Antibody drug conjugates, and methods for preparing and using same

Abstract

Provided in the present application are antibody-drug conjugates, and methods for their preparation and use. The antibody-drug conjugates have the formula Ab-[MLED] x and the drug is selected from antitubulin agents, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors. The prepared antibody-drug conjugate has a better drug-to-antibody ratio and has good target killing effect on colon cancer and non-small cell lung cancer (e.g., lung adenocarcinoma).

Description

[Technical field]

[0001] This application is based on and claims priority from Chinese Application No. 202110615214.X, filed on June 2, 2021, and Chinese Application No. 202110941134.3, filed on August 16, 2021, the disclosures of which are hereby incorporated in their entireties into this application.

[0002] The present application relates to the field of targeted therapy, in particular to an antibody-drug conjugate for treating cancers with high expression of ROR1. In particular, the present application provides a humanized ROR1 antibody that has excellent binding activity to ROR1-positive cells and can efficiently deliver drugs to cells with high expression of ROR1 in an individual. The present application also provides a drug-linker molecule for binding to the antibody, where the drug is selected from the group consisting of antitubulin agents, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors. The prepared antibody-drug conjugate has a better drug-to-antibody ratio and has good target killing effect on colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), and lymphoma. Thus, the present application further provides a method for preparing the antibody-drug conjugate and its use in treating cancers with high expression of ROR1. [Background technology]

[0003] Cancer is a class of diseases caused by the malignant transformation of healthy cells resulting from genetic alterations such as chromosomal translocations, mutations in tumor suppressor genes and growth factor receptors, which lead to malignant proliferation of cells. Defective apoptosis or programmed cell death further promotes the malignant transformation of cells leading to cancer.

[0004] ROR1 (receptor tyrosine kinase-like orphan receptor 1), or neurotrophic tyrosine kinase receptor-related 1 (NTRKR1), is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family, also known as tyrosine-protein kinase transmembrane receptors, and belongs to type I membrane proteins.

[0005] The extracellular domain contains an immunoglobulin (IgG)-like domain, a CRD or frizzled domain, and a kringle domain. From the membrane to the intracellular region, the intracellular domains are a pseudokinase domain, a serine / threonine-rich domain 1, a proline-rich domain, and a serine / threonine-rich domain 2, respectively.

[0006] Under physiological conditions, ROR1 is highly expressed during embryonic development and plays an important role in controlling embryonic muscle and bone development. In recent years, studies have found that ROR1 is highly expressed in various tumor tissues, while ROR1 is also expressed in small amounts in adipose tissue, pancreas, lung, and a small amount of B-cell precursor cells. Studies have shown that ROR1 can bind to ligand molecules Wnt5a (mainly) or EGF, and may be involved in the control of cell proliferation, survival and migration. Overexpression of ROR1 is associated with poor tumor prognosis, and studies have found that targeting ROR1 can effectively inhibit the growth of transplanted tumors.

[0007] Currently, ROR1-targeted indications under clinical investigation cover hematological and solid tumors. The main therapeutic approaches focus on monoclonal antibodies, CAR-T, and antibody-drug conjugates (ADC). Among them, three anti-ROR1 antibody-conjugate drugs are currently under international investigation, two of which have entered clinical research phase for the treatment of MCL, BCL, NHL, NSCLC, TNBC, etc. (one in Phase II (VLS-101) and one in Phase I / II (NBE-002)), and three are in preclinical / discovery phase. Companies involved in the research include VelosBio, NBE-Therapeutics, LegoChem Biosciences, Almac, etc.

[0008] Here, the ADC drug is composed of an antibody, a bioactive molecule and a linker. The bioactive molecule is covalently linked to the antibody through the linker; the antibody (e.g., monoclonal antibody) can specifically recognize a specific target on the surface of cancer cells, then guide the ADC to the surface of cancer cells and enter the cancer cells through endocytosis; the bioactive molecule is then released into the cancer cells to kill the cancer cells, without damaging normal tissue cells as much as possible.

[0009] Regarding the international clinical progress of anti-ROR1 antibody-drug conjugates, VLS-101 developed by VelosBio has the fastest clinical progress, which employs a maleimide connector (MC) and an enzyme-cleavable linker valine-citrulline (Val-Cit), and the tubulin inhibitor MMAE is used as the toxin, and VLS-101 is generated by non-specific binding and has a DAR of about 4. It has been reported in the literature that under physiological conditions, the MC connector is easily exchanged with thiol through a reverse Michael reaction, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 30(2):184-9; Bioconjugate Chem. 2015, 26, 145-152).

[0010] NBE-002 was developed by NBE-Therapeutics, which employs SarA sortase linker technology, using five glycines as an enzyme-cleavable linker and topoisomerase PNU-159682 as a toxin. According to literature reports, the linker has multiple potential enzyme-cleavable sites, and PNU-159682 is sensitive to light and acidic conditions and has some cardiotoxicity under physiological conditions (Bioorg. Med. Chem. Lett. 30 (2020) 127640). Summary of the Invention

[0011] The present application relates to antibody-drug conjugates for the treatment of cancers with high expression of ROR1, which have the general formula Ab-[MLED], using the humanized antibody 19F6-Hu35V1 as the targeting moiety. x The present application exemplarily discloses an antibody-drug conjugate having a structure represented by the following formula: The results show that the conjugate has a better drug-to-antibody ratio (e.g., 1.5-2.5, 3.5-4.5, 6.5-8.5), and the conjugate has excellent binding activity to ROR1-positive cells and good target killing effect on ROR1-positive cancers, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma. Thus, the present application provides an antibody-drug conjugate for treating cancers with high expression of ROR1, a pharmaceutical composition comprising the antibody-drug conjugate, and its use in treating cancers with high expression of ROR1.

[0012] Antibody-drug conjugates In one embodiment, the present application relates to a compound of formula Ab-[MLED] x (In the formula, Ab refers to an antibody or antigen-binding fragment thereof that specifically binds to Receptor Tyrosine Kinase-Like Orphan Receptor (ROR) Family Member 1 (ROR1): M represents a linking site connecting the antibody or antigen-binding fragment thereof; L represents a connector connecting linkage sites M and E; E represents a structural fragment connecting L and D; D represents a cytotoxic drug moiety; and x is selected from 1 to 10.

[0013] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof through a linker (eg, the "MLE" fragment shown in the present application).

[0014] In some embodiments, M has the structure: [ka] is selected from.

[0015] In some embodiments, M is [ka] It is.

[0016] In some embodiments, L is the following group: 1~6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys( Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] where R' is hydrogen, C 1~6 Alkyl or -(CH2CH2O) r -containing alkyl; r is an integer selected from 1 to 10; and s is an integer selected from 1 to 10.

[0017] In some embodiments, L is the following group: 1~6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe- Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] where R' is hydrogen, C 1~6 Alkyl or -(CH2CH2O) r -containing alkyl; r is an integer selected from 1 to 10; and s is an integer selected from 1 to 10.

[0018] In some embodiments, L is the following group: 1~6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys( Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] where R' is hydrogen, C 1~6 Alkyl or -(CH2CH2O) r -containing alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20; preferably, s is an integer selected from 1 to 10.

[0019] In some embodiments, L is selected from the group consisting of Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] wherein s is an integer selected from 1 to 10.

[0020] In some embodiments, L has the structure: [ka] is selected from.

[0021] In some embodiments, L has the structure: [ka] is selected from.

[0022] In some embodiments, L has the structure: [ka] is selected from.

[0023] In some embodiments, L has the structure: [ka] is selected from.

[0024] In some embodiments, L has the structure: [ka] is selected from.

[0025] In some embodiments, L has the structure: [ka] is selected from.

[0026] In some embodiments, L has the structure: [ka] where s is an integer selected from 1 to 20.

[0027] In some embodiments, L has the structure: [ka] wherein s is an integer selected from 1 to 10; preferably, s is selected from 1, 2, and 3.

[0028] In some embodiments, L has the structure: [ka] is selected from.

[0029] In some embodiments, E is a single bond or -NH-CH-, or the following structure: [ka] is selected from.

[0030] In some embodiments, E is a single bond, -NH-CH2-, or [ka] It is.

[0031] In some embodiments, E is -NH-CH2- or [ka] It is.

[0032] In some embodiments, M is [ka] and; L has the following structure: [ka] where s is an integer selected from 1 to 10; E is -NH-CH2- or [ka] It is.

[0033] In some embodiments, [ka] has the following structure: [ka] is selected from.

[0034] In some embodiments, [ka] has the following structure: [ka] is selected from.

[0035] In some embodiments, the cytotoxic agent is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor. In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansine compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharma-ceutically acceptable salt, ester, or analog thereof.

[0036] The cytotoxic drugs disclosed in the present application typically contain various functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amine (-NR1H), tertiary amine (-NR2R3) (where R1, R2, R3 here represent only non-hydrogen substituents on N), or sulfhydryl (-SH), which can react with a suitable functional group on the remainder of the conjugate to achieve attachment to the drug molecule.

[0037] In some embodiments, the cytotoxic drug is attached to E in the antibody-drug conjugate through an -OH, a primary amino group, a secondary amino group, or a tertiary amino group, or -SH thereon.

[0038] In some embodiments, the cytotoxic agent is the following compound: [ka] is selected from.

[0039] In some embodiments, D has the structure: [ka] [ka] is selected from.

[0040] Those skilled in the art will understand that the antibody-drug conjugates described herein can be prepared in modules. For example, the free form of the "drug-linker" (which can be understood as M'-LED, where M' is the form of M before it is covalently bound to an antibody or an antigen-binding fragment thereof) can be obtained first, and then the "drug-linker" is covalently bound to an antibody or an antigen-binding fragment thereof to obtain the antibody-drug conjugates described herein. Correspondingly, the free form M' of the "drug-linker" is bound to one or more sulfhydryl (-SH), amino (-NH2), or carboxyl (-COOH) groups of an antibody or an antigen-binding fragment thereof through a substitution reaction (e.g., removal of a structure such as -SO2Me or -Br thereon) or through an addition reaction.

[0041] In some embodiments, the free form of the Drug-Linker is selected from A-1 to A-31 and B-1 to B-3 below: A-1: [ka] , A-2: [ka] , A-3: [ka] , A-4:

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[0042] In some embodiments, the antibody or antigen-binding fragment thereof is: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 3, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 4, and CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 6, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 7, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 8; or (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 11, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 12, and CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 14, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 15, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; or (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 19, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 20, CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 14, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 15, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; wherein the variant according to any one of items (1a), (1b) and (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; a heavy chain variable region (VH) and / or a light chain variable region (VL); Or, (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined by the AbM numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 29, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 30, and CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 6, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 7, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 8; or (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 36, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 37, and CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 14, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 15, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; or (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 45, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 46, CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 14, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 15, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; wherein the variant according to any one of items (2a), (2b) and (2c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; a heavy chain variable region (VH) and / or a light chain variable region (VL); Or, (3) The following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 31, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 32, and CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 6, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 7, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 8; or (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 38, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 39, CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 14, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 15, CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; or (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 47, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 48, CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 14, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 15, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; wherein the variant according to any one of items (3a), (3b) and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; a heavy chain variable region (VH) and / or a light chain variable region (VL); Or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined by the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 24, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 25, and CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 26; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 27, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 28, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 8; or (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 33, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 34, CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 35; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 43, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 44, CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; or (4c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the sequence set forth in SEQ ID NO: 40, CDR-H2 or a variant thereof having the sequence set forth in SEQ ID NO: 41, CDR-H3 or a variant thereof having the sequence set forth in SEQ ID NO: 42; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the sequence set forth in SEQ ID NO: 43, CDR-L2 or a variant thereof having the sequence set forth in SEQ ID NO: 44, and CDR-L3 or a variant thereof having the sequence set forth in SEQ ID NO: 16; wherein the variant according to any one of items (4a), (4b) or (4c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. Includes.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof is: (a) a VH as set forth in SEQ ID NO: 1 or a variant thereof, and / or a VL as set forth in SEQ ID NO: 2 or a variant thereof; (b) a VH as set forth in SEQ ID NO: 9 or a variant thereof, and / or a VL as set forth in SEQ ID NO: 10 or a variant thereof; or (c) VH set forth in SEQ ID NO: 17 or a variant thereof, and / or VL set forth in SEQ ID NO: 18 or a variant thereof. Includes; A variant herein has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or a variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof is: (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and (b) a human immunoglobulin light chain constant region (CL) or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; Further includes:

[0045] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 22 or a variant thereof, wherein the variant has one or more conservative substitutions of amino acids compared to SEQ ID NO: 22 (e.g., up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0046] In some embodiments, the light chain constant region is a kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO:23 or a variant thereof, wherein the variant has one or more conservative amino acid substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO:23.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:22 and a light chain constant region (CL) set forth in SEQ ID NO:23.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof is: (1) a heavy chain comprising a VH having the sequence set forth in SEQ ID NO:1 and a heavy chain constant region (CH) set forth in SEQ ID NO:22, and a light chain comprising a VL having the sequence set forth in SEQ ID NO:2 and a light chain constant region (CL) set forth in SEQ ID NO:23; (2) a heavy chain comprising a VH having the sequence set forth in SEQ ID NO:9 and a heavy chain constant region (CH) set forth in SEQ ID NO:22, and a light chain comprising a VL having the sequence set forth in SEQ ID NO:10 and a light chain constant region (CL) set forth in SEQ ID NO:23; or (3) A heavy chain comprising a VH having the sequence set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) set forth in SEQ ID NO: 22, and a light chain comprising a VL having the sequence set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 23. Includes.

[0049] In some embodiments, the antibody-drug conjugates are ADC A-1 through ADC A-31 and ADC B-1 through ADC B-3 shown below: ADC A-1: [ka] , ADC A-2: [ka] , ADC A-3: [ka] , ADC A-4: [ka] , ADC A-5: [ka] , ADC A-6: [ka] , ADC A-7: [ka] , ADC A-8:

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[0050] In some embodiments, the antibody-drug conjugate may be 1-10, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5 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 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 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. and having a DAR value (drug-to-antibody ratio) of 3 to 8, such as 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, 6.0-8.0, 6.5-7.0, 6.5-7.5, 6.5-8.0, 7.0-7.5, 7.0-8.0, or 7.5-8.0.

[0051] Drug-Linker Conjugates In another aspect, the present application provides a drug-linker that can be used to prepare the above-mentioned antibody-drug conjugates, the drug-linker having the formula M'-LED, M' represents the structure of M before being attached to said antibody or antigen-binding fragment thereof, and M, L, E and D are as defined in any one of the preceding items of the first aspect.

[0052] In some embodiments, M' has the structure: [ka] is selected from.

[0053] In some embodiments, M' is [ka] It is.

[0054] In some embodiments, L is the following group: 1~6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys( Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] where R' is hydrogen, C 1~6 Alkyl or -(CH2CH2O) r -containing alkyl; r is an integer selected from 1 to 10; and s is an integer selected from 1 to 10.

[0055] In some embodiments, L is the following group: 1~6Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe- Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] where R' is hydrogen, C 1~6 Alkyl or -(CH2CH2O) r -containing alkyl; r is an integer selected from 1 to 10; and s is an integer selected from 1 to 10.

[0056] In some embodiments, L is the following group: 1~6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys( Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] where R' is hydrogen, C 1~6 Alkyl or -(CH2CH2O) r -containing alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20; preferably, s is an integer selected from 1 to 10.

[0057] In some embodiments, L is selected from the group consisting of Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] wherein s is an integer selected from 1 to 10.

[0058] In some embodiments, L has the structure: [ka] is selected from.

[0059] In some embodiments, L has the structure: [ka] is selected from.

[0060] In some embodiments, L has the structure: [ka] is selected from.

[0061] In some embodiments, L has the structure: [ka] is selected from.

[0062] In some embodiments, L has the structure: [ka] is selected from.

[0063] In some embodiments, L has the structure: [ka] is selected from.

[0064] In some embodiments, L has the structure: [ka] where s is an integer selected from 1 to 20.

[0065] In some embodiments, L has the structure: [ka] wherein s is an integer selected from 1 to 10; preferably, s is selected from 1, 2, and 3.

[0066] In some embodiments, L has the structure: [ka] is selected from.

[0067] In some embodiments, E is a single bond or -NH-CH-, or the following structure: [ka] is selected from.

[0068] In some embodiments, E is a single bond, -NH-CH2-, or [ka] It is.

[0069] In some embodiments, E is -NH-CH2- or [ka] It is.

[0070] In some embodiments, M' is [ka] and; L has the following structure: [ka] where s is an integer selected from 1 to 10; E is -NH-CH2- or [ka] It is.

[0071] In some embodiments, [ka] has the following structure: [ka] is selected from.

[0072] In some embodiments, [ka] has the following structure: [ka] is selected from.

[0073] In some embodiments, the cytotoxic agent is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor. In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansine compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharma-ceutically acceptable salt, ester, or analog thereof.

[0074] The cytotoxic drugs disclosed in the present application typically contain various functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amine (-NR1H), tertiary amine (-NR2R3) (where R1, R2, R3 here represent only non-hydrogen substituents on N), or sulfhydryl (-SH), which can react with a suitable functional group on the remainder of the conjugate to achieve attachment to the drug molecule.

[0075] In some embodiments, the cytotoxic drug is attached to E in the antibody-drug conjugate through an -OH, a primary amino group, a secondary amino group, or a tertiary amino group, or -SH thereon.

[0076] In some embodiments, the cytotoxic agent is the following compound: [ka] is selected from.

[0077] In some embodiments, D has the structure: [ka] [ka] is selected from.

[0078] In some embodiments, the Drug-Linker is selected from A-1 through A-31 and B-1 through B-3 shown below: A-1: [ka] , A-2: [ka] , A-3: [ka] , A-4: [ka] , A-5: [ka] , A-6: [ka] , A-7:

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[0079] Pharmaceutical Compositions In another aspect, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate as described in any one of the preceding sections and one or more excipients.

[0080] The antibody-drug conjugates described herein are generally formulated with a pharma- ceutically acceptable parenteral vehicle to form a unitary injectable form for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, and the like. Optionally, the antibody-drug conjugates having the desired purity are mixed with a pharma- ceutical acceptable diluent, carrier, excipient, or stabilizer in the form of a lyophilizate or solution (Remington's Pharmaceutical Sciences (1980) 16th Edition, Osol, A. Ed.). The antibody-drug conjugates described herein, or pharmaceutical compositions comprising the antibody-drug conjugates, can be administered via any route appropriate to the individual to be treated.

[0081] use The antibody-drug conjugates or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as cancers associated with high expression of ROR1, including solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

[0082] Accordingly, the present application provides the use of an antibody-drug conjugate described in any one of the preceding items or a pharmaceutical composition comprising the same in the manufacture of a medicament for the treatment of cancer associated with high expression of ROR1.

[0083] At the same time, the present application also provides a method for treating cancer associated with high expression of ROR1, comprising administering a therapeutically effective amount of the antibody-drug conjugate described in any one of the preceding items or a pharmaceutical composition comprising the same to a subject in need thereof.

[0084] definition Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including those variations in techniques or equivalent technical substitutions that are obvious to those skilled in the art. Furthermore, the laboratory operation steps of genomics, nucleic acid chemistry, and molecular biology used herein are all routine steps widely used in the corresponding fields. Although 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.

[0085] The term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε heavy chains, and the antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into hypervariable regions (called complementarity determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form an antigen-binding site, respectively. The assignment of amino acids to regions or domains may follow various numbering systems known in the art.

[0086] 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 of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2 and CDR3. The exact boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol 27:55-77, 2003) or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, one of skill in the art will readily identify the CDRs defined by each numbering system, and the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol 27:55-77, 2003).

[0087] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be identified according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined according to the Chothia numbering system.

[0088] The term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0089] The term "antigen-binding fragment" of an antibody refers to a polypeptide that is an antibody fragment, such as a polypeptide that is a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or the ability to compete with the full-length antibody for specific binding to an antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety and for all purposes. Antigen-binding fragments of antibodies can be produced using recombinant DNA techniques or enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, F(ab')3, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and such polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding capability to the polypeptide. Engineered antibody variants are reviewed by Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0090] The term "Fd" refers to an antibody fragment composed of the VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab')2 fragment consisting of an intact light chain and a heavy chain Fd fragment (consisting of the VH and CH1 domains).

[0091] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be 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 the variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind an antigen, although the affinity may be lower than that of the complete binding site.

[0092] The term "Fc" refers to an antibody fragment produced by combining the second and third constant regions of a first heavy chain of an antibody with the second and third constant regions of a second heavy chain via disulfide bonds. Antibody Fc fragments have a variety of different functions, but are not involved in antigen binding.

[0093] The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, where VL and VH are connected by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Roseburg and Moore, 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 linkers in the art may consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, although variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement, for example, there is an scFv comprising NH2-VH-VH-COOH, NH2-VL-VL-COOH.

[0094] The term "single domain antibody (sdAb)" has its commonly understood meaning by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind the same antigen that a full-length antibody binds (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single domain antibodies are also called nanobodies.

[0095] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or the ability to compete with the full-length antibody for specific binding to antigen.

[0096] As used herein, unless the context clearly dictates otherwise, when the term "antibody" is referred to it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0097] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided in the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of an antibody can be screened for specificity in the same way as intact antibodies.

[0098] The term "mouse antibody" refers to antibodies obtained by the following method: fusing B cells of an immunized mouse with myeloma cells, selecting mouse hybrid fusion cells that are both immortalized and secrete antibodies, and then performing screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by differentiation and proliferation of B cells after an antigen enters the mouse's body.

[0099] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence 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 FR and / or constant region) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune responses, and homology. The donor antibody can be an antibody from a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) that has the desired properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune responses).

[0100] The term "identity" refers to the degree of match between two polypeptides or two nucleic acids. When two sequences for comparison have the same monomer subunit of a base or amino acid at a particular site (e.g., two DNA molecules each have an adenine at a particular site, or two polypeptides each have a lysine at a particular site), the two molecules are identical at that site. The percent identity between two sequences is a function of the number of identical sites shared by the two sequences to the total number of sites for comparison times 100. For example, if 6 out of 10 sites of two sequences match, the two sequences have 60% identity. For example, the DNA sequences: CTGACT and CAGGTT share 50% identity (3 out of 6 sites match). In general, the comparison of two sequences is performed in a manner that produces maximum identity. Such alignments can be performed using computer programs such as the Align program (DNAstar, Inc.), which is based on the method of Needleman et al. (J. Mol. Biol 48:443-453, 1970). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residual table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0101] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). A family of amino acid residues with similar side chains has been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, valine, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace 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).

[0102] The terms "antibody-drug conjugate" and "ADC" have the same meaning and refer to an antibody, a cytotoxic drug, and a drug made with a connector to connect the antibody and the cytotoxic drug.

[0103] The twenty conventional amino acids involved herein are represented in the usual manner. For example, see Immunology-A Synthesis (2nd Edition, ES Golub and DR 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 as known in the art. For example, alanine can be represented by A or Ala.

[0104] As used herein, the terms "comprising," "including," "having," "containing," or "involving," and other variations thereof, are inclusive or open-ended and do not exclude other unrecited elements or method steps.

[0105] The term "alkyl" means "C 1~20 Alkyl, C 1~10 Alkyl, C 1~6 Alkyl, C 1~4 Alkyl, C 1~3 "alkyl" refers to a group derived by removing one hydrogen atom from a linear or branched hydrocarbon group such as "alkyl" and the like, specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, and the like.

[0106] The drawings described herein are used to allow a further understanding of the present invention and constitute a part of this application. The schematic examples of the present invention and their explanations are used to explain the present invention and do not constitute an undue limitation of the present invention. [Brief description of the drawings]

[0107] [Figure 1] FIG. 1 shows identification of Ba / F3 cells overexpressing human ROR1 by flow cytometry. [Diagram 2] This is a diagram showing changes in tumor volume in mice in each group of human gastric cancer cell NCI-N87 CDX model. Note: Arrows indicate administration, same below. [Diagram 3] FIG. 1 shows changes in mouse body weight in each group of the human gastric cancer cell NCI-N87 CDX model. [Figure 4] FIG. 1 shows changes in tumor volume in mice in each group of the human lung adenocarcinoma cell NCI-H1975 CDX model. [Diagram 5] FIG. 1 shows changes in mouse body weight in each group of the human lung adenocarcinoma cell NCI-H1975 CDX model. [Figure 6] FIG. 1 shows changes in tumor volume in mice in each group of the human colon cancer cell line HT-29 CDX model. [Figure 7] FIG. 1 shows changes in mouse body weight in each group of the human colon cancer cell HT-29 CDX model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] Specific Models for Carrying Out the Invention The technical solutions in the examples of the present invention will be clearly and completely described by referring to the drawings of the examples of the present invention. Obviously, the described examples are only some, but not all, examples of the present invention. The following description of at least one illustrative example is merely of an illustrative nature and is in no way considered as any limitation of the present invention, its application or use. All other examples obtained by those skilled in the art based on the examples of the present invention without creative efforts fall within the protection scope of the present invention.

[0109] The following description of specific examples further illustrates the present invention, but is not intended to limit it. Those skilled in the art can make various modifications or improvements in accordance with the teachings of the present invention without departing from the basic concept and scope of the present invention.

[0110] The sequence information involved in the present invention is set out in the following table: [Table 1]

[0111] The abbreviations used in this application have the following meanings: [Table 2]

[0112] The structures of the compounds described in the following examples were analyzed by nuclear magnetic resonance ( 1 The compounds were identified by 1 H-NMR or mass spectrometry (MS).

[0113] Nuclear magnetic resonance ( 1 1 H-NMR was determined by using a Bruker 400 MHz nuclear magnetic resonance spectrometer; the deuteration reagent was hexadeuteriodimethylsulfoxide (DMSO-d6); the internal standard was tetramethylsilane (TMS).

[0114] Abbreviations used in the examples in the interpretation of nuclear magnetic resonance (NMR) spectra are given below. s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethylsulfoxide. δ values ​​are expressed in ppm.

[0115] Mass spectrometry (MS) was determined by using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0116] Example 1: 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-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (A-1) [ka]

[0117] Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide Prop-2-yn-1-amine (189 mg, 3.4 mmol) and compound IM-1 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL) at 25° C., and N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) were added in sequence, stirred, and reacted for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by flash silica gel column (ethyl acetate / petroleum ether=3 / 1) to obtain 700 mg of the title compound. ESI-MS (m / z): 306.1 [M+H]+ .

[0118] Step 2: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate At 25° C. under nitrogen protection, A-1-1 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL), cooled to 0° C., and added to a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL), then slowly added dropwise to a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL). After addition, the reaction was stirred at 0° C. for 20 minutes, and the reaction solution was blown with nitrogen for 20 minutes. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)benzhydryl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added, and after the addition, the reaction was stirred at 0° C. for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high performance liquid chromatography (conditions as below) to give 500 mg of the title compound. ESI-MS (m / z): 1597.5 [M+H] + . Chromatography column: Daisogel C18 10μm 100×250mm Mobile phase A: water; Mobile phase B: acetonitrile [Table 3]

[0119] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-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)carbonate At room temperature, compound A-1-2 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and added to copper bromide (11 mg, 0.08 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (IM-2, 18.8 mg, 0.06 mmol), stirred, and reacted for 1 hour. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 30 mg of the title compound. ESI-MS (m / z): 815.9 [(M-273) / 2+H] + . Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water [Table 4]

[0120] Step 4: 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-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate Compound A-1-3 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), added to trifluoroacetic acid (0.2 mL), and reacted at room temperature for 30 minutes. After purification by preparative high performance liquid chromatography (under the following conditions), 20.0 mg of the trifluoroacetate salt of the title compound was obtained. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 5] The structural characterization data was as follows: 1HNMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.10 (s, 2H),8.38 (t, J = 5.56 Hz, 1H), 8.32 (d, J = 8.40 Hz, 1H), 8.22-8.20 (m, 2H), 8.09(t, J = 5.68 Hz, 1H), 7.91-7.87 (m, 2H), 7.82-7.78 (m, 1H), 7.69 (brs, 3H),7.61 (d, J = 8.56 Hz, 2H), 7.32 (d, J = 8.56 Hz, 2H), 7.06 (s, 1H), 5.56 (d, J= 16.96 Hz, 1H), 5.51 (d, J = 16.96 Hz, 1H), 5.47 (d, J = 19.28 Hz, 1H), 5.42(d, J = 19.28 Hz, 1H), 5.14 (d, J = 12.20 Hz, 1H), 5.07 (d, J = 12.16 Hz, 1H),4.48 (t, J = 5.24 Hz, 2H), 4.46-4.43 (m, 1H), 4.29 (d, J = 5.60 Hz, 2H),4.08-3.95 (m, 5H), 3.79 (t, J = 5.28 Hz, 2H), 3.51-3.43 (m, 32H), 3.40 (s, 3H),3.39-3.35 (m, 2H), 3.30-3.26 (m, 2H), 3.00 (s, 3H), 2.82-2.74 (m, 2H), 2.56 (t,J = 7.08 Hz, 2H), 2.29 (t, J = 7.36 Hz, 2H), 2.23-2.13 (m, 2H), 1.82 (p, J =7.24 Hz, 2H), 1.78-1.63 (m, 2H), 1.61-1.49 (m, 2H), 1.42-1.27 (m, 2H), 1.15 (d,J = 6.80 Hz, 3H), 1.13 (d, J = 6.76 Hz, 3H), 0.90 (t, J = 7.32 Hz, 3H). ESI-MS(m / z): 816.0[M / 2+H] + .

[0121] Example 2: 4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl))pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butyrylamido)butyrylamido)-5-ureidopentanamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate (A-2) [ka]

[0122] Step 1: Synthesis of tert-butyl 4-(4-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)piperidine-1-carboxylate At 25° C., compound Val-Cit-PABC (A-2-1, 1 g, 2.64 mmol), 4-(N-Boc-4-piperidinyl)butanoic acid (929.65 mg, 3.43 mmol) and EEDQ (977.55 mg, 3.95 mmol) were dissolved in a mixture of methanol (20 mL) and dichloromethane (20 mL), heated to 45° C. and reacted for 2 hours. The reaction solution was subjected to rotary evaporation, poured into methyl tert-butyl ether (50 ml), stirred for 30 minutes, and a cloudy precipitate was formed, which was filtered by suction to obtain 1.3 g of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 633.2[M+H] + .

[0123] Step 2: Synthesis of (S)-N-(4-(hydroxymethyl)phenyl)-2-((S)-3-methyl-2-(4-(piperidin-4-yl)butanamido)butanamido)-5-ureidopentanamide At 25°C, compound A-2-2 (1.5g, 2.37mmol) was dissolved in dichloromethane (3.00mL) and added to trifluoroacetic acid (1.5mL) in one portion and reacted at 25°C for 2 hours; the raw material was consumed as monitored by liquid chromatography-mass spectrometry. The reaction solution was evaporated to dryness under reduced pressure. The crude product was dissolved in methanol (3.00mL) and added to potassium carbonate (1.64g, 11.85mmol) and stirred for 30 minutes. The reaction solution was purified by silica gel column (dichloromethane:methanol=10:1) to obtain 500mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 533.3[M+H] + .

[0124] Step 3: Synthesis of (S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butyrylamido)-3-methylbutyrylamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide At 25°C, 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl 4-methylbenzenesulfonate (668.72mg, 1.13mmol) and compound A-2-3 (1g, 1.88mmol) were dissolved in N,N-dimethylformamide (5mL), added in one portion to potassium carbonate (518.89mg, 3.75mmol), heated to 80°C for 2 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by a silica gel column (dichloromethane:methanol=10:1) to obtain 600mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 954.5[M+H] + .

[0125] Step 4: Synthesis of 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butyrylamido)-3-methylbutyrylamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbamate At 25°C, compound A-2-4 (733mg, 0.77mmol) was dissolved in N,N-dimethylformamide (2mL) and added to DIPEA (396.40mg, 3.07mmol), then added dropwise to a solution of di(p-nitrophenyl)carbonate (701.10mg, 2.30mmol) in N,N-dimethylformamide (1mL), stirred, and reacted at 25°C for 16 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 400mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 6] The structural characterization data was as follows: ESI-MS (m / z): 1119.5[M+H] + .

[0126] Step 5: Synthesis of 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butyrylamido)-3-methylbutyrylamido)-5-ureidopentanamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate At 25°C, belotecan (20mg, 0.046mmol) and compound A-2-5 (46.47mg, 0.042mmol) were dissolved in N,N-dimethylformamide (2mL), and added to HOBT (9.35mg, 69.20μmol) and DIPEA (11.90mg, 0.092mmol), and the reaction system was stirred at 25°C and reacted for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 41mg of the title compound. Chromatography column: SunFire Prep C18 ODS 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 7] The structural characterization data was as follows: ESI-MS (m / z): 1413.5[(M+H] + .

[0127] Step 6: Synthesis of 4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butyrylamido)butyrylamido)-5-ureidopentanoylamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate At 25°C, 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (IM-2, 12.96mg, 0.042mmol) and compound A-2-6 (40mg, 0.028mmol) were dissolved in dimethylsulfoxide (1mL) and water (0.25mL), and added to cuprous bromide (8.20mg, 0.057mmol), and then reacted at 25°C for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 29mg of the title compound. Chromatography column: SunFire Prep C18 ODS 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 8] The structural characterization data was as follows: ESI-MS (m / z): 1718.8[(M+H] + .

[0128] Example 3: (S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butanamido)butanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-3) [ka]

[0129] Step 1: Synthesis of 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butyrylamido)-3-methylbutyrylamido)-5-ureidopentanylamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate At 25 ° C., (1S, 9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano [3 ', 4': 6,7] indolizino [1,2-b] quinoline-10,13-dione (exatecan, 15 mg, 0.034 mmol) and compound A-2-5 (34.70 mg, 0.031 mmol) were dissolved in N, N-dimethylformamide (1 mL), and added to HOBT (6.98 mg, 51.67 μmol) and DIPEA (8.89 mg, 68.89 μmol), stirred, and reacted at 25 ° C. for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography (under the conditions below), and the fractions were lyophilized to obtain 30 mg of the title compound. Chromatography column: SunFire Prep C18 ODS 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 9] The structural characterization data was as follows: ESI-MS (m / z): 1415.4[(M+H] + .

[0130] Step 2: (S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butanamide) buta Synthesis of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-473, A-3) At 25°C, 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (IM-2, 9.71mg, 0.032mmol) and compound A-3-1 (30mg, 0.021mmol) were dissolved in dimethylsulfoxide (0.5mL) and water (0.1mL), and then added to cuprous bromide (6.14mg, 0.042mmol), reacted at 25°C for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 15.74mg of the title compound. Chromatography column: SunFire Prep C18 ODS 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 10] The structural characterization data was as follows: ESI-MS (m / z): 861.1[(M / 2+H] + .

[0131] Example 4: 4-((R)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butane Amido)butanamido)propanamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-yl)carbamate (A-4) [ka]

[0132] Step 1: tert-Butyl 4-(4-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)piperidine-1-carboxylate At 25°C, compound A-4-1 (1.96 g, 6.67 mmol), 4-(N-Boc-4-piperidinyl)butanoic acid (2.35 mg, 8.67 mmol) and EEDQ (2.47 mg, 10.0 mmol) were dissolved in a mixed solution of methanol (20 mL) and dichloromethane (20 mL), heated to 45°C, and reacted at 45°C for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry, and the reaction solution was concentrated and purified with a silica gel column (dichloromethane:methanol = 15:1) to obtain 1.5 g of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 547.1[M+H] + .

[0133] Step 2: Synthesis of (S)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methyl-2-(4-(piperidin-4-yl)butyrylamide)butanamide At 25°C, trifluoroacetic acid (1.5mL) was added to A-4-2 (1.25g, 2.37mmol) in dichloromethane (3.00mL) and reacted at 25°C for 2 hours. The raw material was consumed as monitored by liquid chromatography-mass spectrometry, and the reaction solution was concentrated under reduced pressure to obtain a crude product, which was dissolved in methanol (3.00mL), added to potassium carbonate (1.64g, 11.85mmol), stirred for 30 minutes, and then detected by liquid chromatography-mass spectrometry. The reaction solution was purified on a silica gel column to obtain 750mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 447.1[M+H] + .

[0134] Step 3: Synthesis of (S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butylamino)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl-3-methylbutylamine At 25°C, 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl 4-methylbenzenesulfonate (1.4mg, 2.36mmol) and A-4-3 (700mg, 1.57mmol) were dissolved in N,N-dimethylformamide (5mL), and added to potassium carbonate (433.9mg, 3.14mmol) in one portion. The reaction system was heated to 80°C and reacted for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry, and the reaction solution was purified by a silica gel column (petroleum ether: ethyl acetate = 1:3) to obtain 500mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 868.2[M+H] + .

[0135] Step 4: 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl)carbonate At 25°C, A-4-4 (500mg, 0.58mmol) was dissolved in N,N-dimethylformamide (2mL) and added to DIPEA (297.77mg, 2.30mmol), then added dropwise to di(p-nitrophenyl)carbonate (525.67mg, 1.73mmol) in N,N-dimethylformamide (1mL), and reacted for 16 hours at 25°C under stirring, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 360mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 11] The structural characterization data was as follows: ESI-MS (m / z): 1017.0[M+H] + .

[0136] Step 5: Synthesis of 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butyrylamido)-3-methylbutyrylamido)propionamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate At 25°C, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (exatecan, 20.0mg, 0.038mmol) and compound A-4-5 (42.10mg, 41.4μmol) were dissolved in DMF (1mL) and added to HOBT (6.1mg, 45.2μmol) and DIPEA (9.7mg, 75.3μmol). After the addition, the reaction system was reacted at room temperature under stirring for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography (under the following conditions), and the fraction was lyophilized to obtain 32mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid solution) [Table 12] The structural characterization data was as follows: ESI-MS (m / z): 1635.1[M+H] + .

[0137] Step 6: Synthesis of 4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butanamido)butanamido)propanamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-yl)carbamate At 25°C, compound A-4-6 (30mg, 0.023mmol) was dissolved in DMSO (0.5mL) and water (0.1ml), and added to 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (IM-2, 10.3mg, 0.034mmol) and cuprous bromide (6.5mg, 0.045mmol), reacted at room temperature for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was filtered and then purified by high performance liquid chromatography (under the following conditions). The fraction was lyophilized to obtain 15.40mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA) [Table 13] The structural characterization data was as follows: ESI-MS (m / z): 1635.1[M+H] + .

[0138] Example 5: (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-((18-(2-(methylsulfonyl)pyrimidin-5-yl)-13-oxo-3,6,9-trioxa-12-azaoctadec-17-yn-1-yl)carbamoyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-5) [ka]

[0139] Step 1: Synthesis of tert-butyl (1-(5-formyl-2-hydroxyphenyl)-1-oxo-5,8,11-trioxo-2-azatridecan-13-yl)carbamate 5-Formyl-2-hydroxybenzoic acid (A-5-2, 625.04 mg, 3.76 mmol) was dissolved in dichloromethane (20 mL), added to a drop of DMF, then added dropwise to thionyl chloride (2 mL), heated to reflux, and reacted for 2 hours. The reaction solution was concentrated under reduced pressure, then dissolved in dichloromethane (5 mL) to obtain an acyl chloride intermediate solution for later use. tert-Butoxycarbonyltriethyleneglycolamine (A-5-1, 1 g, 3.42 mmol) was dissolved in dichloromethane (20 mL), cooled to 0 ° C, then added dropwise to the above acyl chloride solution and diisopropylethylamine (36.47 mg, 0.282 mmol), the reaction system was slowly heated to room temperature, reacted for 12 hours, then concentrated under reduced pressure, and purified (under the conditions as follows) to obtain A-5-3 (271 mg). ESI-MS (m / z): 441.2[M+1] + . Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [Table 14]

[0140] Step 2: Synthesis of (2R,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate A-5-3 (270 mg, 0.613 mmol) and (2R,3R,5S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (A-5-4, 267.80 mg, 0.674 mmol) were dissolved in acetonitrile (30 mL), and added to silver oxide (568.18 mg, 2.45 mmol) and 4A molecular sieve powder (1 g), and reacted for 16 hours under stirring and nitrogen protection. The reaction was filtered and then concentrated under reduced pressure to give 460 mg of crude product A-5-5, which was used directly in the next reaction. ESI-MS (m / z): 757.4[M+1] + .

[0141] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate A-5-5 (460 mg, 0.608 mmol) was dissolved in dichloromethane (5 mL) and isopropanol (5 mL), and added to silica gel powder (1 g) and sodium borohydride (11.50 mg, 0.304 mmol) under stirring, reacted for 2 hours, filtered, and the filtrate was concentrated under reduced pressure and purified (under the conditions below) to obtain A-5-6 (343 mg). ESI-MS (m / z): 759.3[M+1] + . Chromatography column: Waters XBridge Prep C18OBD (8μm * 45mm * 450mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [Table 15]

[0142] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate A-5-6 (343 mg, 0.452 mmol) was dissolved in anhydrous dichloromethane (25 mL), added to diisopropylethylamine (175.27 mg, 1.36 mmol), and added dropwise to p-nitrophenyl chloroformate (273.35 mg, 1.36 mmol, dissolved in 25 mL of dichloromethane) under stirring, reacted at room temperature for 12 hours, concentrated the reaction system under reduced pressure, and purified with a silica gel column (eluent: 6% methanol / dichloromethane) to obtain compound A-5-7 (342 mg).

[0143] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate A-5-7 (53.93 mg, 0.058 mmol), (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A-5-8, 20 mg, 0.039 mmol) and HOBt (15.77 mg, 0.117 mmol) was dissolved in DMF (1 mL), added dropwise to diisopropylethylamine (15.09 mg, 0.117 mmol), stirred, reacted for 12 hours, added to water and ethyl acetate, stirred, allowed to stand, separated the liquid, washed the organic phase with saturated brine, dried, and concentrated under reduced pressure to give 49 mg of crude product A-5-9, which was used directly in the next reaction. ESI-MS (m / z): 1262.5[M+1] + .

[0144] Step 6: Synthesis of (2S,3S,4S,5R,6S)-6-(2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)-4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A-5-9 (49mg, 0.039mmol) was dissolved in methanol (2mL), added dropwise to lithium hydroxide (13.03mg, 0.311mmol, dissolved in 0.5mL water) aqueous solution, stirred and reacted for 1 hour, concentrated under reduced pressure, then added dropwise to trifluoroacetic acid (2mL), stirred and reacted for 1 hour, monitored by liquid chromatography-mass spectrometry until it was completed. The reaction system was concentrated under reduced pressure and purified (under the following conditions) to obtain compound A-5-10 (23mg). ESI-MS (m / z): 1022.0[M+1] + . Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA) [Table 16]

[0145] Step 7: Synthesis of (2S,3S,4S,5R,6S)-6-(4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-((18-(2-(methylsulfonyl)pyrimidin-5-yl)-13-oxo-3,6,9-trioxa-12-azaoctadec-17-yn-1-yl)carbamoyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A-5-10 (23 mg, 0.020 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-hex-5-ynoic acid 2,5-dioxopyrrolidin-1-yl ester (IM-3, 8.14 mg, 0.022 mmol) were dissolved in DMF (1 mL), and added dropwise to diisopropylethylamine (7.85 mg, 0.061 mmol), and reacted under stirring for 2 hours, and the raw material was essentially consumed as monitored by TLC. The reaction system was purified (under the following conditions) to obtain compound A-5 (5.10 mg). ESI-MS (m / z): 1272.3[M+1] + . Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 17]

[0146] Example 6: (2S,3S,4S,5R,6S)-6-(4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)ethoxy)acetamido)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-6) [ka]

[0147] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Compound (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetic acid triester (A-5-4, 12.32 g, 31.02 mmol) and 4-hydroxy-3-nitrobenzyl alcohol (A-6-1, 5 g, 29.56 mmol) were dissolved in acetonitrile (200 mL) and added to silver oxide (27.40 g, 118.25 mmol) under stirring, and reacted at room temperature for 12 hours in the dark after nitrogen replacement. The reaction was monitored by liquid chromatography-mass spectrometry until it was completed. The reaction system was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:3) to obtain 12.8 g of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 503[M+18] + .

[0148] Step 2: Synthesis of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Compound A-6-2 (2.2 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50.00 mL each) and added to PtO2 (200 mg), and then the reaction system was subjected to hydrogen balloon replacement three times and reacted under hydrogen atmosphere for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry, the reaction system was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 2.02 g of the crude product of the title compound, which was used directly in the next reaction. The structural characterization data was as follows: ESI-MS (m / z): 456.1[M+1] + .

[0149] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Compound A-6-3 (456 mg, 1.00 mmol) and (2-(2-(Fmoc-amino)ethoxy)ethoxy)acetic acid (A-6-4, 385.91 mg, 1.00 mmol) were dissolved in dichloromethane (10 mL), added to 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 2.00 mmol), and reacted under stirring for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (methanol:dichloromethane=1:20) to obtain 507 mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 823.3[M+1] + .

[0150] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-amido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Nitrophenyl chloroformate (372.60 mg, 1.85 mmol) in dichloromethane (1 mL) was slowly added dropwise to compound A-6-5 (507 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) in dichloromethane (20 mL), and after addition, the reaction system was reacted at room temperature for 15 hours. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (methanol:dichloromethane=1:20) to obtain 496 mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 988.5[M+1] + .

[0151] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-amido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Compound A-6-6 (165.51 mg, 0.168 mmol), compound A-5-8 (40 mg, 0.084 mmol) and 1-hydroxybenzotriazole (33.96 mg, 0.251 mmol) were dissolved in DMF (4 mL), added dropwise to diisopropylethylamine (32.48 mg, 0.251 mmol) and reacted under stirring for 12 hours. The reaction was monitored by liquid chromatography-mass spectrometry, and the reaction system was added to an appropriate amount of water and ethyl acetate, stirred, and then allowed to stand to separate the liquid. The organic phase was washed with saturated brine, dried, and then filtered and concentrated under reduced pressure to obtain 100 mg of the crude product of the title compound, which was used directly in the next reaction. The structural characterization data was as follows: ESI-MS (m / z): 1326.2[M+1] + .

[0152] Step 6: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid Compound A-6-7 (100 mg, 0.075 mmol) in MeOH (5 mL) was added with a drop of dichloromethane and lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) in water (1 mL) and reacted under stirring for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was then added to a 3N aqueous hydrochloric acid solution to adjust the pH to 4, and the reaction system was concentrated under reduced pressure. The mixture was then purified by high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 27.0 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 18] The structural characterization data was as follows: ESI-MS (m / z): 964.2[M+1] + .

[0153] Step 7: Synthesis of (2S,3S,4S,5R,6S)-6-(4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)ethoxy)acetamido)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid Compound A-6-8 (27 mg, 0.028 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-hex-5-inoic acid 2,5-dioxopyrrolidin-1-yl ester (IM-3, 11.26 mg, 0.031 mmol) were dissolved in DMF (1 mL), and added dropwise to diisopropylethylamine (3.62 mg, 0.028 mmol) under stirring, reacted at room temperature for 4 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 11.7 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 19] The structural characterization data was as follows: ESI-MS (m / z): 1214.4[M+1] + .

[0154] Example 7: (2S,3S,4S,5R,6S)-6-(4-(((((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-7) [ka]

[0155] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-amido)-4-((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate A-6-6 (185.85 mg, 0.188 mmol), exatecan (50 mg, 0.094 mmol) and HOBt (38.13 mg, 0.282 mmol) were dissolved in DMF (4 mL) and added dropwise to diisopropylethylamine (36.47 mg, 0.282 mmol) and reacted under stirring for 12 hours. The raw material was consumed as monitored by LCMS. The reaction was extracted with water and ethyl acetate, and the organic phase was washed with saturated brine, then dried and concentrated under reduced pressure to give 120 mg of crude yellow oil, which was used directly in the next reaction. ESI-MS (m / z): 1285.4[M+1] +

[0156] Step 2: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A-7-1 (120 mg, 0.093 mmol) was dissolved in methanol (5 mL) and dichloromethane (approximately 50 uL) and added dropwise to lithium hydroxide aqueous solution (31.37 mg, 0.747 mmol, dissolved in 1 mL water) and reacted under stirring for 2 hours. The raw material was consumed as monitored by LCMS. The reaction was added dropwise to 3N hydrochloric acid aqueous solution to adjust pH=4, concentrated under reduced pressure, and then purified to obtain 32 mg of A-7-2 as a white solid. ESI-MS (m / z): 922.3[M+1] +

[0157] Step 3: Synthesis of (2S,3S,4S,5R,6S)-6-(4-(((((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A-7-2 (30 mg, 0.033 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-hex-5-ynoic acid 2,5-dioxopyrrolidin-1-yl ester (IM-3, 13.08 mg, 0.036 mmol) were dissolved in DMF (1 mL) and added dropwise to diisopropylethylamine (4.21 mg, 0.033 mmol) under stirring, and reacted at room temperature for 4 hours. The raw material was consumed as monitored by LCMS. The reaction was purified to give 18.54 mg of A-7 as a white solid. ESI-MS (m / z): 1171.8[M+1] + . Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 20]

[0158] Example 8: (9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-4) [ka]

[0159] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene At 25 ° C., compound 1-4-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL), added to concentrated sulfuric acid (25 mL), heated to 50 ° C., added to NBS (6.22 g, 34.97 mmol) in batches at 50 ° C., reacted at 50 ° C. for 2 hours, monitored by thin layer chromatography (ethyl acetate: petroleum ether = 1: 10), cooled the reaction solution to room temperature, added dropwise to ice water, extracted with toluene, combined organic phases, washed with sodium sulfite solution, water and saturated brine successively, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the crude product was purified by preparative high performance liquid chromatography, and the fraction was freeze-dried to obtain 4.88 g of the title compound. Chromatography column: C18 ODS 45mm x 450mm x 8.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 21]

[0160] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline At 25°C, compound 1-4-2 (4.88g, 19.48mmol) was dissolved in ethyl acetate (100mL), added to platinum on carbon (2.00g, 19.48mmol, content 5%), subjected to hydrogen exchange, and then reacted under protection of hydrogen balloon at 60°C for 4 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to obtain 3.68g of the crude product of the title compound, which was used directly in the next reaction without further purification.

[0161] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide At 20° C., compound 1-4-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL), and added to triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol), and reacted at 20° C. for 20 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was extracted by adding water and ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was slurried in a mixed solvent of ethyl acetate:petroleum ether=1:5 to obtain 2.86 g of the title compound.

[0162] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid At 20°C, compound 1-4-4 (1.80g, 6.86mmol) was dissolved in THF (20mL) and water (5mL), and added to vinyl acetic acid (708.31mg, 8.23mmol), DIPEA (1.95g, 15.08mmol), tri(o-methylphenyl)phosphorus (62.60mg, 0.20mmol), the reaction system was replaced with nitrogen, then heated to 70°C, reacted for 5 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was added to 1N sodium hydroxide solution to adjust pH=8, and added to ethyl acetate for extraction. The remaining aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate, the organic phase was combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82g of the title compound, which was used directly in the next reaction.

[0163] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butanoic acid At 20°C, compound 1-4-5 (2.60g, 9.71mmol) was dissolved in THF (50mL) and added to Pd / C (0.52g, content 10%), the reaction system was replaced with hydrogen, and then reacted under the protection of a hydrogen balloon at 40°C for 2 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.43g of the title compound, which was used directly in the next reaction without further purification.

[0164] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide Compound 1-4-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5 ° C., and added dropwise to trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL), reacted at 5 ° C. for 4 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was added to water, adjusted to pH = 9 with 10N sodium hydroxide, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by flash silica gel column (ethyl acetate: petroleum ether = 0-20%) to obtain 1.53 g of the title compound.

[0165] Step 7: Synthesis of (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide At 5° C., potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL), and added dropwise to a solution of compound 1-4-7 (1.53 g, 6.08 mmol) in THF (16 mL), and after 10 minutes, added dropwise to amyl nitrite (1.14 g, 9.73 mmol), and reacted at 5° C. for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was adjusted to pH=5 with 1N hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was slurried with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0166] Step 8: N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide At 20°C, compound 1-4-8 (0.50g, 1.78mmol) was dissolved in methanol (8mL) and 2N hydrochloric acid (8mL), added to Pd / C (0.15g, content 10%), the system was subjected to hydrogen substitution, the reaction was carried out under the protection of a hydrogen balloon at 5°C for 2 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 0.52g of the hydrochloride salt of the title compound, which was used directly in the next reaction without further purification.

[0167] Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide At 20°C, compound 1-4-9 (0.52g, 1.70mmol) was dissolved in pyridine (5mL) and added to acetic anhydride (2mL), and reacted at 20°C for 2 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was added to water, extracted with ethyl acetate, the organic phase was washed with water, combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the concentrate was purified by flash silica gel column (ethyl acetate:petroleum ether = 0-30%) to obtain 0.22g of the title compound.

[0168] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide At 20°C, compound 1-4-10 (0.45g, 1.46mmol) was dissolved in methanol (16mL), added to 2N hydrochloric acid (16mL), heated to 60°C, reacted for 2 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was cooled, adjusted to pH=8 by adding saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.23g of the title compound, which was used directly in the next step without further purification.

[0169] Step 11: Synthesis of N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide Compound 1-4-11 (0.23 g, 0.78 mmol) was dissolved in toluene (10 mL) and added to (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (0.23 g, 0.87 mmol) and p-toluenesulfonic acid (26.73 mg, 0.16 mmol), heated to 140° C. for 5 hours, and the reaction was monitored by LC-MS. The reaction solution was concentrated, and the crude product was purified by flash silica gel column (methanol:dichloromethane=0-10%) to obtain 0.15 g of the title compound.

[0170] Step 12: Synthesis of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione Compound 1-4-12 (40.00 mg, 0.08 mmol) was added to concentrated hydrochloric acid (1 mL), heated to 100° C., and reacted for 5 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was filtered, the filtrate was purified by preparative high performance liquid chromatography, and the fraction was lyophilized to obtain 12.00 mg of the trifluoroacetate salt of the title compound 1-4. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 22] The structural characterization data was as follows: ESI-MS (m / z): 452.1[M+H] + .

[0171] Example 9: N-((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)-2-hydroxyacetamide and N-((1R,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)-2-hydroxyacetamide (1-7-A and 1-7-B) [ka]

[0172] Step 1: Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-N-((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)acetamide and 2-((tert-butyldiphenylsilyl)oxy)-N-((1R,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)acetamide At 25°C, the trifluoroacetate salt of compound 1-4 (40.00 mg, 81.91 μmol) was dissolved in N,N-dimethylformamide (1 mL), and the solution was added sequentially to 2-((tert-butyldiphenylsilyl)oxy)acetic acid (30.91 mg, 98.29 μmol), HATU (62.25 mg, 163.81 μmol) and N,N-diisopropylethylamine (42.34 mg, 327.63 μmol), and the reaction was carried out at 25°C for 0.5 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was added to water and extracted with dichloromethane / methanol (v / v=10 / 1), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified and separated by preparative thin layer chromatography (dichloromethane:methanol=20:1) to obtain two isomers, which were named as 1-7-1-A (15.00 mg, Rf value was 0.3) and 1-7-1-B (12.00 mg, Rf value was 0.35) according to their Rf values.

[0173] Step 2: Synthesis of N-((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)-2-hydroxyacetamide and N-((1R,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)-2-hydroxyacetamide At 25°C, 1-7-1-A (15.00mg) and 1-7-1-B (12.00mg) were dissolved in tetrahydrofuran (1mL) in two reaction flasks, respectively, and added dropwise to a mixture (50uL) of tetrabutylammonium fluoride (1M in tetrahydrofuran) / glacial acetic acid (v / v=13 / 1), respectively, and reacted at 25°C for 0.5 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography, and the fraction was lyophilized to obtain compounds 1-7-A (6.94mg) and 1-7-B (4.00mg). Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 23] The structural characterization data for 1-7-A was as follows: 1 HNMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.8 Hz, 1H), 8.16(s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.36 (m, 4H), 5.21 (q, J = 19.0 Hz,2H), 3.95 (d, J = 5.7 Hz, 2H), 3.26-3.11 (m, 2H), 2.53 (s, 3H), 2.30-2.08 (m,2H), 1.94-1.79 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 510.1[M+H] + . The structural characterization data for 1-7-B was as follows: 1HNMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 8.9 Hz, 1H), 8.15(s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.64-5.35 (m, 4H), 5.19 (q, J = 19.0 Hz,2H), 3.97 (d, J = 5.2 Hz, 2H), 3.27-3.10 (m, 2H), 2.51 (s, 3H), 2.27-2.10 (m,2H), 1.93-1.80 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 510.1[M+H] + .

[0174] Example 10: (2S,3S,4S,5R,6S)-6-(4-((((((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-acetamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid and (2S,3 S,4S,5R,6S)-6-(4-((((((((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-acetamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-8 / 9-A and A-8 / 9-B) [ka]

[0175] Step 1: Synthesis of (2S,3S,4S,5R,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-amido)-4-(((((((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Starting materials A-6-6 (303 mg, 0.307 mmol), 1-4 (100 mg, 0.205 mmol) and HOBt (83 mg, 0.614 mmol) were dissolved in DMF (5 mL) and added dropwise to diisopropylethylamine (79 mg, 0.614 mmol), stirred and reacted for 12 hours. Water and ethyl acetate were stirred, allowed to settle and the liquid separated. The organic phase was washed with saturated brine, dried and concentrated under reduced pressure to give 260 mg of crude product A-8 / 9-1, which was used directly in the next reaction. ESI-MS (m / z): 1300.7[M+1] + .

[0176] Step 2: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-4-((((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A-8 / 9-1 (260 mg, 0.200 mmol) was dissolved in methanol (5 mL), then added to 2 drops of dichloromethane for dissolution, added dropwise to lithium hydroxide aqueous solution (67 mg, 1.60 mmol, dissolved in 0.5 mL of water), stirred and reacted for 2 hours. The raw material was consumed as monitored by LCMS. The reaction was added to 3N hydrochloric acid aqueous solution, adjusted to pH=4, concentrated under reduced pressure, and purified (conditions as follows) to obtain A-8 / 9-2 (63 mg). ESI-MS (m / z): 938.3[M+1] + . Column: Waters XBridge Prep C18OBD (5 μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 24]

[0177] Step 3: (2S,3S,4S,5R,6S)-6-(4-((((((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-acetamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carvone Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((((((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-acetamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A-8 / 9-2 (63 mg, 0.067 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-hex-5-ynoic acid 2,5-dioxopyrrolidin-1-yl ester (IM-3, 27 mg, 0.074 mmol) were dissolved in DMF (1 mL) and added dropwise to diisopropylethylamine (9 mg, 0.067 mmol) under stirring, and reacted at room temperature for 4 hours. The raw material was consumed as monitored by LCMS. The reaction solution was separated and purified (under the conditions as follows) to obtain 10.14 mg of A-8 / 9-A and 14.60 mg of A-8 / 9-B. Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 25] A-8 / 9-A peak retention time: 8.90 minutes. ESI-MS (m / z):1188.6 [M+1] + . A-8 / 9-B peak retention time: 9.10 minutes. ESI-MS (m / z):1188.6 [M+1] + .

[0178] Example 11: N-((10S)-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]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-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide and N-((10S)-10 -benzyl-1-(((1R,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)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide (A-10 / 11-A and A-10 / 11-B) [ka]

[0179] Step 1: Synthesis of (S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxa-5,8,11,14,17-pentaazoctadec-22-ynoic acid Compound IM-4 (30.00 mg, 70.00 μmol) was dissolved in N,N-dimethylformamide (1 mL) and added to 6-(2-methylsulfonylpyrimidin-5-yl)-hex-5-inoic acid 2,5-dioxopyrrolidin-1-yl ester (IM-3, 28.00 mg, 77.00 μmol), reacted at room temperature for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was directly purified by preparative high performance liquid chromatography, and the fraction was lyophilized to obtain the title compound IM-5 (20.00 mg). Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 26] The structural characterization data was as follows: ESI-MS (m / z): 691.0[M+18] + .

[0180] Step 2 At 25°C, the hydrochloride salt of 1-4 (30 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL), and added sequentially to IM-5 (49.66 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol), and reacted at 25°C for 0.5 h, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (under the conditions as follows) to separate the two isomers, which were named A-10 / 11-A (11.04 mg, retention time was 7.5 min) and A-10 / 11-B (19.42 mg, retention time was 8.0 min) according to their retention times. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 27] The structural characterization data was as follows: A-10 / 11-A: ESI-MS (m / z): 1107.3[M+H] + . A-10 / 11-B: ESI-MS (m / z): 1107.3[M+H] + .

[0181] Example 12: (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-10-A and 1-10-B) [ka]

[0182] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline Compound 1-10-1 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then slowly added to N-chlorosuccinimide (1.69 g, 12.63 mmol). After the addition, the reaction was carried out at room temperature for 16 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=0-25%) to obtain 0.95 g of the title compound. The structural characterization data was as follows: 1HNMR (400 MHz, DMSO-d6) δ 6.77 (dd, J = 2.5, 1.4 Hz,1H), 6.51 (dd, J = 11.7, 2.5 Hz, 1H), 5.84 (s, 2H).

[0183] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide Compound 1-10-2 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL) and added to acetic anhydride (648.13 mg, 6.35 mmol) under nitrogen protection. After the addition, the temperature was raised to 50° C. and the reaction was carried out for 15 h and monitored by liquid chromatography-mass spectrometry. The reaction solution was quenched with methanol (5 mL) and directly evaporated to dryness under reduced pressure to obtain the crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=0-40%) to obtain 1.01 g of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 265.9[M+H] + .

[0184] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)but-3-enoic acid Compound 1-10-3 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane (24 mL) and water (8 mL), and then added to N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tris(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol) and palladium acetate (42.12 mg, 187.62 μmol), the reaction system was subjected to nitrogen purge three times, heated to 100 ° C., and reacted under nitrogen atmosphere for 16 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N aqueous sodium hydroxide solution (60 mL) and ethyl acetate (50 mL) were added, shaken, and the layers were separated. The lower aqueous phase was separated and the pH was adjusted to about 3 with 4 mol / L hydrochloric acid aqueous solution, then it was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to give 1.00 g of crude product of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 272.0[M+H] + .

[0185] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butanoic acid The crude product of compound 1-10-4 (1.00g, 3.68mmol) was dissolved in tetrahydrofuran (15mL) and then added to 10% palladium on carbon (0.10g); after addition, the reaction system was subjected to hydrogen balloon replacement, and the reaction was carried out under hydrogen atmosphere for 4 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00g of the crude product of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 274.0[M+H] + .

[0186] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide The crude product of compound 1-10-5 (1.00g, 3.65mmol) was dissolved in trifluoroacetic acid (5mL), cooled to 5°C, and slowly added to trifluoroacetic anhydride (3.84g, 18.27mmol, 2.54mL); after addition, the reaction was carried out at 5°C for 2 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, then filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain the crude product, which was purified by flash silica gel column to obtain 0.43g of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 256.1[M+H] + .

[0187] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide Tetrahydrofuran (16mL) and tert-butanol (4mL) were added to the reaction flask, cooled to 5°C in an ice bath, and potassium tert-butoxide (415.18mg, 3.70mmol) was added. Compound 1-10-6 (0.43mg, 1.68mmol) was then dissolved in tetrahydrofuran (1mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (315.24mg, 2.69mmol) was added. After the addition, the reaction was carried out at 5°C for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was quenched with saturated ammonium chloride aqueous solution, and then it was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered, and the filtrate was concentrated under reduced pressure to obtain 455.00mg of the crude product of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 285.0[M+H] + .

[0188] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide The crude product of compound 1-10-7 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), and then added to 3 mol / L aqueous hydrochloric acid (1 mL) and 10% palladium on carbon (40.00 mg); after addition, the reaction was subjected to hydrogen balloon replacement three times, and the reaction was carried out at room temperature under hydrogen atmosphere for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.43 g of crude hydrochloride of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 271.0[M+H] + .

[0189] Step 8: Synthesis of (9H-fluoren-9-yl)methyl (8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate The crude hydrochloride salt of compound 1-10-8 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL) and then added to sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol), after the addition, the reaction was stirred at room temperature for 2 hours and monitored by liquid chromatography-mass spectrometry. The reaction solution was poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reverse phase column (acetonitrile:0.05% formic acid in water=20%-100%) to obtain 301.00 mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 493.2[M+H] + .

[0190] Step 9: Synthesis of (9H-fluoren-9-yl)methyl (8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate Compound 1-10-9 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL) and added to 12 mol / L concentrated hydrochloric acid (1 mL); after the addition, the temperature was raised to 60° C., and the reaction was carried out for 2 hours and monitored by liquid chromatography-mass spectrometry. The reaction solution was poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash silica gel column (ethyl acetate:petroleum ether=0-50%) to obtain 198.00 mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 451.1[M+H] + .

[0191] Step 10: Synthesis of (9H-fluoren-9-yl)methyl ((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (138.72mg, 526.96μmol) and compound 1-10-10 (198.00mg, 439.13μmol) were added to toluene (10mL), and then added to p-toluenesulfonic acid (75.53mg, 439.13μmol). After addition, the temperature was raised to 140°C, and the reaction was carried out for 4 hours. The reaction solution was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column (methanol:dichloromethane=0-5%) to obtain 256.00mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 678.1[M+H]+ .

[0192] Step 11: Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione Compound 1-10-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL) and then added to diethylamine (108.49 mg, 1.48 mmol), after which the reaction was carried out at room temperature for 0.5 h and monitored by liquid chromatography-mass spectrometry. After diethylamine was evaporated from the reaction solution under reduced pressure, the pH was adjusted to 2-3 with 1 mol / L aqueous hydrochloric acid solution, and the reaction solution was directly purified by preparative high performance liquid chromatography to give the title compounds 1-10-A (44.00 mg) and 1-10-B (43.00 mg). Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 28] The structural characterization data for 1-10-A (6 min LCMS, earlier peak with retention time of 1.276 min) was as follows: 1HNMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 10.3 Hz, 1H),7.33 (s, 1H), 6.54 (s, 1H), 5.62 (d, J = 19.3 Hz, 1H), 5.44 (s, 2H), 5.38 (d, J= 19.3 Hz, 1H), 4.43-4.38 (m, 1H), 3.28-3.10 (m, 2H), 2.22-2.12 (m, 1H),2.12-2.02 (m, 1H), 1.93-1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 456.1[M+H] + . The structural characterization data for 1-10-B (6 min LCMS, later peak with retention time of 1.300 min) was as follows: 1 HNMR (400 MHz, DMSO-d6) δ7.98 (d, J = 10.3 Hz, 1H), 7.32(s, 1H), 5.61 (d, J = 19.4 Hz, 1H), 5.44 (s, 2H), 5.32 (d, J = 19.4 Hz, 1H),4.44-4.36 (m, 1H), 3.33-3.25 (m, 1H), 3.22-3.11 (m, 1H), 2.23- 2.13 (m, 1H),2.11- 2.03 (m, 1H), 1.96-1.82 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 456.1[M+H] + . 6 min LCMS conditions: Chromatography column: Waters SunFire C18 OBD 4.6mm x 50mm x 5.0μm Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 29]

[0193] Example 13: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-acetamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-12) [ka]

[0194] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-amido)-4-((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Starting material A-6-6 (56.6 mg, 0.057 mmol), compound 1-11 (30.0 mg, 0.048 mmol) and HOBt (12.9 mg, 0.095 mmol) were dissolved in DMF (1 mL), added dropwise to diisopropylethylamine (18.5 mg, 0.143 mmol), and reacted at 25° C. for 4 hours under stirring. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 29.0 mg of the title compound. ESI-MS (m / z): 1304.3[M+1] + . Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm *150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 30]

[0195] Step 2: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-4-((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid Compound A-12-1 (29.0 mg, 0.022 mmol) was dissolved in methanol (1 mL) / tetrahydrofuran (1 ml), added dropwise to lithium hydroxide aqueous solution (7.7 mg, 0.184 mmol, dissolved in 0.5 mL of water), and reacted at 25 ° C for 2 hours under stirring; the reaction system was added to water (5 ml), adjusted to pH = 2-3 with 3N hydrochloric acid, extracted with ethyl acetate (5 mL) to remove impurities, and the aqueous phase was freeze-dried to obtain a crude product, which was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 5.0 mg of the title compound. ESI-MS (m / z): 942.2[M+1] + . Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 31]

[0196] Step 3: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydroxybenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-acetamido)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid Compound A-12-2 (5.0 mg, 0.0053 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-hex-5-inoic acid 2,5-dioxopyrrolidin-1-yl ester (IM-3, 2.3 mg, 0.0064 mmol) were dissolved in DMF (0.5 mL), added dropwise to diisopropylethylamine (2.0 mg, 0.016 mmol), and reacted at 25° C. for 4 hours. The reaction solution was directly purified by preparative high performance liquid chromatography (under the following conditions) to obtain 0.90 mg of the title compound. ESI-MS (m / z): 1192.0[M+1] + . Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 32]

[0197] Example 14: N-((S)-10-benzyl-1-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide and N-((S)-1 Synthesis of 0-benzyl-1-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide (A-14 / 15-A and A-14 / 15-B) [ka]

[0198] The single-configuration compound 1-10-A (36.00 mg, 79.70 μmol) and compound IM-5 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then added to 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol). After the addition, the reaction was carried out at room temperature for 1 hour and monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography to obtain the single-configuration title compound A-14 / 15-A (51.00 mg). Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 33] The structural characterization data was as follows: ESI-MS (m / z): 1111.0[M+H] + .

[0199] The single-configuration compound 1-10-B (36.00 mg, 79.70 μmol) and compound IM-5 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then added to 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol). After the addition, the reaction was carried out at room temperature for 1 hour and monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by high performance liquid chromatography to obtain the single-configuration title compound A-14 / 15-B (52.00 mg). Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 34] The structural characterization data was as follows: ESI-MS (m / z): 1111.0[M+H] + .

[0200] Example 15: N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-13-A and 1-13-B) [ka]

[0201] Compound A-14 / 15-A (40.00 mg, 35.99 μmol) was dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), and then added to hydrogen chloride in ethyl acetate solution (4 mol / L, 1 mL). After the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography to obtain single-configuration compound 1-13-A (4.75 mg). Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 35] The structural characterization data was as follows: 1HNMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 8.9 Hz, 1H), 8.05(d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.67-5.60 (m, 1H), 5.49 (t, J= 5.8 Hz, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.32-3.22(m, 2H), 2.28-2.15 (m, 2H), 1.93-1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 514.0[M+H] + .

[0202] Compound A-14 / 15-B (40.00 mg, 35.99 μmol) was dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), and then added to hydrogen chloride in ethyl acetate solution (4 mol / L, 1 mL). After the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography to obtain single-configuration compound 1-13-B (8.24 mg). Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 36] The structural characterization data was as follows: 1HNMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 9.0 Hz, 1H), 8.05(d, J = 10.3 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H), 5.68-5.58 (m, 1H), 5.53 (t, J= 5.8 Hz, 1H), 5.43 (d, J = 2.9 Hz, 2H), 5.20 (d, J = 7.3 Hz, 2H), 3.97 (d, J =5.7 Hz, 2H), 3.31-3.21 (m, 2H), 2.26-2.15 (m, 2H), 1.92-1.82 (m, 2H), 0.87 (t,J = 7.3Hz, 3H). ESI-MS (m / z): 514.0[M+H] + .

[0203] Example 16: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadeca-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-(isopropylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (A-26) [ka]

[0204] Step 1: Synthesis of tert-butyl (S)-(2-(4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate At 20°C, compound 2-1 (500.00 mg, 1.15 mmol) was dissolved in anhydrous dichloromethane (15 mL), and added to tert-butyl tert-butyloxycarbonyl carbonate (276.90 mg, 1.27 mmol) and DIPEA (447.21 mg, 3.46 mmol). The mixture was reacted at 20°C for 16 hours under stirring. The reaction was monitored by liquid chromatography-mass spectrometry, and the reaction solution was purified by silica gel column chromatography (mobile phase: dichloromethane / methanol = 50 / 1) to obtain 400 mg of the title compound. The structural characterization data was as follows: ESI-MS (m / z): 534.0 [M+H] + .

[0205] Step 2: Synthesis of tert-butyl (2-((S)-4-(((4-((S)-35-azido-2-(4-((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl)oxy)carbonyl)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate At 0°C, compound A-26-1 (30.00 mg, 0.056 mmol) was dissolved in anhydrous dichloromethane (1 mL) and added to DMAP (54.95 mg, 0.450 mmol) under nitrogen protection, then added dropwise to a solution of triphosgene (16.68 mg, 0.056 mmol) in dichloromethane (1 mL), reacted at 0°C for 30 minutes, and the reaction solution was subjected to nitrogen replacement under reduced pressure, and (S)-2-(32-azido-5-oxo)-1,2-dihydro ... -3,9,12,15,18,21,24,27,30-nonaoxa-6-azapentatriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)benzhydryl)amino)caproamide (117.39 mg, 0.111 mmol) solution was added dropwise, the reaction solution was stirred at 20° C. for 60 minutes, and the reaction was monitored by liquid chromatography-mass spectrometry; the reaction solution was used directly in the next step after concentration. The structural characterization data was as follows: ESI-MS (m / z): 1620.2[M+H] + .

[0206] Step 3: Synthesis of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-4-ethyl-11-(2-(isopropylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-194-03) At 25°C, compound A-26-2 (80.00mg, 0.049mmol) was dissolved in acetonitrile (1.0mL), added to trifluoroacetic acid (0.4mL), and reacted at 20°C for 1 hour; the reaction was monitored by liquid chromatography-mass spectrometry; the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 41.0mg of the trifluoroacetate salt of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 37] The structural characterization data was as follows: ESI-MS (m / z): 1248.2[M+H] + .

[0207] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecan-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-(isopropylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate At 25°C, compound A-26-3 (40.00mg, 0.032mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (IM-2, 14.69mg, 0.048mmol) were dissolved in a mixed solution of DMSO (0.5mL) and HO (0.1mL), and added to cuprous bromide (9.20mg, 0.064mmol), and the reaction solution was reacted at 20°C for 2 hours under the protection of nitrogen; the reaction was monitored by high performance liquid chromatography-mass spectrometry; the reaction solution was purified by preparative high performance liquid chromatography (under the conditions as follows), and the fraction was freeze-dried to obtain 20.0mg of the trifluoroacetate salt of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 38] The structural characterization data was as follows: ESI-MS (m / z): 1552.6 [M+H] + .

[0208] Example 17: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-acetamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxo-3,9-diazapentatriacontanamido)benzyl ((1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-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 (A-28) [ka]

[0209] Step 1: Synthesis of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-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 At 0°C, compound 1-2 (20.00 mg, 0.040 mmol) was dissolved in anhydrous dichloromethane (1 mL), and added to DMAP (39.10 mg, 0.320 mmol) under nitrogen protection, and added dropwise to a solution of triphosgene (11.87 mg, 0.040 mmol) in dichloromethane (1 mL), and the reaction solution was reacted at 0°C for 30 minutes, and the reaction solution was subjected to nitrogen replacement under reduced pressure, and (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24) in anhydrous dichloromethane (20 mL) was dissolved in anhydrous dichloromethane (20 mL). ,27,30-Nonaoxa-6-azapentatriacontanamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)benzhydryl)amino)hexanamide (84.66 mg, 0.080 mmol) solution was added dropwise, the reaction solution was stirred at 20° C. for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry; the reaction solution was purified by preparative high performance liquid chromatography (under the conditions as follows), and the fraction was freeze-dried to obtain 40.0 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 39] The structural characterization data was as follows: ESI-MS (m / z): 1550.2[M+H] + .

[0210] Step 2: Synthesis of (1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-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 (4-((S)-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadeca-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)carbonate At 25°C, compound A-28-1 (30.00mg, 0.019mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (IM-2, 8.87mg, 0.029mmol) were added to a mixture solution of DMSO (0.5mL) and HO (0.1mL), and copper(I) bromide (5.55mg, 0.039mmol) was added, and the reaction solution was reacted at 20°C for 1 hour under the protection of nitrogen; the reaction was monitored by liquid chromatography-mass spectrometry; the reaction solution was purified by preparative high performance liquid chromatography (under the conditions as follows), and the fraction was freeze-dried to obtain 25.0mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 40] The structural characterization data was as follows: ESI-MS (m / z): 1855.2[M+H] + .

[0211] Step 3: 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 (1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-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 At 25° C., compound A-28-2 (15.00 mg, 0.017 mmol) was dissolved in acetonitrile (0.5 mL) and added to trifluoroacetic acid (0.2 mL), and the reaction solution was reacted at 20° C. for 0.5 hours. The reaction was monitored by liquid chromatography-mass spectrometry; the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the fraction was freeze-dried to obtain 9.0 mg of the trifluoroacetate salt of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 41] The structural characterization data was as follows: ESI-MS (m / z): 1583.1[M+H] + .

[0212] Example 18: 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-nonaoxo-3,9-diazapentatriacontanamido)benzyl ((1S,9R)-9-ethyl-5-fluoro-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 (A-29) [ka]

[0213] Step 1: Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide At 25°C, the mesylate of 1-1 (30.00mg, 56.44μmol) was dissolved in N,N-dimethylformamide (1mL), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (58.74mg, 112.88μmol), N,N-diisopropylethylamine (43.76mg, 338.63μmol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (26.62mg, 84.66μmol) were added in sequence, and the reaction was carried out at 25°C for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was added to water, extracted with ethyl acetate, the organic phases were combined, dried over sodium sulfate, concentrated under reduced pressure, and the crude product was separated by thin layer chromatography (dichloromethane:methanol=15:1) to obtain 27.00mg of the title compound.

[0214] Step 2: Synthesis of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((1S,9R)-1-(2-((tert-butyldiphenylsilyloxy)acetamido)-9-ethyl-5-fluoro-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 At 0°C, A-29-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL) and added successively to a solution of 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and triphosgene (8.11 mg, 27.33 μmol) in dichloromethane (0.5 mL), and the reaction was carried out at 0°C for 0.5 hours; the residual triphosgene was replaced with nitrogen, and (S)-2-(3-(4-methylphenyl)-1,4-dihydro-1,5-trifluoro ... This was followed by the addition of a solution of 2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-3,9-diazapentatriacontanamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)benzhydryl)amino)caproamide (43.46 mg, 40.99 μmol) and reacted at 0° C. for 0.5 hours; the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated and the crude product was separated and purified by thin layer chromatography (dichloromethane:methanol=15:1) to obtain 30.00 mg of the title compound.

[0215] Step 3: (1S,9R)-1-(2-((tert-butyldiphenylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-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(4-((4-methoxyphenyl Synthesis of 5-(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)carbonate At 25°C, A-29-2 (250.00mg, 137.51μmol) was dissolved in a mixed solvent of DMSO (2mL) and water (0.4mL), and added to 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (IM-2, 62.98mg, 206.26μmol) and cuprous bromide (39.45mg, 275.01μmol), and reacted at 25°C for 1 hour; the reaction was monitored by liquid chromatography-mass spectrometry; after the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (under the conditions as follows), and the fraction was lyophilized to obtain 150.00mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 42]

[0216] Step 4: Synthesis of (1S,9R)-9-ethyl-5-fluoro-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 (4-((S)-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-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-diazapentatriacontanamide)carbonate At 25°C, A-29-3 (150mg, 49.45μmol) was dissolved in tetrahydrofuran (1mL), added dropwise to a mixture solution (50uL) of tetrabutylammonium fluoride (1M in tetrahydrofuran) / glacial acetic acid (v / v=13 / 1), reacted at 25°C for 0.5 hours, and the reaction was monitored by liquid chromatography-mass spectrometry; after the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (under the conditions as follows), and the fraction was freeze-dried to obtain 50.00mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 43]

[0217] Step 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 ((1S,9R)-9-ethyl-5-fluoro-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 At 25°C, A-29-4 (50mg, 26.52μmol) was dissolved in dichloromethane (1mL) and added to trifluoroacetic acid (60.49mg, 530.49μmol), and reacted at 25°C for 0.5 hours; the reaction was monitored by liquid chromatography-mass spectrometry; after the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative high performance liquid chromatography (under the conditions as follows), and the fraction was freeze-dried to obtain 23.69mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 44] Structural characterization data for A-29 was as follows: ESI-MS (m / z): 1613.6[M+H] + .

[0218] Example 19: 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (B-1) [ka]

[0219] Step 1: Synthesis of 4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutylamino)-5-ureidopentanamido)benzyl((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate At 25°C, (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide (MM AE, 10 mg, 0.014 mmol) and compound B-1-1 (12.82 mg, 0.017 mmol) were dissolved in DMF (1.0 mL), and added to HOBt (2.82 mg, 0.021 mmol) and DIPEA (3.60 mg, 0.028 mmol), and reacted at 25° C. for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry, and the reaction solution was used directly in the next reaction without workup. The structural characterization data was as follows: ESI-MS (m / z): 1345.2 [M+H] + .

[0220] Step 2: Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutylamino)-5-ureidopentanamido)benzyl((S)-1-((S))-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl(methyl)carbamate At 25°C, diethylamine (0.1mL) was added to the reaction solution of compound B-1-2, and reacted at 25°C for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry, and the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product, which was used directly in the next reaction. The structural characterization data was as follows: ESI-MS (m / z): 1123.2 [M+H] + .

[0221] Step 3: Synthesis of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate At 25°C, the crude product of B-1-3 and 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (IM-1, 7.16 mg, 0.027 mmol) were dissolved in DMF (0.5 mL), and the solution was added to HATU (10.15 mg, 0.027 mmol) and DIPEA (3.45 mg, 0.027 mmol), and reacted at 25°C for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry, and the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 5.0 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 45] The structural characterization data was as follows: ESI-MS (m / z): 1373.2 [M+H] + .

[0222] Example 20: N-((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptazaheptanoic acid-27-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl (B-3) [ka]

[0223] Step 1: Synthesis of (9H-fluoren-9-yl)methyl ((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptylheptanediamido-27-yl)carbamate Compound IM-6 (50.0 mg, 0.077 mmol) and (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyryla (Mido)butanamide (MMAE, 55.60 mg, 0.077 mmol) was weighed and dissolved in DMF (1 mL), then added to HATU (32.37 mg, 85.18 μmol) and DIPEA (20.02 mg, 154.88 μmol); after addition, the reaction was carried out at room temperature for 1 hour and monitored by liquid chromatography-mass spectrometry, and the reaction solution was purified by preparative high performance liquid chromatography (under the conditions as follows) to obtain 35.0 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 46] The structural characterization data was as follows: ESI-MS (m / z): 1345.1[M+H] + .

[0224] Step 2: Synthesis of (S)-2-((2S,13S)-19-amino-13-benzyl-2-isopropyl-3-methyl-4,9,12,15,18-pentaoxo-6-oxa-3,8,11,14,17-heptazaone)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide Compound B-3-1 (20.00 mg, 0.015 mmol) was dissolved in dichloromethane (2 mL), and then added to diethylamine (1 mL), and reacted at room temperature for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain 20.0 mg of crude product. The structural characterization data was as follows: ESI-MS (m / z): 1123.1[M+H] + .

[0225] Step 3: Synthesis of N-((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptazaheptanoic acid-27-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl Compound B-3-2 (20.00 mg, 0.015 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (IM-1, 17.00 mg, 0.015 mmol) were dissolved in DMF (1 mL), and then added to HATU (6.33 mg, 16.65 μmol) and DIPEA (3.91 mg, 30.27 μmol). After the addition, the mixture was reacted at room temperature for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 3.52 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 47] The structural characterization data was as follows: ESI-MS (m / z): 1374.1[M+H] + .

[0226] II. Preparation of antibodies and determination of binding activity 1. Antibody Preparation and Purification In the initial stage, mouse antibodies 3D8, 19F6 and 38F8 were obtained through hybridoma screening by immunizing Balb / c, C57Bl / 6, NZB and A / J mice, and then humanized to obtain humanized antibody sequences 3D8_HuC24 (heavy chain variable region, SEQ ID NO: 9; light chain variable region, SEQ ID NO: 10), 19F6_Hu35v1 (heavy chain variable region, SEQ ID NO: 1; light chain variable region, SEQ ID NO: 2), 38F8_Hu57 (heavy chain variable region, SEQ ID NO: 17; light chain variable region, SEQ ID NO: 18); the heavy chain constant regions of the above humanized antibodies were all human IgG1 heavy chain constant region (SEQ ID NO: 22), and the light chain constant regions were all human kappa light chain constant region (SEQ ID NO: 23). The sequence information of 3D8_HuC24, 19F6_Hu35v1, 38F8_Hu57 is summarized in Table 1 below. After synthesis and codon optimization of the coding DNA sequences of the above humanized antibodies, they were cloned into pcDNA3.4 plasmid, and the pcDNA3.4 plasmids corresponding to the heavy and light chains of each humanized antibody were simultaneously transfected into Expi293F cells, and the expressed antibodies in the supernatant were purified using protein A to obtain the corresponding antibodies. [Table 48]

[0227] 2. Affinity Determination The affinity of 19F6_Hu35V1, 3D8_HuC24 and 38F8_Hu57 for ROR1 on the surface of human cells was determined by flow cytometry.

[0228] Construction of human cell line with stable expression of ROR1: Ba / F3 cells were infected with lentivirus (G&P Biosciences) containing the complete coding sequence of human ROR1 (gene number: Q01973), positively transduced cells were screened by puromycin, and monoclonal BA / F3-ROR1 stable cell line was obtained by monoclonal screening by limiting dilution. Expression of ROR1 was confirmed by flow cytometry (Luminex, Guava easyCyte HT). Anti-human ROR1 antibody D10 (patent sequence: US Patent No. 9217040) was used as the detection antibody. As shown in Figure 1, the flow cytometry results showed that the positive rate of Ba / F3-ROR1 was very high (close to 100%), which could be used in subsequent experiments.

[0229] In addition to Ba / F3-ROR1 overexpressing cells, human mantle cell lymphoma cell line Jeko-1 (Nanjing Cobioer Biosciences), human colon cancer cell line HT-29 (the Cell Bank of the Chinese Academy of Sciences), and human lung cancer cell line A549 (the Cell Bank of the China Academy of Sciences) were also used for flow cytometry analysis. Anti-ROR1 antibody UC961 (VH and VL of antibody UC961 were SEQ ID NOs: 5 and 6, respectively, disclosed in WO2018237335) and anti-TNP antibody were used as positive and negative controls, respectively. Ba / F3-ROR1, Jeko-1, HT-29 and A549 cells were respectively incubated with different concentration gradients of purified humanized antibodies for 30 min on ice, washed twice with flow buffer (PBS+2% FBS), then incubated with goat anti-human fluorescent secondary antibody (Jackson ImmunoResearch) for 30 min on ice, and finally loaded onto the machine for detection.

[0230] The results are shown in Tables 2-1, 2-2 and 2-3. In the tables, the affinity EC50 values ​​of 19F6_Hu35V1, 3D8_HuC24 and 38F8_Hu57 in all four types of cells were lower than the affinity EC50 value of the positive control antibody UC961, indicating that the above-mentioned anti-human ROR1 humanized antibodies have excellent binding activity against ROR1-positive cells.

[0231] [Table 49] [Table 50] [Table 51]

[0232] The kinetic affinity of 19F6_Hu35V1, 3D8_HuC24, 38F8_Hu57 and positive control antibody UC961 to human ROR1 ECD protein was determined by Octet ForteBio method. The specific experimental steps were as follows: anti-human IgG Fc AHC probe (ForteBio) was first bound to the antibody to be tested to a response signal value of 0.8-1.2 nM, and the kinetic affinity was measured by immersing the antibody-coated probe in wells containing different concentrations (16, 8, 4, 2, 1, 0.5, 0.25, and 0 μg / mL) of human ROR1 ECD protein, binding was carried out for 2-4 min, followed by dissociation for 4-6 min, and a 1:1 kinetic binding model was used for all fitting analyses.

[0233] As shown in Table 3, 19F6_Hu35V1 had a significantly slower dissociation rate (as indicated by the K value) than that of the control antibody UC961, and a 5.4-fold stronger affinity (as indicated by the K value) than that of the control antibody UC961. 3D8_HuC24 and 38F8_Hu57 showed affinities comparable to that of the control antibody UC961.

[0234] [Table 52]

[0235] III. Conjugation of Compounds Containing Cellular Bioactive Molecules and Linkers to Antibodies The antibody 19F6 involved in the antibody-drug conjugates prepared in the following examples was the 19F6_Hu35v1 antibody described in Section II above.

[0236] Conjugation and preparation of samples was as follows: 0.46ml of 19F6_Hu35v1 antibody (anti-ROR1, 11.0mg / mL) was taken, diluted with 0.1M edetate disodium solution (pH 7.7), then adjusted to pH 7.7 with 1M Na2HPO4 solution, added to 10mM TCEP (tris(2-carboxyethyl)phosphine) solution, mixed well, and left at room temperature for 90 minutes. 4.0-10 times the molar amount of "drug-linker" compound dissolved in dimethylsulfoxide was added to the above system, mixed well, and left at room temperature for 2 hours, then replaced the buffer solution with 10mM histidine buffer solution having a pH of 6.0 using NAP-5 gel column (Cytiva), then added sucrose and Tween 20, mixed well, and obtained antibody-drug conjugates (i.e., ADC compounds) as shown in Table 4.

[0237] The drug-to-antibody ratios (DAR values) of the conjugation samples were determined as follows: The molecular weights of ADC samples were determined by LC-MS, and drug-to-antibody ratios, i.e., DAR values, were calculated; The molecular weight of the ADC samples obtained by conjugation was analyzed by LC-MS.

[0238] Chromatographic conditions for determination: Liquid chromatography column: Thermo MAbPac RP 3.0 * 100mm; Mobile phase A: 0.1%FA / H2O; Mobile phase B: 0.1%FA / ACN; Flow rate: 0.25 ml / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 2 μl; [Table 53] Mass spectrometry conditions for determination: Mass spectrometer model: AB Sciex Triple TOF 5600+; GS1 35;GS2 35;CUR 30;TEM 350;ISVF 5500;DP 250;CE 10;cumulative time 0.5 seconds; m / z 600–4000; a total of 40 time bins.

[0239] [Table 54]

[0240] IV. Detection of the inhibitory effect of antibody-drug conjugates on cell viability in vitro Inhibitory effect of ADC compounds on cell proliferation (1) Cell plating: First, tumor cells HCC827, NCI-H1975, HT-29 and NCI-N87 were cultured in the corresponding medium, digested with trypsin, resuspended, and counted after centrifugation, and the cells were adjusted to the appropriate concentration for plating. The sources of tumor cells are shown in Table 5.

[0241] [Table 55]

[0242] Co-incubation of compounds of the invention and tumor cells: After the cells attached to the wall, the medium was removed and diluted antibody-drug conjugates (ADC compounds of the invention) were added to the wells of the top plate and incubated for 96 hours.

[0243] In vitro cell viability detection: After the incubation was completed, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme / Novazyme) was added to each well, shaken and mixed well in the dark, and reacted for 10 minutes, then detection was performed and the readings were collected on a microplate reader (manufacturer: BMG, model: PHERAStar-FS). By adding Cell Counting-Lite™, background RLU was obtained from the culture wells without cells, and control RLU was obtained from the culture wells with cells but without compounds. Cell inhibition rate=1-(sample RLU-background RLU) / (control RLU-background RLU)×100%; the half inhibitory concentration (IC) of the compound was calculated according to the four-parameter model fitting curve. 50 ) was calculated.

[0244] (2) Data results: The detection results are shown in Tables 6 to 9.

[0245] [Table 56] [Table 57] [Table 58] [Table 59]

[0246] The test results show that the ADC molecules obtained by the new conjugation method had tumor cell killing effects.

[0247] It indicates that the ADC molecules produced by the new conjugation method can kill tumor cells, and the application of the new conjugation method to the ADC molecules was effective.

[0248] V. Evaluation of tumor growth inhibitory effects of antibody-drug conjugates against ROR1-expressing human tumor cell lines in a mouse subcutaneous xenograft tumor model A formulation containing the ADC of the present invention was administered by tail vein injection to mouse CDX models subcutaneously implanted with human gastric cancer cells NCI-N87, human lung adenocarcinoma cells H1975, and human colon cancer cells HT-29. Changes in tumor volume and animal body weight were determined twice a week, and the tumor inhibitory effect of the ADC of the present invention on tumor-bearing mice was calculated.

[0249] Drugs being tested Drug name, source and preparation method: An appropriate amount of the ADC of the present invention (Sichuan Kelun Bio-tech Biopharmaceutical Co., Ltd.) was taken, and the mother solution was diluted with saline to obtain a dosing solution according to a dose of 10 μl / g. Saline was used as the vehicle control (vehicle).

[0250] Experimental animals and cell lines Balb / c-New Mouse (Chengdu Gempharmatech Biotechnology Co., Ltd., Production License Number: SCXK(Sichuan)2020-034, Animal Certificate Number: 202112622, 202109635, 202106975) Human gastric cancer cell line NCI-N87 (ATCC) Human lung adenocarcinoma cell line NCI-H1975 (Nanjing Cobioer Biosciences) Human colon cancer cells HT-29 (Cell Bank, Chinese Academy of Sciences)

[0251] Grouping and evaluation method 100~200mm 3Tumor-bearing mice with an average tumor volume of 100 μg / g were selected for random grouping (the number of groups was determined according to the number of samples). Physiological saline (hereinafter referred to as vehicle control, vehicle) and the ADC of the present invention were administered according to the groups. The frequency of administration was indicated in the specific experimental protocol. The administration method was tail vein injection, and the administration volume was 10 μl / g. After administration, the tumor diameter was measured twice a week with a caliper, and the tumor volume was calculated according to the following formula: V=0.5a×b 2 (wherein a and b represent the long and short diameters of the tumor, respectively) The death of the animals was observed and recorded daily.

[0252] The following formula was used to calculate the tumor growth inhibition rate TGI (%), which was used to evaluate the inhibitory effect of the ADCs of the invention on tumors: V T最後 >V T0 , TGI(%)=[1-(V T最後 -V T0 ) / (V C最後 -V C0 )] * 100%; or V T最後 ≦V T0 , TGI(%)=[1-(V T最後 -V T0 ) / V T0 ] * 100%. During the ceremony, V T最後 : Mean tumor volume of treatment group at the end of the experiment V T0 : Mean tumor volume of the first treatment group administered V C最後 : Mean tumor volume of the vehicle control group at the end of the experiment V C0 : Mean tumor volume of the vehicle control group at the start of dosing

[0253] The following formula was used to calculate the relative tumor proliferation rate T / C (%), which was used to evaluate the inhibitory effect of the ADCs of the invention on tumors: T / C=(V T最後 / V T0 ) / (V C最後 / V C0 ).

[0254] (1) Evaluation of the pharmacological effects of anti-human ROR1 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. NCI-N87 cells in the exponential growth phase were harvested, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female Balb / c-new mice to establish a gastric cancer model. The average tumor volume was approximately 160 mm 3 At that time, the mice were randomly divided into groups according to tumor size, including a vehicle control group (i.e., negative control, vehicle group), a 10 mg / kg group of 19F6-A-1 of the present invention, a 10 mg / kg group of 19F6-A-6, and a 10 mg / kg group of 19F6-A-10, in that order. Each group was administered by injection (iv) through the tail vein on days 0, 3, and 7, i.e., three times in total. After administration, the mice were weighed twice a week, and the long and short diameters of the tumors were measured with calipers, and the tumor volumes were calculated according to the following formula: V=0.5a×b 2 (wherein a and b represent the long and short diameters of the tumor, respectively) The death of the animals was observed and recorded daily.

[0255] The ADC of the present invention had a significant tumor growth inhibition effect in the NCI-N87 gastric cancer xenograft model. Compared with the vehicle group, the tumor growth inhibition (TGI) rates of the ADC 19F6-A-1 10mg / kg group, 19F6-A-6 10mg / kg group and 19F6-A-10 10mg / kg group of the present invention were 51.08%, 131.40% and 50.92%, respectively. No animal deaths, significant weight loss and obvious drug toxicity reactions were observed in each treatment group on day 28, and the mice had good tolerance to the ADC of the present invention during the treatment period. Specific results are shown in Table 10, Figure 2 and Figure 3.

[0256] [Table 60]

[0257] (2) Evaluation of the pharmacological effects of anti-human ROR1 antibody-drug conjugates in the NCI-H1975 model NCI-H1975 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-H1975 cells in the exponential growth phase were harvested, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female Balb / c-new mice to establish a human lung adenocarcinoma cell model. The average tumor volume was approximately 120 mm 3 At that time, the mice were randomly divided into groups according to tumor size, including a vehicle control group (vehicle), a 10 mg / kg group of the ADC 19F6-A-6 of the present invention, and a 10 mg / kg group of the ADC 19F6-A-10 of the present invention (6 mice per group), and each group was dosed by tail vein injection (iv) on days 0, 4, 7, 10 and 14, i.e., five doses in total. After dosing, the mice were weighed, and the long and short diameters of the tumors were measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula: V=0.5a×b 2 (wherein a and b represent the long and short diameters of the tumor, respectively) The death of the animals was observed and recorded daily.

[0258] Compared with the vehicle control group, the data on day 28 after five doses showed that the ADC 19F6-A-6 10 mg / kg group and the 19F6-A-10 10 mg / kg group of the present invention significantly inhibited tumor growth in the NCI-H1975 model, with tumor growth inhibition rates (TGI) of 196.35% and 52.52%, respectively, showing significant tumor growth inhibition effects in the NCI-H1975 small cell lung cancer xenograft model; all tumors in the mice were completely regressed in the ADC 19F6-A-6 10 mg / kg group of the present invention. On day 28, no animal deaths, significant animal weight loss, and obvious drug toxicity reactions were observed in each treatment group. During the treatment period, the mice had good tolerance to the ADC of the present invention. Specific results are shown in Table 11, Figure 4, and Figure 5.

[0259] [Table 61]

[0260] (3) Evaluation of the pharmacological effects of anti-human ROR1 antibody-drug conjugates in the HT29 model Human colon cancer cell line HT29 was cultured in McCoy's 5a medium containing 10% fetal bovine serum at 37°C and 5% CO2. HT29 cells in exponential growth phase were harvested and resuspended to an appropriate concentration by adding PBS and Matrigel at a final concentration of 50%, and inoculated subcutaneously into female Balb / c-new mice to establish a human colon cancer xenograft model. The average tumor volume was approximately 112 mm 3 At that time, the mice were randomly divided into a vehicle control group (vehicle) and a group of 10 mg / kg of the ADC 19F6-A-1 of the present invention according to the tumor size. After grouping, each group was administered by injection (iv) through the tail vein on days 0, 4, 7, 11, 14 and 18, i.e., six times in total. After administration, the mice were weighed, and the long and short diameters of the tumors were measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula: V=0.5a×b 2 (wherein a and b represent the long and short diameters of the tumor, respectively) The deaths of the animals were observed and recorded daily.

[0261] The ADC 19F6-A-1 of the present invention at 10 mg / kg had a significant inhibitory effect on tumor growth in a human colon cancer HT29 xenograft tumor model. Data on the 32nd day after administration showed that the 19F6-A-1 10 mg / kg group had a TGI of 59.14% compared with the vehicle control group. No animal deaths, significant animal weight loss, or obvious drug toxic reactions were observed in each treatment group. During the treatment period, the mice tolerated the ADC of the present invention well. Specific results are shown in Table 12, Figure 6, and Figure 7.

[0262] [Table 62]

[0263] Although a particular model of the invention has been described in detail, one skilled in the art will appreciate that, in accordance with all the teachings disclosed, various modifications and substitutions can be made to those details, all of which are within the scope of the invention, the full scope of the invention being given by the appended claims and any equivalents thereof.

Claims

1. Formula Ab-[MMME� ｘ (In the formula, Ab represents an antibody or antigen-binding fragment thereof that specifically binds to receptor tyrosine kinase-like orphan receptor (ROR) family member 1 (ROR1): M represents a linking site connecting the antibody or antigen-binding fragment thereof; L represents a connector connecting the linking sites M and E; E represents a structural fragment connecting L and D; D represents a cytotoxic drug moiety; x is selected from 1 to 10. An antibody-drug conjugate having a structure represented by:

2. M has the following structure: 【Chemistry 1】 The antibody-drug conjugate of claim 1, selected from

3. M is the following 【Chemistry 2】 The antibody-drug conjugate of claim 1, which has the structure:

4. L is the following group: C １～６ alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, 【Chemistry 3】 where R′ is hydrogen, C １～６ Alkyl or -(CH ２ CH ２ O) ｒ -containing alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20; Preferably, L has the following structure: 【Chemistry 4】 2. The antibody-drug conjugate of claim 1, wherein s is selected from:

5. E is a single bond or -NH-CH ２ - or has the following structure: 【Chemistry 5】 Selected from: Preferably, E is a single bond, -NH-CH ２ - or 【Chemistry 6】 The antibody-drug conjugate of claim 1, 【Request 6】 【Chemical 7】 has the following structure: 【Chemistry 8】 The antibody-drug conjugate of claim 1, selected from

7. the cytotoxic agent is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor; Preferably, the tubulin inhibitor is an auristatin compound or a maytansine 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, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); the RNA polymerase inhibitor is α-amanitin or a pharma-ceutically acceptable salt, ester, or analogue thereof; Preferably, the cytotoxic agent is one of the following compounds: 【Chemistry 9】 selected from the group consisting of: The antibody-drug conjugate of claim 1, wherein the cytotoxic drug is preferably connected to E in the antibody-drug conjugate through an -OH, a primary amino group, a secondary amino group or a tertiary amino group thereon.

8. The antibody or antigen-binding fragment thereof: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined by the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 3 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 5 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 8 or a variant thereof; or (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 11 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 12 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; or (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 20 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; wherein said variant according to any one of items (1a), (1b) and (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or said variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, said substitutions are conservative substitutions, a heavy chain variable region (VH) and / or a light chain variable region (VL); Or, (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined by the AbM numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO:29 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO:30 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO:5 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO:6 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO:7 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO:8 or a variant thereof; or (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 37 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; or (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 46 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; wherein said variant according to any one of items (2a), (2b) and (2c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or said variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, said substitutions are conservative substitutions, a heavy chain variable region (VH) and / or a light chain variable region (VL); Or, (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, with the CDRs defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 31 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 32 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 5 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 8 or a variant thereof; or (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 38 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 39 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; or (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 47 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 48 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; wherein said variant according to any one of items (3a), (3b) and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or said variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, said substitutions are conservative substitutions, a heavy chain variable region (VH) and / or a light chain variable region (VL); Or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined by the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 26 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 28 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 8 or a variant thereof; or (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 33 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 34 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 35 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 43 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; or (4c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 40 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 41 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 42 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 43 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 44 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 16 or a variant thereof; wherein said variant according to any one of items (4a), (4b) or (4c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or said variant has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, said substitutions are conservative substitutions. The antibody-drug conjugate of claim 1, comprising:

9. The antibody or antigen-binding fragment thereof: (a) a VH as set forth in SEQ ID NO: 1 or a variant thereof, and / or a VL as set forth in SEQ ID NO: 2 or a variant thereof; (b) a VH as set forth in SEQ ID NO: 9 or a variant thereof, and / or a VL as set forth in SEQ ID NO: 10 or a variant thereof; or (c) VH set forth in SEQ ID NO: 17 or a variant thereof, and / or VL set forth in SEQ ID NO: 18 or a variant thereof. Including; The antibody-drug conjugate of claim 1, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

10. The antibody or antigen-binding fragment thereof: (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and (b) a human immunoglobulin light chain constant region (CL) or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; Further comprising: Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO:22 or a variant thereof, wherein the variant has one or more conservative amino acid substitutions compared to SEQ ID NO:22 (e.g., up to 20, up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions); Preferably, the light chain constant region is a kappa light chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO:23 or a variant thereof, wherein the variant has one or more conservative substitutions of amino acids compared to SEQ ID NO:23 (e.g., up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 22 and a light chain constant region (CL) set forth in SEQ ID NO:

23. The antibody-drug conjugate of claim 8 or 9.

11. The antibody or antigen-binding fragment thereof: (1) A heavy chain comprising a VH having a sequence set forth in SEQ ID NO:1 and a heavy chain constant region (CH) set forth in SEQ ID NO:22, and a light chain comprising a VL having a sequence set forth in SEQ ID NO:2 and a light chain constant region (CL) set forth in SEQ ID NO:23; (2) A heavy chain comprising a VH having the sequence set forth in SEQ ID NO:9 and a heavy chain constant region (CH) set forth in SEQ ID NO:22, and a light chain comprising a VL having the sequence set forth in SEQ ID NO:10 and a light chain constant region (CL) set forth in SEQ ID NO:23; or (3) A heavy chain comprising a VH having the sequence set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) set forth in SEQ ID NO: 22, and a light chain comprising a VL having the sequence set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO:

23. The antibody-drug conjugate of claim 1, comprising:

12. M is a sulfhydryl group (-SH) or an amino group (-NH ２ 2. The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate is linked to

13. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof of claim 11; -M-LE-D is selected from the structures defined in any one of claims 1 to 7; and x is 1 to 10; preferably, x is 1 to 8 or x is 1 to 4.

14. ADC A-1: 【Chemistry 10】 、 ADC A-2: 【Chemistry 11】 、 ADC A-3: 【Chemistry 12】 、 ADC A-4: 【Chemistry 13】 、 ADC A-5: 【Chemistry 14】 、 ADC A-6: 【Chemistry 15】 、 ADC A-7: 【Chemistry 16】 、 ADC A-8: 【Chemistry 17】 、 ADC A-9: 【Chemistry 18】 、 ADC A-10: 【Chemistry 19】 、 ADC A-11: 【Chemistry 20】 、 ADC A-12: 【Chemistry 21】 、 ADC A-13: 【Chemical 22】 、 ADC A-14: 【Chemistry 23】 、 ADC A-15: 【Chemistry 24】 、 ADC A-16: 【Chemistry 25】 、 ADC A-17: 【Chemistry 26】 、 ADC A-18: 【Chemical 27】 、 ADC A-19: 【Chemistry 28】 、 ADC A-20: 【Chemical 29】 、 ADC A-21: 【Chemistry 30】 、 ADC A-22: 【Chemistry 31】 、 ADC A-23: 【Chemistry 32】 、 ADC A-24: 【Chemical 33】 、 ADC A-25: 【Chemical 34】 、 ADC A-26: 【Chemistry 35】 、 ADC A-27: 【Chemical 36】 、 ADC A-28: 【Chemical 37】 、 ADC A-29: 【Chemical Formula 38】 、 ADC A-30: 【Chemical 39】 、 ADC A-31: 【Chemistry 40】 、 ADC B-1: 【Chemistry 41】 、 ADC B-2: 【Chemistry 42】 , and ADC B-3: 【Chemistry 43】 is selected from the group consisting of wherein HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO:1 and a VL set forth in SEQ ID NO:2, e.g., 19F6_Hu35v1 (comprising a heavy chain comprising a VH set forth in SEQ ID NO:1 and a CH set forth in SEQ ID NO:22, and a light chain comprising a VL set forth in SEQ ID NO:2 and a CL set forth in SEQ ID NO:23); Where: 【Chemistry 44】 The antibody-drug conjugate of claim 1, wherein: represents a specific connection mode between a sulfhydryl group of the antibody or antigen-binding fragment thereof and the connector.

15. 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8 2. The antibody-drug conjugate of claim 1, having a DAR value (drug to antibody ratio) of 3 to 8, such as 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 7.0-7.5, 7.0-8.0 or 7.5-8.

0.

16. Formula M'-L-ED-D (In the formula, M' represents the structure of M before being connected to the antibody or antigen-binding fragment thereof according to claim 8, and M, L, E and D are as defined in claim 1. A drug-linker having a structure represented by: Preferably, M' has the following structure: 【Chemistry 45】 The drug-linker is selected from:

17. A-1: 【Chemistry 46】 、 A-2: 【Chemistry 47】 、 A-3: 【Chemistry 48】 、 A-4: 【Chemistry 49】 、 A-5: 【Chemistry 50】 、 A-6: 【Chemistry 51】 、 A-7: 【Chemistry 52】 、 A-8: 【Chemistry 53】 、 A-9: 【Chemical 54】 、 A-10: 【Chemistry 55】 、 A-11: 【Chemistry 56】 、 A-12: 【Chemistry 57】 、 A-13: 【Chemistry 58】 、 A-14: 【Chemistry 59】 、 A-15: 【Chemistry 60】 、 A-16: 【Chemistry 61】 、 A-17: 【Chemistry 62】 、 A-18: 【Chemistry 63】 、 A-19: 【Chemistry 64】 、 A-20: 【Chemistry 65】 、 A-21: 【Chemistry 66】 、 A-22: 【Chemistry 67】 、 A-23: 【Chemistry 68】 、 A-24: 【Chemistry 69】 、 A-25: 【Chemistry 70】 、 A-26: 【Chemistry 71】 、 A-27: 【Chemical 72】 、 A-28: 【Chemical 73】 、 A-29: 【Chemical Formula 74】 、 A-30: 【Chemistry 75】 、 A-31: 【Chemical 76】 、 B-1: 【Chemical Formula 77】 、 B-2: 【Chemical 78】 、 B-3: 【Chemical Formula 79】 The drug-linker of claim 16, selected from the group consisting of:

18. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1, optionally a drug-linker of claim 16, and one or more excipients.

19. 20. Use of the pharmaceutical composition according to claim 18 in the manufacture of a medicament for the treatment of cancer associated with high expression of ROR1.

20. 20. The use of claim 19, wherein the cancer is selected from the group consisting of solid tumors and hematological malignancies; for example, selected from the group consisting of colon cancer, gastric cancer, breast cancer, lung cancer (such as non-small cell lung cancer, in particular lung adenocarcinoma), and lymphoma.

21. The pharmaceutical composition according to claim 18, which is for treating cancer associated with high expression of ROR1.

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