Heterocyclic compounds, and methods for producing and using the same

Abstract

The present application relates to the pharmaceutical field, specifically to a drug-linker compound. The drug may include a DNA topoisomerase inhibitor. Conjugates, such as antibody-drug conjugates, prepared using the drug-linker compound have excellent drug-antibody binding ratios and excellent target killing effects against tumors such as lung cancer, melanoma, breast cancer, gastric cancer, and colon cancer. The present application also provides a method for preparing the antibody-drug-linker molecule and its use.

Description

[Technical Field]

[0001] This application relates to the field of chemical compounds, and in particular to heterocyclic compounds that can be used in the preparation of conjugates, such as antibody drug conjugates. [Background technology]

[0002] In the field of anticancer drug research and development, ADC drugs have attracted worldwide attention due to their targeting properties. ADCs are a new type of anticancer drug in which a payload is linked to an antibody, and typically consist of an antibody, payload, and linker. The target recognition properties of antibodies and the high activity of toxins result in antitumor effects with higher specificity and efficacy than conventional small molecule drugs.

[0003] After entering the body, ADCs deliver toxins to tumor tissues via antigen-targeting antibodies. After ADCs bind to antigens on the surface of tumor cells and are phagocytosed by the cells, they are degraded in the lysosomes, releasing cytotoxins that damage DNA or block cell division, thereby killing the cells. Summary of the Invention [Problem to be solved by the invention]

[0004] The linker ensures the stability of the ADC in the blood and allows the toxin to kill after reaching the target. ADC toxins mainly include microtubule inhibitors, DNA damaging agents, and RNA polymerase inhibitors. Linkers also include various types of structures, such as cleavable and non-cleavable. The payload and linker structures are crucial to the efficacy and safety of ADCs. Therefore, the research and development of drug-linker compounds with excellent activity and safety remains a challenge in the field. [Means for solving the problem]

[0005] This application relates to drug-linker compounds that can be used to form conjugates with other molecules, such as antibodies, and methods for their preparation and use.

[0006] In one aspect, the present invention provides a compound of formula GM-[LED] x The present invention provides a drug-linker compound having the structure: G is a functional group or leaving group capable of reacting with a specific amino acid or glycosyl group. M is a connecting site that connects to G, and said M is [ka] is. In the formula, ring A is a 5- to 6-membered aliphatic heterocycle or a 5- to 20-membered aromatic ring system, and the aliphatic heterocycle and aromatic ring system may optionally contain an oxygen group (=O), a halogen, a cyano group, an amino group, a carboxyl group, a thiol group, and C 1~6 alkyl group, and M is a single bond and C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 an alkynylene group or an amine group; 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 The alkynylene or amine group is optionally substituted with one or more suitable substituents. L is a linker connecting the connecting sites M and E, and L is C 1~6 an alkylene group, -N(R')-, a carbonyl group, -O-, a natural or unnatural amino acid and its analog, and a short peptide chain consisting of amino acids; [ka] The structure is selected from one or more of: In the formula, R' is hydrogen, C 1~6 It represents an alkyl group or a polyethylene glycol fragment having 1 to 10 EO units, and s is selected from integers of 1 to 20. E is a structural fragment connecting L and D, E is a single bond, -NHCH2-, or [ka] The structure is selected from: D is a payload fragment; and / or x is selected from 1 to 10.

[0007] In some embodiments, G is a functional group or leaving group that can undergo reaction with a specific amino acid or glycosyl group in an antibody.

[0008] In some embodiments, G is a halogen, a halogenated C 1~6 Alkyl group, C 1~6 Sulfonyl group, halogenated C 1~6 Sulfonyl group, halogenated sulfonyl group, C 1~6 Sulfonate ester group, halogenated C 1~6 Sulfonate ester group, C 1~6 Sulfinate ester group, C 1~6 a halogenated C selected from a sulfoxide group, a nitro group, an azido group, a cyano group, an alkenyl group, an alkynyl group, and an alkynyl group-containing structural fragment; 1~6 Alkyl group, C 1~6 Sulfonyl group, halogenated C 1~6 Sulfonyl group, halogenated sulfonyl group, C 1~6 Sulfonate ester group, halogenated C 1~6 Sulfonate ester group, C 1~6 Sulfinate ester group, C 1~6 The sulfoxide groups, alkenyl groups, alkynyl groups and alkynyl group-containing structural fragments are optionally substituted with one or more suitable substituents.

[0009] In some embodiments, G is a halogen, a halogenated C 1~6 Alkyl group, C 1~6 Sulfonyl group, halogenated C 1~6 Sulfonyl group, halogenated sulfonyl group, C 1~6 Sulfonate ester group, halogenated C 1~6 Sulfonate ester group, C 1~6 Sulfinate ester group, C1~6 It is selected from sulfoxide groups, nitro groups, azido groups, cyano groups, alkenyl groups, alkynyl groups and alkynyl group-containing structural fragments.

[0010] In some embodiments, M is [ka] In the formula, ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycycle formed by connecting one or more (for example, two) 6-membered aromatic heterocycles with a benzene ring or a 6-membered heteroaromatic ring via a single bond, and the aliphatic heterocycle may optionally contain an oxygen group (═O), a halogen, and C 1~4 alkyl group, and M1 is a single bond, C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 an alkynylene group or an amine group; 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 The alkynylene or amine group is optionally substituted with one or more suitable substituents.

[0011] In some embodiments, M is [ka] In the formula, ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycycle formed by connecting one or more (for example, two) 6-membered aromatic heterocycles with a benzene ring or a 6-membered heteroaromatic ring via a single bond, and the aliphatic heterocycle may optionally contain an oxygen group (═O), a halogen, and C 1~4 alkyl group, and M1 is a single bond, C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 It is selected from an alkynylene group or an amine group.

[0012] In some embodiments, M is [ka] wherein ring A is [ka] M is selected from a single bond and C 1~6 Alkylene group, C 2~6 Alkenylene group, C 2~6 an alkynylene group or an amine group; 1~6 Alkylene group, C 2~6 Alkenylene group, C 2~6 The alkynylene or amine group is optionally substituted with one or more suitable substituents.

[0013] In some embodiments, M is [ka] is selected from.

[0014] In some embodiments, M is [ka] is selected from.

[0015] In some embodiments, M is [ka] is selected from.

[0016] In some embodiments, M is [ka] is selected from.

[0017] In some embodiments, L is C 1~6Alkylene group, -N(R')-, carbonyl group, -O-, Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) r OCH3)), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, V al-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gl y-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys -Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, [ka] The structure is selected from one or more of: In the formula, R' is hydrogen, C 1~6 represents an alkyl group or a polyethylene glycol fragment having 1 to 10 EO units, and s is selected from integers of 1 to 20, for example, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0018] In some embodiments, L is C 1~6Alkylene group, carbonyl group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gl y-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, G ly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, [ka] wherein s is selected from an integer of 1 to 20.

[0019] In some embodiments, L is selected from the structure consisting of one or more of the following: [ka] [ka]

[0020] In some embodiments, L is selected from the following structures: [ka]

[0021] In some embodiments, L is selected from the following structures: [ka]

[0022] In some embodiments, L is selected from the following structures: [ka]

[0023] In some embodiments, L is selected from the following structures: [ka]

[0024] In some embodiments, E is a single bond, —NHCH 2 —, [ka] is.

[0025] In some embodiments, E is a single bond.

[0026] In some embodiments, E is —NHCH 2 —.

[0027] In some embodiments, E is [ka] is.

[0028] In some embodiments, E is [ka] is.

[0029] In some embodiments, [ka] is selected from the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0030] In some embodiments, [ka] is selected from the following structures: [ka] [ka] [ka]

[0031] In some embodiments, the payload is selected from a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0032] In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansine compound, the DNA intercalator is a pyrrolobenzodiazepine PBD, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., adriamycin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide), and the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

[0033] The payloads disclosed herein typically include, for example, a hydroxyl group (-OH), a carboxyl group (-COOH), a thiol group (-SH), a primary amino group (-NH), a secondary amine group (-NR A H) or tertiary amine groups (-NR B R C )(wherein, R A , R B , R C represents a non-hydrogen substituent on N), and the payload can be linked to a linker in the conjugate by these functional groups.

[0034] In some embodiments, the payload is linked to E in the antibody-drug conjugate through an -OH, -SH, primary amino group, secondary amine group, or tertiary amine group thereon.

[0035] In some embodiments, the payload is selected from a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of a compound of Formula I or Formula II. [ka] In the formula, R1 and R2 are each independently C1~6 It is selected from alkyl groups and halogens. R3 is selected from H and -CO-CH2OH. R4 and R5 are each independently selected from H, halogen and hydroxyl groups, or R4 and R5 combine with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle. R6 is hydrogen or -C 1~4 Alkylene-NR a R b is selected from. R7 is C 1~6 Alkyl groups and -C 1~4 Alkylene-NR a R b is selected from. In the formula, R a , R b are independently H, C for each occurrence. 1~6 Alkyl group, -SO2-C 1~6 Alkyl groups and -CO-C 1~6 The alkyl group is selected from the group consisting of:

[0036] In some embodiments, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: [ka]

[0037] In some embodiments, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: [ka]

[0038] In some embodiments, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: [ka]

[0039] In some embodiments, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: [ka]

[0040] In some embodiments, the fragment corresponding to the payload obtained after the payload is linked to a linker is D in the general formula, and preferably, D is a monovalent structure obtained by losing one H from an -OH, -NH, or secondary amine group on the payload.

[0041] In some embodiments, D is selected from the following structures: [ka]

[0042] In another embodiment, the drug linker compounds provided herein have the formula GM-[LED] x wherein: G is a functional group or leaving group capable of reacting with specific amino acids or glycosyl groups in the antibody or antigen-binding fragment, G is a halogen, halogenated C 1~6 Alkyl group, C 1~6 Sulfonyl group, halogenated C 1~6 Sulfonyl group, halogenated sulfonyl group, C 1~6 Sulfonate ester group, halogenated C 1~6 Sulfonate ester group, C 1~6 Sulfinate ester group, C 1~6 It is preferably selected from sulfoxide groups, nitro groups, azide groups, cyano groups, alkenyl groups, alkynyl groups and alkynyl group-containing structural fragments. M is a connecting site that connects to G, and M is [ka] is. In the formula, ring A is a 5- to 6-membered aliphatic heterocycle or a 5- to 20-membered aromatic ring system, and the aliphatic heterocycle and aromatic ring system may optionally contain an oxygen group (=O), a halogen, a cyano group, an amino group, a carboxyl group, a thiol group, and C 1~6 alkyl group, and M is a single bond and C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 It is selected from an alkynylene group or an amine group. Preferably, M is [ka] In the formula, ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycycle formed by connecting one or more (for example, two) 6-membered aromatic heterocycles with a benzene ring or a 6-membered heteroaromatic ring via a single bond, and the aliphatic heterocycle may optionally contain an oxygen group (═O), a halogen, and C 1~4 alkyl group, and M1 is a single bond, C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 It is selected from an alkynylene group or an amine group. Preferably, M is [ka] wherein ring A is [ka] M is selected from a single bond and C 1~6 Alkylene group, C 2~6 Alkenylene group, C 2~6 It is selected from an alkynylene group or an amine group. Preferably, M is [ka] is selected from. Preferably, M is [ka] is selected from. Preferably, M is [ka] is selected from. Preferably, M is [ka] is selected from. L is a linker connecting the connecting sites M and E, and L is C 1~6 Alkylene group, -N(R')-, carbonyl group, -O-, natural amino acids or unnatural amino acids and their analogs (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) r OCH3), as well as short peptides consisting of amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly- Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Va l-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), [ka] The structure is selected from one or more of: In the formula, R' is hydrogen, C 1~6 It represents an alkyl group or a polyethylene glycol fragment having 1 to 10 EO units, and s is selected from integers of 1 to 20. Preferably, L is C 1~6 Alkylene group, carbonyl group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gl y-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, G ly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, [ka] In the formula, s is selected from an integer of 1 to 20. Preferably, L is selected from the structure consisting of one or more of the following: [ka] [ka] Preferably, L is selected from the following structures: [ka] Preferably, L is selected from the following structures: [ka] Preferably, L is selected from the following structures: [ka] Preferably, L is selected from the following structures: [ka] E is a structural fragment connecting L and D, where E is a single bond, -NHCH2-, or is selected from the following structures: [ka] Preferably, E is a single bond, -NHCH2-, [ka] is. Preferably, E is -NHCH2- or [ka] is. Preferably, E is -NHCH2-. Preferably, E is a single bond. Preferably, E is [ka] is. D is a payload fragment, wherein the payload is selected from a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor; preferably, the tubulin inhibitor is an auristatin-based compound or a maytansine-based compound; preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., adriamycin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); and preferably, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof. Preferably, the payload is selected from a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of a compound of Formula I or Formula II. [ka] In the formula, R1 and R2 are each independently C 1~6 It is selected from alkyl groups and halogens. R3 is selected from H and -CO-CH2OH. R4 and R5 are each independently selected from H, halogen and hydroxyl groups, or R4 and R5 combine with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle. R6 is hydrogen or -C 1~4 Alkylene-NR a R b is selected from. R7 is C 1~6 Alkyl groups and -C 1~4 Alkylene-NR a R b is selected from. In the formula, R a , Rb are independently H, C for each occurrence. 1~6 Alkyl group, -SO2-C 1~6 Alkyl groups and -CO-C 1~6 The alkyl group is selected from the group consisting of: Preferably, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of said compounds: [ka] [ka] The fragment corresponding to the payload obtained after the payload is linked to a linker is D in the general formula, and preferably, D is a monovalent structure obtained by losing one H from an -OH, -NH2, or secondary amine group on the payload. Preferably, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of said compounds: [ka] and / or x is selected from 1 to 10. Preferably, [ka] is selected from the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Preferably, [ka] is selected from the following structures: [ka] [ka] [ka] [ka]

[0043] In another aspect, the present invention provides a compound of formula GM-[LED] x The present invention provides a drug-linker having the structure: wherein x is selected from 1 to 10. GM is [ka] and G is a leaving group for a nucleophilic substitution reaction (e.g., Halogen, mesyl group, fluorophenol group or [ka] ), or a hydroxyl group (-OH), a thiol group (-SH), or an amino group (-NH2), or G forms an unsaturated double bond with an adjacent atom on ring A, and ring A is a 5- to 6-membered aliphatic heterocycle or a 5- to 20-membered aromatic ring system, and the aliphatic heterocycle and aromatic ring system optionally contain an oxygen group (=O), a halogen, a cyano group, an amino group, a carboxyl group, a thiol group, and C 1~6alkyl group, and M1 is a single bond, C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 It is selected from an alkynylene group or an amine group. The structures of L, E and D are as defined above.

[0044] In some embodiments, GM is [ka] G is a mesyl group, or G forms a carbon-carbon double bond with an adjacent atom on ring A, ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycycle formed by connecting one or more 6-membered aromatic heterocycles with a benzene ring or a 6-membered heteroaromatic ring via a single bond, and the aliphatic heterocycle optionally contains an oxygen group (═O), a halogen, and C 1~4 alkyl group, and M1 is a single bond, C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~10 It is selected from an alkynylene group or an amine group.

[0045] In some embodiments, GM is [ka] and [ka] M1 is selected from a single bond, C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 It is selected from an alkynylene group or an amine group.

[0046] In some embodiments, GM is [ka] is.

[0047] In some embodiments, GM is [ka] is selected from.

[0048] In some embodiments, GM is [ka] is selected from.

[0049] In some embodiments, x is selected from 1-10.

[0050] In some embodiments, the drug linker compound is selected from the following: A-01 to A-34, B-01 to B-07, C-01 to C-28: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0051] In some embodiments, the drug linker compound of the present invention is selected from the following: [ka]

[0052] In some embodiments, the drug linker compound is optionally substituted with one or more suitable substituents.

[0053] In some embodiments, the Drug Linker Compound has the following structure: [ka] R 10 , R 11 , R 12 are independently hydrogen, C 1~6 Alkyl group, C 3~6 Cycloalkyl group, 5-12 membered heterocyclic group, C 6~10 Aryl group, 5- to 12-membered heteroaryl group, -C 1~6 Alkyl-C 6~10 Aryl groups and -C 1~6 alkyl-5 to 12 membered heteroaryl groups, wherein the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are optionally selected from the group consisting of hydroxyl, CN, halogen, C 1~6 Alkyl group, C 1~6 Halogenated alkyl groups, C 1~6 Alkoxy group, C 6~10 It is substituted with one or more substituents selected from an aryl group and a 5- to 12-membered heteroaryl group. R 13 and R 14 are each independently hydrogen, C1~6 Alkyl group, C 3~6 The alkyl group, the cycloalkyl group and the heterocyclic group are selected from hydroxyl group, CN, halogen, C 1~6 Alkyl group, C 1~6 Halogenated alkyl groups, C 1~6 Alkoxy group, C 6~10 It is substituted with one or more substituents selected from an aryl group and a 5- to 12-membered heteroaryl group. R 15 is hydrogen, C 1~6 Alkyl group, C 3~6 Cycloalkyl groups, halogenated C 1~6 Alkyl group, C 1~6 Alkyl-OC 1~6 Alkyl group, C 2~6 Alkenyl group, C 2~6 R is selected from an alkynyl group and a 3- to 6-membered heterocycloalkyl group; 16 is H, or R 15 , R 16 and the atoms connecting thereto jointly form a 4- to 7-membered ring, and the 4- to 7-membered ring optionally contains a hydroxyl group, CN, a halogen, C 1~6 Alkyl group, C 1~6 Halogenated alkyl groups, C 1~6 Alkoxy group, C 6~10 It is substituted with one or more substituents selected from an aryl group and a 5- to 12-membered heteroaryl group.

[0054] In some embodiments, R 10 , R 11 , R 12 are independently hydrogen, C 1~6 Alkyl group, C 3~6 Cycloalkyl groups, C 6~10 Aryl, benzyl, hydroxyl-substituted benzyl and indolyl-C 1~6 The alkyl group is selected from the group consisting of: R 13 and R 14 are each independently hydrogen, C 1~6 Alkyl group, C 3~6It is selected from a cycloalkyl group and a 4- to 6-membered heterocyclic group. R 15 is hydrogen, C 1~6 Alkyl group, C 3~6 Cycloalkyl groups, halogenated C 1~6 Alkyl group, C 1~6 Alkyl-OC 1~6 Alkyl group, C 2~6 Alkenyl group, C 2~6 R is selected from an alkynyl group and a 3- to 6-membered heterocycloalkyl group; 16 is H, or R 15 , R 16 and the atoms to which they are connected together form a 4- to 7-membered ring.

[0055] In some embodiments, R 10 , R 11 , R 12 are independently hydrogen, C 1~4 Alkyl group, C 3~6 It is selected from a cycloalkyl group, a phenyl group, a benzyl group, a p-hydroxybenzyl group and an indolylmethyl group.

[0056] In some embodiments, R 13 and R 14 are each independently hydrogen, C 1~4 Alkyl group, C 3~6 It is selected from a cycloalkyl group and a 4- to 6-membered heterocyclic group.

[0057] In some embodiments, R 13 and R 14 are each independently selected from hydrogen, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and a 4- to 6-membered heterocyclic group.

[0058] In some embodiments, R 15 is hydrogen, C 1~4 Alkyl group, C 3~6 Cycloalkyl groups, halogenated C 1~4 Alkyl group, C1~4 Alkyl group -OC 1~4 Alkyl group, C 2~6 Alkenyl group, C 2~6 alkynyl groups and 3- to 6-membered heterocycloalkyl groups, or R 15 , R 16 and the atoms to which they are connected jointly form a 4- to 7-membered heterocycloalkyl group or a 4- to 7-membered heteroaryl group.

[0059] In some embodiments, R 15 is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, halogenated C 1~4 Alkyl group, C 1~4 Alkyl-OC 1~4 Alkyl group, C 2~6 Alkenyl group, C 2~6 alkynyl groups and 3- to 6-membered heterocycloalkyl groups, or R 15 , R 16 and the atoms to which they are connected jointly form a 4- to 7-membered heterocycloalkyl group or a 4- to 7-membered heteroaryl group.

[0060] In some embodiments, R 10 , R 11 , R 12 are independently hydrogen, C 1~4 Alkyl group, C 3~6 It is selected from a cycloalkyl group, a phenyl group, a benzyl group, a p-hydroxybenzyl group and an indolylmethyl group. R 13 and R 14 are each independently selected from hydrogen, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and a 4- to 6-membered heterocyclic group. R 15is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, halogenated C 1~4 Alkyl group, C 1~4 Alkyl-OC 1~4 Alkyl group, C 2~6 Alkenyl group, C 2~6 alkynyl groups and 3- to 6-membered heterocycloalkyl groups, or R 15 , R 16 and the atoms to which they are connected jointly form a 4- to 7-membered heterocycloalkyl group or a 4- to 7-membered heteroaryl group.

[0061] In another aspect, the present invention provides an intermediate compound having the following structure: [ka] [ka] In the formula, X is selected from a benzyloxycarbonyl group, a tert-butoxycarbonyl group, a fluorenylmethyloxycarbonyl group, an allyloxycarbonyl group, a trimethylsilylethoxycarbonyl group, a methoxycarbonyl group, an ethoxycarbonyl group, a phthalic group, a tosyl group, a trifluoroacetyl group, a nitrobenzenesulfonyl group, a benzoyl group, a pivaloyl group, a trityl group, a 4-methoxyphenyldiphenylmethyl group, a dimethoxytrityl group, a 2,4-dimethoxybenzyl group, a p-methoxybenzyl group, and a benzyl group; a is an integer of 1 to 10, preferably an integer of 3 to 8; and R1, R2, and D are as defined above.

[0062] In some embodiments, X is selected from a fluorenylmethyloxycarbonyl group (Fmoc).

[0063] In some embodiments, D is selected from: [ka] In the formula, R1 and R2 are each independently C 1~6 It is selected from alkyl groups and halogens. R3 is selected from H and -CO-CH2OH. R4 and R5 are each independently selected from H, halogen and hydroxyl groups, or R4 and R5 combine with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle. R6 is hydrogen or -C 1~4 Alkylene-NR a R b is selected from. R7 is C 1~6 Alkyl groups and -C 1~4 Alkylene-NR a R b is selected from. In the formula, R a , R b are independently H, C for each occurrence. 1~6 Alkyl group, -SO2-C 1~6 Alkyl groups and -CO-C 1~6 The alkyl group is selected from the group consisting of:

[0064] In another aspect, the present invention provides an intermediate compound having the following structure: [ka] [ka] [ka] [ka]

[0065] In another embodiment, the present invention provides a compound of formula Ab-[MLED] x wherein M, L, E, D, and x are as defined above, and Ab is an antibody or antigen-binding fragment thereof that specifically binds to an antigen.

[0066] In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-34, ADC B-01 to ADC B-07, and ADC C-01 to ADC C-28 shown below. The Abs shown in the diagram below are defined as above, and the thiol group on the antibody forms a sulfide bond with the linker compound of the drug through an addition or substitution reaction to obtain a complete antibody-drug conjugate. x represents the drug-antibody ratio. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, [ka] represents a specific linking scheme between a thiol group in an antibody or antigen-binding fragment thereof and a linker.

[0067] composition In another aspect, the present application provides a composition that may comprise a plurality of ADCs described herein. Each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compounds described herein. As such, the composition is characterized by a "drug-antibody ratio" (DAR) in the range of about 1 to about 10. Methods for measuring the DAR are well known to those skilled in the art and include methods using reverse-phase chromatography or HPLC-MS.

[0068] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the antibody-drug conjugate is 1 to 10, for example, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 to 9, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 ~5.5, 3.5~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0 ~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0069] In some embodiments, the antibody-drug conjugate has a DAR value of 4 to 8. Those skilled in the art will appreciate that the antibody-drug conjugates described herein may be prepared by modularizing the drug-linker (drug-linker compound). For example, first, a free "drug-linker" (GM-[LED] xwhere GM is the structural form prior to covalent binding to an antibody or antigen-binding fragment thereof), and then covalently binds to the antibody or antigen-binding fragment thereof to obtain the antibody-drug conjugate described herein. Accordingly, GM in the free "drug-linker" is linked to one or more thiol groups (-SH), amino groups (-NH), or carboxyl groups (-COOH) on the antibody or antigen-binding fragment thereof by a method such as a substitution reaction (e.g., removal of a structure such as -SOMe or -Br thereon) or an addition reaction. x is selected from 1 to 10.

[0070] Pharmaceutical Composition Pharmaceutical Compositions of Antibody-Drug Conjugates and / or Drug-Linkers In another aspect, the present application provides a pharmaceutical composition comprising any one of the antibody-drug conjugates or any one of the drug-linkers described above, and one or more pharmaceutical excipients.

[0071] The antibody-drug conjugates described herein are typically prepared in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle and used parenterally, for example, by bolus administration, intravenous injection, intratumoral injection, etc. Optionally, the antibody-drug conjugates having the desired purity, in the form of a lyophilized or solution, are mixed with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1980) 16). th (Edition, Osol, A. Ed.) The antibody-drug conjugates described herein or pharmaceutical compositions containing said antibody-drug conjugates may be administered to an individual in need of treatment by any suitable route.

[0072] In some embodiments, the pharmaceutical composition may further comprise another pharmacologically active agent.

[0073] In some embodiments, the additional pharmacologically active agent is a drug with anti-tumor activity, hi some embodiments, the additional pharmacologically active agent is selected from a B7-H3 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic agent, or any combination thereof.

[0074] Purpose 1. Therapeutic uses of antibody-drug conjugates and / or drug-linkers The antibody-drug conjugates, drug-linkers or pharmaceutical compositions described herein can be used to treat several diseases or conditions, for example, B7-H3-positive tumors, Her2-positive tumors, and the like.

[0075] Therefore, the present application provides a use of any one of the antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same described above in the manufacture of a drug for preventing and / or treating and / or supporting the treatment of positive tumors.

[0076] The present application provides a use of any one of the antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same described above in the manufacture of a drug for preventing and / or treating and / or supporting the treatment of B7-H3-positive tumors.

[0077] The present application provides use of any one of the antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same described above in the manufacture of a drug for the prevention and / or treatment and / or adjuvant treatment of HER2-positive tumors.

[0078] The present application also provides a method for preventing and / or treating and / or supplementary treating a tumor-positive subject, the method comprising administering to a subject in need thereof any one of the antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same described above.

[0079] The present application provides a method for the prevention and / or treatment and / or adjuvant treatment of B7-H3-positive tumors, comprising the step of administering to a subject in need thereof any one of the antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same described above.

[0080] The present application provides a method for the prevention and / or treatment and / or adjuvant treatment of HER2-positive tumors, comprising the step of administering to a subject in need thereof any one of the antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same described above.

[0081] The present application further provides a use of the antibody-drug conjugate, drug linker, or pharmaceutical composition described in any one of the above in inhibiting the proliferation of B7-H3-positive tumor cells.

[0082] The present application further provides a use of the antibody-drug conjugate, drug linker, or pharmaceutical composition described in any one of the above in inhibiting the proliferation of Her2-positive tumor cells.

[0083] In some embodiments, the antibody drug conjugate, drug linker, or pharmaceutical composition is administered to cells in vitro or to cells in vivo in a subject, e.g., in a subject to inhibit the growth of tumor cells in the subject, or to tumor cells in vitro (e.g., a cell line or cells derived from a subject) to inhibit the growth of tumor cells in vitro.

[0084] In the present application, B7-H3-positive tumors include solid tumors or blood cancers such as colorectal cancer, gastric cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, melanoma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, etc.), kidney cancer, bladder cancer, thyroid cancer, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, esophageal cancer, liver cancer, salivary gland cancer, bile duct cancer, and meningioma.

[0085] In the present application, the subject is preferably a mammal, such as, for example, a bovine, equine, porcine, canine, feline, rodent, or primate, eg, a human.

[0086] In some embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjunctive therapy, and any combination thereof. In some embodiments, the second therapy can be administered simultaneously, separately, or sequentially with the method.

[0087] In some embodiments, the tumor affected by the pharmaceutical composition of the present invention is selected from breast cancer, colorectal cancer, head and neck cancer, clear cell renal cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer, or any combination thereof.

[0088] 2. Use of drug-linker in conjugate preparation The drug linkers described in the present invention are used to prepare conjugates, including antibody-drug conjugates.

[0089] 3. Use of intermediate compounds in the production of drug linker compounds The intermediate compounds described in this invention are used in the preparation of drug linker compounds.

[0090] Manufacturing method The present invention further provides a method for preparing a drug-linker compound, comprising the step of deprotecting an intermediate compound.

[0091] Method 1 In some embodiments, the method 1 comprises a method for preparing compound A-05a, the method comprising combining with a compound of Formula I. [ka] In the formula, R1 and R2 are each independently C 1~6 It is selected from alkyl groups and halogens. R3 is selected from H and -CO-CH2OH. R4 and R5 are each independently selected from H, halogen and hydroxyl groups, or R4 and R5 combine with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle. R6 is hydrogen or -C 1~4 Alkylene-NR a R b is selected from. R7 is C 1~6 Alkyl groups and -C 1~4 Alkylene-NR a R b is selected from. In the formula, R a , R b are independently H, C for each occurrence. 1~6 Alkyl group, -SO2-C 1~6 Alkyl groups and -CO-C 1~6 The alkyl group is selected from the group consisting of:

[0092] In some embodiments, R1 and R2 are each independently C 1~4 The alkyl group is selected from F, Cl, Br and I.

[0093] In some embodiments, R1 is selected from halogen, methyl, ethyl, propyl, and butyl, for example, methyl and Cl.

[0094] In some embodiments, R2 is selected from F, Cl, Br, and I, such as F and Cl.

[0095] In some embodiments, R1 is selected from a methyl group and Cl, and R2 is selected from F and Cl.

[0096] Method 2 In some embodiments, the method 2 comprises a method for preparing compound A-14-a, the method comprising deprotecting IM-5-a to provide compound of formula IM-6-a. [ka] wherein X is as defined in the intermediate compound above, and R1 and R2 are as defined in Method 1.

[0097] In some embodiments, the deprotection reaction is as follows: The solvent is selected from N,N-dimethylformamide, and an alkanamine compound such as diethylamine is added.

[0098] In some embodiments, the deprotection is carried out at room temperature for a reaction time of 1 to 5 hours, for example, 1 to 3 hours.

[0099] In some embodiments, the method 2 further comprises reacting IM-6-a with IM-2 to obtain compound A-14-a.

[0100] In some embodiments, the solvent for the reaction is selected from N,N-dimethylformamide and N,N-dimethylacetamide.

[0101] In some embodiments, the reaction comprises adding N,N-diisopropylethylamine.

[0102] Method 3 In some embodiments, the method 3 comprises a method for preparing compound B-02-a, the method comprising deprotecting B-02-4a. [ka] wherein X is as described above for the intermediate compound, and R1, R2, and R3 are as described above for the compound of formula 1.

[0103] Method 4 In some embodiments, Method 4 comprises a method for preparing compound C-07-a, the method comprising reacting C-07-6 with C-07-8a. C-07-a: [ka] wherein R1 and R2 are as defined in Method 1.

[0104] In some embodiments, C-07-8a in Method 4 above can be prepared by deprotecting C-07-7a. C-07-7a: [ka] wherein X is as defined in the intermediate compound, and R1 and R2 are as defined in Method 1.

[0105] Method 5 In some embodiments, the method 5 comprises a method for preparing compound C-10a, the method comprising reacting C-10-5a with C-07-8a. C-10-a: [ka] wherein R1, R2 and a are as defined above.

[0106] In some embodiments, C-10-5a in Method 5 above can be prepared by deprotecting C-10-4a. [ka]

[0107] In some embodiments, C-10-4a in Method 5 above is obtained by preparing C-10-3 with Compound A. [ka]

[0108] Method 6 In some embodiments, the method 6 includes a method for preparing compound C-17a, the method including reacting C-17-3a with C-07-8a. [ka] wherein R1, R2 and a are as defined above, preferably R1 is a methyl group, R2 is Cl, and a is 3 or 8; X is selected from CH or N.

[0109] In some embodiments, C-17-3a in Method 6 is prepared by oxidizing C-17-2a. [ka]

[0110] In some embodiments, C-17-2a in Method 6 above can be prepared by deprotecting C-17-1a. [ka]

[0111] In some embodiments, C-17-1a in Method 6 above can be prepared by coupling C-10-2 or C-19-3 with Compound A. [ka]

[0112] definition Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. The technical terms used herein refer to techniques commonly understood in the art and include modifications of techniques or equivalent technical substitutions that are obvious to those skilled in the art. Furthermore, all laboratory procedures used herein, such as genomics, nucleic acid chemistry, and molecular biology, are conventional procedures widely used in the corresponding fields. While the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to better explain the present invention.

[0113] The term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains are classified as κ (kappa) light chains and λ (lambda) light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected via a "J" region of about 12 or more amino acids, and heavy chains further contain a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 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. The constant regions are not directly involved in antibody-antigen binding but perform 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 of the classical complement system (C1q). The VH and VL regions can be subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), interspersed with relatively invariant regions called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL) form the respective antigen-binding sites. The amino acid sequences of each region or domain can be numbered according to various schemes known in the art.

[0114] The term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Each heavy and light chain variable region contains three CDRs, designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be determined, for example, using the Kabat numbering scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering scheme (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering scheme (Lefranc et al., Dev. Compare. Immunol. 27:55-77, 2003) or the AbM numbering scheme (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA CDRs can be defined according to various numbering schemes known in the art, such as those defined in [End Page 110] (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art can readily identify the CDRs defined in each numbering scheme. Furthermore, the correspondence between different numbering schemes is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0115] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be defined according to various numbering schemes known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering schemes. In some embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined according to the Chothia numbering scheme.

[0116] The term "framework region" or "FR" residues refers to amino acid residues other than the above-defined CDR residues in antibody variable regions.

[0117] The term "antigen-binding fragment" of an antibody refers to a polypeptide that is a fragment of an antibody, e.g., a polypeptide that is a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen as the full-length antibody and / or specifically binds to the antigen in competition with the full-length antibody, also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), the entire contents of which are incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' fragments, F(ab)' fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides comprising at least a portion of an antibody sufficient to confer polypeptide-specific antigen-binding ability. Genetically engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0118] The term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; and the term "Fab' fragment" refers to a fragment obtained after reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, and consists of one complete light chain and an Fd fragment of a heavy chain (consisting of the VH and CH1 domains).

[0119] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of one arm of an antibody. An Fv fragment is generally considered the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can identify and bind to an antigen, although possibly with lower affinity than the complete binding site.

[0120] The term "Fc" refers to an antibody fragment formed by disulfide bonding between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has several different functions but is not involved in antigen binding.

[0121] The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (e.g., 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, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, although variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described in 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 circumstances, a disulfide bond may additionally be present between the VH and VL of the scFv. In some embodiments, the VH and VL domains may be positioned opposite each other in any suitable arrangement, e.g., NH2-VH-VH-COOH, NH 2- It is an scFv containing VL-VL-COOH.

[0122] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single domains are also called nanobodies.

[0123] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and / or specifically binds to the antigen in competition with the full-length antibody.

[0124] In this specification, unless the context clearly indicates otherwise, reference to the term "antibody" includes not only complete antibodies but also antigen-binding fragments of antibodies.

[0125] Those skilled in the art can obtain antigen-binding fragments of antibodies (e.g., the antibody fragments described above) from a given antibody (e.g., an antibody provided by the present invention) using known conventional techniques (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can screen for specific antigen-binding fragments of antibodies in the same manner as with intact antibodies.

[0126] The term "mouse antibody" refers to an antibody obtained by fusing B cells and myeloma cells from an immunized mouse, screening for mouse hybridomas that can grow indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or an antibody secreted and produced by plasma cells that are formed by the differentiation and proliferation of B cells after an antigen has entered the mouse body.

[0127] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered to modify its amino acid sequence to increase its homology with that of a human antibody. Typically, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain the expected properties of the donor antibody, such as antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to enhance an immune response. The donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody that has the expected properties (e.g., antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and / or the ability to enhance an immune response).

[0128] The term "identity" refers to the sequence identity between two polypeptides or two nucleic acids. When a position in two compared sequences 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 molecules are identical at that position. The "percent 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, and multiplied by 100. For example, if six of ten positions in two sequences are identical, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three of six total positions are identical). Generally, the comparison is performed when the two sequences are compared to produce the greatest identity. Such comparisons can be accomplished, for example, using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). Additionally, the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which is integrated into the ALIGN program (version 2.0), may be used to measure percent identity between two amino acid sequences using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) in the GAP program of the GCG software package (available from www.gcg.com) may also be used to measure percent identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0129] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide that contains an amino acid sequence that has been modified by introducing substitutions, deletions, or alterations into the amino acid sequence by introducing amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been further modified (i.e., by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Derivatives of polypeptides or peptides may also be produced by chemical modification using techniques known to those of skill in the art, including specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Variants also have similar, identical, or improved functions as the polypeptide or peptide from which they are derived.

[0130] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (KD) or half maximal effective concentration (EC 50 ) can be expressed as

[0131] The specific binding properties between two molecules can be measured using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. The "association rate constant" (k or k) and the "dissociation rate constant" (k or k) can both be calculated from the concentrations and the actual rates of binding and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is equal to the dissociation constant, K (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of K, k, and k may be measured by any valid method. In some embodiments, dissociation constants may be measured using biolayer interferometry (e.g., ForteBio Octet). Alternatively, dissociation constants may be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0132] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide containing the amino acid sequence. Conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacing an amino acid residue with an amino acid residue having a similar side chain, such as substituting a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0133] The descriptions of the 20 conventional amino acids referred to herein follow conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0134] The terms "comprise," "have," "contain," or "such as," and other similar forms herein, are inclusive or open-ended and do not exclude unrecited elements or method steps.

[0135] The term "alkyl group" refers to groups such as "C 1~20 alkyl group," "C 1~10 alkyl group," "C 1~6 alkyl group," "C 1~4 alkyl group," "C 1~3 and the like. Specific examples include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a 2-methylbutyl group, a neopentyl group, a 1-ethylpropyl group, an n-hexyl group, an isohexyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, a 2-ethylbutyl group, a 1,2-dimethylpropyl group, and the like.

[0136] The term "alkylene group" refers to, for example, "C 1~20 alkylene group," "C 1~10 alkylene group," "C 3~10 alkylene group," "C 5~8alkylene group," "C 1~6 alkylene group," "C 1~4 alkylene group," "C 1~3 It represents a group obtained by removing two hydrogen atoms from a straight-chain or branched-chain hydrocarbon group, such as an alkylene group, and specific examples include a methylene group, an ethylene group, a 1,3-propylene group, a 1,4-butylene group, a 1,5-pentylene group, and a 1,6-hexylene group.

[0137] The term "alkenylene group" refers to a divalent group obtained by the loss of two hydrogen atoms from a straight-chain or branched-chain hydrocarbon group containing at least one carbon-carbon double bond, and examples thereof include "C 2~20 alkenylene group," "C 3~10 alkenylene group," "C 5~8 Examples thereof include vinylene group, 1-propenylene group, 2-propenylene group, 1-butenylene group, 2-butenylene group, 1,3-butadienylene group, 1-pentenylene group, 2-pentenylene group, 3-pentenylene group, 1,3-pentadienylene group, 1,4-pentadienylene group, 1-hexenylene group, 2-hexenylene group, 3-hexenylene group, 1,4-hexadienylene group, and the like.

[0138] The term "alkynylene group" refers to a divalent group obtained by the loss of two hydrogen atoms from a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond. For example, "C 2~20 alkynylene group," "C 3~10 alkynylene group," "C 5~8 Examples thereof include ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadinylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadinylene, 1,4-pentadinylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadinylene, and the like.

[0139] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one (e.g., 1, 2, or 3) ring member selected from N, O, and S. Specific examples include 5- to 6-membered aliphatic heterocycles, 5- to 6-membered nitrogen-containing aliphatic heterocycles, and 5- to 6-membered oxygen-containing aliphatic heterocycles, such as tetrahydrofuran, pyrrolidine, piperidine, and tetrahydropyran.

[0140] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O, and S. Specific examples include 5- to 6-membered aromatic heterocycles, 5- to 6-membered nitrogen-containing aromatic heterocycles, and 5- to 6-membered oxygen-containing aromatic heterocycles, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, and 1,2,4,5-tetrazine.

[0141] The term "aromatic ring system" refers to a monocyclic or polycyclic ring system containing at least one aromatic ring (e.g., a benzene ring, etc.) or heteroaromatic ring (e.g., a 5- to 6-membered aromatic heterocycle, such as a 5- to 6-membered nitrogen-containing aromatic heterocycle, such as a pyrimidine ring), where two or more aromatic and / or heteroaromatic rings form a fused ring or are connected by a single bond (e.g., a dipyrimidine phenyl group, etc.). The aromatic ring system may be divalent or higher (e.g., trivalent or tetravalent), for example, a 5- to 20-membered aromatic ring system.

[0142] As used herein, the term "suitable substituents" refers to modifications that one skilled in the art can make to a compound as needed to substitute the compound. "Suitable substituents" include oxygen groups (=O), halogens, cyano groups, NR 8 R 9, carboxyl groups, thiol groups, hydroxyl groups, ester groups (e.g., -C 1~6 Alkyl-C(=O)-OC 1~6 alkyl group), C 1~6 Alkyl group, C 2~6 Alkenyl group, C 2~6 Alkynyl group, C 1~6 Alkyl-OC 1~6 Alkyl group, C 1~6 Halogenated alkyl groups, C 1~6 Alkoxy group, C 3~6 Cycloalkyl groups, 3- to 10-membered heterocyclic groups, 5- to 10-membered heteroaryl groups, C 6~10 Aryl groups, benzyl groups, hydroxyl-substituted benzyl groups, indolylmethylene groups, and C 1~6 Contains halogenated alkoxy groups, R 8 , R 9 are independently H, C 1~6 Alkyl group, C 3~6 Cycloalkyl groups, 3- to 10-membered heterocyclic groups, 5- to 10-membered heteroaryl groups, C 6~10 Aryl group, C 1~6 Alkoxy group, C 1~6 Halogenated alkyl groups, 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 groups).

[0143] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to carriers and / or excipients known in the art that are pharmacologically and / or physiologically compatible with the subject and the active ingredient (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parahydroxybenzoates, chlorobutanol, phenol, and sorbic acid. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parahydroxybenzoates, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, glucan, or glucose), amino acids (such as glutamic acid and glycine), proteins (such as dried whey, albumin, or casein), or their degradation products (such as lactalbumin hydrolysate).

[0144] As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or condition (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method performed to achieve a beneficial or desired clinical result. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or mitigation of disease progression, improvement or reduction in the disease state, and remission of symptoms (whether partial or complete). "Treatment" can also refer to prolonging survival as compared to expected survival (if not receiving treatment).

[0145] As used herein, the term "subject" refers to a mammal, e.g., a primate mammal, such as a human. In some embodiments, the subject (e.g., a human) has a tumor or is at risk of having such a disease.

[0146] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired effect, or at least partially achieve it. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. Determining such effective amounts is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use depends on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the method of drug administration, and other concurrent treatments.

[0147] The terms "cancer" and "tumor" are used interchangeably and refer to a large category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant tumors as well as dormant tumors or micrometastases. Cancer also includes blood cancers.

[0148] The term "blood cancer" includes lymphoma, leukemia, myeloma or malignant lymphoma, as well as splenic cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphoma and T-cell lymphoma. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Blood cancers further include leukemias, such as, for example, secondary leukemia or acute lymphocytic leukemia. Blood cancers further include myeloma (e.g., multiple myeloma) and other cancers associated with the blood and / or B-cells or T-cells. DETAILED DESCRIPTION OF THE INVENTION

[0149] The present invention will be further explained below through the description of specific embodiments, which are not intended to limit the present invention. Those skilled in the art can make various modifications or variations in accordance with the teachings of the present invention without departing from the spirit and scope of the present invention.

[0150] The sequence information of the present invention is set forth in the following table.

[0151] [Table 1]

[0152] [Table 2]

[0153] [Table 3]

[0154] [Table 4]

[0155] [Table 5]

[0156] [Table 6]

[0157] The abbreviations used herein have the following meanings: Abbreviation Meaning Complementarity-determining regions in immunoglobulin variable regions FR Antibody framework region: Amino acid residues other than CDR residues in the antibody variable region VH antibody heavy chain variable region VL antibody light chain variable region IgG immunoglobulin G IMGT: A numbering scheme based on the international ImMunoGeneTics information system® (IMGT) initiated by Lefranc et al. See Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. The immunoglobulin comparison and numbering scheme proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The immunoglobulin numbering scheme proposed by Chothia et al. is a classical rule for identifying the boundaries of CDR regions based on the location of structural ring regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). A CDR definition method derived from related work by AbM Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). mAb monoclonal antibody EC 50 Concentration that produces 50% efficacy or binding I C 50 Concentration producing 50% inhibition ELISA Enzyme-linked immunosorbent assay PCR polymerase chain reaction HRP Horseradish Peroxidase K D equilibrium dissociation constant Ka binding rate constant Kd dissociation rate constant ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity FACS flow cytometry CDR-H1 Complementarity-determining region 1 in the immunoglobulin heavy chain variable region CDR-H2 Complementarity-determining region 2 in the immunoglobulin heavy chain variable region CDR-H3 Complementarity-determining region 3 in the immunoglobulin heavy chain variable region CDR-L1 Complementarity-determining region 1 in the immunoglobulin light chain variable region CDR-L2 Complementarity-determining region 2 in the immunoglobulin light chain variable region CDR-L3 Complementarity-determining region 3 in the immunoglobulin light chain variable region

[0158] [Table 7]

[0159] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 1 H NMR) or mass spectrometry (MS). [Example]

[0160] Nuclear magnetic resonance ( 1 H NMR measurements were performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) as the deuterium reagent and tetramethylsilane (TMS) as the internal standard.

[0161] The abbreviations used in the examples for nuclear magnetic resonance (NMR) spectra are as follows: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0162] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0163] Example 1: N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (M-01) [ka] Compound IM-1 (0.40 g, 640.59 μmol; see Patent Document CN111936169A for its synthesis) and exatecan mesylate (0.37 g, 704.65 μmol) were dissolved in DMF (8 mL), and HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added. The mixture was allowed to react at 25°C for 4 hours. The DIPEA was removed under reduced pressure, and the mixture was lyophilized after adding water. The majority of the DMF was removed to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography (conditions are as follows) to obtain 273 mg of compound M-01. Column: Waters XBridge Prep C18 OBD 45mm x 450mm x 8.0μm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% trifluoroacetic acid)

[0164] [Table 8]

[0165] The structural property data of M-01 was as follows: ESI-MS(m / z):1034.4[M+H] + .

[0166] Example 2: N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxy-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-05) [ka] Under nitrogen gas protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (IM-2, 0.66 g, 1.80 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxa-3-oxa-5,8,11,14-tetraazahexadecanoic acid (IM-3, 0.75 g, 1.77 mmol) were added to DMF (19 mL), and the temperature was raised to 35°C and the reaction was carried out for 16 hours. After that, (1S,9S )-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-4, 1.00 g, 1.77 mmol) was added, and the mixture was cooled to 5-15 °C with ice water. DMTMM (0.98 g, 3.53 mmol) was added, followed by dropwise addition of DIPEA (1.14 g, 8.84 mmol), and the mixture was allowed to react at 25 °C for 16 h. The reaction mixture was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL). The mixture was stirred for 10 min. The DCM phase was separated, washed with brine (100 mL), and concentrated to give the crude product. After purification by preparative high performance liquid chromatography, the product was freeze-dried to obtain 0.98 g of compound A-05.

[0167] The separation and purification method for A-05 was as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0168] [Table 9]

[0169] The structural property data of A-05 was as follows: MS m / z (ESI): 1107.3 [M+H]+ 1H NMR (400 MHz, DMSO) δ 9.10 (s, 2H), 8.66 - 8.63 (m, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.34 - 8.31 (m, 1H), 8.21 - 8.19 (m, 1H), 8.17 - 8.09 (m, 2H), 8.08 - 8.04 (m, 1H), 7.30 (s, 1H), 7.26 - 7.15 (m, 5H), 6.55 (s, 1H), 5.56 - 5.55 (m, 1H), 5.48 - 5.35 (m, 2H), 5.25 - 5.10 (m, 2H), 4.64 (d, J = 6.4 Hz, 2H), 4.45 - 4.44 (m, 1H), 4.06 - 3.98 (m, 2H), 3.77 - 3.52 (m, 6H), 3.41 (s, 3H), 3.25 - 3.12 (m, 2H), 3.03 - 3.00 (m, 1H), 2.83 - 2.72 (m, 1H), 2.58 - 2.56 (m, 2H), 2.48 (s, 3H), 2.33 - 2.30 (m, 2H), 2.21 - 2.13 (m, 2H), 1.91 - 1.76 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H).

[0170] Example 3: N-((S)-10-benzyl-1-(((1S,9S)-5-fluoro-9-ethyl-9-hydroxy-4-chloro-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxy-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-07) [ka] Under nitrogen gas protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (IM-2, 21.6 mg, 0.059 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxa-3-oxa-5,8,11,14-tetraazahexadecanoic acid (IM-3, 24.5 mg, 0.058 mmol) were added to DMF (1 mL), and the mixture was heated to 35°C and reacted for 16 hours. (1S,9S)-1-amino-5-fluoro-9-ethyl-9-hydroxy-4-chloro-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione trifluoroacetate (30.0 mg, 0.053 mmol), HATU (30 mg, 0.079 mmol), and DIPEA (27.2 mg, 0.21 mmol) were added to the reaction mixture, and the reaction mixture was allowed to react for 16 hours at 25°C. The reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 26.4 mg of A-07 compound.

[0171] The separation and purification method for A-07 was as follows. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0172] [Table 10]

[0173] The structural property data of A-07 was as follows: ESI-MS (m / z): 1111.3 [M + H] + .

[0174] Example 4: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxa-3-oxa-5,8,11-triazatetradecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-14) [ka] Step 1: Compound IM-4 (657 mg, 1.22 mmol) and compound 1-4 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol) and stirring at room temperature for 1 hour. After completion of the reaction, the reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 700 mg of compound IM-5.

[0175] The manufacturing method is as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0176] [Table 11]

[0177] Step 2: Compound IM-5 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 307 mg of compound IM-6.

[0178] The manufacturing method is as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0179] [Table 12]

[0180] Step 3: IM-6 (170 mg, 0.226 mmol) and compound IM-2 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 50.56 mg of compound A-14.

[0181] Its structural property data were as follows: MS m / z(ESI):1002.4[M+H]+

[0182] The manufacturing method is as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0183] [Table 13]

[0184] 1 H NMR (400 MHz, DMSO) δ 9.11 (s, 2H), 8.68 (t, J = 6.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.55 (m, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 4.67-4.55 (m, 2H), 4.29-4.15 (m, 3H), 3.98 (s, 2H), 3.41 (s, 3H), 3.25-3.15 (m, 2H), 2.57-2.56 (m, 2H), 2.35-2.27 (m, 2H), 2.22-2.12 (m, 2H), 1.91-1.75 (m, 4H), 1.23-1.09 (m, 9H), 0.87 (t, J = 7.2 Hz, 3H).

[0185] Example 5 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01) [ka] Step 1: At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis see Patent No. CN111295389B specification) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), copper bromide (I) (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added, and the mixture was stirred for 1 hour, then filtered. The filtrate was purified by preparative high performance liquid chromatography (conditions are as follows), obtaining 30.00 mg of compound B-01-2. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water

[0186] [Table 14]

[0187] Step 2: Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and trifluoroacetic acid (0.2 mL) was added. The mixture was allowed to react at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure and then purified by preparative high-performance liquid chromatography (conditions are as follows) to obtain 20.00 mg of the trifluoroacetate salt of compound B-01. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% trifluoroacetic acid)

[0188] [Table 15]

[0189] The structural characterization data was as follows: ESI-MS (m / z): 1631.7 [M+H] + , 816.0[M / 2+H] + .

[0190] Example 6: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxy-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((1S,9R)-9-ethyl-5-fluoro-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-02) [ka] Step 1: At 25 ° C., the mesylate salt of 1-1 (30.00 mg, 56.44 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (58.74 mg, 112.88 μmol), N,N-diisopropylethylamine (43.76 mg, 338.63 μmol), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (26.62 mg, 84.66 μmol) were added in this order. The temperature was maintained at 25 ° C. for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, water was added to the reaction solution, extracted with ethyl acetate, and the organic phases were combined, dried over sodium sulfate, and then concentrated under reduced pressure. The crude product and crude raw materials were separated by thin layer chromatography (dichloromethane:methanol = 15:1) to obtain 27.00 mg of B-02-1 compound.

[0191] Step 2: At 0°C, B-02-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL), and the solution was added in this order to a dichloromethane solution (0.5 mL) of 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and triphosgene (8.11 mg, 27.33 μmol). The mixture was allowed to react for 0.5 hours while maintaining the temperature at 0°C. After purging the residual triphosgene with nitrogen gas, a dichloromethane solution (1 mL) of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-3,9-diazapentatriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (43.46 mg, 40.99 μmol) was added dropwise and the reaction was continued for 0.5 hours at 0 °C. The reaction was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, the reaction solution was concentrated, and the crude product and raw materials were separated by thin-layer chromatography (dichloromethane:methanol = 15:1) and purified to obtain 30.00 mg of B-02-2 compound.

[0192] Step 3: At 25 ° C, B-02-2 (250.00 mg, 137.51 μmol) was dissolved in a mixed solvent of DMSO (2 mL) and water (0.4 mL), and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (62.98 mg, 206.26 μmol) and copper(I) bromide (39.45 mg, 275.01 μmol) were added. The temperature was maintained at 25 ° C and the reaction was continued for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry, and after completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography (conditions are as follows), and the aliquot was lyophilized to obtain 150.00 mg of B-02-3 compound. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0193] [Table 16]

[0194] Step 4: At 25°C, B-02-3 (150 mg, 49.45 μmol) was dissolved in tetrahydrofuran (1 mL), and a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) and glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was carried out for 0.5 hours while maintaining the temperature at 25°C. The reaction was monitored by liquid chromatography-mass spectrometry, and after completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography (conditions are as follows), and the collected solution was lyophilized to obtain 50.00 mg of B-02-4 compound. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0195] [Table 17]

[0196] Step 5: B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL) at 25°C, trifluoroacetic acid (60.49 mg, 530.49 μmol) was added, and the reaction was carried out for 0.5 hours while maintaining the temperature at 25°C. The reaction was monitored by liquid chromatography-mass spectrometry, and after completion of the reaction, the reaction solution was concentrated, and the crude product was purified by preparative high-performance liquid chromatography (conditions are as follows), and the collected solution was freeze-dried to obtain 23.69 mg of B-02 compound. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0197] [Table 18]

[0198] The structural property data of B-02 was as follows: ESI-MS(m / z):1613.6[M+H] + .

[0199] Example 7: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxa-3,17,20,23-tetraoxa-5,8,11,14-tetraazapentan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzeneamide (C-07) [ka] [ka] Step 1: The raw material C-07-1 (4.80 g, 16.33 mmol), tributyl(2-methylsulfanylpyrimidin-4-yl)tin (16.27 g, 39.19 mmol), and dichlorobis(triphenylphosphine)palladium(II) (2.29 g, 3.27 mmol) were dissolved in 1,4-dioxane (100 mL), and the reaction mixture was stirred at 110 °C for 5 hours under a nitrogen gas atmosphere. The reaction was monitored by LC-MS, and the reaction mixture was concentrated and purified by column chromatography (EA / PE = 0 to 50%) to obtain 1.36 g of C-07-2 compound.

[0200] Step 2: Compound C-07-2 (510 mg, 1.33 mol), NaOH (212.24 mg, 5.31 mmol) were dissolved in THF (12.5 mL), MeOH (12.5 mL), and HO (2.5 mL). The mixture was stirred at 25 °C for 2 hours and monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 3N HCl to precipitate a large amount of solid, which was then filtered. The filter cake was collected and dried to obtain 380 mg of compound C-07-3.

[0201] Step 3: Compound C-07-3 (315 mg, 850.32 μmol), tert-butyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate (246.31 mg, 935.35 μmol), HATU (484.99 mg, 1.28 mmol), and DIPEA (329.69 mg, 2.55 mmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 40 mg of compound C-07-3.

[0202] The manufacturing method is as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0203] [Table 19]

[0204] Step 4: Compound C-07-4 (40 mg, 64.96 μmol) was dissolved in DCM (3 mL) and TFA (1.5 mL) and reacted at 25° C. for 1.5 hours. The reaction was monitored by LC-MS, and the reaction mixture was concentrated to dryness to give 36 mg of compound C-07-5.

[0205] Step 5: Compound C-07-5 (26 mg, 46.46 μmol), sodium periodate (99.37 mg, 464.57 μmol), and RuCl3·H2O (9.64 mg, 46.46 μmol) were dissolved in ACN (15 mL) and water (7.5 mL) and reacted at 25 °C for 40 minutes. The reaction was monitored by LC-MS, and the mixture was extracted with water and ethyl acetate. The ethyl acetate layer was concentrated to give 28 mg of compound C-07-6.

[0206] Step 6: Compound exatecan mesylate (600 mg, 1.13 mmol), (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxy-2,15-dioxy-4,7,10,13-tetraazaheptan-17-oic acid (IM-4, 610.17 mg, 1.13 mmol), HATU (643.81 mg, 1.69 mmol), and DIPEA (437.65 mg, 3.39 mmol) were added to DMF (6 mL) and reacted at 25 °C for 16 h. The reaction was monitored by LC-MS. Water was added to the reaction mixture to precipitate a large amount of solid. The solid was collected by filtration, dissolved in DCM, and concentrated to obtain the crude product. The residue was purified by column chromatography (DCM / MeOH=0 to 10%) to obtain 660 mg of C-07-7 compound.

[0207] Step 7: Compound C-07-7 (660 mg, 688.94 μmmol) was dissolved in N,N-dimethylformamide (6 mL), and diethylamine (251.94 mg, 3.44 mmol) was added and reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 325 mg of compound C-07-8.

[0208] The manufacturing method is as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0209] [Table 20]

[0210] Step 8: Compounds C-07-6 (15.95 mg, 25.58 μmol), C-07-8 (20 mg, 25.58 μmol), HATU (14.59 mg, 38.37 μmol), and DIPEA (9.92 mg, 76.75 μmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 7 mg of compound C-07.

[0211] Its structural property data were as follows: ESI-MS(m / z):1342.4[M+H] + .

[0212] The manufacturing method is as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0213] [Table 21]

[0214] Example 8: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxa-3,17,20,23-tetraoxa-5,8,11,14-tetraazapentan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzeneamide (C-10) [ka] Step 1: The starting material C-10-1 (720 mg, 2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and HO (4 mL). The reaction mixture was stirred at 90 °C under a nitrogen atmosphere for 3 h. The reaction was monitored by LC-MS, filtered through diatomaceous earth, and the filtrate was extracted with water and ethyl acetate. The crude product was concentrated to give 710 mg of C-10-1. The crude product was purified by column chromatography (EA / PE = 0-25%).

[0215] Step 2: Compound C-10-1 (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and HO (2 mL). The mixture was stirred at 25 °C for 2 hours and monitored by LC-MS. The pH of the system was adjusted to about 2 with 1N HCl to precipitate a large amount of solid, which was then filtered. The filter cake was collected and dried to obtain 560 mg of compound C-10-2.

[0216] Step 3: Compound C-10-2 (450.80 mg, 1.22 mmol) was dissolved in DCM (10 mL), and m-CPBA (2.46 g, 12.1 mmol, 85% purity) was added to the reaction mixture. The mixture was reacted at 25 °C for 12 hours and monitored by LC-MS. The solvent was dried under a stream of nitrogen gas to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 153 mg of compound C-10-3.

[0217] The manufacturing method is as follows. Column: Phenomenex Luna C18 200 x 40 mm x 10 um. Mobile phase A: acetonitrile; Mobile phase B: water (0.05% hydrochloric acid) Mobile phase: [water(HCl)-ACN]; B%: 13%~43%, 10min).

[0218] [Table 22]

[0219] Step 4: Compound C-10-3 (140 mg, 322.25 μmol), tert-butyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate (84.86 mg, 322.25 μmol), HATU (183.80 mg, 483.37 μmol), and DIPEA (124.94 mg, 966.75 μmol) were added to DMF (4 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 51 mg of compound C-10-4.

[0220] The manufacturing method is as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0221] [Table 23]

[0222] Step 5: Compound C-10-4 (50 mg, 73.56 μmol) was added to DCM (2 mL) and TFA (1 mL) and reacted at 25 °C for 1 h. The reaction was monitored by LC-MS, and the reaction mixture was concentrated to dryness to give 45 mg of compound C-10-5.

[0223] Step 6: Compound C-10-5 (31.91 mg, 51.17 μmol), C-07-8 (40 mg, 51.17 μmol), HATU (29.18 mg, 76.75 μmol), and DIPEA (19.84 mg, 153.50 μmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 13 mg of compound C-10.

[0224] Its structural property data were as follows: ESI-MS(m / z): 1342.5 [M+H]+.

[0225] The manufacturing method is as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0226] [Table 24]

[0227] Example 9: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxa-3,17,20,23-tetraoxa-5,8,11,14-tetraazapentan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzeneamide (C-17) [ka] Step 1: C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-propionate (2.25 g, 8.10 mmol) were added to DMF (3 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in this order. The mixture was heated to 60 °C and reacted for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give crude C-17-1 (3.8 g, 4.75 mmol). This was used directly in the next step without further purification.

[0228] Step 2: C-17-1 (3.40 g, 5.40 mmol) was dissolved in dichloromethane (30 mL) and trifluoroacetic acid (10.8 g, 94.2 mmol, 7 mL) was added. The reaction mixture was stirred at 25 °C for 2 hours, concentrated, purified by preparative high-performance liquid chromatography, and lyophilized to obtain C-17-2 (2.09 g, 3.64 mmol).

[0229] The manufacturing method is as follows. Column: Phenomenex luna C18 (250 mm x 70 mm x 10 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0230] [Table 25]

[0231] Step 3: C-17-2 (56 mg, 97.61 μmol) was added to acetonitrile (6 mL) and water (3 mL), and then sodium periodate (208.79 mg, 976.15 μmol) and ruthenium(III) chloride hydrate (8.10 mg, 39.05 μmol) were added to the reaction system, and the mixture was stirred at 25°C for 30 minutes. The reaction was monitored by LC-MS, and the mixture was extracted with water and ethyl acetate and concentrated to give C-17-3 (60 mg).

[0232] Step 4: IM-6 (20 mg, 25.06 μmol), C-17-3 (16 mg, 25.06 μmol), HATU (19.05 mg, 50.11 μmol), and DIPEA (16.19 mg, 125.28 μmol) were added to DMF (3 mL) in this order, and the reaction system was reacted at 25°C for 1 hour. The reaction solution was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain C-17 (16 mg).

[0233] Its structural property data were as follows: ESI-MS(m / z):1371.4[M+H] + .

[0234] The manufacturing method is as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0235] [Table 26]

[0236] Example 10: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24-tetraoxo-5,8,11,14-tetraazahexan-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-19) [ka] Step 1: C-19-1 (5.00 g, 16.9 mmol), (2-(methylthio)pyrimidin-5-yl)boronic acid (6.34 g, 37.3 mmol), XPhos Pd G3 (1.44 g, 1.70 mmol), and potassium phosphate (10.80 g, 50.9 mmol) were added to 1,4-dioxane (51.0 mL) and water (17.0 mL). The reaction mixture was purged with nitrogen gas three times and then reacted at 100 °C for 5 hours. After cooling to room temperature, water (50.0 mL) was added to the reaction mixture, which was then filtered. The filtrate was concentrated to obtain the crude product. The mixture was triturated with petroleum ether, filtered again, and the filter cake was dried under vacuum to obtain C-19-2 (5.65 g).

[0237] Step 2: C-19-2 (5.26 g, 13.7 mmol) was dissolved in THF (30 mL), MeOH (30 mL), and water (30 mL), and LiOH·HO (1.72 g, 40.9 mmol) was added and stirred at 25 °C for 2 h. The pH of the reaction mixture was adjusted to 3 with 1N aqueous hydrochloric acid, and the precipitated solid was filtered. The filter cake was dried under vacuum to obtain C-19-3 (4.20 g).

[0238] Step 3: C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 3-(2-(2-aminoethoxy)ethoxyethoxyethyl)propionate (1.12 g, 4.04 mmol) were dissolved in DMF (20 mL). HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIPEA (2.09 g, 16.2 mmol) were added in this order, and the mixture was heated to 60 °C and stirred for 2 h. The reaction mixture was cooled to room temperature, and water (10 mL) and ethyl acetate (20 mL) were added. The aqueous phase was extracted twice with ethyl acetate (25 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude C-19-4 (2.50 g). This was used directly in the next step without further purification.

[0239] Step 4: C-19-4 (2.50 g, 3.96 mmol) was dissolved in dichloromethane (3.00 mL), and TFA (4.61 g, 40.4 mmol) was added. The reaction mixture was stirred at 25° C. for 12 hours. The reaction mixture was concentrated, purified by preparative high-performance liquid chromatography, and lyophilized to obtain C-19-5 (1.20 g).

[0240] The manufacturing method is as follows. Column: Phenomenex luna C18 (150 mm x 25 mm x 10 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0241] [Table 27]

[0242] Step 5: C-19-5 (1.10 g, 1.91 mmol) was dissolved in a mixed solvent of acetonitrile (30 mL) and water (15 mL), and ruthenium(III) chloride hydrate (39.70 mg, 0.19 mmol) and sodium periodate (4.09 g, 19.14 mmol) were added. The reaction mixture was incubated at 25 °C for 1 h, then extracted with water (50 mL) and ethyl acetate (80 mL). The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to give C-19-6 (130 mg).

[0243] Step 6: IM-6 (20.0 mg, 0.025 mmol) and C-19-6 (16.0 mg, 0.025 mmol) were added to DMF (1 mL) and dissolved by stirring. HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain C-19 (20.4 mg).

[0244] Its structural property data were as follows: ESI-MS(m / z):1372.4[M+H] + . Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0245] [Table 28]

[0246] Example 11: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazabutan-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-21) [ka] Step 1: C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptan-27-oate (4.03 g, 8.10 mmol) were added to DMF (40 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in that order. The reaction mixture was stirred at 60 °C for 2 h. The reaction mixture was extracted with water (100 mL) and ethyl acetate (60 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give C-21-1 (4.20 g, 4.14 mmol). This was used directly in the next step without further purification.

[0247] Step 2: C-21-1 (3.60 g, 4.24 mmol) was dissolved in dichloromethane (30 mL), TFA (15.3 g, 134 mmol, 10 mL) was added, and the reaction mixture was stirred at 25 °C for 6 hours. The reaction mixture was extracted with water (60 mL) and ethyl acetate (40 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified by preparative high-performance liquid chromatography and then lyophilized to give C-21-2 (2.93 g, 3.63 mmol).

[0248] The manufacturing method is as follows. Column: Phenomenex luna C18 (250 mm x 70 mm x 10 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0249] [Table 29]

[0250] Step 3: C-21-2 (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and then sodium periodate (398.71 mg, 1.86 mmol) and ruthenium(III) chloride hydrate (15.47 mg, 74.56 μmol) were added to the reaction mixture, which was stirred for 30 minutes at 25° C. The reaction mixture was extracted with water and ethyl acetate and concentrated to give C-21-3 (155 mg).

[0251] Step 4: IM-6 (27.91 mg, 34.97 μmol), C-21-3 (30 mg, 34.97 μmol), HATU (26.59 mg, 69.93 μmol), and DIPEA (22.60 mg, 174.84 μmol) were added to DMF (3 mL), and the reaction mixture was incubated at 25°C for 1 hour. The reaction mixture was purified by high-performance liquid chromatography and then lyophilized to obtain C-21 (15 mg).

[0252] Its structural property data were as follows: ESI-MS(m / z):1591.7[M+H] + .

[0253] The manufacturing method is as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0254] [Table 30]

[0255] Example 12: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazabutan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-23) [ka] Step 1: C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptan-27-oate (2.01 g, 4.04 mmol) were added to DMF (20 mL), and HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIEA (2.09 g, 16.2 mmol) were added in that order. The mixture was heated to 60 °C and stirred for 2 h. After cooling to room temperature, the reaction mixture was separated with water (10 mL) and ethyl acetate (20 mL). The aqueous phase was extracted twice with ethyl acetate (25 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give crude product C-23-1 (3.00 g), which was used directly in the next step.

[0256] Step 2: C-23-1 (3.00 g, 3.53 mmol) was added to dichloromethane (10.0 mL), and TFA (15.4 g, 134 mmol) was added, followed by stirring at 25 °C for 12 hours. The reaction mixture was concentrated directly to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography and then lyophilized to obtain C-23-2 (1.20 g).

[0257] The manufacturing method is as follows. Column: Welch Ultimate C18 (150 mm x 25 mm x 5 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0258] [Table 31]

[0259] Step 3: C-23-2 (500 mg, 0.63 mmol) was added to acetonitrile (10 mL) and water (5 mL), followed by the addition of ruthenium(III) chloride hydrate (13.0 mg, 0.063 mmol) and sodium periodate (1.35 g, 6.29 mmol). The mixture was reacted at 25 °C for 1 hour, then extracted with water (10 mL) and ethyl acetate (40 mL). The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to give C-23-3 (350 mg).

[0260] Step 4: IM-6 (20.0 mg, 0.025 mmol) and C-23-3 (21.5 mg, 0.025 mmol) were dissolved in DMF (1 mL), and HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added. The mixture was then reacted at room temperature for 2 hours. The reaction mixture was purified by preparative high-performance liquid chromatography and lyophilized to give C-23 (17.0 mg).

[0261] Its structural property data were as follows: ESI-MS(m / z):1592.6[M+H] + .

[0262] The manufacturing method is as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0263] [Table 32]

[0264] Example 13: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl ((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03) [ka] Step 1: Preparation of 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (B-03-1) (1S,9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) was dissolved in DMF (50 mL), DIPEA (1.18 g, 9.12 mmol, 1.59 mL) was added dropwise, and acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) was added dropwise under stirring in an ice bath. The reaction mixture was stirred for 1 hour. The reaction solution was added to 0.1 M dilute aqueous hydrochloric acid, and the precipitated solid was filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The residue was purified on a silica gel column (methanol / dichloromethane = 0% to 5%) and concentrated again to give the title compound (1.7 g, 3.077 mmol).

[0265] Its structural property data were as follows: ESI-MS(m / z): 552.2 [M+1] + .

[0266] Step 2: Preparation of 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidine[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (B-03-2) Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL). The solution was cooled to 0°C under nitrogen gas protection, and a dichloromethane solution (5 mL) of triphosgene (268.81 mg, 0.905 mmol) was added dropwise. The mixture was then stirred for 0.5 hours while maintaining the temperature. A solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azotrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise, allowed to warm to room temperature, and reacted for 4 hours. The reaction was quenched by adding water, extracted three times with dichloromethane (100 ml × 3), and the combined organic phases were washed with saturated brine, dried, and concentrated. Purification using a silica gel column (MeOH / DCM = 0% to 5%) afforded the title compound (498 mg, 0.304 mmol).

[0267] Its structural property data were as follows: ESI-MS(m / z): 1352.8[M+1] + .

[0268] Step 3: Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl ((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-3) 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamide)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,1 Ethyl 3,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidine[1,2-b]quinolin-1-yl)amino-2-oxoacetate (200 mg, 0.122 mmol) was dissolved in THF (3 mL) and MeOH (3 mL), and aqueous sodium carbonate (25.88 mg, 0.224 mmol) (1 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. The reaction mixture was neutralized by adding dilute hydrochloric acid dropwise, concentrated under reduced pressure, and then directly carried on to the next step.

[0269] Its structural property data were as follows: ESI-MS(m / z):1596.7[M+1] + .

[0270] Step 4: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl ((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-4) 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl ((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl) carbonate (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (0.5 mL) was added, followed by continued reaction for 1 hour. The reaction mixture was neutralized by adding saturated aqueous sodium bicarbonate solution, and the organic phase was concentrated to give the crude product, which was purified by reverse-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and then lyophilized to give the title compound (95 mg, 69.35 μmol).

[0271] Its structural property data were as follows: ESI-MS(m / z):1323.6[M+1] + .

[0272] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl ((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03) 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamide)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4' [6,7] Indolizino[1,2-b]quinolin-9-yl)carbonate (90 mg, 0.066 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL), and copper(I) bromide (9.42 mg, 0.066 mmol) was added and stirred for 2 hours. The reaction mixture was filtered, and the crude product was concentrated and purified by preparative high-performance liquid chromatography. The product was then lyophilized to give the title compound (42.2 mg, 24.69 μmol).

[0273] Its structural property data were as follows: ESI-MS(m / z): 1628.7[M+1]+.

[0274] The separation method for high performance liquid chromatography was as follows. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0275] [Table 33]

[0276] II. Antibody Preparation We immunized fully humanized mice with human B7-H3-4Ig-His protein and measured serum titers by ELISA and flow cytometry. Based on the titer results, we selected optimal mice. Spleen cells were then fused, screened, and subcloned to measure the activity of various monoclonal human / monkey proteins and cells. The optimal clone, 20G11G6 / 2#, was obtained. After modifying the antibody sequence to eliminate PTM sites, reduce PI, and eliminate ADCC activity in the heavy chain constant region, we finally obtained fully human antibody 2#8890 (heavy chain variable region, SEQ ID NO: 3; light chain variable region, SEQ ID NO: 4), which contains a sequence (SEQ ID NO: 31) for modifying the human IgG1 heavy chain constant region to enhance ADCC activity, and a human κ light chain constant region (SEQ ID NO: 32). This fully humanized antibody was constructed (see Table 1). The antibody was codon-optimized and gene-synthesized by Nanjing GenScript Biotechnology Co., Ltd., and constructed into the pTT5 plasmid. The heavy and light chain plasmids were co-transfected into CHO-S cells, and the expressed antibody in the supernatant was purified using Protein A to obtain the corresponding antibody protein 2#8890. The heavy and light chain amino acid sequences of 2#8890 are set forth in SEQ ID NO:42 and SEQ ID NO:43, respectively.

[0277] hIgG1 is an anti-chicken lysozyme antibody, whose heavy chain variable region was fused to a mutated human IgG1 heavy chain constant region (SEQ ID NO: 31) and whose light chain variable region was fused to a wild-type human κ light chain constant region (SEQ ID NO: 32). The antibody hIgG1 was obtained by expression and purification using the method described above.

[0278] The B7-H3 control antibody DS7300 was derived from Patent No. CN103687945A. After codon optimization, the antibody heavy chain variable region nucleotide sequence was synthesized and cloned into the human IgG1 heavy chain constant region (sequence number 31) containing mutations. The light chain variable region nucleotide sequence was synthesized into a pTT5 vector containing the wild-type kappa light chain constant region (sequence number 32). The antibody DS7300 was obtained by expressing and purifying it using the above method.

[0279] [Table 34]

[0280] 3. Conjugation of antibodies with compounds containing cellular biologically active molecules and linkers The antibodies 2#8890, DS7300, and hIgG1 for which antibody-drug conjugates are prepared in the following examples are the corresponding antibodies described in Part 2 above.

[0281] 1. Preparation of ADC 1 (DS7300-M-01, DAR 4) 38.041 ml of homemade DS7300 antibody (26.287 mg / mL) was taken, the pH adjusted to 7.4 with 1 M NaHPO, and the antibody was diluted to 3 mg / mL with 20 mM PB. 4 mM ZnCl (3.413 mL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 4.096 mL, pH 7.4) solutions were added in that order in an ice bath, mixed evenly, and left to stand at 4 °C overnight. A 6-fold volume of M-01 (4.18 mL, 10 mM) solution containing dimethyl sulfoxide was added and mixed evenly. The mixture was incubated at 4 °C for 4 hours, after which 6.826 mL of 10 mM cysteine ​​solution was added. After 1.5 hours of incubation, the reaction mixture was transferred to room temperature and 6.826 mL of 10 mM EDTA solution was added. After 30 minutes, additional DHAA solution (10 mM, 6.826 mL) was added and the reaction continued for 30 minutes. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 1 (DS7300-M-01). The DAR value measured by mass spectrometry was 3.8.

[0282] 2. Preparation of ADC 2 (hIgG1-M-01, DAR 4) 1.2245 ml of hIgG1 antibody (24.5 mg / mL) was taken, the pH adjusted to 7.3 with 1 M NaHPO, and the antibody was diluted to 3 mg / mL with 20 mM PB. 4 mM ZnCl (52.04 μL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.89 μL, pH 7.3) solutions were added in this order in an ice bath, mixed evenly, and left to stand overnight at 4°C. A 6-fold volume of 171 (127.44 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added and mixed evenly. The mixture was incubated at 4°C for 4 hours, after which cysteine ​​solution (10 mM, 208.15 μL) was added. After incubation for 1.5 hours, the reaction mixture was transferred to room temperature and EDTA solution (10 mM, 208.15 μL) was added. After 30 minutes, additional DHAA solution (10 mM, 208.15 μL) was added and the reaction continued for 30 minutes. Upon completion, the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 2 (i.e., hIgG1-M-01). The DAR value measured by mass spectrometry was 4.46.

[0283] 3. Preparation of ADC 3 (2#8890-M-01, DAR 4) 9.36 ml of antibody 2#8890 (3.205 mg / mL) was taken, the pH adjusted to 7.4 with 1 M NaHPO, and the antibody was diluted to 3 mg / mL with 20 mM PB. 4 mM ZnCl (57.31 μL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.9 μL, pH 7.4) solutions were added in that order in an ice bath, mixed evenly, and left to stand at 4°C overnight. A 6-fold volume of M-01 (124.9 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added and mixed evenly. The mixture was incubated at 4°C for 4 hours, after which 208.2 μL of 10 mM cysteine ​​solution was added. After 1.5 hours of incubation, the reaction mixture was transferred to room temperature and 208.2 μL of 10 mM EDTA solution was added. After 30 minutes, 208.2 μL of 10 mM DHAA solution was added and the reaction continued for another 30 minutes. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 3 (2#8890-M-01). The DAR value measured by mass spectrometry was 3.89.

[0284] 4. Preparation of ADC 4 (hIgG1-A-05, DAR 8) 0.943 ml of hIgG1 antibody (11 mg / mL) was diluted with 47 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 57 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-05 (103 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., ADC 4 (hIgG1-A-05). The DAR measured by mass spectrometry was 8.03.

[0285] 5. Preparation of ADC 5 (2#8890-A-05, DAR 8) 3.052 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 153 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 114.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-05 (219.1 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 5 (2#8890-A-05). The DAR measured by mass spectrometry was 7.90.

[0286] 6. Preparation of ADC 6 (hIgG1-A-07, DAR 8) 0.518 ml of hIgG1 antibody (19.3 mg / mL) was diluted with 25.9 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 38.1 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-07 (69.2 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 6 (hIgG1-A-07). The DAR measured by mass spectrometry was 8.04.

[0287] 7. Preparation of ADC 7 (2#8890-A-07, DAR 8) 1.017 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 50.85 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 38.2 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 12-fold volume of A-07 (87.6 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 7 (2#8890-A-07). The DAR measured by mass spectrometry was 7.76.

[0288] 8. Preparation of ADC 8 (hIgG1-A-14, DAR 8) 1.9126 mL of hIgG1 antibody (18.3 mg / mL) was diluted with 95.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 66.86 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-14 (260.53 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., ADC 8 (hIgG1-A-14). The DAR measured by mass spectrometry was 8.0.

[0289] 9. Preparation of ADC 9 (2#8890-A-14, DAR 8) 3.6788 ml of 2#8890 antibody (10.873 mg / mL) was diluted with 183.94 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 152 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-14 (292.26 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 9 (2#8890-A-14). The DAR value determined by mass spectrometry was 7.22.

[0290] 10. Preparation of ADC 10 (hIgG1-B-01, DAR 8) 1.533 mL of hIgG1 antibody (19.57 mg / mL) was diluted with 76.65 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 114.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of B-01 (212.4 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., ADC 10 (hIgG1-B-01). The DAR measured by mass spectrometry was 8.03.

[0291] 11. Preparation of ADC 11 (2#8890-B-01, DAR 8) 3.052 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 152.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 114.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of B-01 (212.4 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 11 (2#8890-B-01). The DAR value determined by mass spectrometry was 7.75.

[0292] The conjugated ADC samples were subjected to LC-MS molecular weight analysis.

[0293] Chromatographic measurement conditions: Liquid phase column: Thermo MAbPac RP 3.0 × 100 mm; Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN; Flow rate: 0.25 ml / min; chamber temperature: 8°C; column temperature: 60°C; sample injection volume: 2 μl;

[0294] [Table 35]

[0295] Mass spectrometry measurement conditions: Mass spectrometry model: AB Sciex Triple TOF 5600+; GS1 35;GS2 35;CUR 30;TEM 350;ISVF 5500;DP 200;CE 10;Cumulative time 0.5s; m / z 600–4000; total time bin 40.

[0296] 12. Preparation of ADC 12 (2#8890-C-07, DAR 4) 0.2274 ml of 2#8890 antibody (10.994 mg / mL) was diluted with 11.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 9.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. An 8-fold volume of C-07 (14.6 μL, 10 mM) solution dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 12 (2#8890-C-07). The DAR value determined by mass spectrometry was 4.12.

[0297] 13. Preparation of ADC 13 (2#8890-C-10, DAR 4) 0.2274 mL of 2#8890 antibody (10.994 mg / mL) was diluted with 11.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 9.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. An 8-fold volume of C-10 (14 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 13 (2#8890-C-10). The DAR measured by mass spectrometry was 4.17.

[0298] The conjugated ADC samples were subjected to LC-MS molecular weight analysis.

[0299] Chromatographic measurement conditions: Liquid phase column: ACQUITY UPLC MAbPac BEH SEC; Mobile phase A: 20mM NH4Ac; Flow rate: 0.1 ml / min; chamber temperature: 8°C; column temperature: 60°C; sample injection volume: 2 μl;

[0300] [Table 36]

[0301] Mass spectrometry measurement conditions: Mass spectrometry model: AB Sciex Triple TOF 5600+; GS1 55;GS2 55;CUR 30;TEM 450;ISVF 5500;DP 75;CE 5;Cumulative time 0.5s; m / z 900–7000; total time bin 40.

[0302] 14. Preparation of ADC 14 (2#8890-C-17, DAR 4) 0.292 ml of 2#8890 antibody (8.565 mg / mL) was diluted with 14.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 4.77 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 5-fold volume of C-17 (8.76 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 14 (2#8890-C-17). The DAR value measured by mass spectrometry was 3.86.

[0303] 15. Preparation of ADC 15 (2#8890-C-19, DAR 4) 0.584 ml of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 9.54 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 5.5-fold volume of C-19 (17.9 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 15 (2#8890-C-19). The DAR value measured by mass spectrometry was 4.05.

[0304] 16. Preparation of ADC 16 (2#8890-C-21, DAR 4) 0.584 ml of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 9.54 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 5.5-fold volume of C-21 (17.9 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 16 (2#8890-C-21). The DAR value measured by mass spectrometry was 3.92.

[0305] 17. Preparation of ADC 17 (2#8890-B-03, DAR 8) 0.867 ml of 2#8890 antibody (34.6 mg / mL) was diluted with 93.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 112.65 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. An 11-fold volume of B-03 (227.58 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 17 (2#8890-B-03, DAR 8). The DAR value determined by mass spectrometry was 7.82.

[0306] 18. Preparation of ADC 18 (2#8890-B-03, DAR 8) 0.867 ml of 2#8890 antibody (34.6 mg / mL) was diluted with 58.35 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 112.65 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of B-03 (211.1 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 18 (2#8890-B-03, DAR 8). The DAR value determined by mass spectrometry was 7.23.

[0307] 19. Preparation of ADC 19 (19F6-B-02) The antibody 19F6_Hu35v1 in the following examples is the 19F6_Hu35v1 antibody described in WO 2022253035A1, and was prepared using the method described in Example 2 of this patent.

[0308] The sample was bound and prepared as follows. 0.46 ml of 19F6_Hu35v1 antibody (11.0 mg / mL) was diluted with 0.1 M edetate disodium solution (pH 7.7) and the pH was adjusted to 7.7 with 1 M NaHPO. 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added, mixed evenly, and allowed to stand at room temperature for 90 min. B-02, dissolved in dimethyl sulfoxide (DMSO) at a 10-fold volume, was added to the solution, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva). Sucrose and Tween 20 were added, mixed evenly, and the antibody-drug conjugate, ADC 19 (19F6-B-02), was obtained. The DAR measured by mass spectrometry was 7.92.

[0309] 20, ADC 20 (trastuzumab-A-05, DAR 8) A 2.469 mL sample of trastuzumab antibody (16.2 mg / mL) was diluted with 123 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 151.57 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and left at room temperature for 1.5 h. A 10-fold volume of A-05 (290.09 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and left at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-05). The DAR measured by mass spectrometry was 8.04.

[0310] 21, ADC 21 (trastuzumab-A-14, DAR 8) A 2.564 mL sample of trastuzumab antibody (15.6 mg / mL) was diluted with 128.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution, and 151.57 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added. The mixture was mixed evenly and left at room temperature for 1.5 h. A 10-fold volume of A-14 (290.1 ​​μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and left at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-14). The DAR measured by mass spectrometry was 8.02.

[0311] 22, ADC 22 (trastuzumab-A-24, DAR 8) A 0.617 mL portion of trastuzumab antibody (16.2 mg / mL) was diluted with 30.86 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 37.89 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and left at room temperature for 1.5 h. A 14-fold volume of A-24 (98.42 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and left at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-24). The DAR value measured by mass spectrometry was 7.37.

[0312] 23, ADC23 (trastuzumab-A-32, DAR 8) A 0.423 mL sample of trastuzumab antibody (4.73 mg / mL) was diluted with 21.17 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution, and 7.58 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added. The mixture was mixed evenly and left at room temperature for 1.5 h. A 10-fold volume of A-32 (13.92 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and left at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-32). The DAR measured by mass spectrometry was 6.77.

[0313] 24, ADC24 (trastuzumab-C-21, DAR 4) A 0.617 mL sample of trastuzumab antibody (16.2 mg / mL) was diluted with 30.86 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 19.1 μL of 19.83 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and left at room temperature for 1.5 h. A 5.5-fold volume of C-21 (38.28 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and left at room temperature for 18 h. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-C-21). The DAR value measured by mass spectrometry was 4.41, of which DAR4 accounted for 60.06%.

[0314] 4. Antibody-drug conjugate activity detection 1. Dynamic affinity detection of anti-human B7-H3 antibody complexes The dynamic affinity of the anti-human B7-H3 antibody complex with human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins was determined using ForteBio (Pall Life Sciences). The antibody complexes were diluted to 5 μg / ml in PBST (0.02% Tween-20). Human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins were serially diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. The antibody complexes were then captured using a Protein A sensor (Pall Life Sciences) in PBST (0.02% Tween-20) for 60 s. The four proteins were then allowed to bind for 60 s and then dissociated for 180 s. The results were analyzed using Data Analysis 11.0 software, 1:1 mode, and global fitting to obtain affinity constants. The results are shown in Table 2, which indicate that the conjugates prepared with the drug linker compounds of the present invention bind to monkey B7-H3 but do not bind to rat B7-H3.

[0315] [Table 37]

[0316] 2. Cell affinity detection of anti-human B7-H3 antibody complexes A flow cytometer (Beckman, model Cytoflex) was used to detect the affinity of the anti-human B7-H3 fully human antibody complex with human colon cancer cells HT29 (Cell Bank of the Chinese Academy of Sciences), human gastric cancer cells NCI-N87 (ATCC), human breast squamous cell carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC). The affinity of the anti-human B7-H3 fully human antibody complex with CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2Ig was also detected. The species cross-reactivity of the anti-human B7-H3 fully human antibody complex with CHOS-rat B7-H3 and CHOS-monkey B7-H3 was also detected. Adherent cells were digested with trypsin-EDTA (0.25%) (Thermo) solution and counted to a cell density of 4.0 × 10 cells. 6 The cells were resuspended in 1% BSA solution, and 50 μl of the cell suspension was added to each well of a 96-well V-bottom plate (2 × 10 cells). 5 (cells / well). The antibody conjugate was diluted with 1% BSA (starting at a final concentration of 10 μg / ml and diluted in 11 three-fold increments), with hIgG1 antibody conjugate as a control (final concentration 10 μg / ml). 50 μl of diluted antibody was added to the V-bottom plate containing the cells and incubated for 60 min at 4°C. The cells were washed twice with 1% BSA, and 50 μl of diluted secondary antibody was added to each well, mixed evenly, and incubated for 30 min at 4°C. The cells were washed twice with 1% BSA, resuspended in 200 μl of 1% BSA, and detected by flow cytometer. Data processing: After the median PE value was calculated, it was entered into GraphPad Prism 6 software and EC 50 was calculated.

[0317] The EC20 affinity of ADC17 to HT29 tumor cells was measured using the anti-human B7-H3 antibody conjugate. 50 The EC 50The affinity of the anti-human B7-H3 fully human antibody conjugate was 8.054 ng / ml, and the affinity results for the other antibody conjugates were as shown in Table 3. The affinity results for the anti-human B7-H3 fully human antibody conjugates to CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2IG cells are as shown in Table 4, and the affinity results for the anti-human B7-H3 antibody conjugates to CHOS-rat B7-H3 and CHOS-monkey B7-H3 cells are as shown in Table 5. These results demonstrate that the conjugates prepared with the drug linker compounds of the present invention bind to monkey B7-H3-overexpressing cells but not to rat B7-H3-overexpressing cells.

[0318] [Table 38]

[0319] [Table 39]

[0320] [Table 40]

[0321] 3. Detection of cellular endocytosis of anti-human B7-H3 antibody complexes Endocytosis of the anti-human B7-H3 antibody complex in human gastric cancer cells NCI-N87 (ATCC), human breast squamous cell carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC) was detected using a flow cytometer (Thermo, model Attune NxT). Adherent cells were digested with trypsin-EDTA (0.25%) (Thermo) solution, counted, and the cell density was adjusted to 1 × 10 using complete medium. 5 The cell suspension was adjusted to 1 × 10 cells / ml, and 100 μl of the cell suspension was added to each well of a 96-well plate (cell count: 1 × 10 4(cells / well). The 96-well plate was incubated at 37°C in a CO2 incubator for 24 h. The 96-well plate was removed, the medium was aspirated and discarded, and 50 μl of fresh complete medium was added to each well. The antibody conjugate to be measured was serially diluted in six steps using complete medium. pHrodo reagent (Thermo, Cat# Z25612) at 300 μg / ml was diluted to 12 μg / ml in complete medium (final pHrodo concentration: 3 μg / ml). The serially diluted antibody to be measured and the diluted pHrodo reagent were mixed equally 1:1 (30 μl:30 μl) and incubated at room temperature, protected from light, for 30 min. 50 μl of the mixture of antibody to be measured and pHrodo reagent was added to the 96-well plate and incubated at 37°C, 5% CO2 for 24 h. The 96-well plate was removed, the medium was aspirated and discarded, and the plate was washed once with sterile PBS. 100 μl of trypsin-EDTA (0.25%) was added to each well to digest the cells, and 100 μl of complete medium was added to neutralize the cells. The cells were dispersed by spraying and then detected by FACS. Data processing: The median YL-1H values ​​were calculated and entered into GraphPad Prism 6 software, and the EC 50 The results are shown in Table 6. All of the antibody-drug conjugates prepared with the drug-linker compounds of the present invention (e.g., ADC 5 and ADC 9) had excellent cellular endocytosis.

[0322] [Table 41]

[0323] 4. In vitro cell killing detection of anti-human B7-H3 antibody-drug conjugates Adherent cells of A375, Calu6-B7-H3, and U87MG-B7-H3 were digested using trypsin-EDTA (0.25%) (Thermo) solution, counted, and cultured at a cell density of 1 × 10 using complete medium. 4 , 5×10 4 , 1×10 4The cell concentration was adjusted to 1000, 5000, or 1000 cells / ml, and 100 μl of the cell suspension was added to each well of a 96-well plate (cell counts of 1000, 5000, and 1000 cells / well, respectively). The 96-well plate was incubated at 37°C in a CO2 incubator and cultured for 24 hours. The ADC to be measured was diluted in complete medium, starting at a final concentration of 3333.3 nM, with 12 four-fold dilutions. 100 μl of the diluted ADC was added to a 96-well plate and cultured at 37°C and 5% CO2 for 4 to 7 days. The 96-well plate was removed, and 20 μl of CCK8 reagent was added to each well. The plate was then incubated at 37°C for 2 to 3 hours. The OD was measured using a microplate reader. 450nm After detecting the signal values, they were input into GraphPad Prism 6 software and calculated as IC 50 was calculated, and the results are shown in Table 7.

[0324] [Table 42]

[0325] As a result, it was demonstrated that the antibody-drug conjugates produced with the drug linker compounds of the present invention have a clear killing effect on tumor cells.

[0326] 5.2 In vivo efficacy of various conjugates of #8890 in HCC1806 model Human breast squamous cell carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was sampled for mycoplasma detection, and the cells were then harvested and counted. 2 × 10 cells were placed on the right scapula of each mouse. 6 HCC1806 cells were subcutaneously inoculated and suspended in 0.1 ml of PBS. The average tumor volume was 100–200 mm. 3When tumors grew to 1000 mg / kg, mice with tumors that were too small or too large were removed. The remaining mice were randomly assigned to six groups, each consisting of six mice, based on tumor volume and body weight. A single dose of the drug was administered via tail vein injection (DAR4 group: 10 mg / kg, DAR8 group: 5 mg / kg). Tumor volume and body weight were measured twice weekly after administration. See Table 8 for specific results.

[0327] [Table 43]

[0328] The results showed that the mice in the treatment group had stable body weight and were well tolerated. 95 days after a single administration, the mice in the ADC 5 group showed complete tumor response. This demonstrates that the antibody-drug conjugates produced with the drug linker compounds of the present invention have significant tumor-suppressing activity.

[0329] 6. Efficacy Measurement of Various Doses of Antibody-Drug Conjugates in the HCC1806 Model Human breast squamous cell carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was sampled for mycoplasma detection, and the cells were then harvested and counted. 2 × 10 cells were placed on the right scapula of each mouse. 6 HCC1806 cells were subcutaneously inoculated and suspended in 0.1 ml of PBS. The average tumor volume was 100–200 mm. 3 When tumors grew to 1000 mcg, mice with tumors that were too small or too large were removed. The remaining mice were randomly assigned to 12 groups, each consisting of 6 mice, based on tumor volume and body weight. A single dose was administered via tail vein injection. Tumor volume and body weight were measured twice weekly after administration. See Table 9 for specific results.

[0330] [Table 44]

[0331] As a result, both the ADC 5 group and the ADC 9 group showed excellent efficacy, and the mice had stable body weight and good tolerability, demonstrating that the antibody-drug conjugates prepared with the drug linker compounds of the present invention have significant tumor-inhibitory activity and good safety.

[0332] 7. Drug efficacy measurement of various antibody-drug conjugates in the NCI-N87 model Human gastric cancer cells NCI-N87 (ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was sampled for mycoplasma detection, and the cells were then harvested and counted. Each mouse was injected with 5 × 10 cells into the right scapula. 6 NCI-N87 cells were subcutaneously inoculated and suspended in 0.1 ml of PBS-based Matrigel. 3 When tumors grew to 100% tumor volume, mice with tumors that were too small or too large were removed. The remaining mice were randomly assigned to eight groups, each consisting of six mice, based on tumor volume and body weight. Administration was via tail vein injection twice daily. Tumor volume and body weight were measured twice weekly after administration. See Table 10 for specific results.

[0333] [Table 45]

[0334] As a result, the mice in each administration group showed stable weight and good tolerability, demonstrating that the antibody-drug conjugates prepared with the drug linker compounds of the present invention have significant tumor-inhibitory activity and good safety.

[0335] 8. Detection of binding activity of anti-human B7-H3 antibody-drug conjugate to Fc receptors The dynamic affinity of the 2#8890 antibody-drug conjugate to the human Fc receptor proteins CD16a, CD32a, CD32b, C1q, and FcRn was measured using ForteBio (Pall Life Sciences). The specific method was as follows: Biotinylated proteins were captured in PBST solution using an SA Sensor (Pall Life Sciences). The antibody and conjugate were diluted to an initial concentration of 5000 nM in PBST, and then diluted two-fold in seven steps to allow binding and dissociation. The measurement results were analyzed in Data Analysis 11.0 software using 1:1 mode and global fitting to obtain the binding rate, dissociation rate, and affinity constant. The results are shown in Table 11.

[0336] [Table 46]

[0337] As a result, the antibody-drug conjugates prepared using the drug linker compounds of the present invention do not bind to the Fc receptors CD16a, CD32a, CD32b, and C1q protein, thereby reducing nonspecific killing mediated by Fc receptors and improving drug safety. At the same time, these conjugates retain FcRn protein binding activity and do not affect the half-life of the drug.

[0338] 9. Pharmacokinetic study of total antibody (Tab), ADC, and payload in serum after multiple intravenous administration of anti-human B7-H3 antibody-drug conjugates to cynomolgus monkeys ELISA and LC-MS / MS were used to quantitatively detect ADCs (ADC5, ADC9), total antibodies (TAb), and payloads in cynomolgus monkey serum. The standard curve quantitative ranges for ADCs (ADC5, ADC9) and total antibodies (TAb) were 11.72–3000.00 ng / mL, and the linear range for payloads was 0.1–40 ng / mL. Both ADCs (ADC5, ADC9) and total antibodies (TAb) used B7-H3 protein as the capture protein. After incorporation into a 96-well microplate, total antibodies (TAb) were detected using goat anti-human IgG-HRP; ADCs (ADC5, ADC9) used anti-toxin mouse antibody and goat anti-mouse IgG as the secondary and detection antibodies, respectively. Color development was achieved by the action of the enzyme and substrate, and the readings were performed using a SpectraMax i3x (Molecular Devices) microplate reader. The concentration of each sample was calculated by fitting a standard curve using the 4-P parameter method. The color depth and the concentration of ADC (ADC 5, ADC 9) and total antibody (TAb) showed a positive correlation. LC-MS / MS analysis was performed using a Shimadzu LC 30-AD flow unit coupled to a SCIEX QTRAP 5500+ (SCIEX) mass spectrometer, using (+)ESI ionization with multiple reaction monitoring (MRM) selected. The column was an Xbridge C18 50 x 4.6 mm, 5 μm column. The analyte compound 1-10 ion pair was 510.2 / 435.2. Sample pretreatment involved protein precipitation using acetonitrile. Results: After multiple intravenous administration of ADC5 and ADC9 to cynomolgus monkeys, measurements showed that the ADC molecules (e.g., ADC5 and ADC9) prepared with the drug linker compounds of the present application exhibited favorable pharmacokinetic properties, were relatively stable in the systemic circulation, and released only small amounts of free toxin.

[0339] [Table 47]

[0340] [Table 48]

[0341] [Table 49]

[0342] 10. Repeated dose toxicity test The repeat-dose toxicity study included a four-dose repeat toxicity study in which ADC5 and ADC9 were administered intravenously to cynomolgus monkeys.

[0343] This study consisted of three groups, one animal per group per sex. Each group received 30 mg / kg of ADC 5, ADC 9, and saline (volume: 10 mL / kg) intravenously, once weekly for a total of two doses. Subsequently, ADC 5, ADC 9, and saline at doses increased to 50 mg / kg were administered once weekly for a total of two doses. No drug-related deaths or moribundity were observed during the study. Animals in the ADC 5 group experienced decreased appetite, alopecia, skin pigmentation, and weight loss during the administration period. Female monkeys experienced decreases in WBC, NEUT, LYM, and MONO. Male monkeys experienced decreases in RBC, HGB, and HCT, and increases in FGB, which tended to recover during the recovery period. Animals in the ADC 9 group experienced decreased appetite during the administration period. Female monkeys experienced decreases in WBC, NEUT, LYM, and MONO, and increases in FGB. In male monkeys, elevated FBG and decreased RBC, HGB, and HCT were observed. During the recovery period, pigmentation and slight hair loss were observed at the injection site, but all other changes showed signs of recovery. Under these experimental conditions, cynomolgus monkeys were intravenously injected with 30 mg / kg of ADC 5 and ADC 9 once a week for two consecutive weeks. The dose was then increased to 50 mg / kg of ADC 5 and ADC 9 once a week for two consecutive weeks. All animals tolerated the treatment. The maximum non-severely toxic dose (HNSTD) was 50 mg / kg.

[0344] 11. Tumor growth inhibition of antibody-drug conjugates in subcutaneously transplanted tumor models in mice Formulations containing the ADC of the present invention were administered via tail vein injection to a mouse CDX model in which human breast squamous cell carcinoma cells HCC1806 had been subcutaneously transplanted. Changes in tumor volume and animal body weight were measured twice a week, and the tumor-suppressing effect of the ADC of the present invention on cancer-bearing mice was calculated.

[0345] Experimental animal: Balb / c Nude mouse (Chengdu Pharmacological Biotechnology Co., Ltd.)

[0346] Cell line: Human breast squamous cell carcinoma cell line HCC1806 (ATCC)

[0347] Experimental Method: HCC1806 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in 5% CO2. HCC1806 cells in the exponential growth phase were harvested, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female Balb / c nude mice to establish a breast squamous cell carcinoma model. The average tumor volume was approximately 200 mm3. 3 At approximately 10 days, mice were randomly assigned according to tumor size and administered the respective drugs. The groups and their doses were as follows: Solvent control group (i.e., negative control, vehicle group): 0.9% NaCl injection was administered. ADC 5: dose 3 mg / kg. ADC 9: dose 3 mg / kg. ADC 11: dose 3.16 mg / kg. ADC 17: dose 3.32 mg / kg. Each group was administered via tail vein injection (iv) on Day 0, for a total of one dose. After administration, the mice were weighed twice a week, and the long and short diameters of the tumor were measured with calipers. The tumor volume was calculated using the following formula: V=0.5a×b 2 In the formula, a and b represent the long and short diameters of the tumor, respectively. The mortality of the animals was observed daily and recorded.

[0348] The tumor growth inhibition rate (TGI) (%) was calculated using the following formula. V T末 >V T0 , TGI(%)=[1-(V T末 -V T0 ) / (V C末 -V C0)] × 100% or V T末 ≦V T0 , TGI(%)=[1-(V T末 -V T0 ) / V T0 ]×100%. In the formula, V T末 : Mean tumor volume at the end of the experiment for each treatment group V T0 : Mean tumor volume at the start of treatment V C末 : Mean tumor volume at the end of the experiment in the negative control group V C0 : Mean tumor volume at the start of administration in the negative control group

[0349] The relative tumor growth rate T / C (%) was calculated using the following formula: T / C=(V T末 / V T0 ) / (V C末 / V C0 )

[0350] The ADCs of the present invention exhibited significant tumor growth inhibition in an HCC1806 breast squamous cell carcinoma xenograft tumor model. On day 14, the tumor growth inhibition rates (TGI) of ADC 5, ADC 9, ADC 11, and ADC 17 of the present invention were 96.68%, 98.50%, 82.61%, and 86.69%, respectively, compared with the vehicle group, demonstrating significant differences compared with the control group. During the treatment period, no deaths or significant weight loss were observed in animals in any treatment group, and no significant drug toxicity was observed. The ADCs of the present invention were well tolerated by mice. See Table 15 for specific results.

[0351] [Table 50]

[0352] 12. Inhibitory effect of antibody-drug conjugates on in vitro cell activity (1) Cell inoculation: First, tumor cells NCI-H1975 and HT-29 were cultured in the corresponding medium, digested with pancreatin, centrifuged, resuspended, counted, and adjusted to a concentration suitable for inoculation onto plates. See Table 16 for tumor cell sources.

[0353] [Table 51]

[0354] After the cells attached, the medium was removed from the cells and diluted antibody drug conjugate ADC 19 was added to the wells of the plate and incubated for 96 hours.

[0355] In vitro cell activity detection: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme) was added to each well and mixed evenly by shaking in the dark. After 10 minutes of reaction, detection was possible and read using a microplate reader (manufacturer: BMG, model: PHERAStar-FS). By adding Cell Counting-Lite™, background RLU was obtained in wells without cells, and control RLU was obtained in wells with cells but no compound. Cell inhibition rate = 1 - (sample RLU - background RLU) / (cell control RLU - background RLU) x 100%. A four-parameter model was used to fit the curve and calculate the half-maximal inhibitory concentration (IC) of ADC 19. 50 ) was calculated.

[0356] The half maximal inhibitory concentration (IC) of ADC 19 against NCI-H1975 cell line 50 ) 5.00 μg / mL, half maximal inhibitory concentration (IC) of ADC 19 against HT-29 cell line 50 )4.56μg / mL.

[0357] As a result, it was demonstrated that antibody-drug conjugates (e.g., ADC19) formed using the drug linkers of the present invention have significant tumor cell-killing activity.

[0358] 13. Efficacy of anti-human Her2 antibody-drug conjugates in the NCI-N87 model NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Exponentially growing NCI-N87 cells were harvested, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female Balb / c-nu mice to establish a gastric cancer model. When the average tumor volume reached approximately 160 mm3, mice were randomly assigned based on tumor size to the following groups: a solvent control group (i.e., negative control, vehicle group), a 1 mg / kg group of the present invention's trastuzumab-A-05, a 1 mg / kg group of trastuzumab-A-14, and a 1 mg / kg group of trastuzumab-A-24. Each group received three doses via tail vein injection (iv) on days 0, 7, and 14. After administration, mice were weighed twice weekly, and the long and short diameters of the tumors were measured with calipers. Tumor volume was calculated using the following formula: V = 0.5a × b2, where a and b represent the major and minor diameters of the tumor, respectively. The mortality of the animals was observed daily and recorded.

[0359] The ADCs of the present invention exhibited significant tumor growth inhibition in an NCI-N87 gastric cancer xenograft model. Compared with the vehicle control group, the tumor growth inhibition rates (TGI) of the 1 mg / kg trastuzumab-A-05 group, the 1 mg / kg trastuzumab-A-14 group, and the 1 mg / kg trastuzumab-A-24 group were 81.62%, 107.38%, and 61.43%, respectively. By day 30, no deaths or significant weight loss were observed in any of the treatment groups, and no significant drug toxicity was observed. During the treatment period, mice tolerated the ADCs prepared with the drug linker compounds of the present invention well. See Table 17 for specific results.

[0360] [Table 52]

[0361] Although the specific embodiments of the present invention have been described in detail above, it is understood that those skilled in the art can make various modifications and substitutions to the details according to all the teachings already disclosed, and all of these modifications fall within the protection scope of the present invention. The full scope of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. Formula GM-[L-ED] ｘ wherein G is a functional group or leaving group capable of reacting with a specific amino acid or glycosyl group; M is a connecting site that connects to G, and said M is 【Chemistry 1】 wherein ring A is a 5- to 6-membered aliphatic heterocycle or a 5- to 20-membered aromatic ring system, and the aliphatic heterocycle and aromatic ring system optionally contain oxygen groups (═O), halogens, cyano groups, amino groups, carboxyl groups, thiol groups, and C １～６ substituted with one or more groups selected from alkyl groups, M １ is a single bond and C １～２０ Alkylene group, C ２～２０ Alkenylene group, C ２～２０ an alkynylene group or an amine group; １～２０ Alkylene group, C ２～２０ Alkenylene group, C ２～２０ the alkynylene or amine group being optionally substituted by one or more suitable substituents; L is a linker connecting the connecting sites M and E, and L is C １～６ an alkylene group, —N(R′)—, a carbonyl group, —O—, natural or unnatural amino acids and analogs thereof, and short peptides consisting of amino acids; 【Chemistry 2】 (Wherein R' is hydrogen, C １～６ represents an alkyl group or a polyethylene glycol fragment having 1 to 10 EO units, and s is selected from an integer of 1 to 20; E is a structural fragment connecting L and D, E is a single bond, -NHCH ２ - or 【Transformation 3】 is selected from the structure D is a payload fragment, and / or x is selected from 1 to 10 A drug-linker compound having the structure:

2. The G is a halogen, a halogenated C １～６ Alkyl group, C １～６ Sulfonyl group, halogenated C １～６ Sulfonyl group, halogenated sulfonyl group, C １～６ Sulfonate ester group, halogenated C １～６ Sulfonate ester group, C １～６ Sulfinate ester group, C １～６ a halogenated C selected from a sulfoxide group, a nitro group, an azido group, a cyano group, an alkenyl group, an alkynyl group, and an alkynyl group-containing structural fragment; １～６ Alkyl group, C １～６ Sulfonyl group, halogenated C １～６ Sulfonyl group, halogenated sulfonyl group, C １～６ Sulfonate ester group, halogenated C １～６ Sulfonate ester group, C １～６ Sulfinate ester group, C １～６ 10. The drug linker compound of claim 1, wherein the sulfoxide group, alkenyl group, alkynyl group, and alkynyl group-containing structural fragment are optionally substituted with one or more suitable substituents.

3. The M is 【Chemistry 4】 (wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycycle formed by connecting one or more (for example, two) 6-membered aromatic heterocycles with a benzene ring or a 6-membered heteroaromatic ring via a single bond, and the aliphatic heterocycle may optionally contain an oxygen group (═O), a halogen atom, and a C １～４ substituted with one or more groups selected from alkyl groups, M １ is a single bond, C １～２０ Alkylene group, C ２～２０ Alkenylene group, C ２～２０ an alkynylene group or an amine group; １～２０ Alkylene group, C ２～２０ Alkenylene group, C ２～２０ The drug linker compound of claim 1 or 2, wherein the alkynylene group or the amine group is optionally substituted with one or more suitable substituents.

4. The M is 【Transformation 5】 (Wherein, ring A is 【Transformation 6】 is selected from M １ is a single bond and C １～６ Alkylene group, C ２～６ Alkenylene group, C ２～６ an alkynylene group or an amine group; １～６ Alkylene group, C ２～６ Alkenylene group, C ２～６ The drug linker compound of any one of claims 1 to 3, wherein the alkynylene group or the amine group is optionally substituted with one or more suitable substituents.

5. The M is 【Transformation 7】 The drug linker compound of any one of claims 1 to 4, selected from:

6. The M is 【Transformation 8】 6. The drug linker compound of claim 5, selected from:

7. The M is 【Chemistry 9】 The drug linker compound of any one of claims 1 to 4, selected from:

8. The M is 【Chemistry 10】 The drug linker compound of any one of claims 1 to 4, selected from:

9. The L is C １～６ an alkylene group, -N(R')-, a carbonyl group, -O-, Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH ２ CH ２ (OCH ２ CH ２ ) ｒ OCH ３ ), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, 【Chemistry 11】 (Wherein R' is hydrogen, C １～６ represents an alkyl group or a polyethylene glycol fragment having 1 to 10 EO units, and s is selected from the group consisting of one or more of the following structures:

10. The above L is C １～６ an alkylene group, a carbonyl group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, 【Chemistry 12】 The drug linker compound according to any one of claims 1 to 9, wherein the compound is selected from the structure consisting of one or more of the following: (wherein s is selected from an integer of 1 to 20).

11. The L is 【Chemistry 13】 【Chemistry 14】 The drug linker compound of any one of claims 1 to 10, wherein the drug linker compound is selected from the structure consisting of one or more of:

12. The L is 【Chemistry 15】 The drug linker compound according to any one of claims 1 to 11, wherein the compound is selected from the structure:

13. The L is 【Chemistry 16】 The drug linker compound according to any one of claims 1 to 12, wherein the compound is selected from the structure:

14. The L is 【Chemistry 17】 The drug linker compound according to any one of claims 1 to 13, wherein the compound is selected from the structure:

15. The L is [Chemistry 18] The drug linker compound according to any one of claims 1 to 12, wherein the compound is selected from the structure:

16. The E is a single bond, —NHCH ２ -, 【Chemistry 19】 The drug linker compound of any one of claims 1 to 15, wherein

17. The E is —NHCH ２ The drug linker compound of claim 16, wherein 【Request Item 18】 【Chemistry 20】 teeth, 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 【Chemistry 28】 The drug linker compound of any one of claims 1 to 17, selected from the structure:

19. The aforementioned 【Chemistry 29】 teeth, 【Transformation 30】 【Chemistry 31】 【Chemistry 32】 The drug linker compound of any one of claims 1 to 18, selected from the structure:

20. The drug linker compound of any one of claims 1 to 19, wherein the payload is selected from a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

21. The drug linker compound of any one of claims 1 to 20, wherein the tubulin inhibitor is an auristatin compound or a maytansine compound, the DNA intercalator is a pyrrolobenzodiazepine PBD, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., adriamycin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide), and the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

22. The payload may be a compound of Formula I, Formula II 【Transformation 33】 (In the formula, R １ , R ２ are each independently C １～６ selected from alkyl groups and halogens; R ３ is H and —CO—CH ２ OH, R ４ and R ５ are each independently selected from H, halogen and hydroxyl groups, or R ４ and R ５ forms a 5- to 6-membered oxygen-containing heterocycle together with the carbon atom to which it is attached, R ６ is hydrogen or -C １～４ Alkylene -NR ａ R ｂ is selected from R ７ is C １～６ Alkyl group and -C １～４ Alkylene -NR ａ R ｂ (In the formula, R ａ , R ｂ are independently H, C for each occurrence. １～６ Alkyl group, —SO ２ -C １～６ Alkyl group and —CO—C １～６ The drug linker compound according to any one of claims 1 to 21, wherein the compound is selected from a compound represented by Formula I or Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of a compound represented by Formula I or Formula II.

23. The payload is 【Transformation 34】 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of said compound.

24. The payload is 【Chemistry 35】 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of said compound.

25. D is 【Transformation 36】 The drug linker compound of any one of claims 1 to 24, selected from the structure:

26. The following A-01 to A-34, B-01 to B-07, C-01 to C-28: 【Chemistry 37】 【Transformation 38】 【Chemistry 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 The drug linker compound of any one of claims 1 to 25, selected from:

27. The drug linker compound of any one of claims 1 to 26, wherein the drug linker compound is optionally substituted with one or more suitable substituents.

28. The following structure: 【Chemistry 48】 (In the formula, R １０ , R １１ , R １２ are independently hydrogen, C １～６ Alkyl group, C ３～６ Cycloalkyl group, 5- to 12-membered heterocyclic group, C ６～１０ an aryl group, a 5- to 12-membered heteroaryl group, —C １～６ Alkyl-C ６～１０ Aryl group and —C １～６ alkyl-5 to 12 membered heteroaryl groups, wherein the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are optionally selected from hydroxyl groups, CN, halogen, C １～６ Alkyl group, C １～６ Halogenated alkyl group, C １～６ Alkoxy group, C ６～１０ substituted by one or more substituents selected from an aryl group, a 5- to 12-membered heteroaryl group; R １３ and R １４ are each independently hydrogen, C １～６ Alkyl group, C ３～６ The alkyl, cycloalkyl and heterocyclic groups are selected from hydroxyl groups, CN, halogen, C １～６ Alkyl group, C １～６ Halogenated alkyl group, C １～６ Alkoxy group, C ６～１０ substituted by one or more substituents selected from an aryl group, a 5- to 12-membered heteroaryl group; R １５ is hydrogen, C １～６ Alkyl group, C ３～６ Cycloalkyl groups, halogenated C １～６ Alkyl group, C １～６ Alkyl-O-C １～６ Alkyl group, C ２～６ Alkenyl group, C ２～６ alkynyl groups and 3- to 6-membered heterocycloalkyl groups; R １６ is H, or R １５ , R １６ and the atoms connecting them together form a 4- to 7-membered ring, which optionally contains a hydroxyl group, CN, a halogen, C １～６ Alkyl group, C １～６ Halogenated alkyl group, C １～６ Alkoxy group, C ６～１０ aryl group, 5- to 12-membered heteroaryl group) 28. The drug linker compound of claim 27, having the formula:

29. It has the following structure: R １０ , R １１ , R １２ are independently hydrogen, C １～６ Alkyl group, C ３～６ Cycloalkyl group, C ６～１０ Aryl groups, benzyl groups, hydroxyl-substituted benzyl groups, and indolyl-C １～６ alkyl groups, R １３ and R １４ are each independently hydrogen, C １～６ Alkyl group, C ３～６ selected from a cycloalkyl group and a 4- to 6-membered heterocyclic group; R １５ is hydrogen, C １～６ Alkyl group, C ３～６ Cycloalkyl groups, halogenated C １～６ Alkyl group, C １～６ Alkyl-O-C １～６ Alkyl group, C ２～６ Alkenyl group, C ２～６ alkynyl groups and 3- to 6-membered heterocycloalkyl groups; R １６ is H, or R １５ , R １６ and the atoms to which it is linked jointly form a 4- to 7-membered ring.

30. 30. Use of a drug linker compound according to any one of claims 1 to 29 in the manufacture of a conjugate (e.g., an antibody-drug conjugate). 【Request Item 31】 【Chemistry 49】 [Transformation 50] 【Chemistry 51】 (wherein X is selected from a benzyloxycarbonyl group, a tert-butoxycarbonyl group, a fluorenylmethyloxycarbonyl group, an allyloxycarbonyl group, a trimethylsilylethoxycarbonyl group, a methoxycarbonyl group, an ethoxycarbonyl group, a phthalic group, a tosyl group, a trifluoroacetyl group, a nitrobenzenesulfonyl group, a benzoyl group, a pivaloyl group, a trityl group, a 4-methoxyphenyldiphenylmethyl group, a dimethoxytrityl group, a 2,4-dimethoxybenzyl group, a p-methoxybenzyl group, and a benzyl group; a is an integer of 1 to 10, preferably an integer of 3 to 8; R １ , R ２ and D is as defined in any one of claims 1 or 20 to 25), or a pharmaceutically acceptable salt thereof. 【Request Item 32】 【Chemistry 52】 【Chemistry 53】 【Chemistry 54】 【Transformation 55】 A compound having the structure:

33. Use of the compound of claim 32 in the manufacture of a drug linker compound of any one of claims 1 to 29.

34. IM-5-a is deprotected to give compound of formula IM-6-a: 【Transformation 56】 (In the formula, X, R １ and R ２ is as defined in claim 31 or 32) A method for producing a compound, comprising the step of obtaining

35. The deprotection reaction is as follows: (1) The solvent is selected from N,N-dimethylformamide; (2) adding an alkanamine compound to the reaction system; (3) Performed under room temperature conditions; (4) The reaction time is 1 to 5 hours.

35. The method of claim 34, wherein the method is carried out under one or more of the following conditions:

36. IM-6-a and IM-2 were reacted to give compound A-14-a: 【Chemistry 57】 (In the formula, R １ and R ２ is as defined in claim 31 or 32) The method of claim 34 further comprising obtaining:

37. The reaction is as follows: (1) The solvent for the reaction is selected from N,N-dimethylformamide and N,N-dimethylacetamide; (2) the reaction includes adding N,N-diisopropylethylamine The method according to claim 36, wherein the reaction is carried out under one or two of the following conditions: [Request Item 38] [Transformation 58] (In the formula, R １ and R ２ as defined in claim 31 or 32, wherein a is an integer of 1 to 10, preferably 3 or 8.

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