Hydrophilic linker and conjugate thereof, and related preparation method and application

Abstract

A class of compounds containing a hydrophilic group, a preparation method therefor and an application thereof. Specifically, the present invention relates to compounds represented by formulas (I) and (IV) or pharmaceutically acceptable forms thereof, pharmaceutical compositions thereof, preparation methods therefor, and uses thereof. The compounds can be prepared as a biologically active conjugate to treat diseases associated with abnormal cell proliferation.

Description

Hydrophilic linkers and their conjugates, as well as related preparation methods and applications

[0001] This application is based on the following applications filed on November 1, 2024: CN application number 202411554776.8; CN application number 202411554395.X; CN application number 202411554571.X; CN application number 202411582899.2; CN application number 202411858078.7; and CN application number 202411859198. Based on and claiming priority to the following applications, the disclosures of the aforementioned applications are incorporated herein by reference in their entirety: .9 Application dated December 17, 2024; PCT application number PCT / CN2025 / 074725 application dated January 24, 2025; PCT application number PCT / CN2025 / 074728 application dated January 24, 2025; CN application number 202510163776.3 application dated February 14, 2025; and CN application number 202510784034.2 application dated June 12, 2025. Technical Field

[0002] This invention relates to a class of hydrophilic linker compounds and their conjugates, as well as their preparation methods and applications in the pharmaceutical field. Background Technology

[0003] Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies targeting specific antigens and effector molecules linked together by a linker. An ADC consists of three main parts: an antibody responsible for selectively recognizing antigens on the surface of target cells, a payload responsible for killing or modulating target cell function, and a linker connecting the antibody and the payload. The linker, acting as a bridge for the ADC drug, needs to possess good stability and hydrophilicity to prevent the effector molecule from breaking off or accumulating and being cleared in the bloodstream; simultaneously, the breakable linker must also be capable of specific release at specific sites, such as the tumor microenvironment.

[0004] To enhance the hydrophilicity of ADCs, sugar, polyethylene glycol, and polysarcosine structures have been introduced into linkers. Among these, β-glucuronic acid is a typical structure of hydrophilic sugar linkers and is also a substrate for β-glucuronidase. Tumor cell lysosomes contain abundant β-glucuronidase, therefore using β-glucuronic acid as a linker is a feasible pathway for releasing effector molecules. Furthermore, due to the hydrophilic properties of β-glucuronic acid, linking it to some hydrophobic effector molecules can improve the hydrophilicity of ADCs. In 2006, Seattle Genetics reported a structure that carries oligristatin effector molecules via a β-glucuronic acid linker and its ADC (Bioconjugate Chem. 2006, 17, 3, 831-840. WO2007011968). In addition, carbohydrate fragments are embedded in the side chains of the structure between the linker coupling head and the enzyme digestion site to enhance the hydrophilicity of the ADC (WO2023280227 A2, WO2023208168 A1). To enhance the shielding or masking effect of the hydrophilic linker on the hydrophobicity of the effector molecule, polysarcosine structures have also been introduced into the linker (WO2019081455 A1, WO2022228493). Summary of the Invention

[0005] This invention provides a novel drug linker containing hydrophilic hydroxyl, amino, and carboxylic acid fragments, which is expected to be used in the research of antibody-drug conjugates, small molecule conjugates, etc.

[0006] The first aspect of the present invention provides a compound or thereof, a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, and prodrug, wherein the compound has the following structure:

[0007] in,

[0008] Each time XG appears, it is independently selected from H and C. 1-6 Alkyl groups, or substituents containing hydrophilic hydroxyl, glycosyl, amino, quaternary ammonium salt, sulfonyl, and carboxylic acid segments, and their derivatives;

[0009] Y is selected from the segment connecting XG and the benzene ring;

[0010] Z is selected from the portion of a biologically active compound;

[0011] CM is selected from structures that can react with specific functional groups in the target structure (such as an antibody);

[0012] L1 is selected from the fragment connecting CM and the aniline moiety;

[0013] a can be 1, 2, or 3.

[0014] In some embodiments, the compound has the following structure:

[0015] in,

[0016] Each time XG appears, it is independently selected from H and C. 1-6 Alkyl groups, or substituents containing hydrophilic hydroxyl, glycosyl, amino, quaternary ammonium salt, sulfonyl, and carboxylic acid segments, and their derivatives;

[0017] Y is selected from the segment connecting XG and the benzene ring;

[0018] Z is selected from the portion of a biologically active compound;

[0019] CM is selected from structures that can react with specific functional groups in the target structure (such as an antibody);

[0020] L1 is selected from the fragment connecting CM and the aniline moiety;

[0021] a can be 1, 2, or 3.

[0022] In some embodiments, the compound has the following structure:

[0023] in,

[0024] Each time XG appears, it is independently selected from H and C. 1-6 Alkyl groups, or substituents containing hydrophilic hydroxyl, glycosyl, amino, quaternary ammonium salt, sulfonyl, and carboxylic acid segments, and their derivatives;

[0025] Y is selected from the segment connecting XG and the benzene ring;

[0026] Z is selected from the portion of a biologically active compound;

[0027] CM is selected from structures that can react with specific functional groups in the target structure (such as an antibody);

[0028] L1 is selected from the fragment connecting CM and the aniline moiety;

[0029] a can be 1, 2, or 3.

[0030] In some implementations, XG is independently selected from H and C each time it appears. 1-6 Alkyl groups, or substituents containing hydrophilic hydroxyl, glycosyl, amino, sulfonyl, and carboxylic acid segments, and their derivatives.

[0031] In some embodiments, the compound has a structure as shown in any one of formula (Ia), formula (Ib), or formula (Ic):

[0032] In some embodiments, the compound has the structure shown in formula (Id):

[0033] In some implementations, XG is selected independently each time it appears from fragments containing polyhydroxyl groups and their derivatives;

[0034] In some implementations, XG is selected independently each time it appears, from segments containing carbohydrate fragments and their derivatives;

[0035] In some implementations, XG is selected independently each time it appears from fragments containing amino groups and carboxylic acids and their derivatives;

[0036] In some implementations, XG is selected independently each time it appears from a fragment containing an amino group and its derivatives;

[0037] In some implementations, XG is selected independently each time it appears, from segments containing carboxylic acids and their derivatives;

[0038] In some implementations, XG is selected independently from the following structural segments each time it appears: H, C 1-6 Alkyl (e.g., methyl), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), Where m is selected from integers from 1 to 30; for example, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30;

[0039] In some implementations, XG is selected independently from the following structural segments each time it appears: H, C 1-6 Alkyl (e.g., methyl), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), Where m is selected from integers from 1 to 30; for example, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30;

[0040] In some implementations, XG is selected independently from the following structural segments each time it appears: H, C 1-6 Alkyl (e.g., methyl), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), Where m is selected from integers from 1 to 30; for example, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30;

[0041] In some implementations, XG is selected from the following structural fragments:

[0042] Where m is selected from integers from 1 to 30;

[0043] In some implementations, XG is selected from the following structures:

[0044] (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ) (For example: (Specifically: )) (For example: (Specifically: )) (For example: (specifically) )) (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ); where m is independently selected from integers from 1 to 30 each time it appears; for example, m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 each time it appears; preferably, m is 3, 4, or 5;

[0045] In some implementations, XG is selected from the following structures:

[0046] (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ) (For example: (Specifically: )) (For example: (Specifically: )) (For example: (specifically) )) (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ); where m is independently selected from integers from 1 to 30 each time it appears; for example, m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 each time it appears; preferably, m is 3, 4, or 5;

[0047] In some implementations, XG is selected from the following structures:

[0048] Each time m appears, it is independently selected from an integer between 1 and 30; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; preferably, m is 3.

[0049] In some implementations, when Z is Y is -CH2-N(CH3)-, where the methylene group is attached to the benzene ring, the nitrogen atom is attached to XG, a is 1, L1 is Val-Ala, and CM is... At that time, XG was not for

[0050] In some implementations, XG is selected independently from the following structural fragments each time it appears:

[0051] Each time m appears, it is independently selected from an integer from 1 to 10; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; preferably, m is 3, 4, or 5.

[0052] In some implementations, XG is selected independently from the following structural fragments each time it appears:

[0053] In some implementations, XG is selected independently from the following structural fragments each time it appears:

[0054] In some implementations, Y is selected from chemical bonds, or is composed of one or more C atoms. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1- 6-alkylene, C 2-6 imidene group, C 2-6 A group consisting of alkynyl, -O-, -NR1-, quaternary ammonium salt, carbonyl, sulfonyl, sulfinyl, phosphoryl, aryl, heteroaryl, heterocyclic, and / or cycloalkyl groups; R1 is selected from one or more hydrogens, C 1-6 Alkyl, C 1- A structure consisting of 6 alkylene groups, hydroxyl groups, -O- groups, carbonyl groups, carboxyl groups, and / or sulfonic acid groups; preferably, R1 is C 1-6 alkyl;

[0055] In some implementations, Y is selected from one or more Cs. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 A group consisting of alkynyl, -O-, -NR1-, quaternary ammonium salt, carbonyl, sulfonyl, sulfinyl, phosphoryl, aryl, heteroaryl, heterocyclic, and / or cycloalkyl groups; R1 is selected from one or more hydrogens, C 1-6 Alkyl, C 1-6 A structure composed of alkylene, hydroxyl, -O-, carbonyl, carboxyl, and / or sulfonic acid groups; preferably, R1 is C 1-6 alkyl;

[0056] In some implementations, Y is selected from chemical bonds, or is composed of one or more C atoms. 1-6 Alkyl, C 1-6 Alkylene, -O-, -NR1-, A group consisting of a carbonyl group, a heteroaryl group, and / or a heterocyclic group; R1 is selected from hydrogen, C 1-6 Alkyl, sulfonic acid groups;

[0057] In some implementations, Y is selected from one or more Cs. 1-6 Alkyl, C 1-6 Alkylene, -O-, -NR1-, A group consisting of a carbonyl group, a heteroaryl group, and / or a heterocyclic group; R1 is selected from hydrogen, C 1-6 Alkyl, sulfonic acid groups;

[0058] In some implementations, Y is selected from chemical bonds, or from one or more C atoms. 1-6 Alkyl, C 1-6 Alkylene A group consisting of - and / or -NR1-; R1 is selected from hydrogen, C 1-6 alkyl;

[0059] In some implementations, Y is selected from one or more Cs. 1-6 Alkyl, C 1-6 Groups composed of alkylene, -O-, -NR1-, carbonyl, heteroaryl, and heterocyclic groups; R1 is selected from hydrogen, C 1-6 Alkyl, sulfonic acid groups;

[0060] In some implementations, Y is selected from one or more Cs. 1-6 Alkyl, C 1-6 Alkylene A group consisting of - and / or -NR1-; R1 is selected from hydrogen, C 1-6 alkyl;

[0061] In some implementations, Y is selected from chemical bonds, or Y is selected from one or more C atoms. 1-6 A group consisting of alkylene and / or -NR1-; R1 is selected from hydrogen, C 1-6 alkyl;

[0062] In some implementations, Y is selected from one or more Cs. 1-6 Alkyl, C 1-6 A group consisting of alkylene groups and -NR1-; R1 is selected from hydrogen and C. 1-6 alkyl;

[0063] In some implementations, Y is selected from one or more Cs. 1-6 A group consisting of alkylene and / or -NR1-; R1 is selected from hydrogen, C 1-6 alkyl;

[0064] In some implementations, Y is selected from chemical bonds, and is composed of methylene and The group consisting of methylene groups or groups consisting of methylene and -N(CH3)-;

[0065] In some implementations, Y is selected from methylene and The group consisting of methylene and -N(CH3)-;

[0066] In some embodiments, Y is selected from chemical bonds or groups consisting of methylene and -N(CH3)-;

[0067] In some embodiments, Y is selected from a group consisting of a methylene group and a -N(CH3)- group;

[0068] In some implementation schemes, Y is selected from chemical bonds, Or -CH2-N(CH3)-;

[0069] In some implementation schemes, Y is selected from Or -CH2-N(CH3)-;

[0070] In some implementations, Y is a chemical bond or Position 1 is connected to the benzene ring, and positions 2 and 3 are connected to XG; or Y is -CH2-N(CH3)-, where the methylene group is connected to the benzene ring and the nitrogen atom is connected to XG.

[0071] In some implementation schemes, Y is Position 1 is connected to the benzene ring, and positions 2 and 3 are connected to XG; or Y is -CH2-N(CH3)-, where the methylene group is connected to the benzene ring and the nitrogen atom is connected to XG.

[0072] In some implementations, Y is selected from chemical bonds or -CH2-N(CH3)-.

[0073] In some implementations, Y is selected from -CH2-N(CH3)-.

[0074] In some embodiments, the bioactive compound is selected from topoisomerase inhibitors, tubulin inhibitors, DNA damaging agents, and RNA damaging agents;

[0075] In some embodiments, the biologically active compound is selected from topoisomerase I inhibitors, topoisomerase II inhibitors, tubulin polymerization inhibitors, tubulin depolymerization inhibitors, DNA interference agents, DNA alkylating agents, DNA cross-linking agents, DNA polymerase inhibitors, DNA damage repair inhibitors, RNA polymerase inhibitors, thymidine synthase inhibitors, poly-ADP-ribose polymerase inhibitors, apoptosis promoters, cell cycle arrestors, protein degrading agents, synthetic lethal agents, kinase inhibitors, and glucocorticoid receptor modulators.

[0076] In some embodiments, the biologically active compound is selected from camptothecin compounds, olritatin compounds, maytansine compounds, eribulin compounds, hammetrine compounds, epothilone compounds, kazimycin compounds, anthracycline compounds, benzodiazepine compounds, α-amaminoid compounds, sucrose compounds, alkaloid compounds, tripterygium compounds, nucleoside compounds, glucocorticoid compounds, rapamycin compounds, and lithylamine compounds;

[0077] In some embodiments, the biologically active compound is selected from topoisomerase I inhibitors (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, ixotecan, topotecan, belototecan, rubotecan, diflomotecan, lurtotecan, Karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or elomotecan).

[0078] In some embodiments, the biologically active compound is selected from compounds with the following structures or their pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds:

[0079] in,

[0080] Existence or non-existence;

[0081] R5 is either fluorine or chlorine.

[0082] R4 is selected from methyl, chlorine, hydroxyl, or amino;

[0083] Alternatively, R4 and R5 form with the bonded carbon atoms.

[0084] R3 is independently selected from hydrogen, C 1-6 Alkyl, halogen, hydroxyl, =O, C 1-6 Hydroxyalkyl, C 1-6 Aminealkyl, C 1-6 Alkoxy or C 1- 6-Hydroalkyl group, or two R3 atoms on adjacent atoms connected to the linked atoms to form a ring;

[0085] Ring A is selected from:

[0086] G is selected from oxygen or sulfur;

[0087] b is selected from 1-5;

[0088] when When R5 is fluorine, R4 is methyl, R3 is hydrogen, and G is oxygen, ring A is not morpholino.

[0089] In some embodiments, the biologically active compound is a compound with the structure shown below, or a pharmaceutically acceptable salt, stereoisomer, or isotopically labeled compound thereof:

[0090] in,

[0091] R5 is either fluorine or chlorine.

[0092] R4 is selected from methyl, chlorine, hydroxyl, or amino;

[0093] Alternatively, R4 and R5 form with the bonded carbon atoms.

[0094] R3 is independently selected from hydrogen, C 1-6 Alkyl, halogen, hydroxyl, =O, C 1-6 Hydroxyalkyl, C 1-6 Aminealkyl, C 1-6 Alkoxy or C 1- 6-Hydroalkyl group, or two R3 atoms on adjacent atoms connected to the linked atoms to form a ring;

[0095] Ring A is selected from:

[0096] Preferred

[0097] G is selected from oxygen or sulfur;

[0098] b is selected from 1-5;

[0099] When R5 is fluorine, R4 is methyl, R3 is hydrogen, and G is oxygen, ring A is not morpholino.

[0100] In some embodiments, in the compounds of formulas (ZI) to (Z-II) above, G is selected from oxygen.

[0101] In some embodiments, in the compounds of formulas (ZI) to (Z-II) above, R3 is independently selected from hydrogen, C, and C. 1-6 Alkyl, halogen, hydroxyl, =O, C 1-6 Hydroxyalkyl, C 1-6 Aminealkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkyl groups, or two R3 atoms on adjacent atoms connected to a 3-6 membered carbon ring or a 3-6 membered heterocycle.

[0102] In some embodiments, R3 is hydrogen in the compounds of formulas (ZI) to (Z-II).

[0103] In some embodiments, in the compounds of formulas (ZI) to (Z-II) above, ring A is selected from...

[0104] In some embodiments, in the compounds of formulas (ZI) to (Z-II) above, ring A is...

[0105] In some embodiments, in the compounds of formulas (ZI) to (Z-II) above, Selected from

[0106] In some embodiments, in the compounds of formulas (ZI) to (Z-II) above, for

[0107] In some embodiments, the biologically active compound is selected from compounds with the following structures or their pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds:

[0108] Among them, R9 and R8 are each independently selected from H, OH, -NH2, and -NH(C). 1-6 Alkyl), C 1-6 Alkyl and halogen; the C 1-6 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-6 Haloalkyl, C 3-6 The cycloalkyl group is substituted; or, R9 and R8 together with the adjacent carbon atoms (connected to the cycloforming carbon atoms of R9 and R8, respectively) form a five-membered oxygen-containing heterocycle.

[0109] R7 is independently selected from H, halogen, -OH, -NH2, -NH(C) 1-6 alkyl), and -NH-CO-(C 1-6 alkylene)-OH; the C 1-6 Alkyl and C 1-6 Alkylene is optionally further subjected to one or more elements selected from halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Substituents of cycloalkyl groups;

[0110] p is 1 or 2;

[0111] q is 0, 1, or 2;

[0112] G represents oxygen or sulfur;

[0113] T represents carbon or oxygen.

[0114] In some embodiments, the biologically active compound is selected from compounds with the following structures or their pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds:

[0115] in,

[0116] R3, R4, R5 and b are as defined in any of the above;

[0117] A1 is C 1-6 Alkylene, the C 1-6 Alkylene is optionally further subjected to one or more elements selected from halogen, hydroxyl, C 1-6 Haloalkyl, C 3-6 Substituents of cycloalkyl groups;

[0118] G is selected from oxygen or sulfur.

[0119] In some embodiments, the bioactive compound is selected from:

[0120] In some embodiments, the bioactive compound is selected from:

[0121] The biologically active compounds disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), and secondary amino (-NR). 1 H), tertiary amino group (-NR) 2 R 3 ), where R 1 R 2 R 3 In this context, it refers only to non-hydrogen substituents on N, or mercapto groups (-SH), which can react with suitable functional groups in the rest of the compound to achieve linkage.

[0122] In some embodiments, the biologically active compound is linked to a carbonyl group in the compound via a -OH, primary amino, secondary or tertiary amino group, or -SH group. In some embodiments, Z is a monovalent structure obtained by losing an H group from the -OH, primary amino, secondary or tertiary amino group, or -SH group on the biologically active compound.

[0123] In some implementations, Z is selected from the following structures:

[0124] Among them, R 4A Selected from -O- or -N-;

[0125] R3, R4, R5, rings A, b, and G are as defined in the preceding term.

[0126] In some implementations, Z is selected from the following structures:

[0127] Where R 9ASelected from chemical bonds, -O- or -NH-; R 7A Selected from chemical bonds, -O-, -NH-, -N(C) 1-6 alkyl)- and -NH-CO-(C 1- 6-alkylene)-O-; the C 1-6 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 3- Substituents of 6-cycloalkyl groups;

[0128] R7, R8, R9, G, T, p, q are defined as any of the above.

[0129] In some implementation schemes, R 7A Selected from chemical bonds, -O-, -NH-, and -NH-CO-(C 1-4 (alkylene)-O-; the C 1-4 The alkylene group is optionally further divided by one or more molecules selected from hydroxyl, C 1-4 Alkyl, C 3-6 The cycloalkyl group is substituted. In some embodiments, R 7A Selected from chemical bonds, -NH-, -NH-CO-CH2-O-, -NH-CO-CH(CH3)-O-, -NH-CO-CH(cyclopropyl)-O-, -NH-CO-C(CH3)2CH2-O-, -NH-CO-CH(OH)CH2-O-.

[0130] In some implementations, Z is selected from:

[0131] In some implementations, Z is selected from:

[0132] In some implementations, Z is

[0133] In some implementations, Z is

[0134] In some implementations, Z is

[0135] In some implementations, CM is selected from functional groups or structural fragments that can react with cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs contained in the target structure.

[0136] In some embodiments, CM is selected from haloacetyl, substituted or unsubstituted maleimide, alkylsulfonyl heteroaryl, and fluorophenol ester.

[0137] In some embodiments, CM is selected from iodoacetyl, maleimide, bromomaleimide, thiomaleimide, alkylsulfonylpyrimidinyl, and fluorophenol ester.

[0138] In some implementations, CM is selected from the following substituted or unsubstituted structural segments:

[0139] In some implementations, CM is selected from the following substituted or unsubstituted structural segments:

[0140] In some implementation schemes, CM is

[0141] In some implementations, L1 is selected from segments consisting of one or more of the following structures, including but not limited to C. 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl, carbonyl, sulfonyl, amino, hydroxyl, -O-, heterocyclic, heteroaryl, aryl, natural or non-natural amino acids and their polypeptides, polyethylene glycol, polysarcosine, carboxylic acid, glycosyl and their derivatives or quaternary ammonium salts.

[0142] In some embodiments, L1 is selected from one or more of the following substituted or unsubstituted structural segments: C 1-6Alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic groups (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(R'), Glu(R'), Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (e.g., Gly-Lys, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID)). NO: 41), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO: 42), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Va l-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-G ly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu- Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:43), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:44), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:45), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:46), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:47)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene -CO2H, -C 1-6 Alkylene groups -SO3H, -SO3H, -PO3H2, -C 1-6Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)-C 1-6 alkylene-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1- 6-alkylene-SO3H)2、-CH2N(C 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O)rC 1-6 Alkyl group, -CH2N(C) 1-6Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol fragments containing 1-10 EO units (i.e., -(CH2CH2O)r'-C) 1-6 Alkyl group, r' = 1-10), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residue), EDTA (ethylenediaminetetraacetic acid residue), -C 1-6 Alkylene-N(C) 1- 6-alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkyl-N(C) 1- 6-alkyl)-EDTA, wherein r is selected from an integer from 1 to 20; s is selected from an integer from 1 to 20.

[0143] In some embodiments, L1 is selected from one or more of the following substituted or unsubstituted structural segments: C 1-6Alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic groups (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(R'), Glu(R'), Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (e.g., Gly-Lys, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID)). NO: 41), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO: 42), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Va l-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-G ly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu- Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:43), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:44), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:45), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:46), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:47)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene -CO2H, -C 1-6 Alkylene groups -SO3H, -SO3H, -PO3H2, -C1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)-C 1-6 alkylene-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1- 6-alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O)rC 1-6 Alkyl group, -CH2N(C)1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol fragments containing 1-10 EO units (i.e., -(CH2CH2O)r'-C) 1-6 Alkyl group, r' = 1-10), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residue), EDTA (ethylenediaminetetraacetic acid residue), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-EDTA, wherein r is selected from an integer from 1 to 20; s is selected from an integer from 1 to 20.

[0144] In some implementations, "-EDTA" refers to

[0145] In some implementations, "-NOTA" refers to

[0146] In some implementations, "-DOTA" refers to

[0147] In some implementations, "-DOTAGA" refers to

[0148] In some implementations, Lys(R') refers to the following structure

[0149] In some implementations, Glu(R') refers to the following structure

[0150] In some implementations, r is selected from integers from 1 to 15, such as 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, and 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0151] In some implementations, s is selected from integers from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0152] In some implementations, L1 is selected from short peptides composed of amino acids.

[0153] In some implementations, L1 is Val-Ala.

[0154] Another aspect of the present invention provides a compound or its salt, ester, stereoisomer, or isotope label thereof, wherein the compound has a structure as shown in formula (II-a) or formula (II-b):

[0155] Among them, XG, L1 and CM are each described independently as any one of the above items.

[0156] Another aspect of the present invention provides compounds or their salts, esters, stereoisomers, or isotope labels, wherein the compounds have the structure shown in formula (II-c) or formula (II-d):

[0157] Among them, XG, L1 and CM are each described independently as any one of the above items.

[0158] Another aspect of the present invention provides a compound or its salt, ester, stereoisomer, or isotope label thereof, wherein the compound has a structure as shown in formula (III-a) or formula (III-b):

[0159] XG is as described in any of the above items.

[0160] Another aspect of the present invention provides compounds or their salts, esters, stereoisomers, or isotopic labels, wherein the compounds have the structure shown in formula (III-c) or formula (III-d):

[0161] XG is as described in any of the above items.

[0162] Another aspect of the present invention provides compounds, or their salts, esters, stereoisomers, or isotope labels, as shown below:

[0163] Another aspect of the present invention provides a compound or its salt, ester, stereoisomer, or isotope label thereof, wherein the compound has the following structure:

[0164] in,

[0165] XG is selected from fragments containing hydrophilic hydroxyl groups, glycosyl groups, amino groups, and carboxylic acid groups and their derivatives;

[0166] Z is selected from the biologically active compound portion;

[0167] E is selected from the segment connecting L2 and Z;

[0168] CM is selected from structures that can react with specific functional groups in the target structure (such as an antibody);

[0169] L2 is selected from the segment connecting parts CM, XG, and E;

[0170] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, n is 1, 2, 3, 4, 5, and more preferably 2, 3, or 4.

[0171] In some implementations, XG is selected from fragments containing polyhydroxy groups and their derivatives.

[0172] In some embodiments, XG is selected from fragments containing amino groups and carboxylic acids and their derivatives.

[0173] In some embodiments, XG is selected from fragments containing amine groups and their derivatives.

[0174] In some implementations, XG is selected from segments containing carboxylic acid fragments and their derivatives.

[0175] In some implementations, XG is as described in any of the above.

[0176] In some implementations, XG is selected from the following structural fragments:

[0177] Where m is selected from integers from 1 to 30;

[0178] In some implementations, XG is selected from the following structural fragments:

[0179] (For example: ), (For example: );

[0180] In some implementations, XG is selected from the following structural fragments:

[0181] In some implementations, XG is selected from the following structural fragments:

[0182] In some implementations, XG is selected from the following structural fragments:

[0183] In some implementations, Z is as described in any of the above descriptions.

[0184] In some implementations, the CM is as described in any of the above descriptions.

[0185] In some implementations, L2 is selected from segments consisting of one or more of the following structures, including but not limited to C. 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl, carbonyl, sulfonyl, amino, hydroxyl, -O-, heterocyclic, heteroaryl, aryl, natural or non-natural amino acids and their polypeptides, polyethylene glycol, polysarcosine, carboxylic acid, glycosyl and their derivatives or quaternary ammonium salts.

[0186] In some embodiments, L2 is selected from substituted or unsubstituted structural fragments of one or more of the following: C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic group (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Ly). s, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(R'), Glu(R'), Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly(DGGFG), Glu-Gly-Gly-Phe-Gly(EGGFG), Ala-Ala, Ala-Lys, Ala-Ly s(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala ,Ala-D-Ala-Ala,Ala-Ala-Asn,Ala-Ala-Gly,D-Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Ser- Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGF G), Gly-Gly-Val-Ala(GGVA), Gly-Phe-Leu-Gly(GFLG), Glu-Ala-Ala-Ala(EAAA), Gly-Gly-Gly-Gly-Gly(GGGGG)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C1-6 alkylene (CO2H), -C 1-6 Alkylene groups SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 alkylene-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O)rC 1-6 Alkyl group, -CH2N(C) 1-6 alkyl)-C(=O)-(OCH2CH2)r-OC 1-6 Alkyl group, -(CH2N(Me)-C(=O))rC1-6 Alkyl groups, polyethylene glycol fragments containing 1-10 EO units (i.e., -(CH2CH2O)r'-C) 1-6 Alkyl group, r' = 1-10), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residue), EDTA (ethylenediaminetetraacetic acid residue), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-EDTA, wherein r is selected from an integer from 1 to 20; s is selected from an integer from 1 to 20.

[0187] In some implementations, "-EDTA" refers to

[0188] In some implementations, "-NOTA" refers to

[0189] In some implementations, "-DOTA" refers to

[0190] In some implementations, "-DOTAGA" refers to

[0191] In some implementations, Lys(R') refers to the following structure

[0192] In some implementations, Glu(R') refers to the following structure

[0193] In some implementations, XG is linked to lysine in L2.

[0194] In some implementations, XG is linked to the amino group on the lysine residue in L2.

[0195] In some implementations, XG forms the following structure with lysine in L2:

[0196] In some implementations, XG forms the following structure with lysine in L2:

[0197] In some implementations, L2 is Val-Lys-Gly.

[0198] In some implementations, L2-XG is

[0199] In some implementations, L2-XG is

[0200] In some implementations, L2 is Val-Lys.

[0201] In some implementations, L2-XG is

[0202] In some implementations, L2-XG is

[0203] In some implementations, r is selected from integers from 1 to 15, such as 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, and 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0204] In some implementations, s is selected from integers from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0205] In some implementations, m is independently selected from an integer from 1 to 30 each time it appears, for example, m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 each time it appears.

[0206] In some implementations, E is selected from the self-elimination structure;

[0207] In some implementations, E is selected from single bonds, -NHCH2-, and -NHBn-O(=O)-.

[0208] In some implementations, E is selected from a single bond or -NHCH2-.

[0209] In some implementations, E is a single bond.

[0210] Another aspect of the present invention provides the following structure:

[0211] Another aspect of the present invention provides a bioactive conjugate having the following structure:

[0212] Wherein: XG, Y, Z, and L1 are each independently as described in any of the above items;

[0213] CM' is selected from the structure connecting L1 and A;

[0214] A is selected from antibodies or their antigen-binding fragments, peptides or small molecule fragments that have a targeting effect;

[0215] a can be 1, 2, or 3;

[0216] x is between 1 and 10.

[0217] In some embodiments, the coupling has a structure shown in any one of formulas (I-A1), (I-A2), or (I-A3):

[0218] In some implementation schemes, Further for Among them -Q] x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment that is linked to CM'.

[0219] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment that is linked to CM'.

[0220] Another aspect of the present invention provides bioactive conjugates having the structure shown in formula (II-A1) or formula (II-A2):

[0221] Wherein: XG and L1 are each independently as described in any of the above items;

[0222] CM' is selected from the structure connecting L1 and A;

[0223] A is selected from antibodies or their antigen-binding fragments, peptides or small molecule fragments that have a targeting effect;

[0224] x is between 1 and 10.

[0225] Another aspect of the present invention provides bioactive conjugates having the structure shown in formula (II-B1) or formula (II-B2):

[0226] Wherein: XG and L1 are each independently as described in any of the above items;

[0227] CM' is selected from the structure connecting L1 and A;

[0228] A is selected from antibodies or their antigen-binding fragments, peptides or small molecule fragments that have a targeting effect;

[0229] x is between 1 and 10.

[0230] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment that is linked to CM'.

[0231] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment that is linked to CM'.

[0232] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment that is linked to CM'.

[0233] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment that is linked to CM'.

[0234] Another aspect of the present invention provides bioactive conjugates having the structure shown in formula (III-A1) or formula (III-A2):

[0235] Wherein: XG is as described in any of the above items;

[0236] A is selected from antibodies or their antigen-binding fragments, peptides or small molecule fragments that have a targeting effect;

[0237] x is between 1 and 10.

[0238] Another aspect of the present invention provides bioactive conjugates having the structure shown in formula (III-B1) or formula (III-B2):

[0239] Wherein: XG is as described in any of the above items;

[0240] A is selected from antibodies or their antigen-binding fragments, peptides or small molecule fragments that have a targeting effect;

[0241] x is between 1 and 10.

[0242] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment linked to a pyrimidine group.

[0243] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment linked to a pyrimidine group.

[0244] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment linked to a pyrimidine group.

[0245] In some implementation schemes, Further for Among them -Q) x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment linked to a pyrimidine group.

[0246] Another aspect of the present invention provides a bioactive conjugate having the following structure:

[0247] in,

[0248] XG, E, L2, and Z are each described independently as described in any of the above items;

[0249] CM' is selected from the structure connecting L2 and A;

[0250] A is selected from antibodies or their antigen-binding fragments, peptides or small molecule fragments that have a targeting effect;

[0251] x is between 1 and 10.

[0252] In some implementation schemes, Further for Among them -Q] x -Ab is the antibody or its antigen-binding fragment: Q is the portion of the antibody or its antigen-binding fragment that is linked to CM'.

[0253] In some implementations, XG, Y, L1, L2, E, Z each occur independently as described in any of the above.

[0254] In some embodiments, the amino acid residue is a cysteine, lysine, serine, or threonine residue.

[0255] In some implementations, Q is S, NH, or O.

[0256] In some implementations, -Q] x -Ab or -Q) x -Ab is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0257] In some embodiments, CM' is selected from functional groups or structural fragments that have reacted with cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs contained in the structure of the antibody or its antigen-binding fragment.

[0258] In some implementations, CM' is selected from C 1-6 Alkyl-substituted acyl groups, substituted or unsubstituted succinimide groups, substituted or unsubstituted heteroaryl groups, and 3-6 membered heterocyclic acyl groups.

[0259] In some embodiments, CM' is selected from acyl, succinimide, substituted or unsubstituted pyrimidinyl, and piperidinyl.

[0260] In some implementations, CM' is selected from the following substituted or unsubstituted structural segments:

[0261] In some implementation schemes, CM' is

[0262] In some implementations, A is selected from antibodies that target tumor antigens, such as monoclonal or bispecific antibodies that target Her2, Her3, EGFR, TROP2, B7H3, c-Met, CEACAM5, CLDN18.2, FRa, CDH6, CDH3, PTK7, DLL3, and GPC3.

[0263] In some implementations, A is selected from antibodies that target tumor antigens, such as trastuzumab antibody, patocilizumab antibody, etc.

[0264] In some implementations, A is selected from peptides that target tumor antigens, such as somatostatin analogs, GnRH / LHRH analogs, vascular peptide-2, Heptaarginine, TAT47-57, DPV1047 Vectocell peptide, peptides that target EphA2, GPC3, or Nectin-4, etc.

[0265] In some implementations, A is selected from small molecule fragments that target tumor antigens, such as N-acetylgalactosamine (GalNAc) fragments, bisphosphonate fragments with bone targeting, fragments that selectively bind to prostate-specific membrane antigen (PSMA), fragments that selectively bind to somatostatin receptors, etc.

[0266] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0267] (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:

[0268] (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,

[0269] (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;

[0270] 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;

[0271] or,

[0272] (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:

[0273] (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,

[0274] (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;

[0275] 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;

[0276] or,

[0277] (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:

[0278] (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,

[0279] (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;

[0280] 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;

[0281] or,

[0282] (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:

[0283] (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,

[0284] (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;

[0285] 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.

[0286] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0287] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0288] (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,

[0289] (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;

[0290] or,

[0291] (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:

[0292] (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,

[0293] (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;

[0294] or,

[0295] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0296] (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,

[0297] (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;

[0298] or,

[0299] (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:

[0300] (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,

[0301] (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;

[0302] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0303] (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

[0304] (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;

[0305] 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.

[0306] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0307] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0308] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0309] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0310] (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

[0311] (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).

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0317] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0318] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36;

[0319] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0320] (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; or

[0321] (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;

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0327] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine residue.

[0328] In some embodiments, the antibody or its antigen-binding fragment is selected from antibodies or their antigen-binding fragments that specifically bind to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0329] In some implementations, the antibody or its antigen-binding fragment is selected from trastuzumab, pertuzumab, trastuzumab mutant, pertuzumab mutant, or a biepisode antibody or its antigen-binding fragment constructed from trastuzumab and pertuzumab.

[0330] 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.

[0331] Another aspect of the present invention provides antibody-drug conjugates as shown below:

[0332] Another aspect of the present invention provides an antibody-drug conjugate structure as shown below:

[0333] Where Ab-(S-) refers to any of the antibodies or antigen-binding fragments described above;

[0334] This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment.

[0335] In some implementations, x is 1-10, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 6-8.

[0336] intermediate

[0337] In some embodiments, this application provides intermediate compounds having the following structures, or salts, stereoisomers, tautomers, or isotopically labeled compounds thereof:

[0338] Each of the PG groups is independently protected by an H or amino group each time it appears. The amino protecting group is an alkoxycarbonyl amino protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl).

[0339] In some embodiments, this application provides intermediate compounds having the following structures, or salts, stereoisomers, tautomers, or isotopically labeled compounds thereof:

[0340] Connector

[0341] On the other hand, this application provides the following connector fragment:

[0342] Among them, XG, Y, L1, L2, CM' and n are connected to Z through position 1 and connected to the antibody or antigen binding fragment through position 2, as described in any of the above.

[0343] On the other hand, this application provides the following connector fragment:

[0344] Wherein, XG, Y, L1, CM', and a are connected to Z via position 1 and to the antibody or antigen-binding fragment via position 2, as described in any of the above embodiments. In some embodiments, the linker structure is as follows:

[0345] Position 1 is connected to Z, and position 2 is connected to the antibody or its antigen-binding fragment.

[0346] In some implementations, the connector structure is as follows:

[0347] Position 1 is connected to Z, and position 2 is connected to the antibody or its antigen-binding fragment.

[0348] Composition

[0349] On the other hand, this application provides compositions that may comprise a plurality of the ADCs described herein. Each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compounds described herein. Thus, the compositions are characterized by a "drug-antibody ratio" (DAR) in the range of about 1 to about 10. Methods for determining the DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0350] For example, in any embodiment, the composition described herein has a DAR of about 1 to about 10 or any subrange thereof, such as: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 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 10. 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.

[0351] In some embodiments, the DAR of the ADC compositions described herein is about 3 to 9, such as about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8. .0, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 5.0 to 8.0, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 5.5 to 8.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.5, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 7.5, approximately 6.5 to 8.5, approximately 7.0 to 7.5.

[0352] In some embodiments, the DAR of the ADC composition described herein is about 3.5 to 5.0, for example about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0.

[0353] In some embodiments, the DAR of the ADC compositions described herein is about 1.0 to 6.0, for example about 1.0 to 5.5, about 1.0 to 5.0, about 1.5 to 6.0, about 1.5 to about 5.5, about 1.5 to 5.0, 2.0 to 5.5, about 2.0 to about 5.0, for example 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1 .25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1. 46, approximately 1.47, approximately 1.48, approximately 1.49, approximately 1.5, approximately 1.51, approximately 1.52, approximately 1.53, approximately 1.54, approximately 1.55, approximately 1.56, approximately 1.57, approximately 1.58, approximately 1.59, approximately 1.6, approximately 1.61, approximately 1.62, approximately 1.63, approximately 1.64, approximately 1.65, approximately 1.66, approximately 1.6 7, approximately 1.68, approximately 1.69, approximately 1.7, approximately 1.71, approximately 1.72, approximately 1.73, approximately 1.74, approximately 1.75, approximately 1.76, approximately 1.77, approximately 1.78, approximately 1.79, approximately 1.8, approximately 1.81, approximately 1.82, approximately 1.83, approximately 1.84, approximately 1.85, approximately 1.86, approximately 1.87, approximately 1.88 Approximately 1.89, approximately 1.9, approximately 1.91, approximately 1.92, approximately 1.93, approximately 1.94, approximately 1.95, approximately 1.96, approximately 1.97, approximately 1.98, approximately 1.99, approximately 2.0, approximately 2.01, approximately 2.02, approximately 2.03, approximately 2.04, approximately 2.05, approximately 2.06, approximately 2.07, approximately 2.08, approximately 2.09. Approximately 2.1, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.2, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.3, 2. .31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, approximately 2.53, approximately 2.54, approximately 2.55, approximately 2.56, approximately 2.57, approximately 2.58, approximately 2.59, approximately 2.6, approximately 2.61, approximately 2.62, approximately 2.63, approximately 2.64, approximately 2.65, approximately 2.66, approximately 2.67, approximately 2.68, approximately 2.69, approximately 2.7, approximately 2.71, approximately 2.72, approximately 2. 73, approximately 2.74, approximately 2.75, approximately 2.76, approximately 2.77, approximately 2.78, approximately 2.79, approximately 2.8, approximately 2.81, approximately 2.82, approximately 2.83, approximately 2.84, approximately 2.85, approximately 2.86, approximately 2.87, approximately 2.88, approximately 2.89, approximately 2.9, approximately 2.91, approximately 2.92, approximately 2.93, approximately 2.9 4. Approximately 2.95, 2.96, 2.97, 2.98, 2.99, 3.0, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.1, 3.11, 3.12, 3.13, 3.14, 3.1 5. Approximately 3.16, 3.17, 3.18, 3.19, 3.2, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.3, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36 Approximately 3.37, 3.38, 3.39, 3.4, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.5, 3.51, 3.52, 3.53, 3.54, 3.55, 3.56, 3.57 Approximately 3.58, 3.59, 3.6, 3.61, 3.62, 3.63, 3.64, 3.65, 3.66, 3.67, 3.68, 3.69, 3.7, 3.71, 3.72, 3.73, 3.74, 3.75, 3.76, 3.77, 3.78 Approximately 3.79, approximately 3.8, approximately 3.81, approximately 3.82, approximately 3.83, approximately 3.84, approximately 3.85, approximately 3.86, approximately 3.87, approximately 3.88, approximately 3.89, approximately 3.9, approximately 3.91, approximately 3.92, approximately 3.93, approximately 3.94, approximately 3.95, approximately 3.96, approximately 3.97, approximately 3.98, approximately 3.99, approximately 4.0, approximately 4.01, approximately 4.02, approximately 4.03, approximately 4.04, approximately 4.05, approximately 4.06, approximately 4.07, approximately 4.08, approximately 4.09, approximately 4.1, approximately 4.11, approximately 4.12, approximately 4.13, approximately 4.14, approximately 4.15, approximately 4.16, approximately 4.17, approximately 4.18, approximately 4.19, approximately 4.2, approximately 4.21, approximately 4.22, approximately 4.23, approximately 4.24, approximately 4.25, approximately 4.26, approximately 4.27, approximately 4.28, approximately 4.29, approximately 4.3, approximately 4.31, approximately 4.32, approximately 4.33, approximately 4.34, approximately 4.35, approximately 4.36, approximately 4.37, approximately 4.38, approximately 4.39, approximately 4.4, approximately 4.41, approximately 4.42, approximately 4.43, approximately 4.44, approximately 4.45, approximately 4.46, approximately 4.47, approximately 4.48, approximately 4.49, approximately 4.5, approximately 4.51, approximately 4.52, approximately 4.53, approximately 4.54, approximately 4.55, approximately 4.56, approximately 4.57, approximately 4.58, approximately 4.59, approximately 4.6, approximately 4.61, approximately 4.62, approximately 4.63, approximately 4.64, approximately 4.65, approximately 4.66, approximately 4.67, approximately 4.68, approximately 4.69, approximately 4.7, approximately 4.71, approximately 4.72, approximately 4.73, approximately 4.74, approximately 4.75, approximately 4.76, approximately 4.77, approximately 4.78, approximately 4.79, approximately 4.8, approximately 4.81, approximately 4.82, approximately 4.83, approximately 4.84, approximately 4.85, approximately 4.86, approximately 4.87, approximately 4.88, approximately 4.89, approximately 4.9, approximately 4.91, approximately 4.92, approximately 4.93, approximately 4.94, approximately 4.95, approximately 4.96, approximately 4.97, approximately 4.98, approximately 4.99, approximately 5.0.

[0354] In some embodiments, the DAR of the ADC compositions described herein is about 2.4, about 2.5, about 2.56, about 3.9, about 5.56, about 6.4, about 6.7, about 6.8, about 6.9, about 7.08, about 7.1, about 7.29, about 7.33, about 7.5, about 7.6, about 7.75, about 7.76, about 7.87, about 7.9, about 7.99, about 7.81, about 8.0, about 8.03, about 8.1, about 8.12, about 8.19, about 8.2, or about 8.26.

[0355] In some embodiments, the DAR of the ADC compositions described herein is 2.4, 2.5, 2.56, 3.9, 5.56, 6.4, 6.7, 6.8, 6.9, 7.08, 7.1, 7.29, 7.33, 7.5, 7.6, 7.75, 7.76, 7.87, 7.9, 7.99, 7.81, 8.0, 8.03, 8.1, 8.12, 8.19, 8.2, 8.26.

[0356] In some embodiments, the DAR of the ADC compositions described herein is about 4.41, about 4.34, about 4.51, about 4.36, about 4.63, about 4.28, about 4.38, about 3.86, about 4.16, about 3.99, or about 2.45.

[0357] In some embodiments, the DAR of the ADC compositions described herein is 4.41, 4.34, 4.51, 4.36, 4.63, 4.28, 4.38, 3.86, 4.16, 3.99, or 2.45.

[0358] Pharmaceutical Composition

[0359] In another aspect, this application provides a pharmaceutical composition comprising any of the compounds described in any of the preceding claims or their pharmaceutically acceptable salts, any of the conjugates described in any of the preceding claims or any of the compositions described in any of the preceding claims, and one or more pharmaceutical excipients.

[0360] The compounds or conjugates described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, or intratumoral injection. Optionally, antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16). th (ed., Osol, A.Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates may be administered via any route appropriate for the individual to be treated.

[0361] application

[0362] This application provides for the use of the compounds described above, or pharmaceutically acceptable salts, conjugates, compositions, or pharmaceutical compositions thereof, in the preparation of a medicament for treating cancer.

[0363] This application provides the aforementioned compound or its pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition for the treatment of cancer.

[0364] This application provides the compounds described above or their pharmaceutically acceptable salts, conjugates, compositions, or pharmaceutical compositions for the treatment of solid tumors or hematologic malignancies, such as those selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), and urothelial carcinoma.

[0365] This application provides a cancer treatment method comprising, based on a therapeutically effective amount of the aforementioned compound or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, for an individual in need.

[0366] In some embodiments, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

[0367] In some embodiments, the antibody-drug conjugate, compound, drug-linker, or pharmaceutical composition containing it is sufficient (e.g., in a subject):

[0368] (1) Inhibits the proliferation of cells (such as tumor cells);

[0369] (2) Inhibits tumor growth;

[0370] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0371] (4) Suppress signal transduction;

[0372] (5) Prevention and / or treatment of cancer; or

[0373] (6) Any combination of (1)-(5) above.

[0374] In some implementations, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

[0375] All technical features disclosed in this specification, such as the definitions of various functional groups, except for mutually exclusive technical features, can be combined in any way to obtain different general formula ranges or specific solutions. These ranges and solutions are all within the scope of this invention.

[0376] definition

[0377] 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.

[0378] 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 in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The amino acid assignment in each region or domain can follow various numbering systems known in the art. The term "antibody" also includes embodiments where the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or is a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into pyroglutamate. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise N-terminal glutamine or glutamic acid, or N-terminal amino acid cyclized into pyroglutamate.

[0379] 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).

[0380] 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.

[0381] The following general rules (published at www.bioinf.org.uk: Professor Andrew C. Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids that form the antigenic epitope that the antibody binds to. In rare cases, these generally constant features may not appear; however, Cys residues are the most conserved feature.

[0382] V H The complete amino acid sequence is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In some embodiments, V H The amino acid sites in the sequence can be numbered sequentially starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the V mentioned herein... H and V L The amino acid sites in the sequence are defined according to their sequential numbering.

[0383] The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

[0384] 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.

[0385] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0386] 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.

[0387] 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).

[0388] 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.

[0389] 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.

[0390] 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:48) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:49) 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.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] 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.

[0396] 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).

[0397] 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.

[0398] 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).

[0399] 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.

[0400] The term "linker" refers to a structural segment that connects a cytotoxic drug to an antibody or antigen-binding fragment. For example, the formula Ab-[MLED] x The -MLE- structure fragment in the text.

[0401] The term "drug-linker" refers to the structure of the cytotoxic drug and linker described in this invention before they are linked to an antibody or its antigen-binding fragment. For example, "drug-linker" refers to M'-LED, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment. The "drug-linker" is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.

[0402] The "drug-linker" also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the "drug-linker" compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0403] 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.

[0404] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20 Alkyl", C 1-10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3Alkyl 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.

[0405] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched hydrocarbon group, such as "C". 1-6 Alkylene, C 1-5 Alkylene, C 1-4 Alkylene, C 1-3 "alkylene", specific examples include but are not limited to: methylene, ethylene, propylene, butylene, etc.

[0406] The term "aryl" refers to an unsaturated carbocyclic group having a conjugated π-electron system, such as "6-10 aryl", and specific examples include, but are not limited to, phenyl and naphthyl.

[0407] The term "heteroaryl" refers to an unsaturated group having a conjugated π-electron system consisting of ring atoms, with at least one (e.g., 1, 2, 3, or 4) ring atoms being heteroatoms, such as N, O, and S, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized. Examples include "5-12-membered heteroaryl", "5-11-membered heteroaryl", "5-10-membered heteroaryl", "5-9-membered heteroaryl", and "5-6-membered heteroaryl". Specific examples include, but are not limited to: phenyl, naphthylfuranyl, pyrroleyl, thiopheneyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indazoleyl, indolyl, quinolinyl, and isoquinolinyl.

[0408] The term "heterocyclic group" refers to a saturated or partially unsaturated cyclic group consisting of ring atoms, with at least one (e.g., 1, 2, 3, 4, or 5 heteroatoms) being a heteroatom, such as N, O, and S, wherein the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms are optionally oxidized, and the carbon atom is optionally oxidized. The heterocyclic group includes monocyclic, bicyclic, or polycyclic rings, including spirocyclic, fused, or bridged rings. The term "nitrogen-containing heterocyclic group" refers to a group with at least one heteroatom being N, such as "5-12 membered nitrogen-containing heterocyclic group," "5-9 membered nitrogen-containing heterocyclic group," and "9-12 membered nitrogen-containing heterocyclic group," with specific embodiments including but not limited to: pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, and 1,2,3,4-tetrahydroquinolinyl.

[0409] The term "isotopically labeled compound" means that the compound is structurally identical to the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0410] As used herein, the term "suitable substituent" refers to modifications of a compound that can be made by those skilled in the art to suit the needs of the compound's substituents. "Suitable substituents" include oxo (=O), halogen, cyano, and NR. 8 R 9 Carboxyl, thiol, hydroxyl, ester group (e.g., -C) 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 R 9 Each is independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, halogens, hydroxyl groups, carboxyl groups, and ester groups (e.g., -C)1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0411] The term "substitution" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a specified compound or structural segment by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound. In some embodiments, each substituent is independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 (alkylene) group, C1-C6 haloalkyl (alkylene) group, C1-C6 alkoxy group, C2-C6 (alkenyl) group, C2-C6 (alkynyl) group, C3-C8 (cycloalkyl) group, 3-8 membered (heterocyclic) group, C6-C 10 (A)aryl and 5-10 membered (A)heteroaryl, etc. In some embodiments, the substituents are each independently composed of one or more of the following structures: NR 10 R 11 -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6 (alkylene) group, C1-C6 haloalkylene group, C1-C6 alkoxy group, C2-C6 (alkenylene) group, C2-C6 (alkynylene) group, C3-C8 (cycloalkylene) group, 3-10 membered (heterocyclic) group, C6-C 10 (sub-)aryl and 5-10 quinone (sub-)heteroaryl, etc., among which R 10 R 11 R' is as defined above. For example, the substituent can be a suitable substituent as described above.

[0412] If a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted.

[0413] Whether explicitly stated or not, all numerical values ​​in this application are modified by the term "about". The term "about" means within ±20%, ±10%, preferably ±5%, and more preferably ±2% of the stated numerical value. Detailed Implementation

[0414] 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.

[0415] Information about the sequences involved in this invention is described in the table below:

[0416] The abbreviations used in this article have the following meanings:

[0417] 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).

[0418] 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).

[0419] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0420] 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.

[0421] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0422] Example 1: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-1)

[0423] Step 1: Preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-1-2)

[0424] 50.0 g (279 mmol) of 6-nitroisobenzofuran-1(3H)-one was added to a tetrahydrofuran solution of methylamine (2 M, 500 mL), and the mixture was heated to 75 °C and stirred for 5 hours. The reaction solution was directly concentrated to obtain the crude title compound (58.0 g), which was used directly in the next step without purification.

[0425] Its structural characterization data are as follows:

[0426] ESI-MS (m / z): 211.0 [M+H] + .

[0427] Step 2: Preparation of (2-((methylamino)methyl)-4-nitrophenyl)methanol (INT-1-3)

[0428] 2-Hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g, 119 mmol) was dissolved in tetrahydrofuran (500 mL). After cooling to 0 °C, a borane-dimethyl sulfide solution (10 M, 89.3 mL) was added dropwise to the reaction system. After the addition was complete, the temperature was raised to 70 °C and stirred for 5 hours. The temperature was lowered to 0 °C again, and a hydrogen chloride-methanol solution (2 M, 100 mL) was added dropwise to the above reaction solution. The temperature was raised again to 65 °C and stirred for 12 hours. The reaction solution was filtered, and the filtrate was directly concentrated to obtain the crude title compound (44.3 g), which was used directly in the next step without purification.

[0429] Its structural characterization data are as follows:

[0430] ESI-MS (m / z): 197.0 [M+H] + .

[0431] Step 3: Preparation of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (INT-1-4)

[0432] (2-((methylamino)methyl)-4-nitrophenyl)methanol (40.3 g, 205 mmol) was dissolved in dichloromethane (800 mL), cooled to 0°C, and then imidazole (55.9 g, 822 mmol) and tert-butyldiphenylchlorosilane (84.0 g, 308 mmol, 78.8 mL) were added. The mixture was then restored to 25°C and stirred for 1 hour. The reaction was quenched with water (200 mL), and the mixture was extracted three times with dichloromethane (300 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 5 / 1), the product was concentrated again to obtain the title compound (59.7 g, 137 mmol).

[0433] Its structural characterization data are as follows:

[0434] ESI-MS (m / z): 435.1 [M+H] + .

[0435] Step 4: Preparation of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (INT-1-5)

[0436] 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (54.9 g, 126 mmol) was dissolved in dichloromethane (550 mL), cooled to 0 °C, and DIPEA (48.9 g, 379 mmol, 66.0 mL) and allyl chloroformate (30.5 g, 253 mmol, 26.8 mL) were added. The mixture was then restored to 25 °C and stirred for 1 hour. The reaction was quenched with water (300 mL), and the mixture was extracted three times with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 3 / 1), the product was concentrated again to obtain the title compound (65.5 g, 126 mmol).

[0437] Its structural characterization data are as follows:

[0438] ESI-MS (m / z): 519.1 [M+H] + .

[0439] Step 5: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-1)

[0440] (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (62.8 g, 121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL). Iron powder (33.8 g, 605 mmol) and ammonium chloride (64.8 g, 1.21 mol) were added, and the mixture was heated to 80 °C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was extracted three times with dichloromethane (100 mL x 3). The combined organic phases were washed with a saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (49.3 g).

[0441] Its structural characterization data are as follows:

[0442] ESI-MS (m / z): 511.7 [M+H] + .

[0443] Example 2 of intermediate preparation: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-2)

[0444] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-2-1)

[0445] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added. The mixture was stirred at room temperature for 15 hours and concentrated under reduced pressure to obtain the crude product. After purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%), the crude product was concentrated again under reduced pressure to obtain the title compound (194.2 mg, 0.25 mmol).

[0446] Its structural characterization data are as follows:

[0447] MS m / z (ESI): 782.2 [M+H] +

[0448] Step 2: Preparation of (S)-(5-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-2-2)

[0449] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (194.2 mg, 0.25 mmol) was dissolved in DMF (5 mL), and pyridine hydrofluoric acid salt (390.2 mg, 3.93 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 20 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (109.3 mg, 0.21 mmol).

[0450] Its structural characterization data are as follows:

[0451] MS m / z (ESI): 566.1 [M+Na] +

[0452] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamidyl)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-2-3)

[0453] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), DIPEA (81.3 mg, 0.63 mmol) was added, followed by p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol). The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (134.50 mg, 0.19 mmol).

[0454] Its structural characterization data are as follows:

[0455] MS m / z (ESI): 731.2 [M+Na] +

[0456] Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-2)

[0457] Allyl carbamate (134.50 mg, 0.19 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H 165.50 mg, 0.38 mmol of benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (30.80 mg, 0.23 mmol), pyridine (16.5 mg, 0.21 mmol), and DIPEA (73.5 mg, 0.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) and lyophilized to give the title compound (114.70 mg, 0.12 mmol).

[0458] Its structural characterization data are as follows:

[0459] MS m / z (ESI): 1005.2 [M+H] +

[0460] Example 3 of intermediate preparation: (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (INT-3)

[0461] Step 1: Preparation of (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid tert-butyl ester (compound INT-3-2)

[0462] (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione methanesulfonate (80 mg, 150.50 μmol), (S)-3-(tert-butoxycarbonyl)thiazolyl-4-carboxylic acid (70.22 mg, 301.01 μmol), HATU (171.68 mg, 451.5 μmol), and DIPEA (97.25 mg, 752.51 μmol) were added to a DMF (6 mL) reaction system. The reaction mixture was stirred at 25 °C for 18 hours. The reaction solution was prepared by high performance liquid chromatography and then freeze-dried to obtain the title compound (85 mg, 130.63 μmol).

[0463] Its structural characterization data are as follows:

[0464] MS m / z (ESI): 651.3 [M+H] +

[0465] Its preparation method is as follows:

[0466] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0467] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0468] Step 2: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (compound INT-3)

[0469] (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-carboxylic acid tert-butyl ester (60 mg, 92.21 μmol) was dissolved in HCl / 1,4-dioxane (5 mL) and stirred at 25 °C for 1 hour. The reaction solution was concentrated to obtain a crude product, which was then purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (25 mg, 42.16 μmol).

[0470] Its structural characterization data are as follows:

[0471] MS m / z (ESI): 551.2 [M+H] +

[0472] 1 H NMR(400MHz,DMSO)δ8.65(d,J=8.4Hz,1H),7.77(d,J=10.4Hz,1H),7.29(s,1H), 6.53(s,1H),5.60-5.50(m,1H),5.42(s,2H),5.28-5.08(m,2H),4.14(d,J=8.4H z,1H),4.00(d,J=7.6Hz,1H),3.90-3.80(m,1H),3.20-3.10(m,2H),3.08-2.90( m,2H),2.38(s,3H),2.27-2.05(m,2H),1.95-1.75(m,2H),0.87(t,J=6.0Hz,3H).

[0473] Its preparation method is as follows:

[0474] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0475] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0476] Example 4 of intermediate preparation: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0477] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamidyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-1)

[0478] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added. The mixture was stirred at room temperature for 15 hours and concentrated under reduced pressure to obtain the crude product. After purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%), the crude product was concentrated again under reduced pressure to obtain the title compound (194.2 mg, 0.25 mmol).

[0479] Its structural characterization data are as follows:

[0480] MS m / z (ESI): 782.2 [M+H] +

[0481] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-6-2)

[0482] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (194.2 mg, 0.25 mmol) was dissolved in DMF (5 mL), and pyridine hydrofluoric acid salt (390.2 mg, 3.93 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 20 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (109.3 mg, 0.21 mmol).

[0483] Its structural characterization data are as follows:

[0484] MS m / z (ESI): 566.1 [M+Na] +

[0485] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-3)

[0486] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), DIPEA (81.3 mg, 0.63 mmol) was added, followed by p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol). The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (134.50 mg, 0.19 mmol).

[0487] Its structural characterization data are as follows:

[0488] MS m / z (ESI): 731.2 [M+Na] +

[0489] Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazinyl[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-4)

[0490] Allyl carbamate (134.50 mg, 0.19 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H 165.50 mg, 0.38 mmol of benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (30.80 mg, 0.23 mmol), pyridine (16.5 mg, 0.21 mmol), and DIPEA (73.5 mg, 0.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) and lyophilized to give the title compound (114.70 mg, 0.12 mmol).

[0491] Its structural characterization data are as follows:

[0492] MS m / z (ESI): 1005.2 [M+H] +

[0493] Step 5: Preparation of (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-5)

[0494] Dissolve (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (114.70 mg, 0.12 mmol) in DMF (3 mL), add diethylamine (87.8 mg, 1.2 mmol), and stir at room temperature for 1 hour. After the reaction was complete, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) after most of the solvent was removed, and then freeze-dried to obtain the title compound (62.6 mg, 0.08 mmol).

[0495] Its structural characterization data are as follows:

[0496] MS m / z(ESI): 783.1 [M+H] +

[0497] Step Six: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-6)

[0498] (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl) Allyl carbamate (1.0 g, 1.28 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (476.90 mg, 1.41 mmol) were dissolved in DMF (5 mL). HATU (631.06 mg, 1.66 mmol) and DIPEA (495.29 mg, 3.83 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was rapidly purified and then freeze-dried to give the title compound (950 mg, 860.37 μmol).

[0499] Its structural characterization data are as follows:

[0500] MS m / z (ESI): 1104.5 [M+H] +

[0501] Its preparation method is as follows:

[0502] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0503] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0504] Step 7: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0505] (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline) Allyl 1-yl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (950 mg, 860.37 μmol) was dissolved in DMF (3.5 mL), and diethylamine (860.37 μmol) was added. The mixture was stirred at room temperature for 20 min. The reaction solution was concentrated and then 100 mL of a mixed solvent (EA / PE = 1 / 3) was added and stirred overnight. The mixture was filtered and dried to obtain the title compound (750 mg, 850.4 μmol).

[0506] Its structural characterization data are as follows:

[0507] MS m / z (ESI): 882.5 [M+H] +

[0508] Example 5 of intermediate preparation: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0509] Step 1: Preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-5-2)

[0510] 6-Nitroisobenzofuran-1(3H)-one (INT-5-1) (50.0 g, 279 mmol) was added to a tetrahydrofuran solution of methylamine (2 M, 500 mL), and the mixture was heated to 75 °C and stirred for 5 hours. The reaction solution was directly concentrated to obtain the crude title compound (58.0 g), which was used directly in the next step without purification.

[0511] Its structural characterization data are as follows:

[0512] ESI-MS (m / z): 211.0 [M+H] + .

[0513] Step 2: Preparation of (2-((methylamino)methyl)-4-nitrophenyl)methanol (INT-5-3)

[0514] 2-Hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g, 119 mmol) was dissolved in tetrahydrofuran (500 mL). After cooling to 0 °C, a borane-dimethyl sulfide solution (10 M, 89.3 mL) was added dropwise to the reaction system. After the addition was complete, the temperature was raised to 70 °C and stirred for 5 hours. The temperature was lowered to 0 °C again, and a hydrogen chloride-methanol solution (2 M, 100 mL) was added dropwise to the above reaction solution. The temperature was raised again to 65 °C and stirred for 12 hours. The reaction solution was filtered, and the filtrate was directly concentrated to obtain the crude title compound (44.3 g), which was used directly in the next step without purification.

[0515] Its structural characterization data are as follows:

[0516] ESI-MS (m / z): 197.0 [M+H] + .

[0517] Step 3: Preparation of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (INT-5-4)

[0518] (2-((methylamino)methyl)-4-nitrophenyl)methanol (40.3 g, 205 mmol) was dissolved in dichloromethane (800 mL), cooled to 0 °C, and then imidazole (55.9 g, 822 mmol) and tert-butyldiphenylchlorosilane (84.0 g, 308 mmol, 78.8 mL) were added. The mixture was then restored to 25 °C and stirred for 1 hour. The reaction was quenched with water (200 mL), and the mixture was extracted three times with dichloromethane (300 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 5 / 1), the product was concentrated again to obtain the title compound (59.7 g, 137 mmol).

[0519] Its structural characterization data are as follows:

[0520] ESI-MS (m / z): 435.1 [M+H] + .

[0521] Step 4: Preparation of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (INT-5-5)

[0522] 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (54.9 g, 126 mmol) was dissolved in dichloromethane (550 mL), cooled to 0 °C, and DIPEA (48.9 g, 379 mmol, 66.0 mL) and allyl chloroformate (30.5 g, 253 mmol, 26.8 mL) were added. The mixture was then restored to 25 °C and stirred for 1 hour. The reaction was quenched with water (300 mL), and the mixture was extracted three times with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 3 / 1), the product was concentrated again to obtain the title compound (65.5 g, 126 mmol).

[0523] Its structural characterization data are as follows:

[0524] ESI-MS (m / z): 519.1 [M+H] + .

[0525] Step 5: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0526] (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (62.8 g, 121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL). Iron powder (33.8 g, 605 mmol) and ammonium chloride (64.8 g, 1.21 mol) were added, and the mixture was heated to 80 °C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was extracted three times with dichloromethane (100 mL x 3). The combined organic phases were washed with a saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (49.3 g).

[0527] Its structural characterization data are as follows:

[0528] ESI-MS (m / z): 511.7 [M+H] + .

[0529] Example 1: Synthesis of (2R,3R)-4-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2,3-dihydroxy-4-oxobutyric acid (A-2)

[0530] Step 1: Preparation of allyl (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (A-2-2):

[0531] INT-2 (1.13 g, 1.12 mmol) was dissolved in DMF (20 mL), and diethylamine (2 mL) was added dropwise. The mixture was stirred at room temperature for 0.5 hours. After concentration, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 1 / 1) and then freeze-dried to give the title compound (493 mg, 629.78 μmol).

[0532] Its structural characterization data are as follows:

[0533] MS m / z(ESI): 782.8 [M+H]+

[0534] Step 2: Preparation of allyl (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)carbamate (A-2-3):

[0535] Weigh out allyl (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (473 mg, 604.23 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (222.01 mg, 604.23 μmol) were dissolved in DMF (5 mL). Diisopropylethylamine (234.27 mg, 1.81 mmol) and HATU (344.41 mg, 906.35 μmol) were added with stirring, and the reaction was stirred at room temperature for 1 hour. LC-MS monitoring showed a small amount of starting material remaining. Water was added to precipitate the precipitate, which was filtered and dried under vacuum to give the crude title compound (400 mg, 353.29 μmol).

[0536] Its structural characterization data are as follows:

[0537] MS m / z(ESI): 1132.2 [M+H]+

[0538] Step 3: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (A-2-4 / B-22):

[0539] Weigh out allyl(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2- (Methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)carbamate (400 mg, 353.29 μmol) was dissolved in DMF (5 mL). Tetraphenylphosphine palladium (81.65 mg, 70.66 μmol) and 1,3-dimethylbarbituric acid (82.74 mg, 529.93 μmol) were added with stirring. The mixture was purged with nitrogen and protected, and the reaction was stirred at room temperature for 1 hour. After purification by rapid column chromatography (C18, water / acetonitrile = 0.82), the product was lyophilized to give the title compound (135 mg, 128.80 μmol). 15 mg of the reverse-phase purified product was purified by preparative high-performance liquid chromatography and lyophilized to give the title compound (7.87 mg, 7.13 μmol, purity 95%).

[0540] Its structural characterization data are as follows:

[0541] MS m / z(ESI): 1048.1 [M+H]+

[0542] Its preparation method is as follows:

[0543] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0544] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0545] Step 4: Preparation of (2R,3R)-4-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2,3-dihydroxy-4-oxobutyric acid (A-2):

[0546] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3] [',4':6,7]Indolezino[1,2-b]quinoline-1-yl)carbamate (30 mg, 28.62 μmol) and L-tartaric acid (8.59 mg, 57.24 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (11.10 mg, 85.87 μmol) and HATU (16.31 mg, 42.93 μmol). The mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (3.02 mg, 2.30 μmol, 90% purity).

[0547] Its structural characterization data are as follows:

[0548] MS m / z(ESI): 1180.2 [M+H]+

[0549] Its preparation method is as follows:

[0550] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0551] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0552] Example 2: Synthesis of 2,2'-((2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)azinediyl)diacetic acid (A-5)

[0553] Step 1: Preparation of 2-(2,6-dioxomorpholine)acetic acid (A-5-2):

[0554] Weigh out 1 g (5.23 mmol) of triacetic acid and dissolve it in pyridine (3 mL). Add acetic anhydride (2 g, 5.23 mmol) dropwise with stirring, and react at 40 °C for 5 hours. Concentrate the reaction solution under reduced pressure to obtain the target product, which can be used directly in the next reaction step.

[0555] Step 2: Preparation of 2,2'-((2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)azinediyl)diacetic acid (A-5):

[0556] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ [de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (30 mg, 28.62 μmol) and 2-(2,6-dioxomorpholine)acetic acid (9.91 mg, 57.24 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (11.10 mg, 85.87 μmol) was added. The mixture was stirred at room temperature for 1 hour. LCMS monitoring showed a small amount of reactant residue. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (4.75 mg, 3.89 μmol, purity 84%).

[0557] Its structural characterization data are as follows:

[0558] MS m / z(ESI): 1221.2 [M+H]+

[0559] Its preparation method is as follows:

[0560] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0561] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0562] Example 3: Synthesis of 2,2'-((2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)azinediyl)diacetic acid (A-6)

[0563] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3' [4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (30 mg, 28.62 μmol) and 4-[2-(2,6-dioxomorpholin-4-yl)ethyl]morpholin-2,6-dione (14.67 mg, 57.24 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (11.10 mg, 85.87 μmol) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (4.5 mg, 3.06 μmol, purity 90%).

[0564] Its structural characterization data are as follows:

[0565] MS m / z(ESI): 1322.3 [M+H]+

[0566] Its preparation method is as follows:

[0567] Column: Waters SunFire Prep C18 OBD (5μm*45mm*450mm)

[0568] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0569] Example 4: Synthesis of 2,2'-((2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)azinediyl)diacetic acid (A-9)

[0570] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1, 2-[b]quinoline-1-yl)carbamate (30 mg, 28.62 μmol) and 2-[4,7-bis(carboxymethyl)-1,4,7-triazacyclohexane-1-yl]acetic acid (43.41 mg, 143.11 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (27.74 mg, 214.67 μmol) and HATU (40.79 mg, 107.33 μmol). The mixture was stirred at room temperature for 1 hour. LCMS monitoring showed a small amount of reactant residue. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (12.51 mg, 8.73 μmol, purity 93%).

[0571] Its structural characterization data are as follows:

[0572] MS m / z(ESI): 1333.4 [M+H]+

[0573] Its preparation method is as follows:

[0574] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0575] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0576] Example 5: Synthesis of 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid (A-18):

[0577] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4] [6,7]Indolezido[1,2-b]quinoline-1-yl)carbamate (400 mg, 381.63 μmol) and diethylene glycol (102.34 mg, 763.26 μmol) were dissolved in DMF (5 mL), and diisopropylethylamine (147.96 mg, 1.14 mmol) and HATU (217.53 mg, 572.44 μmol) were added. The mixture was stirred at room temperature for 1 hour. The product was clearly observed under LCMS monitoring. The reaction solution was added dropwise to a 1N hydrochloric acid aqueous solution to precipitate the product. The precipitate was filtered, washed with water, and dried under vacuum to obtain the crude title compound (333 mg, 286.03 μmol). 30 mg of the crude product was weighed, purified by preparative high performance liquid chromatography, and freeze-dried to obtain the title compound (12.01 mg, 9.80 μmol, purity 95%).

[0578] Its structural characterization data are as follows:

[0579] MS m / z(ESI): 1164.3 [M+H]+

[0580] Its preparation method is as follows:

[0581] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0582] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0583] Example 6: Synthesis of 2,2'-((2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)azinediyl)diacetic acid (B-4)

[0584] Weigh out 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl) Hexyl-5-acetyleneamide, butyramide, propionamide, benzyl(methyl)amino)-2-oxoethoxy)acetic acid (15 mg, 12.88 μmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (4.06 mg, 25.77 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (5.00 mg, 38.65 μmol) and HATU (7.34 mg, 19.33 μmol). The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (3 mg, 2.32 μmol, purity 98%).

[0585] Its structural characterization data are as follows:

[0586] MS m / z(ESI): 1267.3 [M+H]+

[0587] Its preparation method is as follows:

[0588] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0589] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0590] Example 7: Synthesis of 2-(8-(1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)-14-hydroxy-13,13-bis(hydroxymethyl)-2-methyl-3,7-dioxo-5-oxa-2,8,12-triazatetradecyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-7)

[0591] Weigh out 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyryl (Amine)propionamide)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid (15 mg, 12.88 μmol) and 2,2'-(propane-1,3-diylbis(azanediyl))bis(2-(hydroxymethyl)propane-1,3-diol) (10.91 mg, 38.65 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (5.00 mg, 38.65 μmol) and HATU (7.34 mg, 19.33 μmol). The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (2.36 mg, 1.54 μmol, purity 93%).

[0592] Its structural characterization data are as follows:

[0593] MS m / z (ESI): 1428.5 [M+H]+

[0594] Its preparation method is as follows:

[0595] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0596] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0597] Example 8: Synthesis of 2-((N-methyl-2-(2-oxo-2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-10)

[0598] Weigh out 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl) (15 mg, 12.88 μmol) pyrimidin-5-yl)hex-5-ynylamide)butyramide)propionamide)benzyl)methyl)amino)-2-oxoethoxy)acetic acid) 15 mg, and 4.67 mg, and 25.77 μmol) DMF (1 mL) was dissolved in the solution. Diisopropylethylamine (5.00 mg, and 38.65 μmol) and HATU (7.34 mg, and 19.33 μmol) were added, and the mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (2.5 mg, 1.81 μmol, purity 96%).

[0599] Its structural characterization data are as follows:

[0600] MS m / z(ESI): 1327.4 [M+H]+

[0601] Its preparation method is as follows:

[0602] Chromatographic column: Luna C18(3), 21.2×250mm, 10μm (5μm*19*250mm)

[0603] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0604] Example 9: Synthesis of 2-((N-methyl-2-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-11)

[0605] Weigh out 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin) A mixture of 15 mg (12.88 μmol) of pyridin-5-hexyl-5-ynylamide (butyramide) propionamide (benzyl)(methyl)amino)-2-oxoethoxy)acetic acid and N-methyl-D-glucosamine (5.03 mg, 25.77 μmol) was dissolved in DMF (1 mL). Diisopropylethylamine (5.00 mg, 38.65 μmol) and HATU (7.34 mg, 19.33 μmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction was clearly observed under LC-MS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (1.4 mg, 0.99 μmol, purity 95%).

[0606] Its structural characterization data are as follows:

[0607] MS m / z(ESI): 1341.4 [M+H]+

[0608] Its preparation method is as follows:

[0609] Chromatographic column: Luna C18(3), 21.2×250mm, 10μm (5μm*19*250mm)

[0610] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0611] Example 10: 3-(2-(2-(2-(4-((2-(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl Synthesis of β-cyclodextrin (D-1)

[0612] Step 1: Preparation of 2-((N-methyl-2-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (D-1-1):

[0613] Weigh out 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidine) (-5-yl)hexyl-5-ynylamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid (222 mg, 190.69 μmol) and propargylamine (21.01 mg, 381.37 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (73.93 mg, 572.06 μmol) and HATU (108.69 mg, 286.03 μmol) were added. The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (45 mg, 37.46 μmol).

[0614] Its structural characterization data are as follows:

[0615] MS m / z(ESI): 1201.28 [M+H]+

[0616] Its preparation method is as follows:

[0617] Chromatographic column: Luna C18(3), 21.2×250mm, 10μm (5μm*19*250mm)

[0618] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0619] Step 2: 3-(2-(2-(4-((2-(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl) Preparation of β-cyclodextrin (D-1) (Methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethoxy)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)propionamido-β-cyclodextrin (D-1)

[0620] Take 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propionyl-β-cyclodextrin (34.04 mg, 24.97 μmol) and 2-((N-methyl-2-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butyramide)propionamide)benzyl((1S,9S)-9- Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (30 mg, 24.97 μmol) was dissolved in DMSO (0.8 mL) and water (0.2 mL). Cuprous bromide (3.58 mg, 24.97 μmol) was added with stirring, and the reaction was carried out at room temperature for one hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (5.7 mg, 2.11 μmol, 95%).

[0621] Its structural characterization data are as follows:

[0622] MS m / z (ESI): 1282.4 [1 / 2M+H]+

[0623] Its preparation method is as follows:

[0624] Column: Waters SunFire C18OBD (5μm*19mm*150mm)

[0625] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0626] Example 11: Synthesis of 3,3'-((2-((2-carboxyethoxy)methyl)-2-((3-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (A-4)

[0627] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline 1-(2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (24.29 mg, 57.24 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (11.10 mg, 85.87 μmol) and HATU (16.31 mg, 42.93 μmol). The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (2.60 mg, 1.70 μmol, purity 95%).

[0628] Its structural characterization data are as follows:

[0629] MS m / z (ESI): 1454.5 [M+H]+

[0630] Its preparation method is as follows:

[0631] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0632] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0633] Example 12: Synthesis of 2,2'-(12-carboxy-11-(carboxymethyl)-1-(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)phenyl)-2-methyl-3-oxo-2,5,8,11-tetraazadodecane-5,8-diyl)diacetic acid:(A-7)

[0634] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3] [',4':6,7]Indolezido[1,2-b]quinoline-1-yl)carbamate (30 mg, 28.62 μmol) and 2-(bis(2-(2,6-dioxomorpholine)ethyl)amino)acetic acid (20.45 mg, 57.24 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (11.10 mg, 85.87 μmol) was added. The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and freeze-dried to give the title compound (3.60 mg, 2.28 μmol, purity 90%).

[0635] Its structural characterization data are as follows:

[0636] MS m / z(ESI): 1423.5 [M+H]+

[0637] Its preparation method is as follows:

[0638] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0639] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0640] Example 13: Synthesis of 2,2',2”-(10-(1-carboxy-4-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-11)

[0641] Step 1: Preparation of 1-tert-butyl-5-(4-nitrophenyl)-2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)glutarate (A-11-2):

[0642] Weigh 100 mg (142.67 μmol) of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecano-1-yl)valeric acid and 19.85 mg (142.67 μmol) of 4-nitrophenol and dissolve them in 2 mL of dichloromethane. Add 27.35 mg (142.67 μmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and stir the mixture at room temperature for 4 hours. The product was clearly visible under LCMS monitoring. The reaction solution was extracted with dichloromethane and water, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude title compound (117 mg, 142.34 μmol).

[0643] Its structural characterization data are as follows:

[0644] MS m / z(ESI): 822.5 [M+H]+

[0645] Step 2: Preparation of 2,2',2”-(10-(1-carboxy-4-(4-nitrophenoxy)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-11-3):

[0646] 1-tert-butyl-5-(4-nitrophenyl)-2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)glutarate (117 mg, 142.34 μmol) was added to dichloromethane (1.2 mL) and trifluoroacetic acid (1.8 mL), and the mixture was stirred overnight at 35 °C. The product was clearly visible under LC-MS monitoring. The reaction solution was concentrated under reduced pressure to obtain the crude target product, which was directly used in the next reaction step (73 mg, 122.16 μmol).

[0647] Its structural characterization data are as follows:

[0648] MS m / z (ESI): 1196.4 [2M+H]+

[0649] Step 3: Preparation of 2,2',2”-(10-(1-carboxy-4-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-11):

[0650] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (50 mg, 47.70 μmol) and 2,2' 2”-(10-(1-carboxy-4-(4-nitrophenoxy)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (73 mg, 122.16 μmol) was dissolved in DMF (1 mL), and diisopropylethylamine (18.50 mg, 143.11 μmol) was added. The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and freeze-dried to give the title compound (12.36 mg, 7.96 μmol, purity 97%).

[0651] Its structural characterization data are as follows:

[0652] MS m / z (ESI): 1507.6 [M+H]+

[0653] Its preparation method is as follows:

[0654] Chromatographic column XBridge Prep C18OBD (5μm*19mm*150mm)

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

[0656] Example 14: 17,17-bis((2-carboxyethoxy)methyl)-1-(4-((2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy Synthesis of methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethoxy)acetamido)methyl)-1H-1,2,3-triazol-1-yl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocoane-22-acid (A-12)

[0657] Weigh out 2-((N-methyl-2-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6, 7]Indolezido[1,2-b]quinoline-1-yl)carbamate (50 mg, 41.62 μmol) and 1-azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadococosane-22-acid (25.41 mg, 41.62 μmol) were dissolved in DMSO (0.8 mL) and water (0.2 mL). Cuprous bromide (5.97 mg, 41.62 μmol) was added with stirring, and the reaction was carried out at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. After quenching the reaction solution with water, it was purified by rapid column chromatography (C18, water / acetonitrile = 1 / 1) and freeze-dried to give the title compound (32 mg, 17.66 μmol, purity 99%).

[0658] Its structural characterization data are as follows:

[0659] MS m / z(ESI):906.5[1 / 2M+H]+, 1811.7[M+H]+

[0660] Example 15: 2-(8,8-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-16-hydroxy-15,15-bis(hydroxymethyl)-2-methyl-3,13-dioxo-6,10-dioxa-2,14-diazahexadecyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methyl) Synthesis of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-15)

[0661] Step 1: Preparation of 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (B-15-2):

[0662] 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (2.00 g, 4.71 mmol) was weighed and dissolved in DMF (14 mL). Triethylamine (476.87 mg, 4.71 mmol) was added, followed by 2-(benzyloxy)-1-methylpyridine trifluoromethanesulfonate (1.646 g). The mixture was heated to 80-85 °C and stirred for 16 hours. The product was clearly visible under LCMS monitoring. After concentration, the product was purified by rapid column chromatography (C18, water / acetonitrile = 0.14) and then freeze-dried to give the title compound (2.42 g, 1.44 mmol).

[0663] Its structural characterization data are as follows:

[0664] MS m / z (ESI): 515.2 [M+H]+

[0665] Step 2: Preparation of 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 (B-15-3):

[0666] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (778 mg, 1.51 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (824.25 mg, 6.80 mmol) were dissolved in DMF (15 mL), and diisopropylethylamine (1.17 g, 9.07 mmol) was added. HATU (2.59 g, 6.80 mmol) was added under cold water bath, and the reaction was stirred at room temperature for 2 hours. The product was clearly visible under LCMS monitoring. After concentration, the product was purified by rapid column chromatography (C18, water / acetonitrile = 0.14) and then freeze-dried to give the title compound (1.25 g, 806.19 μmol).

[0667] Its structural characterization data are as follows:

[0668] MS m / z(ESI): 824.4 [M+H]+

[0669] 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 (B-15-4)

[0670] Benzyl 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)propoxy)propionate (738 mg, 806.19 μmol) was dissolved in ethanol (30 mL), 10% palladium on carbon (150 mg, 161.24 μmol) was added, and acetic acid (145.24 mg, 2.42 mmol) was added. Air was removed, hydrogen gas was introduced, and the mixture was stirred at room temperature for 2 hours. The temperature was then increased to 40 °C and stirred for 4 hours. The product was clearly visible under LCMS monitoring. The palladium on carbon was filtered off, and the filtrate was concentrated under reduced pressure. Water (15 mL) and acetonitrile (15 mL) were added to dissolve the product, and the solution was freeze-dried to give the title compound (650 mg, 797.27 μmol).

[0671] Its structural characterization data are as follows:

[0672] MS m / z(ESI): 734.3 [M+H]+

[0673] Step 4: 2-(8,8-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-16-hydroxy-15,15-bis(hydroxymethyl)-2-methyl-3,13-dioxo-6,10-dioxa-2,14-diazahexadecyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methyl) Preparation of sulfonyl(pyrimidin-5-yl)hex-5-ynylamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-15):

[0674] Weigh out 30 mg (28.62 μmol) of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamate. 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 (21.0 mg, 28.62 μmol) was dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (11.10 mg, 85.87 μmol) and HATU (16.31 mg, 42.93 μmol). The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (19.69 mg, 11.05 μmol, purity 99%).

[0675] Its structural characterization data are as follows:

[0676] MS m / z (ESI): 882.6 [1 / 2M+H]+

[0677] Its preparation method is as follows:

[0678] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0679] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0680] Example 16: 2-(9,9-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-17-hydroxy-16,16-bis(hydroxymethyl)-2-methyl-3,7,14-trioxo-5,11-dioxa-2,8,15-triazaheptadecyl)-4-((S)-2-((S)-3-methyl-2-(6-(2- Synthesis of (methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide)butamide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-16)

[0681] Step 1: Preparation of benzyl(9-((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-1,17-dihydroxy-2,2,16,16-tetra(hydroxymethyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecane-9-yl)carbamate (B-16-1):

[0682] 258 mg (266.09 μmol) of bis(perfluorophenyl)-3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate and 128.93 mg (1.06 mmol) of 2-amino-2-(hydroxymethyl)propane-1,3-diol were dissolved in DMF (1 mL). Diisopropylethylamine (103.17 mg (798.27 μmol) was added, and the mixture was stirred overnight at 40 °C. The reaction was monitored by LCMS, and the product was clearly visible. The reaction solution was quenched with water, purified by preparative high-performance liquid chromatography, and freeze-dried to give the title compound (108 mg (138.32 μmol)).

[0683] Its structural characterization data are as follows:

[0684] MS m / z(ESI): 781.4 [M+H]+

[0685] Its preparation method is as follows:

[0686] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0687] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0688] Step 2: Preparation of 3,3'-((2-amino-2-((3-((1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl)propionamide)(B-16-2):

[0689] 108 mg (138.32 μmol) of benzyl (9-((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-1,17-dihydroxy-2,2,16,16-tetra(hydroxymethyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecane-9-yl)carbamate was dissolved in ethanol (4 mL), and palladium on carbon (30.74 mg, 138.32 μmol) was added. The mixture was evacuated, hydrogen gas was introduced, and the reaction was stirred at room temperature for 4 hours. The reaction was monitored by LC-MS, and the product was clearly visible. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the title compound (61 mg, 94.33 μmol).

[0690] Its structural characterization data are as follows:

[0691] MS m / z (ESI): 647.4 [M+H]+

[0692] Step 3: 2-(9,9-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-17-hydroxy-16,16-bis(hydroxymethyl)-2-methyl-3,7,14-trioxo-5,11-dioxa-2,8,15-triazaheptadecyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-( Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-16):

[0693] Weigh out 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid (30m 25.77 μmol of diisopropylethylamine (9.99 mg, 77.31 μmol) and 3,3'-((2-amino-2-((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)propionamide) (16.66 mg, 25.77 μmol) were dissolved in DMF (1 mL), and HATU (14.69 mg, 38.65 μmol) was added. The mixture was stirred at room temperature for 1 hour. The reaction was monitored by LCMS, and the product was obvious. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and freeze-dried to give the title compound (14.00 mg, 7.57 μmol, purity 97%).

[0694] Its structural characterization data are as follows:

[0695] MS m / z(ESI): 1794.4 [M+H]+

[0696] Its preparation method is as follows:

[0697] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0698] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0699] Example 17: Synthesis of (1R,2S,3S,4S,5S,6R)-2,3,4,5-tetrahydroxy-6-methoxycyclohexyl(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)carbamate (B-17)

[0700] Step 1: Preparation of (3aR,4S,5S,5aR,8aR,8bR)-5-methoxy-2,2,7,7-tetramethylhexahydrobenzo[1,2-d:3,4-d']bis([1,3]dioxacyclopentene)-4-yl(4-nitrophenyl) carbonate (B-17-2):

[0701] (3aR,4S,5S,5aR,8aR,8bR)-5-methoxy-2,2,7,7-tetramethylhexahydrobenzo[1,2-d:3,4-d']bis([1,3]dioxacyclopentene)-4-ol (200 mg, 729.10 μmol) and 4-nitrophenyl chloroformate (146.96 mg, 729.10 μmol) were dissolved in dichloromethane (4 mL), and 4-dimethylaminopyridine (133.61 mg, 1.09 mmol) was added. The mixture was stirred at room temperature for two hours. The reaction was monitored by TLC, and the product was clearly visible. After concentration, the product was purified by rapid column chromatography (petroleum ether / ethyl acetate = 3 / 1) to give the title compound (184 mg, 418.74 μmol).

[0702] Step 2: (3aS,4S,5R,5aR,8aR,8bS)-5-methoxy-2,2,7,7-tetramethylhexahydrobenzo[1,2-d:3,4-d']bis([1,3]dioxacyclopentene)-4-yl(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrobenzo[1,2-d:3,4-d']bis([1,3]dioxacyclopentene)-4-yl(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrobenzo[1,2-d:3,4-d']bis([1,3]dioxacyclopentene)-4-yl(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxa ... Preparation of hydrogen-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)carbamate (B-17-3):

[0703] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminomethyl The ester (50 mg, 47.70 μmol) and (3aR,4S,5S,5aR,8aR,8bR)-5-methoxy-2,2,7,7-tetramethylhexahydrobenzo[1,2-d:3,4-d']bis([1,3]dioxane)-4-yl(4-nitrophenyl) carbonate (50 mg, 113.79 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (18.50 mg, 143.11 μmol) was added. The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was added to 0.5 N dilute hydrochloric acid aqueous solution to precipitate the precipitate. The precipitate was filtered, washed with water, and dried under vacuum to obtain the crude title compound (64 mg, 47.46 μmol).

[0704] Its structural characterization data are as follows:

[0705] MS m / z (ESI): 1349.5 [M+H]+

[0706] Step 3: Preparation of (1R,2S,3S,4S,5S,6R)-2,3,4,5-tetrahydroxy-6-methoxycyclohexyl(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)carbamate (B-17):

[0707] Weigh (3aS,4S,5R,5aR,8aR,8bS)-5-methoxy-2,2,7,7-tetramethylhexahydrobenzo[1,2-d:3,4-d']bis([1,3]dioxacyclopentene)-4-yl(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]in A carbamate (64 mg, 47.46 μmol) of dolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)methyl)carbamate (64 mg, 47.46 μmol) was dissolved in methanol (1.5 mL) and dichloromethane (0.5 mL), and 3M hydrochloric acid aqueous solution (1.2 mL) was added dropwise. The mixture was stirred at room temperature for 16 hours. The product was clearly visible under LCMS monitoring. The reaction solution was concentrated under reduced pressure, purified by preparative high performance liquid chromatography, and freeze-dried to obtain the title compound (7.38 mg, 5.53 μmol, purity 95%).

[0708] Its structural characterization data are as follows:

[0709] MS m / z(ESI): 1269.3 [M+H]+

[0710] Its preparation method is as follows:

[0711] Column: Luna C18OBD (5μm*19mm*150mm)

[0712] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0713] Example 18: 2-((N-methyl-2-(2-oxo-2-(((1-((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)-1H-1,2,3-triazol-4-yl) Synthesis of methyl)amino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-29)

[0714] Weigh out 17,17-bis((2-carboxyethoxy)methyl)-1-(4-((2-(2-((2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino) (2-Oxoethoxy)acetamido)methyl)-1H-1,2,3-triazol-1-yl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocoadecan-22-acid (27 mg, 14.90 μmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol (11.64 mg, 59.61 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (17.33 mg, 134.11 μmol) and HATU (25.48 mg, 67.06 μmol). The reaction mixture was stirred at room temperature for 3 hours. The product was clearly visible under LCMS monitoring. The reaction solution was quenched with water, purified by preparative high-performance liquid chromatography, and freeze-dried to obtain the title compound (12.76 mg, 5.28 μmol, purity 97%).

[0715] Its structural characterization data are as follows:

[0716] MS m / z (ESI): 1172.1 [1 / 2M+H]+

[0717] Its preparation method is as follows:

[0718] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0719] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0720] Example 19: 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((17S,18R,19R,20R)-17,18,19,20,21-pentahydroxy-2,15-dimethyl-9,9-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3 Synthesis of (-oxopropoxy)methyl)-3,7,14-trioxo-5,11-dioxa-2,8,15-triazatetradecyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-30)

[0721] Step 1: Preparation of bis(perfluorophenyl)-3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (C-30-2):

[0722] 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (1 g, 2.12 mmol) and 2,3,4,5,6-pentafluorophenol (1.95 g, 10.61 mmol) were dissolved in dichloromethane (16 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.03 g, 10.61 mmol) was added. The mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC, and the product was clearly visible. After concentration, the product was purified by rapid column chromatography (petroleum ether / ethyl acetate = 4 / 1) and concentrated under reduced pressure to give the title compound (2.03 g, 2.10 mmol).

[0723] Step 2: Preparation of benzyl((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 (C-30-3):

[0724] 203 mg (209.36 μmol) of bis(perfluorophenyl)-3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate and 163.48 mg (837.46 μmol) of (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol (163.48 mg (837.46 μmol)) were dissolved in DMF (1 mL), and diisopropylethylamine (81.17 mg (628.09 μmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction was clearly observed by LCMS. The reaction solution was quenched with water, purified by preparative high-performance liquid chromatography, and freeze-dried to obtain the title compound (159 mg (158.52 μmol)).

[0725] Its structural characterization data are as follows:

[0726] MS m / z(ESI): 1004.4 [M+H]+

[0727] Its preparation method is as follows:

[0728] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0729] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0730] 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(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(C-30-4):

[0731] Benzyl((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 (159 mg, 158.52 μmol) was dissolved in ethanol (3 mL) and water (2 mL). Palladium on carbon (50 mg, 224.99 μmol) was added, air was removed, hydrogen gas was introduced, and the reaction was stirred at room temperature for 16 hours. The product was clearly visible under LCMS monitoring. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound (108 mg, 124.29 μmol).

[0732] Its structural characterization data are as follows:

[0733] MS m / z (ESI): 869.4 [M+H]+

[0734] Step 4: 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((17S,18R,19R,20R)-17,18,19,20,21-pentahydroxy-2,15-dimethyl-9,9-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3- Synthesis of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-30):

[0735] Weigh out 30 mg (25.77 μmol) of 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid) 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(oxy))bis(N-methyl-N-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide) (22.39 mg, 25.77 μmol) was dissolved in DMF (1 mL), and diisopropylethylamine (9.99 mg, 77.31 μmol) and HATU (14.69 mg, 38.65 μmol) were added. The mixture was stirred at room temperature for 1 hour. The reaction was clearly observed under LCMS monitoring. The reaction solution was quenched with water, purified by preparative high-performance liquid chromatography, and freeze-dried to obtain the title compound (15.42 mg, 7.58 μmol, purity 99%).

[0736] Its structural characterization data are as follows:

[0737] MS m / z(ESI): 1008.0 [1 / 2M+H]+

[0738] Its preparation method is as follows:

[0739] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0740] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0741] Example 20: 2-((N-methyl-2-(2-oxo-2-(((1-(1-thio-1-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)-5,8,11-trioxa-2-azatridecane-13-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethoxy)acetamido)methyl)-4-((S)-2-( Synthesis of (S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-31)

[0742] Step 1: Preparation of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)thiourea)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (C-31-2):

[0743] (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-isothiocyanate tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (200 mg, 513.64 μmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethylamine (112.10 mg, 513.64 μmol) were dissolved in dichloromethane (4 mL) and reacted overnight at room temperature with stirring. The product was clearly visible under LCMS monitoring. The reaction solution was concentrated under reduced pressure to obtain the target product, which was directly used in the next reaction step.

[0744] Its structural characterization data are as follows:

[0745] MS m / z (ESI): 608.3 [M+H]+

[0746] Step 2: Preparation of 1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thiourea (C-31-3):

[0747] (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)thiourea)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (312.10 mg, 513.64 μmol) was dissolved in methanol (5 mL), and sodium methoxide (221.99 mg, 4.11 mmol) was added with stirring. The reaction mixture was stirred at room temperature for 2 hours. After quenching with water, the reaction solution was purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (101 mg, 229.82 μmol).

[0748] Its structural characterization data are as follows:

[0749] MS m / z(ESI): 440.2 [M+H]+

[0750] Its preparation method is as follows:

[0751] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0752] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0753] Step 3: 2-((N-methyl-2-(2-oxo-2-(((1-(1-thio-1-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)-5,8,11-trioxa-2-azatridecane-13-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethoxy)acetamido)methyl)-4-((S)-2-(( Preparation of (1S,9S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-31):

[0754] Weigh out 1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thiourea (10.24 mg, 23.31 μmol) and 2-((N-methyl-2-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butane A benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (D-1-1) (28 mg, 23.31 μmol) was dissolved in DMSO (0.8 mL) and water (0.2 mL). Cuprous bromide (3.34 mg, 23.31 μmol) was added with stirring, and the reaction was carried out at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and freeze-dried to give the title compound (16.15 mg, 9.35 μmol, purity 95%).

[0755] Its structural characterization data are as follows:

[0756] MS m / z(ESI): 1641.2 [M+H]+

[0757] Its preparation method is as follows:

[0758] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0759] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0760] Example 21: 2-(38-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-23,23-bis((3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide)propionamide pyl)amino)-3-oxopropoxy)methyl)-2-methyl-3,21,28,34-tetraoxo-6,9,12,15, Synthesis of 18,25-hexaoxa-2,22,29,33-tetraazaoctadecyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-32)

[0761] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (30 mg, 28.62 μmol) and 36-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- 21,21-bis((3-((3-(5-((((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)amino)-3-oxopropoxy)methyl)-19,26,32-trioxo-4,7,10,13,16,23-hexaoxa-20,27,31-triazahexahexadecanoic acid (49.69 mg, 28.62 μmol) was dissolved in DMF (1 mL), and HATU (16.31 mg, 42.93 μmol) was added with stirring. Then, diisopropylethylamine (11.10 mg, 85.87 μmol) was added dropwise, and the reaction was carried out at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (17.73 mg, 6.09 μmol, purity 95%).

[0762] Its structural characterization data are as follows:

[0763] MS m / z(ESI):1383.7[1 / 2M+H]+,922.8[1 / 3M+H]+

[0764] Its preparation method is as follows:

[0765] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0766] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0767] Example 22: Synthesis of (R)-4-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)butamido)propamido)benzyl)(methyl)amino)-2-hydroxy-N,N,N-trimethyl-4-oxobutane-1-ammonium (A-13)

[0768] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indole Azido[1,2-b]quinoline-1-yl)carbamate (100 mg, 95.41 μmol) and (S)-3-hydroxy-4-(trimethylamino)butyric acid (15.38 mg, 95.41 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (36.99 mg, 286.22 μmol) and HATU (54.38 mg, 143.11 μmol). The mixture was stirred at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high-performance liquid chromatography, and then freeze-dried to obtain the title compound (46.95 mg, 37.41 μmol, purity 95%).

[0769] Its structural characterization data are as follows:

[0770] MS m / z (ESI): 1192.4 [M]+

[0771] Its preparation method is as follows:

[0772] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0773] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0774] Example 23: Synthesis of 2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)(methyl)amino)acetic acid (A-14)

[0775] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamate (30.00 mg, 28.62 μmol) and 2-iodoacetic acid (5.32 mg, 28.62 μmol), dissolve them in DMF (1 mL), add diisopropylethylamine (7.40 mg, 57.24 μmol), and stir the mixture at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (3.01 mg, 2.59 μmol, purity 95%).

[0776] Its structural characterization data are as follows:

[0777] MS m / z (ESI): 1106.4 [M]+

[0778] Its preparation method is as follows:

[0779] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0780] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% NH4HCO3)

[0781] Example 24: Synthesis of 1-Carboxy-N-(Carboxymethyl)-N-(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)-N-methylmethylammonium (A-15)

[0782] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (30.00 mg, 28.62 μmol) and 2-iodoacetic acid (21.29 mg, 114.49 μmol) and dissolve them in DMF (1 mL). Add diisopropylethylamine (18.50 mg, 143.11 μmol) and stir the mixture at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (4.08 mg, 3.33 μmol, purity 95%).

[0783] Its structural characterization data are as follows:

[0784] MS m / z (ESI): 1106.4 [M]+

[0785] Its preparation method is as follows:

[0786] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0787] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0788] Example 25: Synthesis of 3-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)(methyl)amino)propane-1-sulfonic acid (A-16)

[0789] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (40 mg, 38.16 μmol) and sodium 3-iodopropane-1-sulfonate (10.38 mg, 38.16 μmol), dissolve in DMF (1 mL), add diisopropylethylamine (14.80 mg, 114.49 μmol), and stir the reaction at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (11.40 mg, 9.55 μmol, purity 98%).

[0790] Its structural characterization data are as follows:

[0791] MS m / z (ESI): 1070.4 [M]+

[0792] Its preparation method is as follows:

[0793] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0794] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0795] Example 26: Synthesis of 2-(((2-hydroxyethyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-18)

[0796] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamate (40 mg, 38.16 μmol) and 2-iodoethanol (262.51 mg, 1.53 mmol), dissolve in DMF (1 mL), add diisopropylethylamine (14.80 mg, 114.49 μmol), and stir the reaction at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (14.55 mg, 12.66 μmol, purity 95%).

[0797] Its structural characterization data are as follows:

[0798] MS m / z(ESI): 1093.3 [M+H]+

[0799] Its preparation method is as follows:

[0800] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0801] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0802] Example 27: Synthesis of 1-(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)phenyl)-N,N,N-trimethylmethylammonium (B-19)

[0803] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamate (47 mg, 44.84 μmol) and iodomethane (190.94 mg, 1.35 mmol), dissolve them in DMF (1 mL), add diisopropylethylamine (17.39 mg, 134.52 μmol), and stir the reaction at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (17 mg, 14.99 μmol, purity 95%).

[0804] Its structural characterization data are as follows:

[0805] MS m / z (ESI): 1076.4 [M]+

[0806] Its preparation method is as follows:

[0807] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0808] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0809] Example 28: Synthesis of 2-((dimethylamino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-20)

[0810] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamido)butamido)propamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (50 mg, 47.70 μmol) and paraformaldehyde (4.59 mg, 76.30 μmol), dissolve in THF (3 mL), add sodium cyanoborohydride (14.99 mg, 238.52 μmol), and stir at 45 °C for 4 hours. The product was clearly visible under LCMS monitoring. The reaction solution was quenched with water, purified by preparative high-performance liquid chromatography, and then freeze-dried to obtain the title compound (3.80 mg, 3.40 μmol, purity 95%).

[0811] Its structural characterization data are as follows:

[0812] MS m / z(ESI): 1062.3 [M+H]+

[0813] Its preparation method is as follows:

[0814] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0815] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0816] Example 29: Synthesis of 2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)(methyl)amino)-N,N,N-trimethyl-2-oxoethylammonium (B-21)

[0817] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4'] [6,7]Indolezido[1,2-b]quinoline-1-yl)carbamate (40 mg, 38.16 μmol) and 2-(trimethylammonium)acetate (4.47 mg, 38.16 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (14.80 mg, 114.49 μmol) and HATU (21.75 mg, 57.24 μmol) were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and freeze-dried to obtain the formate of the title compound (14.54 mg, 12.03 μmol, purity 95%).

[0818] Its structural characterization data are as follows:

[0819] MS m / z(ESI): 1148.4 [M+H]+

[0820] Its preparation method is as follows:

[0821] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0822] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0823] Example 30: Synthesis of 2-((methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)butamido)propamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-3)

[0824] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3] [',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (20 mg, 19.08 μmol) and (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (10.31 mg, 57.24 μmol) were dissolved in THF (3 mL), and sodium cyanoborohydride (6.00 mg, 95.41 μmol) was added. The mixture was stirred at 45 °C for 4 hours. The product was clearly visible under LCMS monitoring. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and freeze-dried to give the title compound (5.56 mg, 4.36 μmol, purity 95%).

[0825] Its structural characterization data are as follows:

[0826] MS m / z(ESI): 1213.3 [M+H]+

[0827] Its preparation method is as follows:

[0828] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0829] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0830] Example 31: Synthesis of 2-((N-methyl-2-(2-oxo-2-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (C-35)

[0831] Weigh out 2-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propionamido (40 mg, 34.36 μmol) benzyl(methyl)amino)-2-oxoethoxy)acetic acid and (2S,3R,4R,5S,6R)-2-(aminomethyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (6.64 mg, 34.36 μmol) were dissolved in DMF (1 mL), followed by the addition of diisopropylethylamine (13.32 mg, 103.07 μmol) and HATU (19.58 mg, 51.54 μmol). The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (13.87 mg, 10.04 μmol, purity 97%).

[0832] Its structural characterization data are as follows:

[0833] MS m / z(ESI): 1339.5 [M+H]+

[0834] Its preparation method is as follows:

[0835] Column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0836] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0837] Example 32: 2-(43-methyl-15,22,38,42-tetraoxo-20,20-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azanonadecan-19-yl)-2,5,8,11,18,25,28,31,34-nonaoxa-14,21,37,43-tetraazatetradecane-44-yl)-4-((S)-2-((S)-3-methyl-2 Synthesis of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (A-17)

[0838] Weigh out 30 mg of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (A-2-4). 2,5-dioxopyrrolidone-1-yl15,22,38-trioxo-20,20-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azadecane-19-yl)-2,5,8,11,18,25,28,31,34-nonoxa-14,21,37-triazatetracosan-42-ester (39.03 mg, 28.62 μmol) was dissolved in DMF (1 mL), and diisopropylethylamine (11.10 mg, 85.87 μmol) was added. The mixture was stirred at room temperature for 1 hour. The product was clearly visible under LCMS monitoring. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to give the title compound (30.5 mg, 12.62 μmol, purity 95%).

[0839] Its structural characterization data are as follows:

[0840] MS m / z(ESI): 1148.7 1 / 2[M+H]+

[0841] Its preparation method is as follows:

[0842] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0843] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0844] Example 33: Synthesis of (2S,3S,4S,5R,6S)-6-(4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(3-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)propamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (C-36)

[0845] Step 1: Preparation of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (C-36-2):

[0846] (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (5 g, 10.30 mmol) was dissolved in methanol, and 10% palladium on carbon (1 g, 10.30 mmol) was added. The mixture was stirred overnight under hydrogen purging and protection. The solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure.

[0847] Its structural characterization data are as follows:

[0848] MS m / z(ESI): 456.2 [M+H]+

[0849] Step 2: Preparation of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (C-36-3):

[0850] (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (2.3 g, 5.05 mmol) and 3-(9H-fluorene-9-ylmethoxycarbonylamino)propionic acid (1.89 g, 6.06 mmol) were dissolved in DMF (20 mL). HATU (3.84 g, 10.10 mmol) was added with stirring, followed by dropwise addition of diisopropylethylamine (1.31 g, 10.10 mmol, 1.78 mL). The reaction was continued for 1 hour. The solution was purified by rapid column chromatography (C18, 0-85% acetonitrile / 0.5% trifluoroacetic acid in water) and then freeze-dried to give the title compound (2.0 g, 2.67 mmol).

[0851] Its structural characterization data are as follows:

[0852] MS m / z(ESI): 749.3 [M+H]+

[0853] Step 3: Preparation of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (C-36-4):

[0854] (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (0.5 g, 667.80 μmol) was dissolved in dry dichloromethane (6 mL). The mixture was cooled and stirred to 0 °C under nitrogen purging and protection. Triethylamine (135.15 mg, 1.34 mmol, 193.07 μL) was added dropwise, followed by the slow addition of a solution of (4-nitrophenyl)chloroformate (201.91 mg, 1.00 mmol dissolved in 1 mL of dry dichloromethane). The reaction was continued for 1.5 hours. After purification by rapid column chromatography (silica gel column, 0-100% ethyl acetate / petroleum ether), the compound was concentrated again to give the title compound (390 mg, 426.77 μmol).

[0855] Its structural characterization data are as follows:

[0856] MS m / z(ESI): 914.2 [M+H]+

[0857] Step 4: Preparation of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (C-36-5):

[0858] (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (340 mg, 372.06 μmol) and (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy 4-Methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (217.55 mg, 409.27 μmol, methanesulfonate) was dissolved in DMF (4 mL), and diisopropylethylamine (144.26 mg, 1.12 mmol, 197.07 μL) was added dropwise. The mixture was stirred for 1.5 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was extracted twice with ethyl acetate and the extracts were combined. The organic phase was washed three times with saturated brine, dried, and concentrated under reduced pressure. After purification by rapid column chromatography (silica gel column, 0-100% ethyl acetate / petroleum ether), the mixture was concentrated again to give the title compound (230 mg, 245.42 μmol).

[0859] Its structural characterization data are as follows:

[0860] MS m / z(ESI): 1211.5 [M+H]+

[0861] Step 5: Preparation of (2S,3S,4S,5R,6S)-6-(2-(3-aminopropionamido)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (C-36-6):

[0862] (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7) Indolezino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (130 mg, 107.42 μmol) was dissolved in tetrahydrofuran (1 mL), and lithium hydroxide aqueous solution (22.54 mg, 537.12 μmol, dissolved in 0.1 mL water) was added dropwise. The reaction mixture was stirred for 1 hour. The reaction solution was neutralized by adding 1N hydrochloric acid aqueous solution, and the mixture was purified by rapid column chromatography (C18 column, 0-100% ethyl acetate / petroleum ether) and then concentrated again to give the title compound (18 mg, 21.23 μmol).

[0863] Its structural characterization data are as follows:

[0864] MS m / z (ESI): 848.4 [M+H]+

[0865] Step Six: Preparation of (2S,3S,4S,5R,6S)-6-(4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(3-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamido)propamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (C-36):

[0866] (2S,3S,4S,5R,6S)-6-(2-(3-aminopropionamido)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)- 3,4,5-Trihydroxytetrahydro-2H-pyran-2-carboxylic acid (18 mg, 21.23 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (7.46 mg, 21.23 μmol) were dissolved in DMF (1.00 mL), and DIPEA (8.23 mg, 63.69 μmol, 11.25 μL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (6.6 mg, 11.05 μmol, 99% purity).

[0867] Its structural characterization data are as follows:

[0868] MS m / z(ESI): 1098.4 [M+H]+

[0869] Its preparation method is as follows:

[0870] Column: Fenox C18OBD (5μm*19mm*150mm)

[0871] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0872] Example 34: Synthesis of 2-((2-(dimethylamino)-N-methylacetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (B-23):

[0873] Weigh out 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4': [6,7]Indolezido[1,2-b]quinoline-1-yl)carbamate (50 mg, 47.70 μmol) and dimethylglycine (9.84 mg, 95.41 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (18.50 mg, 143.11 μmol, 24.93 μL) was added dropwise. HATU (27.19 mg, 71.56 μmol) was then added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was quenched with water, purified by preparative high performance liquid chromatography, and freeze-dried to obtain the formate salt of the title compound (21.02 mg, 17.62 μmol, purity 95%).

[0874] Its structural characterization data are as follows:

[0875] MS m / z (ESI): 1133.4 [M+H] +

[0876] Its preparation method is as follows:

[0877] Column: SHIMADZUPrep C18 OBD (5μm*19mm*150mm)

[0878] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% HCOOH)

[0879] Example 35: Preparation of 2,2',2”-(10-(2-(((S)-6-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-4)

[0880] Step 1: Preparation of (S)-(2-((8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)tert-butyl carbamate (G-3-1)

[0881] (tert-Butoxycarbonyl)glycine (62.98 mg, 359.52 μmol) and (S)-3-amino-8-ethyl-8-hydroxy-2,8,11,14-tetrahydro-12H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(1H)-dione (100 mg, 179.76 μmol) were dissolved in DMF (2 mL), DIPEA (58.08 mg, 449.41 μmol) was added dropwise, and HATU (143.45 mg, 377.50 μmol) was added. The mixture was stirred overnight at room temperature. After the reaction was completed, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 50-100%, then ethyl acetate-methanol = 0-10%, and finally dichloromethane-methanol = 10:1 for finishing) and concentrated under reduced pressure again to give the title compound (78 mg, 142.71 μmol) as a yellow solid.

[0882] Its structural characterization data are as follows:

[0883] MS m / z (ESI): 547.2 [M+H] +

[0884] Step 2: Preparation of (S)-2-amino-N-(8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)acetamide (G-3-2)

[0885] Dichloromethane (2 mL) and trifluoroacetic acid (2 mL) were added to (S)-(2-((8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadieno[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)carbamate tert-butyl (78 mg, 142.71 μmol), and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure, and the crude product was lyophilized with water and acetonitrile to give the trifluoroacetate of the title compound (79 mg, 140.95 μmol).

[0886] Its structural characterization data are as follows:

[0887] MS m / z (ESI): 447.1 [M+H] +

[0888] Step 3: Preparation of N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (C-3-3)

[0889] N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (46.96 mg, 62.45 μmol) and (S)-2-amino-N-(8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)acetamide trifluoroacetate (35 mg, 62.45 μmol) were dissolved in DMF (2 mL), DIPEA (48 mg, 371.40 μmol) was added, followed by PyBOP (48.75 mg, 93.67 μmol), and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (150 mg, 50.83 μmol, 40% purity), which was used directly in the next reaction without purification.

[0890] Its structural characterization data are as follows:

[0891] MS m / z (ESI): 1180.3 [M+H] +

[0892] Step 4: Preparation of N-((S)-1-(((S)-6-amino-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (G-3-4)

[0893] To N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-2-oxoethyl Dichloromethane (3 mL) was added to the crude product of (150 mg, 50.83 μmol, 40% purity)-1-oxohexane-2-yl)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide. Then, trifluoroacetic acid (0.5 mL) was added dropwise. The system turned yellow instantly, and the reaction was stirred at room temperature for 1.0 h. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The crude product was purified by high-performance liquid chromatography and then freeze-dried to obtain the trifluoroacetate salt of the title compound (20 mg, 18.88 μmol, 98% purity).

[0894] Its structural characterization data are as follows:

[0895] MS m / z (ESI): 924.4 [M+H] +

[0896] Its preparation method is as follows:

[0897] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0898] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0899] Step 5: Preparation of 2,2',2”-(10-(2-(((S)-6-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-4)

[0900] Dissolve 10 mg (9.63 μmol) of trifluoroacetate of N-((S)-1-(((S)-6-amino-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide in DMF (3 mL), and add 2,2',2”-( 12.08 mg (24.08 μmol) of 10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid was added dropwise, followed by the addition of DIPEA (8 mg, 61.90 μmol), and the mixture was stirred at room temperature for 1 hour. DIPEA (8 mg, 61.90 μmol) was then added again, and the mixture was stirred at room temperature for another hour. After the reaction was complete, the reaction solution was acidified with trifluoroacetic acid, the solvent was removed by lyophilization, and the solution was purified by high-performance liquid chromatography and then freeze-dried to obtain the trifluoroacetate of the title compound (5.43 mg, 3.55 μmol, 93.2% purity).

[0901] Its structural characterization data are as follows:

[0902] MS m / z (ESI): 1312.4 [M+H] +

[0903] Its preparation method is as follows:

[0904] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

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

[0906] Example 36: 2,2',2”-(10-(2-(((S)-6-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline) Preparation of -1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-7)

[0907] Step 1: Preparation of (9H-fluorene-9-yl)methyl(2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (G-7-4)

[0908] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (240 mg, 435.89 μmol) was dissolved in THF (10 mL). After purging with nitrogen three times, lithium hydroxide monohydrate (109.74 mg, 2.62 mmol) was added under ice bath conditions. After stirring at room temperature for 10 minutes, (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (642.29 mg, 1.74 mmol) was added. After the addition was complete, the mixture was stirred at 22 °C for 5 hours. Add 20 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract three times with dichloromethane (10 mL x 3), wash with 10 mL of brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product of the title compound. After purification by preparative high performance liquid chromatography, freeze-dry to obtain the title compound (100 mg, 116.42 μmol).

[0909] Its structural characterization data are as follows:

[0910] MS m / z (ESI): 859.3 [M+H] +

[0911] Its preparation method is as follows:

[0912] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0913] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0914] Step 2: Preparation of (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-7-3)

[0915] (9H-fluorene-9-yl)methyl(2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (40 mg, 46.57 μmol) was dissolved in DMF (2 mL), and diethylamine (17.03 mg, 232.85 μmol) was added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (12 mg, 18.85 μmol).

[0916] Its structural characterization data are as follows:

[0917] MS m / z (ESI): 637.3 [M+H] +

[0918] Its preparation method is as follows:

[0919] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0920] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0921] Step 3: Preparation of (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-7-2)

[0922] (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (100 mg, 133.21 μmol) and N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (110.18 mg, 146.53 μmol) was dissolved in DMF (2 mL), and DIPEA (68.86 mg, 532.82 μmol) and HATU (119.37 mg, 314.12 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (60 mg, 43.78 μmol).

[0923] Its structural characterization data are as follows:

[0924] MS m / z (ESI): 1370.5 [M+H] +

[0925] Its preparation method is as follows:

[0926] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0927] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0928] Step 4: Preparation of (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-7-1)

[0929] (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (80 mg, 58.37 μmol) was dissolved in DCM (5 mL), and formic acid (1 mL) was added. The mixture was stirred at 22 °C for 4 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (11 mg, 8.51 μmol, purity 95%).

[0930] Its structural characterization data are as follows:

[0931] MS m / z (ESI): 1114.4 [M+H] +

[0932] Its preparation method is as follows:

[0933] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0934] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0935] Step 5: 2,2',2”-(10-(2-(((S)-6-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline- Preparation of 1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-7)

[0936] The (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxyl Amine (7 mg, 6.28 μmol) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9.45 mg, 18.85 μmol) were dissolved in DMF (2 mL). DIPEA (1.0 mg, 7.74 μmol) was added, and the mixture was stirred at 22 °C for 2 hours. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (3.2 mg, 2.03 μmol, purity 95%).

[0937] Its structural characterization data are as follows:

[0938] MS m / z (ESI): 1500.6 [M+H] +

[0939] Its preparation method is as follows:

[0940] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0941] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0942] Example 37: 2-((N-methyl-2-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propionamido Preparation of benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-21)

[0943] Step 1: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-21-1)

[0944] INT-6-3 (6.18 g, 8.72 mmol) and (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (1.50 g, 2.18 mmol, 80% purity) were dissolved in DMF (20 mL) and pyridine (10 mL). 1-hydroxybenzotriazole (1.18 g, 0.93 mmol) was added, and the mixture was stirred at 85 °C for 1 hour. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (328 mg, 324.11 μmol).

[0945] Its structural characterization data are as follows:

[0946] MS m / z (ESI): 898.3 [M+H] +

[0947] Its preparation method is as follows:

[0948] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0949] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0950] Step 2: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-21-2)

[0951] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazole Alkyl-3-carboxylic acid ester (225 mg, 222.33 μmol) was dissolved in DMF (20 mL), and N,N-diisopropylethylamine (172.41 mg, 1.33 mmol), Fmoc-L-valine (264.10 mg, 778.17 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (262.12 mg, 889.34 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (125 mg, 102.51 μmol).

[0952] Its structural characterization data are as follows:

[0953] MS m / z (ESI): 1219.4 [M+H] +

[0954] Spectral column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0955] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0956] Step 3: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-21-3)

[0957] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (125 mg, 102.51 μmol) was dissolved in DMF (6 mL), and DBU (46.82 mg, 307.54 μmol) was added. The mixture was stirred at room temperature for 20 minutes. After the reaction was complete, the compound was purified by rapid column chromatography (C18, water / acetonitrile = 0-50%, 0.05% TFA) and then freeze-dried to obtain the title compound (91 mg, 81.90 μmol).

[0958] Its structural characterization data are as follows:

[0959] MS m / z(ESI): 998.0 [M+H]+

[0960] Step 4: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-21-4)

[0961] 2-(((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminomethyl Acyl)thiazolidin-3-carboxylic acid ester (55 mg, 49.50 μmol) was dissolved in DMF (5 mL), and N,N-diisopropylethylamine (31.99 mg, 247.50 μmol), 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ethynic acid (26.56 mg, 99.00 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (43.77 mg, 148.50 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (45 mg, 36.08 μmol).

[0962] Its structural characterization data are as follows:

[0963] MS m / z (ESI): 1247.4 [M+H] +

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

[0965] Step 5: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-21-5)

[0966] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ [de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazoline-3-carboxylic acid ester (45 mg, 35.72 μmol) was dissolved in DMF (6 mL), followed by the addition of 1,3-dimethylbarbituric acid (27.88 mg, 178.58 μmol) and tetrakis(triphenylphosphine)palladium (20.64 mg, 17.86 μmol). The mixture was evacuated, purged with nitrogen three times, and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was analyzed by high performance liquid chromatography to obtain the title compound (29 mg, 24.68 μmol).

[0967] Its structural characterization data are as follows:

[0968] MS m / z (ESI): 1163.3 [M+H] +

[0969] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0970] Step Six: Preparation of 2-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamido)butamido)propamido)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid (A-21-6)

[0971] 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano [3',4':6,7]indolazino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-carboxylic acid ester (85 mg, 73.07 μmol) was dissolved in DMF (3 mL), and 2,2'-oxodiacetic acid (15.68 mg, 116.91 μmol), DIPEA (37.77 mg, 292.27 mmol), and HATU (55.56 mg, 146.14 μmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. The title compound (52.05 mg, 40.65 μmol) was obtained by high performance liquid chromatography after the solvent was directly removed from the reaction solution.

[0972] Its structural characterization data are as follows:

[0973] ESI-MS (m / z): 1279.3 (M+H) + .

[0974] The purification method is as follows:

[0975] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0976] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0977] Step 7: 2-((N-methyl-2-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido) Preparation of benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-21)

[0978] 2-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-( 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid (19 mg, 14.85 μmol) was dissolved in DMF (3 mL), and N-methyl-D-glucosamine (2.90 mg, 14.85 μmol), DIPEA (9.60 mg, 74.26 mmol), and HATU (8.47 mg, 22.28 μmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. After the solvent was directly removed from the reaction solution, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (12.9 mg, 8.50 μmol).

[0979] Its structural characterization data are as follows:

[0980] ESI-MS (m / z): 1457.3 (M+H) + .

[0981] The purification method is as follows:

[0982] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0983] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0984] Example 38: 2-((N-methyl-2-(2-oxo-2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)acetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido) Preparation of benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-22)

[0985] 2-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2 -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)(methyl)amino)-2-oxoethoxy)acetic acid (19 mg, 14.85 μmol) was dissolved in DMF (3 mL), and D-glucosamine (4.04 mg, 22.28 μmol), DIPEA (7.68 mg, 59.40 mmol), and HATU (11.29 mg, 29.70 μmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. After the solvent was directly removed from the reaction solution, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (16.60 mg, 11.56 μmol).

[0986] Its structural characterization data are as follows:

[0987] ESI-MS (m / z): 1443.3 (M+H) + .

[0988] The purification method is as follows:

[0989] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0990] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0991] Example 39: 2-(43-methyl-15,22,38,42-tetraoxo-20,20-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azanonadecan-19-yl)-2,5,8,11,18,25,28,31,34-nonaoxa-14,21,37,43-tetraazatetratetradecane-44-yl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl) Preparation of acyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-23)

[0992] 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3 A carboxylic acid ester (5 mg, 4.3 μmol) was dissolved in DMF (3 mL), and 2,5-dioxopyrrolidone-1-yl15,22,38-trioxo-20,20-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azanonadecan-19-yl)-2,5,8,11,18,25,28,31,34-nonaoxa-14,21,37-triazatetrazo-dodecane-42-carboxylic acid ester (5.86 mg, 4.3 μmol) and DIPEA (2.78 mg, 21.49 μmol) were added. The reaction mixture was stirred at 45 °C for 4 hours. After the solvent was directly removed from the reaction solution, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (3.42 mg, 1.38 μmol).

[0993] Its structural characterization data are as follows:

[0994] ESI-MS (m / z): 1206.6 (M+H / 2) + .

[0995] The purification method is as follows:

[0996] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0997] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0998] Example 40: Preparation of 2-((2-(dimethylamino)-N-methylacetamido)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (A-24)

[0999] 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de [16 mg, 13.75 μmol]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester was dissolved in DMF (3 mL), and dimethylglycine (2.13 mg, 20.63 μmol), DIPEA (5.33 mg, 41.26 μmol), and HATU (10.46 mg, 27.51 μmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. After the solvent was directly removed from the reaction solution, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (11.50 mg, 8.75 μmol).

[1000] Its structural characterization data are as follows:

[1001] ESI-MS (m / z): 1248.3 (M+H) + .

[1002] The purification method is as follows:

[1003] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1004] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1005] Example 41: Preparation of 2,2',2”-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-10)

[1006] Step 1: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (A-10-7)

[1007] L-alanine (73.15 mg, 821.92 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (100.0 mg, 273.97 μmol) were dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (106.03 mg, 821.92 μmol) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (60 mg, 163.49 μmol).

[1008] Its structural characterization data are as follows:

[1009] MS m / z(ESI): 367.2 [M+H]+

[1010] Step 2: Preparation of (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)butamido)propamido)benzyl)(methyl)carbamate (A-10-8)

[1011] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (30 mg, 81.74 μmol) and INT-6-5 (63.95 mg, 81.74 μmol) were dissolved in DMF (1 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (34.17 mg, 89.91 μmol) and DIPEA (31.63 mg, 245.22 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 1 / 2) and then freeze-dried to obtain the title compound (45 mg, 39.75 μmol).

[1012] Its structural characterization data are as follows:

[1013] MS m / z(ESI): 1132.2 [M+H] +

[1014] Step 3: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (A-10-9)

[1015] (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl) Amino-butamido-propamido-benzyl-(methyl)carbamate (45 mg, 39.75 μmol) was dissolved in DMF (1 mL), and tetrakis(triphenylphosphine)palladium (4.6 mg, 3.98 μmol) was added, followed by 15 μL of formic acid and 30 μL of N-methylmorpholine. The mixture was stirred at room temperature for 1 hour under nitrogen protection. After the reaction was completed, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 4 / 1) and then freeze-dried to obtain the title compound (27 mg, 25.76 μmol).

[1016] Its structural characterization data are as follows:

[1017] MS m / z (ESI): 1048.4 [M+H] +

[1018] Step 4: Preparation of 2,2',2”-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-10)

[1019] 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (27 mg, 25 mg). 76 μmol) was dissolved in DMF (1 mL), DIPEA (9.97 mg, 77.28 μmol) was added, followed by 2,2',2”-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (38.76 mg, 77.28 μmol). After the reaction was complete, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (3.68 mg, 2.56 μmol).

[1020] Its structural characterization data are as follows:

[1021] MS m / z (ESI): 1434.6 [M+H] +

[1022] Its preparation method is as follows:

[1023] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[1024] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1025] Example 42: 3,3'-((2-((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-2-((11S,14S)-11-((2-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[ Preparation of 1,2-b]quinoline-3-yl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methanesulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazaeicosoe-20-yn-1-yl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)propamide) (E-4)

[1026] Step 1: Preparation of (S)-(2-((8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadieno[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)tert-butyl carbamate (E-4-4)

[1027] (tert-Butoxycarbonyl)glycine (62.98 mg, 359.52 μmol) and (S)-3-amino-8-ethyl-8-hydroxy-2,8,11,14-tetrahydro-12H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(1H)-dione (100 mg, 179.76 μmol) were dissolved in DMF (2 mL), DIPEA (58.08 mg, 449.41 μmol) was added dropwise, and HATU (143.45 mg, 377.50 μmol) was added. The mixture was stirred overnight at room temperature. After the reaction was completed, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 50-100%, then ethyl acetate-methanol = 0-10%, and finally dichloromethane-methanol = 10:1 for finishing) and concentrated under reduced pressure again to obtain the title compound (78 mg, 142.71 μmol).

[1028] Its structural characterization data are as follows:

[1029] MS m / z (ESI): 547.2 [M+H] +

[1030] Step 2: Preparation of (S)-2-amino-N-(8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)acetamide (E-4-3)

[1031] Dichloromethane (2 mL) and trifluoroacetic acid (2 mL) were added to (S)-(2-((8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadieno[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)carbamate tert-butyl (78 mg, 142.71 μmol), and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure, and the crude product was lyophilized with water and acetonitrile to give the trifluoroacetate of the title compound (79 mg, 140.95 μmol).

[1032] Its structural characterization data are as follows:

[1033] MS m / z (ESI): 447.1 [M+H] +

[1034] Step 3: Preparation of N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (E-4-2)

[1035] N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (46.96 mg, 62.45 μmol) and (S)-2-amino-N-(8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)acetamide trifluoroacetate (35 mg, 62.45 μmol) were dissolved in DMF (2 mL), DIPEA (48 mg, 371.40 μmol) was added, followed by PyBOP (48.75 mg, 93.67 μmol), and the reaction was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (150 mg, 50.83 μmol, 40% purity), which was used directly in the next reaction without purification.

[1036] Its structural characterization data are as follows:

[1037] MS m / z (ESI): 1180.3 [M+H] +

[1038] Step 4: Preparation of N-((S)-1-(((S)-6-amino-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (E-4-1)

[1039] To N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-2-oxo Dichloromethane (3 mL) was added to crude ethyl(amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (150 mg, 50.83 μmol, 40% purity), followed by the addition of 0.5 mL of trifluoroacetic acid. The system turned yellow instantly, and the reaction was stirred at room temperature for 1.0 h. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The crude product was purified by high performance liquid chromatography and then freeze-dried to obtain the trifluoroacetate salt of the title compound (20 mg, 18.88 μmol, 98% purity).

[1040] Its structural characterization data are as follows:

[1041] MS m / z (ESI): 924.4 [M+H] +

[1042] Its preparation method is as follows:

[1043] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[1044] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1045] Step 5: 3,3'-((2-((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-2-((11S,14S)-11-((2-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1, Preparation of 2-b]quinoline-3-yl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methanesulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazaeicosoe-20-yn-1-yl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)propamide) (E-4)

[1046] N-((S)-1-(((S)-6-amino-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methylsulfonyl) The trifluoroacetate of pyrimidin-5-yl)hex-5-yneamide (35 mg, 33.72 μmol) and 3-[3-(2-carboxyethoxy)-2,2-bis(2-carboxyethoxymethyl)propoxy]propionic acid (19.29 mg, 45.45 μmol) were dissolved in DMF (2.5 mL), PyBOP (29.57 mg, 56.82 μmol) was added, and then DIPEA (48 mg, 371.40 μmol) was added dropwise. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (4.94 mg, 3.53 μmol, 95% purity).

[1047] Its structural characterization data are as follows:

[1048] MS m / z (ESI): 1330.4 [M+H] +

[1049] Its preparation method is as follows:

[1050] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[1051] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[1052] Example 43: 3,3'-((2-((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-2-((11S,14S)-11-((2-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[ Preparation of 1,2-b]quinoline-3-yl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methanesulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazaeicosoe-20-yn-1-yl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)propamide) (J-4)

[1053] Trifluoroacetate of N-((S)-1-(((S)-6-amino-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (20 mg, 19 mg). 27 μmol) 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 (20.00 mg, 24.53 μmol, 90% purity) was dissolved in DMF (2 mL), and Carter's condensing agent (17.04 mg, 38.53 μmol) was added. DIPEA (32 mg, 247.60 μmol) was added dropwise, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was directly purified by high-performance liquid chromatography and then freeze-dried to obtain the title compound (10.69 mg, 6.00 μmol, 92% purity).

[1054] Its structural characterization data are as follows:

[1055] MS m / z (ESI): 1639.8 [M+H] +

[1056] Its preparation method is as follows:

[1057] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[1058] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1059] Example 44 3,3'-((2-((11S,14S)-11-((2-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-( Preparation of (methylsulfonyl)pyranimidazole-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazaeicosano-20-yn-1-yl)-2-((3-(methyl(2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-(2,3,4,5,6-pentahydroxyhexyl)propionamide)(L-4)

[1060] The trifluoroacetate of N-((S)-1-(((S)-6-amino-1-((2-((((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-2-oxoethyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (20 mg, 19.27 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 (20.47 mg, 19.27 μmol) was dissolved in DMF (2 mL), HATU (8.05 mg, 21.19 μmol) was added, and DIPEA (16 mg, 123.80 μmol) was added dropwise. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (2.45 mg, 1.29 μmol, 98% purity).

[1061] Its structural characterization data are as follows:

[1062] MS m / z (ESI): 1862.6 [M+H] +

[1063] Its preparation method is as follows:

[1064] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[1065] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1066] Example 45: (17S,20S)-6,6-bis((2-carboxyethoxy)methyl)-17-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6, Preparation of [7]indoazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-20-isopropyl-27-(2-(methylsulfonyl)pyrimidin-5-yl)-11,19,22-trioxo-4,8-dioxa-12,18,21-triazaheptadecane-26-alkynic acid (E-5)

[1067] The (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]in Dolino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (20 mg, 16.28 μmol) and 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (27.64 mg, 65.13 μmol) were dissolved in DMF (2 mL). DIPEA (8.42 mg, 65.13 μmol) and HATU (13.64 mg, 35.90 μmol) were added, and the mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (5.0 mg, 3.09 μmol, purity 94%).

[1068] Its structural characterization data are as follows:

[1069] MS m / z (ESI): 1520.4 [M+H] +

[1070] Its preparation method is as follows:

[1071] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1072] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1073] Example 46: 3,3'-((2-((3-((1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)amino)-3-oxopropoxy)methyl)-2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indole) Preparation of azido[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazacotetradecane-20-yn-1-yl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)propamide) (J-5)

[1074] (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)thiazolidin-4-carboxamide Dissolve 6 mg (4.85 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 (5.93 mg, 7.27 μmol) in DMF (1 mL), add 2,6-dimethylpyridine (2.08 mg, 19.38 μmol) and HATU (3.69 mg, 9.69 μmol), and stir at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (3.18 mg, 1.65 μmol, purity 95%).

[1075] Its structural characterization data are as follows:

[1076] MS m / z(ESI): 1829.7 [M+H] +

[1077] Its preparation method is as follows:

[1078] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1079] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1080] Example 47: 3,3'-((2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl- Preparation of 21-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazacotetradecane-20-yn-1-yl)-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)(L-5)

[1081] (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (20 mg, 16.28 μL) 20.24 mg (21.17 μmol) of methylpropoxy ((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 (20.24 mg, 21.17 μmol) was dissolved in DMF (2 mL), followed by the addition of DIPEA (8.42 mg, 65.13 μmol) and HATU (12.37 mg, 32.57 μmol), and the mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (8.8 mg, 4.07 μmol, purity 95%).

[1082] Its structural characterization data are as follows:

[1083] MS m / z (ESI): 1026.9 [M / 2+H] +

[1084] Its preparation method is as follows:

[1085] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1086] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1087] Coupling Examples

[1088] 1. Preparation of Trastuzumab-A-6

[1089] Take 0.957 mL of Trastuzumab antibody (20.9 mg / mL), add 48.0 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, 75.8 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-6 solution dissolved in dimethyl sulfoxide (192.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-A-6). The DAR value was determined by mass spectrometry to be 7.6.

[1090] 2. Preparation of Trastuzumab-B-4

[1091] Take 0.529 mL of Trastuzumab antibody (24 mg / mL), add 26.3 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 48.3 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Then add 12 times the amount of B-4 dissolved in dimethyl sulfoxide (107.0 μL, 10 mM), mix well, and incubate at room temperature for 2 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 (Trastuzumab-B-4). The DAR value was determined by mass spectrometry to be 8.0.

[1092] 3. Preparation of Trastuzumab-C-10

[1093] Take 0.404 mL of Trastuzumab antibody (24 mg / mL), add 20.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 36.9 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 12 times the amount of C-10 solution dissolved in dimethyl sulfoxide (84.0 μL, 10 mM), mix well, and let stand 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 C-10). The DAR value was determined by mass spectrometry to be 8.1.

[1094] 4. Preparation of Trastuzumab-C-11

[1095] Take 0.361 mL of Trastuzumab antibody (24 mg / mL), add 17.9 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 32.9 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 12 times the amount of C-11 solution dissolved in dimethyl sulfoxide (75.0 μL, 10 mM), mix well, and let stand 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-C-11). The DAR value was determined by mass spectrometry to be 8.1.

[1096] 5. Preparation of Trastuzumab-D-1

[1097] Take 0.625 mL of Trastuzumab antibody (24 mg / mL), add 31.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 12 times the amount of D-1 dissolved in dimethyl sulfoxide (144.0 μL, 10 mM) solution and mix well. 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-D-1). The DAR value was determined by mass spectrometry to be 3.9.

[1098] 6. Preparation of Trastuzumab-B-7

[1099] Take 0.164 mL of Trastuzumab antibody (24 mg / mL), add 8.1 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, 15.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-7 solution dissolved in dimethyl sulfoxide (35.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-B-7). The DAR value was determined by mass spectrometry to be 2.4.

[1100] 7. Preparation of Trastuzumab-B-19

[1101] Take 0.781 mL of Trastuzumab antibody (19.2 mg / mL), add 39.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-19 solution dissolved in dimethyl sulfoxide (132.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-B-19). The DAR value was determined by mass spectrometry to be 7.75.

[1102] 8. Preparation of Trastuzumab-B-18

[1103] Take 0.781 mL of Trastuzumab antibody (19.2 mg / mL), add 39.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-18 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-B-18). The DAR value was determined by mass spectrometry to be 5.56.

[1104] 9. Preparation of Trastuzumab-A-16

[1105] Take 0.781 mL of Trastuzumab antibody (19.2 mg / mL), add 39.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-16 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-A-16). The DAR value was determined by mass spectrometry to be 7.76.

[1106] 10. Preparation of Trastuzumab-A-14

[1107] Take 0.781 mL of Trastuzumab antibody (19.2 mg / mL), add 39.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-14 solution dissolved in dimethyl sulfoxide (130.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-A-14). The DAR value was 7.29 by mass spectrometry.

[1108] 11. Preparation of Trastuzumab-A-15

[1109] Take 0.781 mL of Trastuzumab antibody (19.2 mg / mL), add 39.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-15 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-A-15). The DAR value was determined by mass spectrometry to be 7.81.

[1110] 12. Preparation of Trastuzumab-A-13

[1111] Take 0.728 mL of Her2 antibody (20.6 mg / mL), add 36.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-13 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-A-13). The DAR value was determined by mass spectrometry to be 8.19.

[1112] 13. Preparation of Trastuzumab-B-22

[1113] Take 0.847 mL of Trastuzumab antibody (17.7 mg / mL), add 42.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-22 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-B-22). The DAR value was determined by mass spectrometry to be 8.1.

[1114] 14. Preparation of Trastuzumab-C-35

[1115] Take 0.847 mL of Trastuzumab antibody (17.7 mg / mL), add 42.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add C-35 solution dissolved in dimethyl sulfoxide (128.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-C-35). The DAR value was determined by mass spectrometry to be 8.1.

[1116] 15. Preparation of Trastuzumab-C-3

[1117] Take 0.847 mL of Trastuzumab antibody (17.7 mg / mL), add 42.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add C-3 solution dissolved in dimethyl sulfoxide (130.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-C-3). The DAR value was determined by mass spectrometry to be 6.9.

[1118] 16. Preparation of Trastuzumab-B-20

[1119] Take 0.847 mL of Trastuzumab antibody (17.7 mg / mL), add 42.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-20 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-B-20). The DAR value was determined by mass spectrometry to be 6.8.

[1120] 17. Preparation of Trastuzumab-A-4

[1121] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-4 solution dissolved in dimethyl sulfoxide (121.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-A-4). The DAR value was determined by mass spectrometry to be 7.5.

[1122] 18. Preparation of Trastuzumab-A-7

[1123] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-7 solution dissolved in dimethyl sulfoxide (129.0 μL, 10 Mm, 12 times the molar volume of the antibody) and mix well. 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-A-7). The DAR value was determined by mass spectrometry to be 6.7.

[1124] 19. Preparation of Trastuzumab-C-31

[1125] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add C-31 solution dissolved in dimethyl sulfoxide (121.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-C-31). The DAR value was determined by mass spectrometry to be 8.1.

[1126] 20. Preparation of Trastuzumab-A-11

[1127] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-11 solution dissolved in dimethyl sulfoxide (120.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-A-11). The DAR value was determined by mass spectrometry to be 7.9.

[1128] 21. Preparation of Trastuzumab-B-17

[1129] Take 0.622 mL of Her2 antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-17 solution dissolved in dimethyl sulfoxide (122.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-B-17). The DAR value was determined by mass spectrometry to be 8.1.

[1130] 22. Preparation of Trastuzumab-B-15

[1131] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-15 solution dissolved in dimethyl sulfoxide (117.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-B-15). The DAR value was determined by mass spectrometry to be 8.1.

[1132] 23. Preparation of Trastuzumab-C-32

[1133] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add C-32 solution dissolved in dimethyl sulfoxide (122.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-C-32). The DAR value was determined by mass spectrometry to be 8.0.

[1134] 24. Preparation of Trastuzumab-A-12

[1135] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-12 solution dissolved in dimethyl sulfoxide (116.6 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-A-12). The DAR value was determined by mass spectrometry to be 7.9.

[1136] 25. Preparation of Trastuzumab-C-30

[1137] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add C-30 solution dissolved in dimethyl sulfoxide (117.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-C-30). The DAR value was determined by mass spectrometry to be 8.0.

[1138] 26. Preparation of Trastuzumab-B-16

[1139] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add B-16 solution dissolved in dimethyl sulfoxide (119.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-B-16). The DAR value was determined by mass spectrometry to be 8.2.

[1140] 27. Preparation of Trastuzumab-C-29

[1141] Take 0.622 mL of Trastuzumab antibody (22.5 mg / mL), add 31.0 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, 53.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add C-29 solution dissolved in dimethyl sulfoxide (119.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-C-29). The DAR value was determined by mass spectrometry to be 8.0.

[1142] 28. Example: Preparation of Trastuzumab-A-2

[1143] Take 0.957 mL of Trastuzumab antibody (20.9 mg / mL), add 48.0 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, 75.8 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-2 solution dissolved in dimethyl sulfoxide (153.0 μL, 10 mM, 10 times the molar volume of the antibody) and mix well. 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-A-2). The DAR value was determined by mass spectrometry to be 2.5.

[1144] 29. Preparation of Trastuzumab-A-17

[1145] Take 0.781 mL of Trastuzumab antibody (19.2 mg / mL), add 39.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-17 solution dissolved in dimethyl sulfoxide (138.0 μL, 10 mM, 12 times the molar amount of antibody) and mix well. 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-A-17). The DAR value was determined by mass spectrometry to be 8.0.

[1146] 30. Preparation of Trastuzumab-B-21

[1147] Take 0.857 mL of Trastuzumab antibody (17.5 mg / mL), add 43.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 57.0 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Then add B-21 solution dissolved in dimethyl sulfoxide (128.0 μL, 10 mM, 12 times the molar amount of 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-B-21). The DAR value was determined by mass spectrometry to be 8.26.

[1148] 31. Preparation of Trastuzumab-A-18

[1149] Take 0.820 mL of Trastuzumab antibody (18.3 mg / mL), add 41.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add A-18 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-A-18). The DAR value was 7.33 by mass spectrometry.

[1150] 32. Preparation of Trastuzumab-C-36

[1151] Take 0.820 mL of Trastuzumab antibody (18.3 mg / mL), add 41.0 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, 57.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add C-36 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar volume of the antibody) and mix well. 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-C-36). The DAR value was determined by mass spectrometry to be 8.0.

[1152] 33. Preparation of Trastuzumab-B-23

[1153] Take 0.923 mL of Trastuzumab antibody (18.3 mg / mL), add 46.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 68.0 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Then add B-23 solution dissolved in dimethyl sulfoxide (131.0 μL, 10 mM, 12 times the molar volume of the antibody), mix well, and incubate at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-23). ​​The DAR value was determined by mass spectrometry to be 6.4.

[1154] 34. Preparation of Trastuzumab-G-4

[1155] Take 0.833 mL of Trastuzumab antibody (24 mg / mL), add 41.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 10 times the amount of antibody dissolved in dimethyl sulfoxide G-4 (148.0 μL, 10 mM), mix well, and let stand at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-G-4). The DAR value was determined by mass spectrometry to be 8.0.

[1156] 35. Preparation of Trastuzumab-G-7

[1157] Take 1.013 mL of Trastuzumab antibody (14.8 mg / mL), add 51.8 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 15 mM TCEP (tris(2-carboxyethyl)phosphine, 38.0 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 10 times the amount of antibody dissolved in dimethyl sulfoxide B-4 solution (97.3 μL, 10 mM), mix well, and let stand at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-G-7). The DAR value was determined by mass spectrometry to be 6.4.

[1158] 36. Preparation of Trastuzumab-A-21

[1159] 0.789 mL of Trastuzumab antibody (19.0 mg / mL) was diluted with 39.4 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, A-21 solution dissolved in dimethyl sulfoxide (118.4 μL, 10 mM, 11 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-A-21). The DAR value was determined by mass spectrometry to be 8.03.

[1160] 37. Preparation of Trastuzumab-A-22

[1161] 0.789 mL of Trastuzumab antibody (19.0 mg / mL) was diluted with 39.4 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, A-22 solution dissolved in dimethyl sulfoxide (119.6 μL, 10 mM, 11 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. After this, 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-A-22). The DAR value was determined by mass spectrometry to be 7.99.

[1162] 38. Preparation of Trastuzumab-A-23

[1163] Take 0.670 mL of Trastuzumab antibody (19.0 mg / mL), dilute with 33.5 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, 48.2 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Add A-23 solution dissolved in dimethyl sulfoxide (99.5 μL, 10 mM, 11 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 (i.e., Trastuzumab-A-23). ​​The DAR value was determined by mass spectrometry to be 2.56.

[1164] 39. Preparation of Trastuzumab-A-24

[1165] 0.77 mL of Trastuzumab antibody (19.5 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, A-24 solution dissolved in dimethyl sulfoxide (119.6 μL, 10 mM, 11 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-A-24). The DAR value was determined by mass spectrometry to be 8.12.

[1166] 40. Preparation of Trastuzumab-E-4

[1167] Take 0.889 mL of Trastuzumab antibody (22.5 mg / mL), add 45.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in E-4 (174.0 μL, 10 mM) in dimethyl sulfoxide solution, mix well, and let stand 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-E-4). The DAR value was determined by mass spectrometry to be 7.1.

[1168] 41. Preparation of Trastuzumab-J-4

[1169] Take 0.667 mL of Trastuzumab antibody (22.5 mg / mL), add 33.3 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 57.0 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 11 times the amount of antibody dissolved in dimethyl sulfoxide J-4 (124.0 μL, 10 mM), mix well, and let stand at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-J-4). The DAR value was determined by mass spectrometry to be 8.0.

[1170] 42. Preparation of Trastuzumab-L-4

[1171] Take 0.796 mL of Trastuzumab antibody (22.5 mg / mL), add 40.0 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, 67.8 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 11 times the amount of antibody dissolved in dimethyl sulfoxide L-4 (138.4 μL, 10 mM) solution and mix well. Incubate at room temperature for 2 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 (Trastuzumab-L-4). The DAR value was determined by mass spectrometry to be 8.0.

[1172] 43. Preparation of Trastuzumab-E-5

[1173] 0.667 mL of Trastuzumab antibody (22.5 mg / mL) was diluted with 33.3 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, 121.0 μL of E-5 solution dissolved in dimethyl sulfoxide (10 mM, 11 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-E-5). The DAR value was 7.08 as determined by mass spectrometry.

[1174] 44. Preparation of Trastuzumab-J-5

[1175] Take 0.667 mL of Trastuzumab antibody (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, 57 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 11 times the amount of antibody dissolved in dimethyl sulfoxide J-5 (120.0 μL, 10 mM) solution and mix well. 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-J-5). The DAR value was determined by mass spectrometry to be 7.9.

[1176] 45. Preparation of Trastuzumab-L-5

[1177] 0.667 mL of Trastuzumab antibody (22.5 mg / mL) was diluted with 33.3 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) solu...

Claims

1. A compound or thereof, a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled compound, metabolite, or prodrug, wherein said compound has the following structure: in, Each time XG appears, it is independently selected from H and C. 1-6 Alkyl groups, or substituents containing hydrophilic hydroxyl, glycosyl, amino, quaternary ammonium salt, sulfonyl, and carboxylic acid segments, and their derivatives; Y is selected from the segment connecting XG and the benzene ring; Z is selected from the portion of a biologically active compound; CM is selected from structures that can react with specific functional groups in the target structure (such as an antibody); L1 is selected from the fragment connecting CM and the aniline moiety; a can be 1, 2, or 3.

2. The compound of claim 1 or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled compound, metabolite, and prodrug, wherein, Each time XG appears, it is independently selected from the following structural segments: H, C 1-6 Alkyl (e.g., methyl), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), (For example: ), Where m is selected from integers from 1 to 30; for example, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; Alternatively, XG can be selected from the following structures: (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: ) (For example: (Specifically: ))、 (For example: (Specifically: ))、 (For example: (specifically) ))、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: )、 (For example: ); where m is independently selected from integers from 1 to 30 each time it appears; for example, m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 each time it appears; preferably, m is 3, 4, or 5; Preferably, Y is selected from chemical bonds, or is composed of one or more C atoms. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkylene, C 2- 6-eneyl, C 2-6 A group consisting of alkynyl, -O-, -NR1-, quaternary ammonium salt, carbonyl, sulfonyl, sulfinyl, phosphoryl, aryl, heteroaryl, heterocyclic, and / or cycloalkyl groups; R1 is selected from one or more hydrogens, C 1-6 Alkyl, C 1-6 A structure composed of alkylene, hydroxyl, -O-, carbonyl, carboxyl, and / or sulfonic acid groups; preferably, R1 is C 1-6 alkyl; Preferably, the bioactive compound is selected from topoisomerase I inhibitors, topoisomerase II inhibitors, tubulin polymerization inhibitors, tubulin depolymerization inhibitors, DNA interference agents, DNA alkylating agents, DNA cross-linking agents, DNA polymerase inhibitors, DNA damage repair inhibitors, RNA polymerase inhibitors, thymidine synthase inhibitors, poly-ADP-ribose polymerase inhibitors, apoptosis promoters, cell cycle arrestors, protein degrading agents, synthetic lethal agents, kinase inhibitors, and glucocorticoid receptor modulators. Preferably, the bioactive compound is selected from camptothecin compounds, olritatin compounds, maytansine compounds, eribulin compounds, hammetrine compounds, epothilone compounds, kazimycin compounds, anthracycline compounds, benzodiazepine compounds, α-amaminoid compounds, sucrose compounds, alkaloid compounds, tripterygium compounds, nucleoside compounds, glucocorticoid compounds, rapamycin compounds, and lipolytamine compounds; Preferably, CM is selected from functional groups or structural fragments that can react with cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs contained in the target structure. Preferably, CM is selected from halogenated acetyl groups, substituted or unsubstituted maleimide groups, alkyl sulfonyl heteroaryl groups, and fluorophenolic ester groups; Preferably, CM is selected from iodoacetyl, maleimide, bromomaleimide, thiomaleimide, alkylsulfonylpyrimidinyl, and fluorophenol ester. More preferably, CM is selected from the following substituted or unsubstituted structural segments: Preferably, L1 is selected from a segment composed of one or more of the following structures, including but not limited to C. 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl, carbonyl, sulfonyl, amino, hydroxyl, -O-, heterocyclic, heteroaryl, aryl, natural or non-natural amino acids and their polypeptides, polyethylene glycol, polysarcosine, carboxylic acid, glycosyl and their derivatives or quaternary ammonium salts; Preferably, L1 is selected from one or more of the following substituted or unsubstituted structural fragments: C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic group (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, M et, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(R'), Glu(R'), Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly(DGGFG), Glu-Gly-Gly-Phe-Gly(EGGFG), Ala-Ala, Ala-Lys, Ala-Lys( Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-L ys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGF G), Gly-Gly-Val-Ala(GGVA), Gly-Phe-Leu-Gly(GFLG), Glu-Ala-Ala-Ala(EAAA), Gly-Gly-Gly-Gly-Gly(GGGGG)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C1-6 alkylene (CO2H), -C 1-6 Alkylene groups SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 alkylene-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1- (6-alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O)rC 1-6 Alkyl group, -CH2N(C) 1-6 alkyl)-C(=O)-(OCH2CH2)r-OC 1-6 Alkyl group, -(CH2N(Me)-C(=O))rC 1-6 Alkyl groups, polyethylene glycol fragments containing 1-10 EO units (i.e., -(CH2CH2O)r'-C) 1-6 Alkyl group, r' = 1-10), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residue), EDTA (ethylenediaminetetraacetic acid residue), -C 1-6 Alkylene-N(C) 1- 6-alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkyl-N(C) 1- 6-alkyl)-EDTA, wherein r is selected from an integer from 1 to 20; s is selected from an integer from 1 to 20.

3. A compound or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, and prodrug, wherein said compound has a structure as shown in formula (II-a) or (II-b): in, XG, L1, and CM are each independently as described in claim 1 or 2.

4. A compound or its salt, ester, stereoisomer, or isotope label, wherein the compound has the following structure: in, XG is as described in any one of claims 1 to 3.

5. According to any one of claims 1-4, the compound has the following structure:

6. A compound or its salt, ester, stereoisomer, or isotope label, wherein the compound has the following structure: in, XG is selected from fragments containing hydrophilic hydroxyl groups, glycosyl groups, amino groups, and carboxylic acid groups and their derivatives; Z is selected from the biologically active compound portion; E is selected from the segment connecting L2 and Z; CM is selected from structures that can react with specific functional groups in the target structure (such as an antibody); L2 is selected from the segment connecting parts CM, XG, and E; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, n is 1; Preferably, XG is selected from the following structural segments: Where m is selected from integers from 1 to 30; Alternatively, XG is selected from the following structural fragments: (For example: )、 (For example: )； Preferably, the bioactive compound is selected from topoisomerase I inhibitors, topoisomerase II inhibitors, tubulin polymerization inhibitors, tubulin depolymerization inhibitors, DNA interference agents, DNA alkylating agents, DNA cross-linking agents, DNA polymerase inhibitors, DNA damage repair inhibitors, RNA polymerase inhibitors, thymidine synthase inhibitors, poly-ADP-ribose polymerase inhibitors, apoptosis promoters, cell cycle arrestors, protein degrading agents, synthetic lethal agents, kinase inhibitors, and glucocorticoid receptor modulators. Preferably, the bioactive compound is selected from camptothecin compounds, olritatin compounds, maytansine compounds, eribulin compounds, hammetrine compounds, epothilone compounds, kazimycin compounds, anthracycline compounds, benzodiazepine compounds, α-amaminoid compounds, sucrose compounds, alkaloid compounds, tripterygium compounds, nucleoside compounds, glucocorticoid compounds, rapamycin compounds, and lipolytamine compounds; Preferably, CM is selected from functional groups or structural fragments that can react with cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs contained in the target structure. Preferably, CM is selected from the following substituted or unsubstituted structural segments: Preferably, L2 is selected from a segment composed of one or more of the following structures, including but not limited to C. 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl, carbonyl, sulfonyl, amino, hydroxyl, -O-, heterocyclic, heteroaryl, aryl, natural or non-natural amino acids and their polypeptides, polyethylene glycol, polysarcosine, carboxylic acid, glycosyl and their derivatives or quaternary ammonium salts; Preferably, L2 is selected from substituted or unsubstituted structural fragments of one or more of the following: C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic group (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, M et, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(R'), Glu(R'), Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly(DGGFG), Glu-Gly-Gly-Phe-Gly(EGGFG), Ala-Ala, Ala-Lys, Ala-Lys( Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-L ys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGF G), Gly-Gly-Val-Ala(GGVA), Gly-Phe-Leu-Gly(GFLG), Glu-Ala-Ala-Ala(EAAA), Gly-Gly-Gly-Gly-Gly(GGGGG)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C1-6 alkylene (CO2H), -C 1-6 Alkylene groups SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 alkylene-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O)rC 1-6 Alkyl group, -CH2N(C) 1-6 alkyl)-C(=O)-(OCH2CH2)r-OC 1-6 Alkyl group, -(CH2N(Me)-C(=O))rC 1-6 Alkyl groups, polyethylene glycol fragments containing 1-10 EO units (i.e., -(CH2CH2O)r'-C) 1-6 Alkyl group, r' = 1-10), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residue), EDTA (ethylenediaminetetraacetic acid residue), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-EDTA, where r is selected from integers from 1 to 20; s is selected from integers from 1 to 20; Preferably, E is selected from self-eliminating structures; Preferably, E is selected from single bonds, -NHCH2-, and -NHBn-O(=O)-.

7. According to claim 6, the compound has the following structure:

8. A biologically active conjugate having a structure as shown in any one of formulas (IA) to (IV-A): Alternatively, the coupling structure is as follows: Alternatively, the coupling structure is as follows: Alternatively, the coupling structure is as follows: in, CM' is selected from the structure that connects A; A is selected from antibodies or their antigen-binding fragments, peptides or small molecule fragments that have a targeting effect; x is between 1 and 10; Z, E, L1, L2, XG, Y, a, and n are as described in any one of claims 1 to 6; Preferably, CM' is selected from functional groups or structural fragments that have reacted with cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs contained in the Ab structure. Preferably, CM' is selected from C 1-6 Alkyl-substituted acyl groups, substituted or unsubstituted succinimide groups, substituted or unsubstituted heteroaryl groups, and 3-6 membered heterocyclic acyl groups; Preferably, CM' is selected from acyl, succinimide, substituted or unsubstituted pyrimidinyl, and piperidinyl. Preferably, CM' is selected from the following substituted or unsubstituted structural segments: Preferably, the Ab is selected from antibodies that target tumor antigens, such as monoclonal or bispecific antibodies targeting Her2, Her3, EGFR, TROP2, B7H3, c-Met, CEACAM5, CLDN18.2, FRa, CDH6, CDH3, PTK7, DLL3, GPC3, etc. Preferably, A is selected from antibodies that target tumor antigens, such as trastuzumab antibody, patocilizumab antibody, etc. Preferably, A is selected from polypeptides that target tumor antigens, such as somatostatin analogs, GnRH / LHRH analogs, vascular peptide-2, Heptaarginine, TAT47-57, DPV1047 Vectocell peptide, polypeptides that target EphA2, GPC3 or Nectin-4, etc. Preferably, A is selected from small molecule fragments that target tumor antigens, such as N-acetylgalactosamine (GalNAc) fragments, bisphosphonate fragments with bone targeting, fragments that selectively bind to prostate-specific membrane antigen (PSMA), fragments that selectively bind to somatostatin receptors, etc.

9. According to claim 8, the coupling structure is as follows: in, Ab-(S-) indicates an antibody or its antigen-binding fragment. This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment.

10. A composition comprising one or more conjugates as described in claim 8 or 9, wherein the composition has a DAR (drug-antibody conjugate ratio) of 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, the concentration is 3 to 8, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.0, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.0, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 8.0, or 7.5 to 8.0; or, the DAR of the composition is about 6.0 to 9.0, preferably about 6.0 to 8.0, for example, about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.

12. Approximately 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.2, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29, 6.3, 6.31, 6.32, 6.33 Approximately 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 6.4, 6.41, 6.42, 6.43, 6.44, 6.45, 6.46, 6.47, 6.48, 6.49, 6.5, 6.51, 6.52, 6.53, 6.54, approximately 6.55, approximately 6.56, approximately 6.57, approximately 6.58, approximately 6.59, approximately 6.6, approximately 6.61, approximately 6.62, approximately 6.63, approximately 6.64, approximately 6.65, approximately 6.66, approximately 6.67, approximately 6.68, approximately 6.69, approximately 6.7, approximately 6.71, approximately 6.72, approximately 6.73, approximately 6.74, approximately 6.75, approximately 6 .76, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97, approximately 6.98, approximately 6.99, approximately 7.0, approximately 7.01, approximately 7.02, approximately 7.03, approximately 7.04, approximately 7.05, approximately 7.06, approximately 7.07, approximately 7.08, approximately 7.09, approximately 7.1, approximately 7.11, approximately 7.12, approximately 7.13, approximately 7.14, approximately 7.15, approximately 7.16, approximately 7.17, approximately 7. 18, approximately 7.19, approximately 7.2, approximately 7.21, approximately 7.22, approximately 7.23, approximately 7.24, approximately 7.25, approximately 7.26, approximately 7.27, approximately 7.28, approximately 7.29, approximately 7.3, approximately 7.31, approximately 7.32, approximately 7.33, approximately 7.34, approximately 7.35, approximately 7.36, approximately 7.37, approximately 7.38, approximately 7.3 9, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.54, approximately 7.55, approximately 7.56, approximately 7.57, approximately 7.58, approximately 7.59, approximately 7.6 Approximately 7.61, 7.62, 7.63, 7.64, 7.65, 7.66, 7.67, 7.68, 7.69, 7.7, 7.71, 7.72, 7.73, 7.74, 7.75, 7.76, 7.77, 7.78, 7.79, 7.8, 7.

81. Approximately 7.82, 7.83, 7.84, 7.85, 7.86, 7.87, 7.88, 7.89, 7.9, 7.91, 7.92, 7.93, 7.94, 7.95, 7.96, 7.97, 7.98, 7.99, 8.0, 8.01, 8.

02. Approximately 8.03, 8.04, 8.05, 8.06, 8.07, 8.08, 8.09, 8.1, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 8.2, 8.21, 8.22, 8.23, approximately 8.24, around 8.25, around 8.26, around 8.27, around 8.28, around 8.29, around 8.3, around 8.31, around 8.32, around 8.33, around 8.34, around 8.35, around 8.36, around 8.37, around 8.38, around 8.39, around 8.4, around 8.41, around 8.42, around 8.43, around 8.44, around 8 .45, approximately 8.46, approximately 8.47, approximately 8.48, approximately 8.49, approximately 8.5, approximately 8.51, approximately 8.52, approximately 8.53, approximately 8.54, approximately 8.55, approximately 8.56, approximately 8.57, approximately 8.58, approximately 8.59, approximately 8.6, approximately 8.61, approximately 8.62, approximately 8.63, approximately 8.64, approximately 8.65, approximately 8.66, approximately 8.67, approximately 8.68, approximately 8.69, approximately 8.7, approximately 8.71, approximately 8.72, approximately 8.73, approximately 8.74, approximately 8.75, approximately 8.76, approximately 8.77, approximately 8.78, approximately 8.79, approximately 8.8, approximately 8.81, approximately 8.82, approximately 8.83, approximately 8.84, approximately 8.85, approximately 8.86, approximately 8.87, approximately 8.88, approximately 8.89, approximately 8.9, approximately 8.91, approximately 8.92, approximately 8.93, approximately 8.94, approximately 8.95, approximately 8.96, approximately 8.97, approximately 8.98, approximately 8.99, approximately 9.

0.

11. A pharmaceutical composition comprising the antibody-drug conjugate of claim 8 or 9 or the composition of claim 10, and one or more pharmaceutical excipients.

12. Use of the antibody-drug conjugate of claim 8 or 9, the composition of claim 10, or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating cancer.

13. The use according to claim 12, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

14. The antibody-drug conjugate of claim 8 or 9, the composition of claim 10, or the pharmaceutical composition of claim 11, for the treatment of Her2-expressing cancers.

15. The antibody-drug conjugate of claim 8 or 9, the composition of claim 10, or the pharmaceutical composition of claim 11, for the treatment of solid tumors or hematologic malignancies; such as gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) or urothelial carcinoma.

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