] Take 1 mL of Trastuzumab antibody (14.9 mg / mL), dilute with 50 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Add M-24 solution dissolved in dimethyl sulfoxide (147.6 μL, 10 mM, 14 molar equivalents of the antibody), mix well, and incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-M-24). The DAR value was determined by mass spectrometry to be 8.12.

[0823] Other antibody-drug conjugates of the present invention can be synthesized by referring to the methods in the above embodiments.

[0824] Biological evaluation

[0825] I. Evaluation of the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model

[0826] The formulation containing the ADC of the present invention was administered to a subcutaneously transplanted human breast cancer cell JIMT-1 mouse CDX model via tail vein injection. Tumor volume and animal weight changes were measured weekly to calculate the tumor-suppressing efficacy of the ADC of the present invention in tumor-bearing mice.

[0827] test drug

[0828] Take an appropriate amount of ADC and administer it according to the dosing design, for a total of three administrations. Dilute the stock solution to the dosing solution with 0.9% NaCl injection. Use 0.9% NaCl injection as a solvent control (Vehicle).

[0829] Laboratory animals and cell lines

[0830] NOD SCID mice (Sichuan Vital River Laboratory Animal Technology Co., Ltd.)

[0831] Human breast cancer cell line JIMT-1 (Nanjing Kebai)

[0832] Experimental grouping and evaluation methods

[0833] JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. JIMT-1 cells in the exponential growth phase were collected, resuspended in PBS containing 50% matrix gel to a suitable concentration, and subcutaneously inoculated into female NOD SCID mice to establish a breast cancer model. The tumors were cultured until the average volume reached approximately 200-250 mmHg. 3 At approximately 10:00 AM, tumors were randomly assigned to two groups based on tumor size: a solvent control group (i.e., negative control, Vehicle group) and a drug-treated group. Drug administration was administered on Day 0, Day 7, and Day 14, for a total of three doses. The administration method was tail vein injection, with a volume of 10 ml / kg. Tumor diameter was measured weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0834] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0835] V T末 >V T0 TGI(%) = [1-(V T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1-(V T末 -V T0 ) / V T0 ]*100%.

[0836] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0837] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0838] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0839] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0840] Testing showed that the ADC of this invention has a clear inhibitory effect on tumor cells.

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, said compound having the structure shown in formula (I): in: Q is the structure before it is linked to the antibody or antigen-binding fragment; L is the connector sub-part; E is the self-eliminating part; D represents the bioactive molecule portion; n is selected from 1-10.

2. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, Q is selected from the following structure: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears; Preferably, Q is selected from the following structures: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears; Preferably, Q is selected from the following structures: Preferably, Q is selected from the following structures:

3. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, L is a divalent structure composed of one or more of the following substituted or unsubstituted structural segments: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 5-12 heterocyclic, -N(R')-, carbonyl, -O-, glycosyl, tromethamine, 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)) and Lys(R') r and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Al). a-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Ala-Ala-Glu, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:44), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:45), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:46), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:47), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 48), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 49), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 50), EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, NOPO, Wherein Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R' represents hydrogen, C 1-6 Alkyl groups, polyhydroxy fragments, glycosyl groups, polyethylene glycol-containing fragments, -(CH2CH2O) r -C 1-6 Alkyl group, -C(=O)-(CH2CH2O) r -C 1-6 Alkyl, polysarcosine, -(C(=O)-CH2N(Me)) r -C 1-6 Alkyl groups, carboxylic acid-containing fragments, tetracarboxylic acid residues and their derivatives, EDTA and its derivatives, or DOTA and its derivatives; r is independently selected from integers from 1 to 20 each time it appears; Preferably, L is selected from the following structures: Where r is selected independently from the integers 1 to 20 each time it appears; n is selected independently from the integers 1 to 20 each time it appears. Preferably, L is selected from the following structures:

4. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, E are each independently selected from single bonds, -NH-CH2-, Preferably, E is -NH-CH2-; Preferably, E is independently selected from single bonds and 5. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, The bioactive molecules are each independently selected from antitumor drugs or compounds with antitumor effects; Preferably, each of the bioactive molecules is independently selected from cytotoxic compounds or antimetabolites; Preferably, the cytotoxic compound is pyrrolobenzodiazepine. PBD-like compounds; Preferably, D is connected to E via -OH, a primary amino or secondary amino group, or -SH, or by reducing the double bonds in its structure; Preferably, the bioactive molecule is selected from the following compounds: Preferably, the bioactive molecule is selected from the following compounds:

6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound is selected from the structures shown below: D-1: D-2: D-3: D-4: L-1: L-2: L-3: L-4: L-5: L-6: L-7: L-8: L-9: L-10: L-11: L-12: M-1: M-2: M-3: M-4: M-5: M-6: M-7: M-8: M-9: M-10: M-11: M-12: M-13: M-14: M-15： M-16: M-17: M-18： M-19: M-20: M-21: M-22: M-23: M-24:

7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound is coupled to an antibody or antigen fragment by a substitution reaction (e.g., removal of the -SO2Me or pentafluorophenol structure) or by an addition reaction.

8. An antibody-drug conjugate, said conjugate having the following structure: in: L' is as described in any one of claims 1-5; E' is as described in any one of claims 1-5; D' is as described in any one of claims 1-5; A represents an antibody or its antigen-binding fragment; Q' is the structural form of Q as described in any one of claims 1-5 after covalently linking it with an antibody or its antigen-binding fragment; x is selected from 1 to 10; Preferably, in the antibody-drug conjugate, D' can be conjugated to the antibody or its antigen-binding fragment via a linker; Preferably, the antibody or its antigen-binding fragment can specifically bind to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

9. The antibody-drug conjugate of claim 8, wherein, Q' is selected from the following structure: Each time p appears, it is independently selected from an integer between 1 and 12; Preferably, Q' is selected from the following structures: Preferably, Q' has the following structure:

10. The antibody-drug conjugate of claim 8, wherein, The antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system: (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (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: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (3a) 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; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

11. The antibody-drug conjugate according to any one of claims 8-10, wherein, The antibody or its antigen-binding fragment comprises: (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4; The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment comprises: (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO:

4.

12. The antibody-drug conjugate according to any one of claims 8-11, wherein, The antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids). Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region. Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, 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; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 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: 41; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 and a light chain constant region (CL) as shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) A heavy chain comprising the VH 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 of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; (2) A heavy chain comprising the VH region 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 region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (3) 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: 41, 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 (4) 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: 41, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40; (3) The heavy chain comprising the sequence shown in SEQ ID NO: 42, and the light chain comprising the sequence shown in SEQ ID NO: 38; or (4) The heavy chain comprising the sequence shown in SEQ ID NO: 43, and the light chain comprising the sequence shown in SEQ ID NO:

40.

13. The antibody-drug conjugate according to any one of claims 8-12, wherein Q' is linked to a thiol group (-SH) or an amino group (-NH2) on Ab.

14. The antibody-drug conjugate according to any one of claims 8-13, wherein, The antibody or its antigen-binding fragment is selected from the antibody or its antigen-binding fragment according to any one of claims 10-12; x is 1 to 8.

15. The antibody-drug conjugate according to any one of claims 8-14, Selected from: ADC D-1: ADC D-2: ADC D-3: ADC D-4: ADC L-1: ADC L-2: ADC L-3: ADC L-4: ADC L-5: ADC L-6: ADC L-7: ADC L-8: ADC L-9: ADC L-10: ADC L-11: ADC L-12: ADC M-1: ADC M-2: ADC M-3: ADC M-4: ADC M-5: ADC M-6: ADC M-7: ADC M-8: ADC M-9: ADC M-10: ADC M-11: ADC M-12: ADC M-13: ADC M-14: ADC M-15: ADC M-16: ADC M-17: ADC M-18: ADC M-19: ADC M-20: ADC M-21: ADC M-22: ADC M-23: ADC M-24: in, -S in various antibody-drug conjugates x -Ab or -NH) x -Ab represents an antibody or antigen-binding fragment thereof containing VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2; wherein x is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in, This indicates the specific linkage between the thiol and pyrimidine groups in the antibody or its antigen-binding fragment. This indicates the specific linkage between the amino and carbonyl groups in the antibody or its antigen-binding fragment.

16. A composition of an antibody-drug conjugate, said composition comprising one or more antibody-drug conjugates according to any one of claims 8-15; preferably, the DAR value (drug-antibody conjugate ratio) of said 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 3~6, ​​for example, 3.0~3.5, 3.0~4.0, 3.0~4.5, 3.0~5.0, 3.0~5.5, 3.0~6.0, 3.5~4.0, 3.5~4.5, 3.5~5.0, 3.5~5.5, 3.5~6.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 5.0~5.5, 5.0~6.0, 5.5~6.0, 6.0~8.0, 6.5~8.0, 7.0~8.0, 7.5~8.0, or 7.5~8.

5.

17. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 8-15 or a pharmaceutically acceptable salt or stereoisomer thereof, the composition of claim 16, and one or more pharmaceutical excipients.

18. Use of the compound of any one of claims 1-7 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug of any one of claims 8-15, the composition of claim 16, or the pharmaceutical composition of claim 17 in the preparation of a medicament for treating cancer; preferably, the cancer is a HER2-expressing cancer.

19. The use according to claim 18, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

20. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or the antibody-drug conjugate of any one of claims 8-15, the composition of claim 16, or the pharmaceutical composition of claim 17, for the treatment of cancer; Preferably, the cancer is a cancer that expresses HER2; Preferably, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

21. A method of treating cancer, comprising administering to a subject in need an effective amount of the compound of any one of claims 1-7 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or an antibody-drug conjugate of any one of claims 8-15, the composition of claim 16, or the pharmaceutical composition of claim 17; Preferably, the cancer is a cancer that expresses HER2; Preferably, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

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