Camptothecin derivatives and ligand-drug conjugates

Abstract

The present disclosure relates to a ligand-drug complex having a novel structure or a pharmaceutically acceptable salt thereof. Specifically, the present disclosure provides a ligand-drug complex having a general structural formula of Pc-(L-D) n or a pharmaceutically acceptable salt thereof, a method for producing the same, a pharmaceutical composition containing the complex, and its use in the treatment of tumors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from Chinese patent application number 202210515898.0 filed on May 12, 2022, Chinese patent application number 202210999585.7 filed on August 19, 2022, and Chinese patent application number 202310406469.4 filed on April 14, 2023, which are incorporated herein by reference in their entireties for all purposes.

[0002] The present disclosure belongs to the biomedical field and relates to structurally novel ligand-drug conjugates, methods for their preparation, pharmaceutical compositions containing the conjugates, and their use as antitumor drugs. [Background technology]

[0003] Antibody-drug conjugates (ADCs) are novel targeted drugs that combine the tumor targeting properties of antibodies with the highly effective killing properties of toxin molecules by linking monoclonal antibodies that specifically bind to tumor cell surface antigens with biologically active toxin molecules, while avoiding the drawbacks of the former, such as the relatively low therapeutic efficacy of the former, and the latter, such as excessive toxicity, side effects, and poor drug discoverability. Compared with traditional chemotherapy drugs, ADC drugs can precisely target tumor cells while reducing the impact on normal cells, achieving safer and more effective antitumor effects.

[0004] In 2000, the first antibody-drug conjugate, Mylotarg (gemtuzumab ozogamicin), received marketing approval from the U.S. FDA for the treatment of adult acute myeloid leukemia (AML). In 2011, the U.S. FDA approved the marketing of Adcetris (bretuximab vedotin), a novel ADC-targeted drug, for the treatment of Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Both Mylotarg and Adcetris are drugs for the treatment of hematologic malignancies. In 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the U.S. FDA for the treatment of advanced or metastatic breast cancer that is HER2-positive and resistant to trastuzumab and paclitaxel, making it the first approved ADC drug for the treatment of solid tumors.

[0005] ADCs generally contain three components: an antibody, a linker, and a toxin. Camptothecin derivatives are one type of toxin used in ADC development, achieving antitumor effects by inhibiting topoisomerase I. Daiichi Sankyo Company, Limited (Daiichi Sankyo) developed the ADC drug Enhertu (Trastuzumab deruxtecan, DS-8201) using the camptothecin derivative exatecan as a toxin to target HER2, which received marketing approval from the US FDA in 2019. Clinical studies have shown that Enhertu has relatively good therapeutic effects against HER2-positive breast cancer, gastric cancer, and non-small cell lung cancer.

[0006] Although several ADC drugs are currently on the market, some safety issues and drug resistance problems still exist, and unmet clinical needs remain significant. Therefore, there is an urgent need in the art for further development of more effective and safe ADC drugs. Summary of the Invention

[0007] The present disclosure provides a compound having the general structural formula Pc-(LD) nor a pharmaceutically acceptable salt thereof, Among them, Pc is a ligand unit, L is a linker unit, D is a Drug unit represented by the following formula (DI): [ka] Among them, X is selected from NH or O; R 1 is selected from halogen, CN, a C1-C6 alkyl group, a C3-C6 cycloalkyl group, or a C2-C6 alkynyl group, and the C1-C6 alkyl group, C3-C6 cycloalkyl group, or C2-C6 alkynyl group may optionally be selected from one or more R a1 is replaced by X1 is a CR 2 or N, R 2 is selected from H, halogen, CN, or R 1 , R 2 together with the atom to which they are connected form a 5- to 6-membered heterocyclyl group, which may optionally be one or more R a2 is replaced by R 4 is selected from H, a C1-C3 alkyl group, a C3-C6 cycloalkyl group, or a 4- to 7-membered heterocyclyl group, and the C1-C3 alkyl group, C3-C6 cycloalkyl group, or 4- to 7-membered heterocyclyl group may optionally be selected from one or more R a4 is replaced by R 5 is selected from H, halogen, CN, NH2 or NO2, or R 1 , R 5 together with the atoms to which they are linked form a 5- to 6-membered heterocyclyl group, a 5- to 6-membered heteroaryl group, or a C5-C7 cycloalkenyl group, and the 5- to 6-membered heterocyclyl group, the 5- to 6-membered heteroaryl group, or the C5-C7 cycloalkenyl group may optionally be joined by one or more R a5 is replaced by R 6is selected from H or a C1-C3 alkyl group; R 7 is selected from H, a C1-C3 alkyl group, or a C3-C6 cycloalkyl group, or R 6 , R 7 together with the C atom to which they are connected form a C3-C6 cycloalkyl group, and the C3-C6 cycloalkyl group may optionally be one or more R a7 is replaced by Each R a1 , R a2 , R a4 , R a5 , R a7 are independently selected from D, halogen, CN, ═O, OH, NH, C-C alkyl, C-C cycloalkyl, or 4- to 7-membered heterocyclyl groups, and the OH, NH, C-C alkyl, C-C cycloalkyl, or 4- to 7-membered heterocyclyl groups may optionally be joined by one or more R b is replaced by Each R b are independently selected from halogen, CN, ═O, a C1-C3 alkyl group, OH, O(C1-C3 alkyl group), NH2, NH(C1-C3 alkyl group), or N(C1-C3 alkyl group)2; The conditions are: i) R 1 is selected from methyl groups, and R 2 If is chosen from F, then R 6 , R 7 form a cyclopropyl group together with the C atom to which they are attached, and ii) when X is selected from NH, R 5 is not selected from H, and iii) the compound represented by formula (DI) is [ka] Not including, And, n is a real number from 1 to 16.

[0008] In some embodiments, each R a1 , R a2 , R a4 , R a5 , R a7are independently selected from D, halogen, CN, ═O, OH, NH2, a C1-C3 alkyl group, a C3-C6 cycloalkyl group, or a 4- to 7-membered heterocyclyl group.

[0009] In some embodiments, each R a2 and R a7 are independently selected from D.

[0010] In some embodiments, R 1 is selected from halogen, a C1-C3 alkyl group, a C3-C6 cycloalkyl group, or a C2-C3 alkynyl group.

[0011] In some embodiments, R 1 is selected from Cl, Br, a methyl group, a cyclopropyl group, or an ethynyl group.

[0012] In some embodiments, R 1 is selected from Cl, Br or a methyl group.

[0013] In some embodiments, R 2 is selected from H, halogen, CN, or R 1 , R 2 together with the atom to which they are linked form a 5- to 6-membered heterocyclyl group, which contains one or two oxygen atoms as ring atoms, and which is optionally substituted with one or more D atoms.

[0014] In some embodiments, R 2 is selected from H, halogen, CN, or R 1 , R 2 together with the atom to which they are linked form a 5- to 6-membered heterocyclyl group, which contains 1 or 2 oxygen atoms as ring atoms.

[0015] In some embodiments, R 2 is selected from H, F or Cl, or R 1 , R 2together with the atoms connected to them [ka] Form.

[0016] In some embodiments, R 2 is selected from H, F or Cl, or R 1 , R 2 together with the atoms connected to them [ka] Form.

[0017] In some embodiments, R 5 is selected from H, halogen, NH2 or NO2, or R 1 , R 5 together with the atoms to which they are connected form a 5-6 membered heteroaryl group or a C5-C6 cycloalkenyl group, and the 5-6 membered heteroaryl group or the C5-C6 cycloalkenyl group optionally contains one or more R a5 is replaced by

[0018] In some embodiments, R 5 is selected from H, Cl, F, NH2 or NO2, or R 1 , R 5 together with the atoms connected to them [ka] Form.

[0019] In some embodiments, R 4 is selected from H or a C1-C3 alkyl group.

[0020] In some embodiments, R 4 is selected from H.

[0021] In some embodiments, R 6 is selected from H or a methyl group.

[0022] In some embodiments, R 7 is selected from H, a C1-C3 alkyl group or a C3-C6 cycloalkyl group optionally substituted with one or more D, or R 6 , R 7 form a C3-C6 cycloalkyl group together with the C atom to which they are attached.

[0023] In some embodiments, R 7 is selected from H, a methyl group, an isopropyl group, or a cyclopropyl group optionally substituted with one or more D, or R 6 , R 7 form a cyclopropyl group together with the C atom to which they are attached.

[0024] In some embodiments, R 1 , R 2 together with the C atoms connected to each of them. [ka] Form R 6 is selected from H or a methyl group, and R 7 is selected from H, a methyl group, an isopropyl group, or a cyclopropyl group optionally substituted with one or more D, or R 6 , R 7 form a cyclopropyl group together with the C atom to which they are attached.

[0025] In some embodiments, the structural unit [ka] teeth, [ka] Selected from.

[0026] In some embodiments, R 1 is selected from a methyl group, and R 2 is selected from F and R6 , R 7 form a cyclopropyl group together with the C atom to which they are attached.

[0027] In some embodiments, X is selected from NH and R 5 is selected from Cl, F, NH2 or NO2.

[0028] In some embodiments, the Drug Unit of Formula (DI) is selected from the Drug Unit of Formula (D-Ia): [ka] Among them, R 1 , R 2 , R 4 , R 5 , R 6 , R 7 is as defined above.

[0029] In some embodiments, the compound of Formula (DI) is the compound shown below: [ka] Selected from JPEG2025516381000012.jpg230170JPEG2025516381000013.jpg220170JPEG2025516381000014.jpg47169.

[0030] In some embodiments, the present disclosure provides a compound having the general structural formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, Among them, Pc, L, n are as defined above; D is the following compound: [ka] It is chosen from one of JPEG2025516381000016.jpg50169.

[0031] In some embodiments, the linker unit L is [ka] wherein m1 and m2 are independently selected from integers of 2 to 8, m3 is independently selected from integers of 1 to 16, and L 1 , L 2 are each independently selected from peptide residues consisting of 1 to 8 amino acids, and the peptide residues are further optionally substituted with one or more substituents selected from halogen, CN, ═O, C1-C6 alkyl group, OH, O(C1-C6 alkyl group), NH2, NH(C1-C6 alkyl group), N(C1-C6 alkyl group)2, C3-C6 cycloalkyl group, and 4- to 7-membered heterocyclyl group.

[0032] In some embodiments, the L 1 , L 2 are each independently selected from peptide residues consisting of 2, 3, or 4 amino acids, which are further optionally substituted with one or more substituents selected from halogen, CN, ═O, C1-C6 alkyl group, OH, O(C1-C6 alkyl group), NH2, NH(C1-C6 alkyl group), N(C1-C6 alkyl group)2, C3-C6 cycloalkyl group, and 4- to 7-membered heterocyclyl group.

[0033] In some embodiments, the L 1 is a Gly-Gly-Phe-Gly tetrapeptide residue or an Ala-Ala-Ala tripeptide residue.

[0034] In some embodiments, the L 2 is a Gly-Gly-Phe-Gly tetrapeptide residue or a Val-Lys dipeptide residue.

[0035] In some embodiments, m1 is selected from 5.

[0036] In some embodiments, m2 is selected from 2 and m3 is selected from 8.

[0037] In some embodiments, the linker unit L has the following chemical structure: [ka] and its a-terminus is covalently bonded to a ligand unit Pc and its b-terminus is covalently bonded to a drug unit D.

[0038] In some embodiments, the general formula of the present disclosure is Pc-(LD) n or a pharmaceutically acceptable salt thereof, is a ligand-drug conjugate which is the following compound or a pharmaceutically acceptable salt thereof: [ka] Selected from JPEG2025516381000020.jpg235168JPEG2025516381000021.jpg233167JPEG2025516381000022.jpg222162JPEG2025516381000023.jpg219169.

[0039] where Pc and n are as defined above.

[0040] In some embodiments, the general formula is Pc-(LD) n or a pharmaceutically acceptable salt thereof, wherein the ligand unit Pc may be selected from a polypeptide, an antibody, or an antigen-binding fragment thereof.

[0041] In some embodiments, the Ligand unit Pc is capable of specifically binding to one or more antigens selected from the group consisting of HER2, p95HER2, HER3, CD3, CD16, ROR1, DLL3, CDH6, CD70, CD5, CD20, BCMA, EGFR, VEGF, and LIV-1.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof is monospecific, bispecific, trispecific, or tetraspecific.

[0043] In some embodiments, the antibody is selected from Trastuzumab, Pertuzumab, or Rituximab.

[0044] In some embodiments, the Pc is an antibody or antigen-binding fragment thereof that specifically binds to HER2, p95HER2, CDH6, ROR1, or LIV-1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and a light chain variable region (VL); In some embodiments, (1) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 contained in a VH set forth in SEQ ID NO: 1, 3, 19, 21, 37, 46, 54, 56, 71, 80, 82, or 84, or / and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 contained in a VL set forth in SEQ ID NO: 2, 4, 20, 22, 38, 47, 55, 57, 72, 81, 83, or 85, or (2) The heavy chain variable region and / or the light chain variable region comprise an amino acid sequence having at least 80% identity, or at most three insertion, deletion or substitution mutations, with each of the CDRs in HCDR1-3 and / or LCDR1-3 set forth in group (1), and further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. Optionally, the HCDR1-3 and / or LCDR1-3 are numbered according to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system.

[0045] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 to 3 or / and LCDR1 to 3 are: (1) the HCDRs 1 to 3 are SEQ ID NOs: 7 to 9, or / and the LCDRs 1 to 3 are SEQ ID NOs: 10 to 12; (2) the HCDRs 1 to 3 are SEQ ID NOs: 13 to 15, or / and the LCDRs 1 to 3 are SEQ ID NOs: 16 to 18; (3) the HCDRs 1 to 3 are SEQ ID NOs: 23 to 25, or / and the LCDRs 1 to 3 are SEQ ID NOs: 26 to 28; (4) the HCDRs 1 to 3 are SEQ ID NOs: 29 to 31, or / and the LCDRs 1 to 3 are SEQ ID NOs: 32 to 34; (5) the HCDRs 1 to 3 are SEQ ID NOs: 40 to 42, or / and the LCDRs 1 to 3 are SEQ ID NOs: 43 to 45; (6) the HCDRs 1 to 3 are SEQ ID NOs: 48 to 50, or / and the LCDRs 1 to 3 are SEQ ID NOs: 51 to 53; (7) the HCDRs 1 to 3 are SEQ ID NOs: 58 to 60, or / and the LCDRs 1 to 3 are SEQ ID NOs: 61 to 63; (8) the HCDRs 1 to 3 are SEQ ID NOs: 64 to 66, or / and the LCDRs 1 to 3 are SEQ ID NOs: 67 to 69; (9) the HCDRs 1 to 3 are SEQ ID NOs: 74 to 76, or / and the LCDRs 1 to 3 are SEQ ID NOs: 77 to 79; (10) the HCDRs 1 to 3 are SEQ ID NOs: 86 to 88, or / and the LCDRs 1 to 3 are SEQ ID NOs: 89 to 91; (11) the HCDRs 1 to 3 are SEQ ID NOs: 92 to 94, or / and the LCDRs 1 to 3 are SEQ ID NOs: 95 to 97; (12) The HCDRs 1 to 3 are SEQ ID NOs: 98 to 100, or / and the LCDRs 1 to 3 are SEQ ID NOs: 101 to 103, or (13) The HCDRs 1 to 3 and / or the LCDRs 1 to 3 have an amino acid sequence that is at least 80% identical to, or has at most three insertion, deletion, or substitution mutations, as compared with, each CDR in the HCDRs 1 to 3 and / or LCDRs 1 to 3 described in any one of groups (1) to (12). Preferably, the HCDRs 1 to 3 and / or LCDRs 1 to 3 have an amino acid sequence that is at least 80% identical to, each CDR in the HCDRs 1 to 3 and / or LCDRs 1 to 3 described in any one of groups (1) to (12). Furthermore, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. Selected from.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, 19, 21, 37, 46, 54, 56, 71, 80, 82, or 84, or / and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 20, 22, 38, 47, 55, 57, 72, 81, 83, or 85; or the heavy chain variable region and / or the light chain variable region each comprise an amino acid sequence at least 80% identical to any one of the heavy chain variable region and / or light chain variable region, and further wherein the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region sequence and / or a light chain constant region sequence, wherein the heavy chain constant region and / or the light chain constant region are selected from complete constant region sequences or fragments thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3, or Fc; optionally, the heavy chain constant region is selected from a human or mouse IgG1, IgG2, IgG3, or IgG4 constant region; and the light chain constant region is selected from a human or mouse kappa constant region or a lamda constant region. Optionally, the antibody or antigen-binding fragment thereof comprises complete heavy and light chains, wherein the heavy chain consists of the VH and heavy chain constant region, and the heavy chain constant region has the amino acid sequence set forth in SEQ ID NO: 5, 39, or 73; and the light chain consists of the VL and light chain constant region, and the light chain constant region has the amino acid sequence set forth in SEQ ID NO: 6.

[0048] In some embodiments, the general formula is Pc-(LD) n or a pharmaceutically acceptable salt thereof, wherein n is selected from a real number of 1 to 16, for example, n is selected from a real number of 2 to 12, for example, n is selected from a real number of 4 to 10, for example, n is selected from a real number of 5 to 9, for example, n is selected from a real number of 6 to 8.

[0049] In some embodiments, n is selected from a real number between 5 and 9, for example, n is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

[0050] The present disclosure further provides a Drug-Linker Compound having the general structural formula L'-D, or a pharmaceutically acceptable salt thereof, wherein: Drug Unit D is as defined above; L' is [ka] and its b-terminus is covalently attached to a drug unit D; and L 1 , L 2 , m1, m2, m3 are as defined above.

[0051] In some embodiments, L' is [ka] and its b-terminus is covalently bonded to the drug unit D; m1 is selected from 5; and L 1 is selected from Gly-Gly-Phe-Gly tetrapeptide residues or Ala-Ala-Ala tripeptide residues.

[0052] In some embodiments, L' is [ka] wherein the b-terminus is covalently bonded to the drug unit D; m2 is selected from 2; m3 is selected from 8; and L 2 is selected from a Gly-Gly-Phe-Gly tetrapeptide residue or a Val-Lys dipeptide residue.

[0053] In some embodiments, L' has the following chemical structure: [ka] and its b-terminus is covalently attached to the drug unit D.

[0054] In some embodiments, the drug-linker compound of the present disclosure having the general formula L'-D, or a pharmaceutically acceptable salt thereof, is the following compound, or a pharmaceutically acceptable salt thereof: [ka] Selected from JPEG2025516381000029.jpg255156JPEG2025516381000030.jpg216154JPEG2025516381000031.jpg241148JPEG2025516381000032.jpg111169.

[0055] The present disclosure further provides a compound represented by the following formula (DH) or a pharmaceutically acceptable salt thereof: [ka] Among them, R 1 , X1, X, R 4 , R 5 , R 6 , R 7 is as defined above.

[0056] In some embodiments, the compound of formula (DH), or a pharmaceutically acceptable salt thereof, is the compound shown below, or a pharmaceutically acceptable salt thereof: [ka] Selected from JPEG2025516381000035.jpg225170JPEG2025516381000036.jpg254165.

[0057] In another aspect, the present disclosure provides a compound according to the present disclosure, wherein the general formula is Pc-(LD) n or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0058] In another aspect, the present disclosure provides a method of treating a tumor in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of a compound having the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0059] In another aspect, the present disclosure provides a compound having the general formula Pc-(LD) in the manufacture of a medicament for treating a tumor. n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0060] In another aspect, the present disclosure provides a compound of the general formula Pc-(LD) in the treatment of tumors. n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0061] In another aspect, the present disclosure provides a compound having the general formula Pc-(LD) for treating tumors. n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0062] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure represented by formula (DH) above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0063] In another aspect, the present disclosure provides a method for treating a tumor in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of a compound represented by formula (DH) above or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0064] In another aspect, the present disclosure provides the use of a compound represented by the above formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating a tumor.

[0065] In another aspect, the present disclosure provides the use of a compound represented by the above formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the treatment of tumors.

[0066] In another aspect, the present disclosure provides a compound represented by the above formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for treating a tumor.

[0067] In another aspect, the present disclosure provides a compound having the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, the method comprising coupling a drug-linker compound of the present disclosure having the general formula L'-D to the ligand, wherein optionally the ligand is an antibody or an antigen-binding fragment thereof.

[0068] In another aspect, the present disclosure provides a compound having the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, comprising the step of linking the Drug unit D of the present disclosure to the Ligand unit Pc, optionally via the Linker unit L, and optionally wherein the Ligand is an antibody or an antigen-binding fragment thereof.

[0069] In another aspect, the present disclosure provides a compound having the general formula Pc-(LD) n and / or the use of the compound represented by formula (DH) or a pharmaceutically acceptable salt thereof in the manufacture of a drug-linker compound having the general formula L'-D or a pharmaceutically acceptable salt thereof.

[0070] The ligand-drug conjugates provided by the present disclosure have significant anti-inflammatory activity and / or reduced toxicity and side effects. [Brief explanation of the drawings]

[0071] [Figure 1] 1 shows the results of X-ray single crystal diffraction analysis of compound 19-P1. [Figure 2] 1 shows the tumor growth curve of the OVCAR3 subcutaneous tumor model. [Figure 3] 1 shows the tumor growth curve of the OVCAR3 subcutaneous tumor model. [Figure 4]1 shows the tumor growth curve of the OVCAR3 subcutaneous tumor model. [Figure 5] 1 shows the body weight change curve of mice in an OVCAR3 subcutaneous tumor model. [Figure 6] 1 shows the tumor growth curve of the H838 subcutaneous tumor model. [Figure 7] 1 shows the weight change curve of H838 subcutaneous tumor model mice.

[0072] Definitions and explanations of terms Unless otherwise specified, the terms used in this disclosure have the following meanings, and the definitions of groups and terms described in this disclosure, including their illustrative definitions, exemplary definitions, preferred definitions, definitions described in the tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined or combined with each other. Unless specifically defined, a particular term should not be considered indefinite or unclear, but should be understood according to its general meaning in the art. When a trade name appears in this specification, it is intended to refer to the corresponding trade name or its active ingredient.

[0073] The term "ligand" refers to a polymeric compound capable of recognizing or binding to an antigen or receptor associated with a target cell. The function of a ligand is to deliver a drug to a target cell population that is bound by the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells. In embodiments of the present disclosure, a ligand or ligand unit is represented as Pc, and the ligand can form a linking bond with a linker unit via a heteroatom on the ligand. In some embodiments of the present disclosure, the ligand is selected from an antibody or antigen-binding fragment, and the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody. In some embodiments of the present disclosure, the antibody is a monoclonal antibody.

[0074] The term "linker" or "linker unit" refers to a chemical structure fragment or bond that is connected at one end to a ligand and at the other end to a drug.

[0075] The term "drug" refers to a small molecule compound that has biological activity in vivo. In some embodiments of the present disclosure, the drug is a glucocorticoid receptor agonist or its corresponding phosphate ester molecule that has anti-inflammatory function.

[0076] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In some embodiments of the present disclosure, the "ligand-drug conjugate" is an antibody drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active drug via a stable linker unit.

[0077] The term "DAR" or "drug-antibody ratio" refers to the average number of small molecule glucocorticoid receptor agonist drugs linked to each antibody molecule. In the antibody-drug conjugates of the present disclosure, the DAR is defined by the variable "n," which can be an integer or a decimal number.

[0078] The term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region and thereby is capable of specifically binding to an antigen. The term "antibody" as used herein encompasses a variety of forms and structures, so long as they exhibit the desired antigen-binding activity. The term "antibody" as used herein also encompasses alternative protein scaffolds or artificial scaffolds with grafted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which, for example, contain mutations introduced to stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds, for example, comprising biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may further comprise non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used for grafting CDRs, including, but not limited to, tenascin, fibronectin, peptide aptamers, etc.

[0079] As used herein, the term "antibody" includes typical "four-chain antibodies" belonging to the immunoglobulin family consisting of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of, from N- to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. If the full-length antibody is of the IgE isotype, it optionally further contains a heavy chain constant region CH4 domain. The light chain refers to a polypeptide chain consisting of, from N- to C-terminus, a light chain variable region (VL) and a light chain constant region (CL), with the heavy and light chains linked via disulfide bonds to form a "Y" structure. Immunoglobulins differ in the amino acid composition and sequence of the heavy chain constant regions, and therefore their antigenicity also differs. Thus, "immunoglobulin" as used herein can be divided into five classes or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig of the same class can be further divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into κ chains and λ chains based on differences in the constant region. Each of the five Ig classes may have either a κ chain or a λ chain.

[0080] As used herein, "antibody" also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by animals such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe), and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0081] An "antibody" herein may be derived from any animal, including, but not limited to, humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or chondrichthyes (e.g., sharks).

[0082] As used herein, "antibody" includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a single-specific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a complete antibody, a fragment of a complete antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0083] The term "monoclonal antibody" refers to an antibody obtained from an essentially homogeneous antibody population, i.e., except for possible variants (e.g., naturally occurring mutations or produced during the preparation process, such variants typically being present in minor amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. The use of the modifier "monoclonal" herein should not be construed as requiring production of the antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, as well as other methods known in the art.

[0084] The term "natural antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering. Exemplary "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFvs), bispecific antibodies, and the like.

[0085] The term "monospecific" denotes having one or more binding sites, each of which binds to the same epitope on the same antigen.

[0086] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope on the same antigen or a different epitope on a different antigen. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody / antigen-binding molecule can bind.

[0087] The term "valency" refers to the presence of a given number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0088] The terms "full length antibody," "intact antibody," and "complete antibody" may be used interchangeably herein and refer to an antibody having a structure that is essentially similar to that of a natural antibody.

[0089] The terms "antigen-binding fragment" and "antibody fragment" may be used interchangeably herein and do not have the entire structure of a complete antibody, but only include local or regional variants of the complete antibody, which have the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0090] Papain digestion of a complete antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing a heavy and light chain variable domain and further containing the light chain constant domain and the first heavy chain constant domain (CH1). Thus, the term "Fab fragment" herein refers to an antibody fragment containing the light chain VL domain and constant domain (CL) and the heavy chain VH domain and first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few additional residues at the carboxy terminus of the heavy chain CH1 domain, and by the inclusion of one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues in the constant domains bear free thiol groups. Pepsin treatment produces an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0091] An "Fv fragment" is the smallest fragment produced by IgG and IgM and contains a complete antigen-binding site; the Fv fragment has the same binding characteristics and similar three-dimensional binding properties as Fab, and the VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0092] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH domains are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, although variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may be present between the VH and VL of an scFv, forming a disulfide-linked Fv (dsFv).

[0093] The term "diabody" refers to a diabody whose VH and VL domains are expressed on a single polypeptide chain, but which uses a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and generating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0094] The term "chimeric antibody" refers to an antibody in which a portion of its light and / or heavy chains is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of its light and / or heavy chains is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but which retains binding activity for a target antigen (USP 4,816,567 to Cabilly et al., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). For example, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions are derived from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions are derived from a second antibody (e.g., a human antibody) (e.g., a human-mouse chimeric antibody).

[0095] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with human antibodies. Generally speaking, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., FRs and / or constant regions in the variable region) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, etc.

[0096] The term "fully human antibody" refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. A "fully human antibody" herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "fully human antibody" herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have already been grafted onto human framework sequences.

[0097] The term "variable region" refers to the region contained in the heavy or light chain of an antibody that enables the antibody to bind to an antigen; "heavy chain variable region" may be used interchangeably with "VH" and "HCVR," and "light chain variable region" may be used interchangeably with "VL" and "LCVR." The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th edition, W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity-determining region" and "CDR" herein may be used interchangeably and generally refer to the hypervariable region (HVR) of a heavy chain variable region (VH) or a light chain variable region (VL). These regions are also called complementarity-determining regions because they can form precise complementarity with an antigen epitope in their spatial conformation. The CDR of a heavy chain variable region may be abbreviated as HCDR, and the CDR of a light chain variable region may be abbreviated as LCDR. The terms "framework region" and "FR region" herein may be used interchangeably and refer to amino acid residues other than CDRs in the heavy chain variable region or light chain variable region of an antibody. A typical antibody variable region generally consists of four FR regions and three CDR regions in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0098] "CDRs" herein can be represented and defined in any manner known in the art, including, but not limited to, the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and tool websites that may be used include, but are not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlapping and subsets of amino acid residues defined in different ways.

[0099] The term "heavy chain constant region" as used herein refers to the carboxy-terminal portion of an antibody heavy chain, which is not directly involved in binding an antibody to an antigen but exhibits effector functions such as interaction with Fc receptors. Its amino acid sequence is more conserved than that of the antibody variable domain. The "heavy chain constant region" includes at least the CH1 domain, hinge region, CH2 domain, and CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes "full-length heavy chain constant regions" and "heavy chain constant region fragments," the former of which has a structure essentially similar to that of a native antibody constant region, and the latter of which contains only a "portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of the CH1 domain, hinge region, CH2 domain, and CH3 domain. If the antibody is an IgE antibody, it further contains a CH4 domain, whereas if the antibody is a heavy chain antibody, it does not contain the CH1 domain. For example, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domain.

[0100] The term "light chain constant region" as used herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding of the antibody to an antigen, and said light chain constant region may be selected from a constant kappa domain or a constant lambda domain.

[0101] The term "Fc," as used herein, refers to the carboxy-terminal portion of an antibody prepared by papain hydrolysis of a whole antibody; typically, it comprises the CH3 and CH2 domains of the antibody. Fc regions include, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus thereof. The C-terminal lysine (based on residue 447 in the Kabat numbering system) of the Fc region can be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain; thus, the Fc region may or may not include Lys447.

[0102] As used herein, the term "identity" may be calculated to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences by aligning the sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at this position.

[0103] Taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences, the percent identity between the two sequences will vary depending on the changes in the same positions shared by the sequences.

[0104] A mathematical algorithm can be used to compare the sequences and calculate the percent identity between two sequences. For example, the percent identity between two amino acid sequences can be determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm integrated into the GAP program in the GCG software package (available at www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. For example, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at www.gcg.com) using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and the one to be used unless otherwise specified) employs a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0105] In this specification, "n is a real number from 1 to 16" means that n is any real number from 1 to 16.

[0106] In this specification [ka] indicates the linkage site.

[0107] The illustrations of racemic or enantiomerically pure compounds herein are taken from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise noted, the wedge bond and dashed wedge bond are [ka] indicates the absolute configuration of one stereocenter, and the black solid and dashed bonds [ka] indicates the relative configuration of one stereocenter (e.g., the cis-trans configuration of an alicyclic compound).

[0108] The term "tautomer" refers to a functional isomer resulting from the rapid displacement of an atom in a molecule between two positions. Compounds of the present disclosure may exhibit the phenomenon of tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Tautomers generally exist in equilibrium, and isolating a single tautomer usually produces a mixture whose physicochemical properties match those of the compound mixture. The position of the equilibrium is determined by the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present disclosure includes all tautomeric forms of the compounds.

[0109] The term "stereoisomer" refers to isomers resulting from differences in the spatial arrangement of atoms in molecules, and includes cis-trans isomers, enantiomers and diastereomers.

[0110] The compounds of the present disclosure may contain asymmetric atoms, such as carbon, sulfur, nitrogen, phosphorus, or asymmetric double bonds, and therefore may exist in particular geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may include cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic or other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures. All of these isomers and mixtures thereof are within the scope of the definition of the compounds of the present disclosure. Substituents such as alkyl groups may contain other asymmetric carbon atoms, sulfur atoms, nitrogen atoms, or phosphorus atoms, and all of these isomers and mixtures thereof for all substituents are included within the scope of the definition of the compounds of the present disclosure. Compounds of the present disclosure containing an asymmetric atom may be isolated in optically pure or racemic form, or optically pure forms may be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0111] The term "substituted" refers to the replacement of any one or more hydrogen atoms at a particular atom by a substituent, as long as the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), this means that two hydrogen atoms are replaced; oxo does not occur in aromatic groups.

[0112] The term "optionally" or "optionally" refers to the subsequently described event or circumstance that may or may not occur, and the description includes both cases where the event or circumstance occurs and cases where it does not occur. For example, an ethyl group being "optionally" substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, any substitution or substitution pattern that cannot be spatially present and / or cannot be synthesized is not introduced.

[0113] Any variable (e.g., R a , R b When any group appears more than once in the composition or structure of a compound, its definition in each occurrence is independent. For example, if a group is represented by two R b When replaced by, each R b has independent options.

[0114] When the number of one linking group is 0, for example, -(CH2)0-, this indicates that the linking group is a bond.

[0115] When one of the variables is selected from a chemical bond or is absent, it indicates that the two groups being linked are directly linked; for example, when L in ALZ represents a bond, it indicates that the structure is actually AZ.

[0116] In the present specification, when the linking group does not specify the linking direction, the linking direction is arbitrary. For example, the structural unit [ka] When X is selected from "C1-C3 alkylene-O", X may connect Ring A and Ring B from left to right to form "Ring A-C1-C3 alkylene-O-Ring B", or may connect Ring A and Ring B from right to left to form "Ring A-O-C1-C3 alkylene-Ring B".

[0117] C in this specification m -C n indicates that the group has an integer number of carbon atoms ranging from m to n.

[0118] The term "alkyl group" refers to a group having the general formula C n H 2n+1 The term "C1-C6 alkyl group" refers to a hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, wherein the alkyl group may be straight or branched. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl. The term "C1-C3 alkyl group" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, or isopropyl.

[0119] The "C1-C6 alkyl group" described herein may further include a "C1-C3 alkyl group."

[0120] The term "alkynyl group" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond. For example, the term "C2-C6 alkynyl group" should be understood as a straight-chain or branched hydrocarbon group preferably containing one or more triple bonds and having 2, 3, 4, 5, or 6 carbon atoms. Examples of "C2-C6 alkynyl group" include, but are not limited to, ethynyl (-C≡CH), prop-1-ynyl (1-propynyl, -C≡CCH3), prop-2-ynyl (-CH2C≡CH), but-1-ynyl, but-2-ynyl, or but-3-ynyl. The "C2-C6 alkynyl group" may include a "C2-C3 alkynyl group", and examples of a "C2-C3 alkynyl group" include an ethynyl group (-C≡CH), a prop-1-ynyl group (1-propynyl group, -C≡CCH3), and a prop-2-ynyl group (-CH2C≡CH).

[0121] The term "cycloalkyl group" refers to a carbocyclic ring that is fully saturated and exists in the form of a monocyclic, fused, bridged, or spirocyclic ring, etc. The term "C3-C6 cycloalkyl group" should be understood to indicate a saturated monocyclic, fused, spiro, or bridged ring having 3 to 6 carbon atoms, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0122] The term "cycloalkenyl group" refers to a non-aromatic carbocyclic group that is incompletely saturated and exists as a monocyclic, fused, bridged, or spirocyclic ring. Unless otherwise specified, the carbocyclic ring typically has 5 to 8 members. The term "C5-C7 cycloalkenyl group" refers to a cycloalkenyl group having 5, 6, or 7 ring atoms. Specific examples include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. The term "C5-C7 cycloalkenyl group" may include ranges such as "C5-C6 cycloalkenyl group." The term "C5-C6 cycloalkenyl group" refers to a cycloalkenyl group having 5 or 6 ring atoms. Specific examples include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.

[0123] The term "heterocyclyl group" refers to a fully saturated or partially saturated monocyclic, fused, spiro, or bridged ring group containing 1 to 5 heteroatoms or heteroatomic groups (i.e., atomic groups containing heteroatoms) in the ring, where the "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)-, -S(=O)-, -P(=O)-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-. The term "4- to 7-membered heterocyclyl group" refers to a heterocyclyl group containing 4, 5, 6, or 7 ring atoms, and the ring atoms include 1 to 3 heteroatoms or heteroatomic groups independently selected from the above. Examples of 4-membered heterocyclyl groups include, but are not limited to, azetidinyl and oxetanyl groups; examples of 5-membered heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole, or 2,5-dihydro-1H-pyrrolyl groups; examples of 6-membered heterocyclyl groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridyl, or 4H-[1,3,4]thiadiazinyl groups; and examples of 7-membered heterocyclyl groups include, but are not limited to, diazepanyl groups. Preferably, the "4- to 7-membered heterocyclyl group" may include the scope of "4- to 7-membered heterocycloalkyl group", "5- to 6-membered heterocyclyl group", "5- to 6-membered heterocycloalkyl group", and the like.

[0124] The term "5- to 6-membered heteroaryl group" refers to an aromatic ring group having 5 or 6 ring atoms and containing 1 to 3, preferably 1 to 2 heteroatoms independently selected from N, O and S. In particular, the 5- to 6-membered heteroaryl group is selected from a thienyl group, a furanyl group, a pyrrolyl group, an oxazolyl group, a thiazolyl group, an imidazolyl group, a pyrazolyl group, an isoxazolyl group, an isothiazolyl group, an oxadiazolyl group, a triazolyl group, a thiadiazolyl group, a pyridyl group, a pyridazinyl group, a pyrimidinyl group, a pyrazinyl group, a triazinyl group, and the like.

[0125] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0126] The term "treatment" refers to surgical or therapeutic treatment aimed at preventing or slowing (reducing) the progression of undesirable physiological changes or pathologies in a subject, such as cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not getting worse), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include those already suffering from a disease or disorder, as well as those susceptible to a disease or disorder, or those in whom a disease or disorder is to be prevented. References to terms such as delay, alleviation, attenuation, mitigation, and remission are intended to encompass resolution, elimination, non-occurrence, and the like.

[0127] The term "effective amount" refers to the amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating the symptoms of a disease or the progression of that disease. "Effective amount" also refers to the amount of a compound that is sufficient to ameliorate a symptom, e.g., treat, cure, prevent, or ameliorate an associated medical condition, or treat, cure, prevent, or ameliorate an increase in the rate of such a condition. When an active ingredient is administered alone to an individual, the therapeutically effective dose refers to the amount of that ingredient alone. When a combination is applied, the therapeutically effective dose refers to the combined dose of the active ingredients that produces the therapeutic effect, regardless of combined, sequential, or simultaneous administration.

[0128] The term "subject" refers to an organism receiving treatment for a particular disease or condition described in this disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, receiving treatment for a disease or condition.

[0129] The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal being treated, but can be determined routinely by one of ordinary skill in the art according to his or her knowledge and the present disclosure.

[0130] The term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0131] The term "pharmaceutically acceptable salts" refers to salts of pharmaceutically acceptable acids or bases, including salts formed with a compound and an inorganic or organic acid, and salts formed with a compound and an inorganic or organic base.

[0132] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the present disclosure to an organism.

[0133] The term "pharmaceutically acceptable additive" refers to an additive that does not have an obvious irritating effect on an organism and does not impair the biological activity and performance of the active compound. Suitable additives are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0134] The words "comprise" or "comprise" and their English variants, such as comprises or comprising, may be understood in an open and non-exclusive sense, i.e. "including but not limited to."

[0135] The present disclosure further includes isotopically labeled compounds of the present disclosure that are the same as those described herein, except that one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Illustrative isotopes that can be attached to compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0136] Some isotopically labeled compounds of the present disclosure (e.g., 3 H and 14C) may be used in compound and / or substrate tissue distribution analysis. 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred for their ease of preparation and detectability. 15 O. 13 N, 11 C and 18 Positron-emitting isotopes, such as F, may be used in positron emission tomography (PET) studies to measure substrate occupancy. Isotopically labeled compounds of the present disclosure can generally be prepared by the following schemes and / or procedures similar to those disclosed in the Examples, substituting isotopically labeled reagents for non-isotopically labeled reagents.

[0137] The pharmaceutical compositions of the present disclosure can be applied to parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure can be in the form of a sterile aqueous solution for injection for intramuscular or subcutaneous administration. The pharmaceutical compositions of the present disclosure can accept other solvents or vehicles, such as water, Ringer's solution, or isotonic sodium chloride solution, when used.

[0138] In all administration methods of the compounds described herein, the daily dose is 0.001 mg / kg to 600 mg / kg body weight, preferably 0.05 mg / kg to 200 mg / kg body weight, and more preferably 0.1 mg / kg to 100 mg / kg body weight, in single or divided doses.

[0139] The compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitution forms well known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present disclosure.

[0140] The chemical reactions of specific embodiments of the present disclosure are completed in a suitable solvent, which must be suitable for the chemical transformations of the present disclosure and the necessary reagents and materials. To obtain compounds of the present disclosure, one skilled in the art may need to modify or select synthetic steps or reaction processes based on existing embodiments.

[0141] One of the important considerations in planning a synthetic route in the art is the selection of an appropriate protecting group for a reactive functional group (e.g., an amino group, a carboxy group in the present disclosure), see, for example, Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc., all references cited in this disclosure are incorporated herein in their entirety. DETAILED DESCRIPTION OF THE INVENTION

[0142] The present disclosure will be described in detail below with reference to examples, but this does not imply any adverse limitations on the present disclosure. Although the present specification has already described the present disclosure in detail and disclosed specific examples, it will be apparent to those skilled in the art that various modifications can be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure are commercially available and can be used without further purification.

[0143] Unless otherwise specified, the ratio of the mixed solvents is a volumetric mixture ratio.

[0144] Unless otherwise specified, % refers to wt%.

[0145] Compounds are named artificially or by ChemDraw® software, commercially available compounds adopt supplier catalog names.

[0146] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are in units of 10-6 The solvents used for NMR measurements were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard was tetramethylsilane (TMS). 50 " is the half-maximal inhibitory concentration, which refers to the concentration at which half of the maximum inhibitory effect is achieved, and "EC 50 " is the concentration that enables half-maximal effect, which refers to the concentration that produces 50% of the maximum effect.

[0147] Example 1, (S)—N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-11H-[1,4]dioxino[2,3-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 1) [ka] Step 1: Synthesis of 1-(7-amino-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-chloroethan-1-one (Intermediate 1-2) Reactant 1-1 (500 mg, 3.31 mmol) was dissolved in 1,2-dichloroethane (3 mL). The reaction mixture was cooled to 0 °C, and boron trichloride (1 M, 2.65 mL) and aluminum trichloride (573.36 mg, 4.30 mmol) were added. Chloroacetonitrile (299.67 mg, 3.97 mmol) was added to the reaction mixture at 0 °C under nitrogen protection, and the reaction mixture was stirred at 90 °C for 16 h under nitrogen protection. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, ice water (30 mL) and 1 N HCl (10 mL) were added sequentially, followed by stirring for 30 min. The reaction mixture was extracted three times with dichloromethane (30 mL × 3). The combined organic phase was washed with saturated brine (30 mL), and the washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. Concentration to dryness under reduced pressure and the crude product was subjected to preparative thin layer chromatography (silica, petroleum ether:ethyl acetate=9:1) to give the title compound (210 mg). MS m / z (ESI): 228.0 [M + H] + .

[0148] Step 2: Synthesis of (S)-15-(chloromethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxino[2,3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (Intermediate 1-4) Intermediate 1-2 (100 mg, 439.28 μmol) and Intermediate 1-3 (115.64 mg, 439.28 μmol) were dissolved in anhydrous toluene (3 mL), and pyridinium p-toluenesulfonate (22.08 mg, 87.86 μmol) was added. The reaction mixture was stirred at 100°C for 16 h under nitrogen protection. LC-MS confirmed the completion of the reaction. After cooling to room temperature, the reaction mixture was filtered, and the filter cake was washed with ethanol (5 mL x 2) to obtain the title compound (130 mg) as a crude product. MS m / z(ESI):455.1[M+H] + .

[0149] Step 3: Synthesis of (S)-15-(azidomethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxino[2,3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (Intermediate 1-5) Intermediate 1-4 (120 mg, 263.82 μmol) was dissolved in dimethyl sulfoxide (1 mL), sodium azide (25.73 mg, 395.73 μmol) was added, and the reaction mixture was stirred at 25°C for 3 h under nitrogen protection. LC-MS confirmed the completion of the reaction. Ice water (2 mL) was added, and the mixture was stirred for 0.5 h and filtered to obtain the title compound (90 mg) as a crude product. MS m / z(ESI):462.1[M+H] + .

[0150] Step 4: Synthesis of (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxino[2,3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (Intermediate 1-6) Intermediate 1-5 (90 mg, 195.05 μmol) was dissolved in anhydrous toluene (1 mL), triethyl phosphite (81.02 mg, 487.62 μmol) was added, and the reaction mixture was stirred at 100°C for 3 h under nitrogen protection. The reaction mixture was cooled to 25°C, and a solution of hydrochloric acid in methanol (0.5 mL) was added. The reaction mixture was stirred at 85°C for 16 h under nitrogen protection. LC-MS confirmed the completion of the reaction. The reaction mixture was cooled to room temperature and filtered to obtain the title compound (18 mg). MS m / z(ESI):436.1[M+H] + .

[0151] Step 5: Synthesis of (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-11H-[1,4]dioxino[2,3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 1) Intermediate 1-6 (18 mg, 41.34 μmol) and 2-hydroxyacetic acid (15.72 mg, 206.69 μmol) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (23.58 mg, 62.01 μmol) and N,N-diisopropylethylamine (DIEA) (16.03 mg, 124.02 μmol) were added. The reaction mixture was stirred at 25 °C for 3 h. LC-MS showed the reaction was complete. The reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, with a gradient of acetonitrile from 10% to 30%, and an elution time of 12 min) to give the title compound (7 mg). MS m / z(ESI):494.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.69(t,J=6.0 Hz,1H),7.92(s,1H),7.56(s,1H),7.26(s,1H),6.49(s,1H),5.57(t,J=5.7 Hz,1H),5.46(s,2H),5.43(s,2H),4.74(d,J=6.0 Hz,2H),4.44(s,4H),3.82(d,J=5.6 Hz,2H),1.94-1.80(m,2H),0.88(m,3H).

[0152] Example 3, (S)—N-((9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 3) [ka] Step 1: Synthesis of 1-(2-amino-5-chloro-4-fluorophenyl)-2-chloroethan-1-one (Intermediate 3-2) Boron trichloride (1M, 13.74 mL) was dissolved in 1,2-dichloroethane (24 mL). The reaction mixture was cooled to 0 °C, and reactant 3-1 (2 g, 13.74 mmol) and chloroacetonitrile (1.56 g, 20.61 mmol) were added. The reaction mixture was stirred at 0 °C for 10 min. Aluminum trichloride (2.38 g, 17.86 mmol) was then added. The reaction mixture was then heated to 25 °C under nitrogen protection and stirred for 10 min. The reaction mixture was stirred at 90 °C for 18 h under nitrogen protection. LC-MS confirmed the completion of the reaction. After cooling to room temperature, ice water (50 mL) and 5% HCl (10 mL) were added slowly, successively, and the mixture was stirred at 25 °C for 30 min. Dichloromethane (50 mL) was then added, and the organic phase was washed with water (2 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The crude product was concentrated to dryness under reduced pressure and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, with a gradient of acetonitrile from 40% to 60%, and an elution time of 10 min) to give the title compound (320 mg). MS m / z (ESI): 222.0 [M + H] + .

[0153] Step 2: Synthesis of (S)-9-chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Intermediate 3-3) Intermediate 3-2 (220 mg, 990.80 μmol) and intermediate 1-3 (273.86 mg, 1.04 mmol) were dissolved in toluene (2 mL), and pyridinium p-toluenesulfonate (24.90 mg, 99.08 μmol) was added. The reaction mixture was stirred at 100°C for 18 h. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, ethanol (1 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL × 2) to obtain the title compound (270 mg) as a crude product. MS m / z(ESI):449.0[M+H] + .

[0154] Step 3: Synthesis of (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (3-4) Intermediate 3-3 (50 mg, 111.29 μmol) was dissolved in ethanol (1 mL) and urotropine (23.40 mg, 166.94 μmol) was added. The reaction mixture was stirred at 90 °C for 1.5 h. Completion of the reaction was confirmed by LC-MS. The reaction mixture was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a decreasingly polar mixture of water (containing 0.225% formic acid) and methanol as the eluent, with a gradient of 0% to 30% methanol, and a run time of 12 min) to obtain the title compound (22 mg). MS m / z(ESI):430.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.71(d,J=8.0 Hz,1H),8.23(d,J=10.3 Hz,1H),8.14(s,0.3H, HCOOH),7.36(s,1H),6.57(s,1H),5.52(s,2H),5.46(s,2H),4.55(s,2H),1.93-1.84(m,2H),0.90-0.85(m,3H).

[0155] Step 4: Synthesis of (S)-N-((9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 3) 3-4 (22 mg, 51.18 μmol) and 2-hydroxyacetic acid (19.46 mg, 255.92 μmol) were dissolved in anhydrous N,N-dimethylformamide (1 mL). HATU (29.19 mg, 76.77 μmol) and N,N-diisopropylethylamine (19.84 mg, 153.55 μmol) were added, and the reaction mixture was stirred at 25 °C for 1.5 h. LC-MS confirmed the reaction was complete. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a decreasingly polar mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient from 10% to 40%, and an elution time of 12 min) to obtain the title compound (2.20 mg). MS m / z (ESI): 488.1 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.90-8.85(m,2H),8.20(d,J=10.3 Hz,1H),7.35(s,1H),6.55(s,1H),5.60(t,J=5.7 Hz,1H),5.56(s,2H),5.45(s,2H),4.83(d,J=6.0 Hz,2H),3.83(d,J=5.8 Hz,2H),1.93-1.81(m,2H),0.88(m,3H).

[0156] Example 5, (S)—N-((9-bromo-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 5) [ka] Step 1: Synthesis of 1-(2-amino-5-bromo-4-fluorophenyl)-2-chloroethan-1-one (Intermediate 5-2) Boron trichloride (1M, 10.53 mL) was dissolved in 1,2-dichloroethane (24 mL). The reaction mixture was cooled to 0 °C, and intermediate 5-1 (2 g, 10.53 mmol) and chloroacetonitrile (1.19 g, 15.79 mmol) were added. The reaction mixture was stirred at 0 °C for 10 min, and then aluminum trichloride (1.82 g, 13.68 mmol) was added. The reaction mixture was stirred at 25 °C for 10 min under nitrogen protection. The temperature was then raised to 90 °C and stirred for 18 h. LC-MS confirmed the completion of the reaction. After cooling to room temperature, ice water (50 mL) and 5% HCl (10 mL) were added slowly, successively, and the mixture was stirred at 25 °C for 30 min. Dichloromethane (50 mL) was then added, and the organic phase was washed with water (2 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. Purification by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a mixture of decreasing polarity of water (containing 0.05% formic acid) and acetonitrile as eluent, gradient ratio of acetonitrile from 39% to 49%, elution time 12 min) gave the title compound (380 mg). MS m / z(ESI):265.9[M+H] + .

[0157] Step 2: Synthesis of (S)-9-bromo-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Intermediate 5-3) Intermediate 5-2 (200 mg, 750.48 μmol) and intermediate 1-3 (207.44 mg, 788.01 μmol) were dissolved in anhydrous toluene (4 mL), and pyridinium p-toluenesulfonate (22.63 mg, 90.06 μmol) was added. The reaction mixture was stirred at 100°C for 18 h. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, ethanol (1 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL × 2) to obtain the title compound (200 mg) as a crude product. MS m / z (ESI): 493.0 [M + H] + .

[0158] Step 3: Synthesis of (S)-11-(aminomethyl)-9-bromo-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Intermediate 5-4) Intermediate 5-3 (200 mg, 405.10 μmol) was dissolved in ethanol (4 mL) and urotropine (113.58 mg, 810.19 μmol) was added. The reaction mixture was stirred at 90 °C for 1.5 h. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, the reaction mixture was concentrated to dryness under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 6% to 26%, and an elution time of 12 min) to obtain the title compound (30 mg). MS m / z (ESI): 476.0 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.83(d,J=7.4 Hz,1H),8.18(d,J=9.8 Hz,1H),8.14(s,0.4H, HCOOH),7.36(s,1H),6.57(s,1H),5.52(s,2H),5.46(s,2H),4.53(s,2H),1.92-1.85(m,2H),0.88(t,J=7.3 Hz,3H).

[0159] Step 4: Synthesis of (S)-N-((9-bromo-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 5) Intermediate 5-4 (15 mg, 26.88 μmol) and 2-hydroxyacetic acid (10.22 mg, 134.41 μmol) were dissolved in anhydrous N,N-dimethylformamide (1 mL). HATU (15.33 mg, 40.32 μmol) and N,N-diisopropylethylamine (10.42 mg, 80.65 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. LC-MS confirmed the reaction was complete. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length, using a decreasingly polar mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient from 6% to 36%, and an elution time of 12 min) to obtain the title compound (2.09 mg). MS m / z (ESI): 532.0 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=9.00(d,J=7.5 Hz,1H),8.86(t,J=5.9 Hz,1H),8.15(d,J=9.8 Hz,1H),7.35(s,1H),6.54(s,1H),5.60(t,J=5.7 Hz,1H),5.56(s,2H),5.45(s,2H),4.83(d,J=5.8 Hz,2H),3.83(d,J=5.9 Hz,2H),1.94-1.82(m,2H),0.88(t,J=7.4 Hz,3H).

[0160] Example 6, (S)—N-((8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 6) [ka] Step 1: Synthesis of 1-(2-amino-4-chloro-5-methylphenyl)-2-chloroethan-1-one (Intermediate 6-2) Boron trichloride (1 M, 2.82 mL) was dissolved in 1,2-dichloroethane (8 mL). The reaction mixture was cooled to 0 °C, and reactant 6-1 (0.5 g, 3.53 mmol) and chloroacetonitrile (319.91 g, 4.24 mmol) were added. The reaction mixture was stirred at 0 °C for 10 min, and then aluminum trichloride (612.09 mg, 4.59 mmol) was added. The reaction mixture was stirred at 25 °C for 10 min under nitrogen protection. The reaction mixture was then heated to 90 °C and stirred for 18 h. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, ice water (25 mL) and 5% HCl (5 mL) were added slowly, successively, and the mixture was stirred at 25 °C for 30 min. Dichloromethane (20 mL) was then added, and the organic phase was washed with water (20 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. Preparative thin layer chromatography (silica, petroleum ether:ethyl acetate=9:1) gave the title compound (500 mg). MS m / z(ESI):218.0[M+H] +

[0161] Step 2: Synthesis of (S)-8-chloro-11-(chloromethyl)-4-ethyl-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Intermediate 6-3) Intermediate 6-2 (250 mg, 1.15 mmol) and intermediate 1-3 (316.87 mg, 1.20 mmol) were dissolved in toluene (5 mL), and pyridinium p-toluenesulfonate (34.57 mg, 137.56 μmol) was added. The reaction mixture was stirred at 100°C for 18 h. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, ethanol (1 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL × 2) to obtain the title compound (230 mg) as a crude product. MS m / z(ESI):445.1[M+H] + .

[0162] Step 3: Synthesis of (S)-11-(aminomethyl)-8-chloro-4-ethyl-4-hydroxy-9-methyl-1,12-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Intermediate 6-4) Intermediate 6-3 (49.56 mg, 111.29 μmol) was dissolved in ethanol (0.5 mL) and urotropine (23.40 mg, 166.94 μmol) was added. The reaction mixture was stirred at 90 °C for 1.5 h. Completion of the reaction was confirmed by LC-MS. The reaction mixture was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient from 2% to 32%, and an elution time of 12 min) to obtain the title compound (11.0 mg). MS m / z(ESI):426.2[M+H] + .

[0163] Step 4: Synthesis of (S)-N-((8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 6) Intermediate 6-4 (11 mg, 25.83 μmol) and 2-hydroxyacetic acid (9.82 mg, 129.15 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). HATU (14.73 mg, 38.74 μmol) and N,N-diisopropylethylamine (10.01 mg, 77.49 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. LC-MS confirmed the reaction was complete. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length, using a decreasingly polar mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient from 10% to 40%, and an elution time of 12 min) to obtain the title compound (3.00 mg). MS m / z(ESI):484.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.77(t,J=6.1 Hz,1H),8.52(s,1H),8.26(s,1H),7.32(s,1H),6.54(s,1H),5.59(t,J=5.8 Hz,1H),5.52(s,2H),5.44(s,2H),4.85(d,J=6.0 Hz,2H),3.84(d,J=5.6 Hz,2H),2.60(s,3H),1.91-1.82(m,2H),0.88(t,J=7.3 Hz,3H)

[0164] Example 7, (S)—N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopenta[f]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 7) [ka] Step 1: Synthesis of 4,6-dibromo-2,3-dihydro-1H-inden-5-amine (Intermediate 7-2) Intermediate 7-1 (10.0 g) was dissolved in anhydrous acetonitrile, and N-bromosuccinimide (NBS) (27.5 g) was added in portions at 0°C. The mixture was then stirred overnight at room temperature. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 200 mL of ethyl acetate and washed with water (100 mL x 2). The resulting organic phase was dried over anhydrous sodium sulfate, mixed with the sample on silica gel, placed on diatomaceous earth, and rinsed with 500 mL of petroleum ether. The filtrate was concentrated to give the title compound (17.0 g). MS m / z(ESI):289.9[M+H] + .

[0165] Step 2: Synthesis of 4-bromo-2,3-dihydro-1H-inden-5-amine (Intermediate 7-3) Intermediate 7-2 (15.0 g) and stannous chloride (15.0 g) were dissolved in 75 mL of acetic acid, and 140 mL of 6N concentrated hydrochloric acid was added. The mixture was reacted at 90°C for 3 hours. The reaction mixture was cooled to room temperature and concentrated to remove acetic acid. The residue was dissolved in 100 mL of ethyl acetate, the pH was adjusted to approximately 8 with saturated aqueous sodium carbonate, filtered, and the organic phase was separated from the filtrate. The aqueous phase was further extracted with ethyl acetate (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the title compound (10 g). MS m / z (ESI): 212.0 [M + H] + .

[0166] Step 3: Synthesis of N-(4-bromo-2,3-dihydro-1H-inden-5-yl)acetamide (Intermediate 7-4) Intermediate 7-3 (10.0 g) was dissolved in 100 mL of anhydrous dichloromethane, triethylamine (10.6 g) was added, and acetyl chloride (5.5 g) was slowly added dropwise at 0 °C. The reaction mixture was then stirred overnight at room temperature. The reaction mixture was washed with water (100 mL × 2), and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 90:10) to give the title compound (7.1 g). MS m / z (ESI): 254.0 [M + H] +.

[0167] Step 4: Synthesis of N-(4-acetyl-2,3-dihydro-1H-inden-5-yl)acetamide (Intermediate 7-5) Under nitrogen protection, intermediate 7-4 (6.7 g) and tributyl-(2-ethoxyvinyl)tin (10.4 g) were dissolved in 100 mL of anhydrous 1,4-dioxane. Bis(triphenylphosphine)palladium dichloride (1.8 g) was then added, and the reaction mixture was stirred at 100 °C overnight. The reaction mixture was cooled to room temperature, and 30 mL of 3N hydrochloric acid was added. The mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through diatomaceous earth, and the resulting filtrate was diluted with 100 mL of ethyl acetate and washed with water (100 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 85:15) to give the title compound (4.7 g). MS m / z(ESI):218.1[M+H] + .

[0168] Step 5: Synthesis of N-(4-(2-bromoacetyl)-2,3-dihydro-1H-inden-5-yl)acetamide (Intermediate 7-6) Intermediate 7-5 (4.7 g) was dissolved in 50 mL of acetic acid, and 7.3 g of a 33% hydrobromic acid-acetic acid solution was added. Bromine (2.85 g) was slowly added dropwise at room temperature, and the reaction was continued with stirring at room temperature for 3 h. After completion of the reaction, the reaction solution was poured into ice water and stirred until a large amount of solid precipitated. The filter cake was washed with petroleum ether, and the obtained solid was dried to give the title compound (5.0 g). MS m / z (ESI): 296.0 [M + H] + .

[0169] Step 6: Synthesis of 1-(5-amino-2,3-dihydro-1H-inden-4-yl)-2-chloroethan-1-one (Intermediate 7-7) Intermediate 7-6 (5.0 g) was dissolved in 30 mL of ethanol, and 35 mL of 6N concentrated hydrochloric acid was added. The reaction mixture was stirred at 80 °C for 2 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to remove the solvent. The residue was dissolved in 100 mL of dichloromethane and the pH was adjusted to approximately 7 with saturated aqueous sodium bicarbonate. The organic phase was separated, and the aqueous phase was further extracted with dichloromethane (50 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 75:25) to yield the title compound (840 mg). MS m / z(ESI):210.0[M+H] + .

[0170] Step 7: Synthesis of (S)-15-(chloromethyl)-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione (Intermediate 7-8) Intermediate 7-7 (100 mg) and Intermediate 1-3 (125.55 mg) were dissolved in toluene (5 mL), and pyridinium p-toluenesulfonate (5.99 mg) was added. The reaction mixture was stirred at 90°C for 18 hours. After cooling to room temperature, ethanol (1 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered, and the filter cake was washed with petroleum ether (2 mL x 2) to obtain the title compound (180 mg). MS m / z(ESI):437.0[M+H] + .

[0171] Step 8: Synthesis of (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione (Intermediate 7-9) Intermediate 7-8 (50 mg) was dissolved in a mixture of methanol (1 mL) and N,N-dimethylformamide (1 mL), and urotropine (483.13 mg) was added. The reaction mixture was stirred at 50 °C for 4 h. After completion of the reaction, the reaction mixture was cooled to room temperature, concentrated hydrochloric acid (0.5 mL) was added, and the mixture was stirred for 0.5 h. The mixture was then concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity as the eluent, with an acetonitrile gradient of 20% to 40%, and an elution time of 12 min) to obtain the title compound (22.0 mg). MS m / z(ESI):418.2[M+H] + .

[0172] Step 9: Synthesis of (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 7) Intermediate 7-9 (15 mg) and hydroxyacetic acid (10.93 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (27.32 mg) and diisopropylethylamine (4.64 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, with an acetonitrile gradient of 30% to 50%, and an elution time of 12 min) to obtain the title compound (9.0 mg). MS m / z (ESI): 476.2 [M + H] + . 1H NMR(400MHz,DMSO-d6)δ=8.28(t,J=5.1 Hz,1H),8.02(d,J=8.5 Hz,1H),7.78(d,J=8.3 Hz,1H),7.30(s,1H),6.53(s,1H),5.49-5.45(m,1H),5.43(s,2H),5.36(s,2H),4.97(d,J=5.0 Hz,2H),3.88(d,J=5.5 Hz,2H),3.57(t,J=7.2 Hz,2H),3.09(t,J=7.4 Hz,2H),2.23-2.15(m,2H),1.95-1.80(m,2H),0.88(t,J=7.3 Hz,3H).

[0173] Example 9, (S)—N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3”,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (Compound 9) [ka] Intermediate 9-1 (6.00 mg, 14.66 μmol, can be synthesized according to the method reported in patent document WO2020219287) and intermediate 9-2 (4.49 mg, 43.97 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), HATU (8.36 mg, 21.98 μmol) and N,N-diisopropylethylamine (5.68 mg, 43.97 μmol) were added thereto, and the reaction mixture was stirred at 25 ° C. for 1 h. LC-MS showed that the reaction was complete. The reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, with an acetonitrile gradient of 6% to 36%, and an elution time of 12 min) to obtain the title compound (3.00 mg). MS m / z(ESI):494.2[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=8.96(t,J=6.0 Hz,1H),8.50(d,J=8.3 Hz,1H),7.90(d,J=10.9 Hz,1H),7.32(s,1H),6.53(s,1H),6.30(s,1H),5.52(s,2H),5.44(s,2H),4.85(d,J=5.9 Hz,2H),2.53(s,3H),1.92-1.83(m,2H),1.05-1.00(m,2H),0.88(t,J=7.3 Hz,3H),0.85-0.81(m,2H).

[0174] Example 10, (S)—N-((8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (Compound 10) [ka] Intermediate 6-4 (6.24 mg, 14.66 μmol) and intermediate 9-2 (4.49 mg, 43.97 μmol) were dissolved in N,N-dimethylformamide (1 mL), and HATU (8.36 mg, 21.98 μmol) and DIEA (5.68 mg, 43.97 μmol) were added. The reaction mixture was stirred at 25 °C for 1 h. LC-MS confirmed the completion of the reaction. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient from 16% to 46%, and an elution time of 12 min) to obtain the title compound (2.20 mg). MS m / z(ESI):510.1[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=8.96(t,J=5.9 Hz,1H),8.54(s,1H),8.26(s,1H),7.32(s,1H),6.54(s,1H),6.29(s,1H),5.52(s,2H),5.44(s,2H),4.84(d,J=6.0 Hz,2H),2.59(s,3H),1.94-1.80(m,2H),1.01(d,J=3.0 Hz,2H),0.88(t,J=7.3 Hz,3H),0.83(d,J=3.0 Hz,2H).

[0175] Example 11, N-(((S)-8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 11) [ka] Intermediate 6-4 (6.0 mg, 14.09 μmol) and intermediate 11-1 (8.18 mg, 70.45 μmol) were dissolved in N,N-dimethylformamide (0.5 mL), and HATU (8.04 mg, 21.13 μmol) and DIEA (5.46 mg, 42.27 μmol) were added. The reaction mixture was stirred at 25 °C for 1 h. LC-MS confirmed the completion of the reaction. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length, using a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient from 16% to 46%, and an elution time of 12 min) to obtain the title compound (3.0 mg). MS m / z(ESI):524.1[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=8.66(t,J=5.9 Hz,1H),8.47(s,1H),8.26(s,1H),7.32(s,1H),6.54(s,1H),5.53(d,J=5.0 Hz,1H),5.50(s,2H),5.44(s,2H),4.91-4.76(m,2H),3.61-3.55(m,1H),2.59(s,3H),1.94-1.82(m,2H),1.05-0.97(m,1H),0.88(t,J=7.3 Hz,3H),0.34-0.31(m,2H),0.29-0.20(m,2H).

[0176] Example 12, (S)-2-amino-N-((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)acetamide (Compound 12) [ka] Step 1: Synthesis of 1-(2-fluoro-3,4-dimethoxyphenyl)ethan-1-one (Intermediate 12-2) Intermediate 12-1 (25.0 g) was dissolved in 1,2-dichloroethane (DCE, 250 mL). The reaction mixture was cooled to 0 °C, and aluminum trichloride (64.04 g) was slowly added. Acetyl chloride (64.04 g) was then added dropwise to the reaction mixture. The reaction mixture was stirred at 0 °C for 2 h under a nitrogen atmosphere. After completion of the reaction, water (300 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (150 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ISCO®, 120 g SepaFlash® flash silica gel column, 0-50% petroleum ether / ethyl acetate gradient, flow rate 70 mL / min) to give the title compound (21.0 g). MS m / z (ESI): 199.0 [M + H] + .

[0177] Step 2: Synthesis of 1-(2-fluoro-3,4-dihydroxyphenyl)ethan-1-one (Intermediate 12-3) Intermediate 12-2 (15.00 g) was dissolved in anhydrous dichloromethane (DCM, 150 mL). The reaction mixture was cooled to -78 °C, and boron tribromide (56.88 g) was slowly added dropwise. The reaction mixture was stirred at -78 °C under a nitrogen atmosphere for 2 h, then warmed to 0 °C and reacted for 4 h. After completion of the reaction, the reaction mixture was quenched by slowly adding the mixture dropwise to ice water. After quenching, the mixture was extracted with ethyl acetate (150 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ISCO®, 120 g SepaFlash® flash silica gel column, 0-50% petroleum ether / ethyl acetate gradient, flow rate 70 mL / min) to give the title compound (8.50 g). MS m / z (ESI): 171.0 [M + H] + .

[0178] Step 3: Synthesis of 1-(4-fluorobenzo[d][1,3]dioxol-5-yl)ethan-1-one (Intermediate 12-4) Intermediate 12-3 (4.0 g) was dissolved in anhydrous N,N-dimethylformamide (40 mL), and cesium carbonate (11.49 g) and 1,2-diiodomethane (18.89 g) were added. The reaction mixture was stirred at 100 °C for 8 min under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was slowly poured into water and extracted with ethyl acetate (50 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ISCO®, 24 g SepaFlash® flash silica gel column, 0-15% petroleum ether / ethyl acetate gradient, flow rate 60 mL / min) to give the title compound (2.0 g). MS m / z (ESI): 183.0 [M + H] + .

[0179] Step 4: Synthesis of 1-(4-fluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)ethan-1-one (Intermediate 12-5) Intermediate 12-4 (2.0 g) was dissolved in anhydrous dichloromethane (15 mL), concentrated sulfuric acid (5.38 g, mass fraction 98%) was added, and the reaction mixture was cooled to 0 °C. Concentrated nitric acid (3.46 g, mass fraction 68%) was then slowly added dropwise to the reaction mixture. The reaction mixture was stirred at 25 °C for 2 h. After completion of the reaction, the reaction mixture was slowly added dropwise to ice water (50 mL), and ethyl acetate (50 mL) was added. The organic phase was washed with water (50 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (ISCO®, 24 g SepaFlash® flash silica gel column, gradient 0–40% petroleum ether / ethyl acetate, flow rate 60 mL / min) to obtain the title compound (1.8 g). 1 H NMR (400 MHz, deuterated chloroform) δ = 7.51 (s, 1H), 6.26 (s, 2H), 2.63 (s, 3H).

[0180] Step 5: Synthesis of 1-(6-amino-4-fluorobenzo[d][1,3]dioxol-5-yl)ethan-1-one (Intermediate 12-6) Intermediate 12-5 (1.8 g) was dissolved in anhydrous methanol (18 mL) and water (9 mL), and ammonium chloride (635.83 mg) and iron powder (2.21 mg) were added. The reaction mixture was stirred at 80°C for 2 h under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was cooled to room temperature. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate (50 mL). The organic phase was washed with water (50 mL x 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to give the title compound (1.5 g). MS m / z (ESI): 198.0 [M + H] + .

[0181] Step 6: Synthesis of N-(6-acetyl-7-fluorobenzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 12-7) Intermediate 12-6 (500.0 mg) was dissolved in anhydrous dichloromethane (5 mL), pyridine (601.79 mg) was added, and acetyl chloride (398.14 mg) was added dropwise to the reaction mixture under a nitrogen atmosphere. The reaction mixture was stirred at 25°C for 1.5 h under a nitrogen atmosphere. After completion of the reaction, the organic phase was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (ISCO®, 12 g SepaFlash® flash silica gel column, gradient 0-40% petroleum ether / ethyl acetate, flow rate 60 mL / min) to obtain the title compound (380.0 g). 1 H NMR (400MHz, deuterated chloroform) δ = 11.72 (s, 1H), 8.18 (d, J = 1.0 Hz, 1H), 6.10 (s, 2H), 2.64 (d, J = 8.4 Hz, 3H), 2.22 (s, 3H).

[0182] Step 7: Synthesis of N-(6-(2-bromoacetyl)-7-fluorobenzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 12-8) Intermediate 12-7 (380.0 mg) was dissolved in acetic acid (3 mL), and a solution of hydrogen bromide in acetic acid (1.95 g, 33% content) was added thereto. Liquid bromine (256.42 mg) was then slowly added dropwise to the reaction mixture. The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was slowly poured into ice water, stirred for 0.5 hours, filtered, and the filter cake was washed with water (20 mL x 2) and dried to obtain the title compound (400.0 mg). MS m / z(ESI):317.8[M+H] + .

[0183] Step 8: Synthesis of 1-(6-amino-4-fluorobenzo[d][1,3]dioxol-5-yl)-2-chloroethan-1-one (Intermediate 12-9) Intermediate 12-8 (400.0 mg) was dissolved in absolute ethanol (2 mL) and concentrated hydrochloric acid (2 mL), and the reaction mixture was stirred at 60 °C for 3 h. After the reaction was complete, the reaction mixture was cooled to room temperature, and then ice water (20 mL) was slowly added. The pH was adjusted to 8 with saturated sodium bicarbonate, and ethyl acetate (40 mL) was added. The organic phase was washed with water (20 mL × 2), and the washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to give the title compound (220.0 mg). MS m / z (ESI): 232.0 [M + H] + .

[0184] Step 9: Synthesis of (S)-14-(chloromethyl)-7-ethyl-15-fluoro-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 12-10) Intermediate 12-9 (200.0 mg) and intermediate 1-3 (227.32 mg) were dissolved in toluene (3 mL), and pyridinium p-toluenesulfonate (21.70 mg) was added. The reaction mixture was stirred at 90°C for 16 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ethanol (1 mL) was added. The reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered, and the filter cake was washed with ethanol (5 mL x 2) to obtain the title compound (320.0 mg). MS m / z (ESI): 459.0 [M + H] + .

[0185] Step 10: Synthesis of (S)-14-(aminomethyl)-7-ethyl-15-fluoro-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 12-11) Intermediate 12-10 (260.00 mg) was dissolved in anhydrous methanol (1 mL) and anhydrous N,N-dimethylformamide (1 mL), and urotropine (238.32 mg) was added. The reaction mixture was stirred at 50 °C for 3 h. After the reaction was completed, the reaction mixture was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 0% to 30%, elution time 14 min)) to obtain the title compound (85.0 mg). MS m / z(ESI):440.0[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=7.49(s,1H),7.26(s,1H),6.53(s,1H),6.38(s,2H),5.44(s,4H),4.27(s,2H),1.94-1.79(m,2H),0.88(t,J=7.3 Hz,3H).

[0186] Step 11: Synthesis of (S)-2-(((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethyl)carbamate tert-butyl ester (Intermediate 12-12) Intermediate 12-11 (7 mg) and 2-((tert-butoxycarbonyl)amino)acetic acid (5.58 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.11 mg) and diisopropylethylamine (2.06 mg) were added thereto, and the reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was concentrated to dryness to give the title compound (8.00 mg). MS m / z (ESI): 597.3 [M + H] + .

[0187] Step 12: Synthesis of (S)-2-amino-N-((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)acetamide (Compound 12) Intermediate 12-12 (6 mg) was dissolved in dichloromethane (0.5 mL), trifluoroacetic acid (902.27 mg) was added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 2% to 32%, elution time 12 min)) to obtain the title compound (1.3 mg). MS m / z(ESI):497.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.80-8.58(m,1H),7.51(s,1H),7.26(s,1H),6.52(s,1H),6.40(s,2H) ),5.50(s,2H),5.43(s,2H),4.85(s,2H),3.09-2.75(m,2H),1.92-1.79(m,2H),0.87(t,J=7.1 Hz,3H).

[0188] Example 13, 2-cyclopropyl-N-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 13) [ka] Intermediate 7-9 (10 mg) and intermediate 11-1 (8.34 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), HATU (13.66 mg) and diisopropylethylamine (3.10 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, with an acetonitrile gradient of 34% to 54%, and an elution time of 12 min) to obtain the title compound (0.8 mg). MS m / z(ESI):516.2[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.25(t,J=5.0 Hz,1H),8.01(d,J=8.5 Hz,1H),7.78(d,J=8.5 Hz,1H),7.30(s,1H),6.53(s,1H),5.43(s,2H),5.36(s,2H),4.99-4.85(m,2H),3.59(s,1H),3.56(d,J=6.3 Hz,2H),3.08(t,J=7.7 Hz,2H),2.23-2.16(m,2H),1.92-1.72(m,2H),1.15-1.01(m,1H),0.88(t,J=7.4 Hz,3H),0.46-0.19(m,4H)

[0189] Example 14-1, 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14) [ka] Step 1: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethanone (Intermediate 14-2) Intermediate 14-1 (10.0 g, 60.92 mmol) was dissolved in nitromethane (100 mL), and nitric acid (35.43 g, 365.50 mmol, 65% purity) was slowly added. The reaction mixture was stirred at 25 °C for 2.5 h. After the reaction was completed, saturated sodium bicarbonate solution was slowly added to the reaction mixture to adjust the pH to 7-8. Dichloromethane (100 mL) was then added, and the organic phase was washed with water (50 mL × 2). The washed organic phase was dried over anhydrous sodium sulfate. The product was purified by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 1:2) to obtain the title compound (5 g). MS m / z(ESI):210.0[M+H] + .

[0190] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)ethanone (Intermediate 14-3) Intermediate 14-2 (2.37 g, 11.33 mmol) was dissolved in absolute ethanol (25 mL), palladium on carbon (0.2 g, 10% purity) was added, and the reaction mixture was stirred under hydrogen protection at 25° C. for 16 h. After completion of the reaction, the reaction mixture was filtered, the filter cake was washed twice with ethyl acetate, and the filtrate was concentrated to dryness under reduced pressure to give the title compound (1.6 g). MS m / z(ESI):180.1[M+H] + .

[0191] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 14-4) Intermediate 14-3 (1.0 g, 5.58 mmol) was dissolved in dichloromethane (10 mL), and the reaction mixture was cooled to 0° C. N,N-diisopropylethylamine (DIEA) (1.08 g, 8.37 mmol) and acetyl chloride (569.55 mg, 7.26 mmol) were added thereto. The reaction mixture was stirred at 25° C. for 1.5 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure to give the title compound (1.23 g). MS m / z(ESI):222.1[M+H] + .

[0192] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 14-5) Intermediate 14-4 (1.23 g, 5.00 mmol) was dissolved in acetic acid (12 mL), and a solution of hydrogen bromide in acetic acid (1.84 g, 7.51 mmol, purity 33%) was added thereto. Liquid bromine (959.69 mg, 6.01 mmol) was then slowly added thereto, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was poured into ice water and stirred for 10 min. After filtration, the filter cake was washed twice with water and concentrated to dryness under reduced pressure. Ethyl acetate (2 mL) and petroleum ether (10 mL) were added to the residue, and the reaction mixture was stirred at 25 °C for 0.5 h. The reaction mixture was filtered, and the filter cake was dried to give the title compound (500 mg). MS m / z (ESI): 300.0 [M + H] + .

[0193] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)-2-chloroethanone (Intermediate 14-6) Intermediate 14-5 (0.2 g, 666.43 μmol) was dissolved in absolute ethanol (1 mL) and concentrated hydrochloric acid (1 mL), and the reaction mixture was stirred at 60 °C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ice water (10 mL) and saturated sodium bicarbonate (10 mL) were added slowly, successively. Dichloromethane (50 mL) was further added, and the organic phase was washed with water (20 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. Preparative thin-layer chromatography (petroleum ether:ethyl acetate = 6:1) afforded the title compound (160 mg). MS m / z (ESI): 214.0 [M + H] + .

[0194] Step 6: Synthesis of (S)-14-(bromomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 14-7) Intermediate 14-6 (50 mg, 234.06 μmol) and intermediate 1-3 (61.62 mg, 234.06 μmol) were dissolved in toluene (1 mL), and pyridinium p-toluenesulfonate (5.88 mg, 23.41 μmol) was added. The reaction mixture was stirred at 90°C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ethanol (1 mL) was added. The reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL × 2) and then dried to obtain the title compound (60 mg). MS m / z(ESI):441.1[M+H] + .

[0195] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 14-8) Intermediate 14-7 (55.00 mg, 124.76 μmol) was dissolved in ethanol (1 mL) and urotropine (52.47 mg, 374.29 μmol) was added. The reaction mixture was stirred at 80 °C for 1.5 h. After the reaction was completed, the reaction mixture was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.225% formic acid) and methanol with decreasing polarity as the eluent, with a methanol gradient of 0% to 27%, and an elution time of 12 min) to obtain the title compound (10 mg). MS m / z(ESI):422.1[M+H] + .

[0196] Step 8: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14) Intermediate 14-8 (10.00 mg, 20.17 μmol) and Intermediate 11-1 (23.42 mg, 201.71 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (11.50 mg, 30.26 μmol) and N,N-diisopropylethylamine (7.82 mg, 60.51 μmol) were added thereto, and the reaction mixture was stirred at 25°C for 1.5 h. After the reaction was completed, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column 5 μm, diameter 30 mm, length 150 mm, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity as the eluent, the gradient ratio of acetonitrile was 4% to 44%, and the elution time was 9 minutes) to obtain the title compound (3 mg). MS m / z(ESI):520.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.37(t,J=5.8 Hz,1H),7.62(s,1H),7.28(s,1H),7.00(s,1H),6.25(s,1H),6.05(s,2H),5.27(d,J=5.0 Hz,1H),5.23(s,2H),5.18(s,2H),4.48(d,J=5.5 Hz,2H),1.66-1.55(m,2H),0.76-0.40(m,1H),0.63(t,J=7.3 Hz,3H),0.14-0.06(m,2H),0.05-0.04(m,2H). [ka]

[0197] Step 9: Preparation of 2-cyclopropyl-2-hydroxybenzyl acetate (Intermediate 14-9-P1 / P2) Intermediate 14-9 was resolved into isomers 14-9-P1 and 14-9-P2. Intermediate 14-9 (1.3 g) was purified by supercritical fluid chromatography (DAICEL CHIRALPAK AD column, 10 μm silica, 30 mm diameter, 250 mm length, ethanol (containing 0.1% aqueous ammonia) as the eluent) to give intermediate 14-9-P1 (600 mg) and intermediate 14-9-P2 (600 mg). The above two isomers were further analyzed by the following chiral supercritical fluid chromatography conditions.

[0198] [Table 1] Intermediate 14-9-P1: Under the chiral supercritical fluid chromatography conditions described above, its retention time is 2.990 minutes. 1 H NMR (400MHz, METHANOL-d4) δ7.43-7.29(m,5H),5.29-5.16(m,2H),3.67(d,J=7.6 Hz,1H),1.19-1.07(m,1H),0.58-0.38(m,4H). Intermediate 14-9-P2: Under the chiral supercritical fluid chromatography conditions described above, its retention time is 2.661 minutes. 1 H NMR (400MHz,METHANOL-d4)δ7.46-7.28(m,5H),5.30-5.16(m,2H),3.67(d,J=7.6 Hz,1H),1.21-1.03(m,1H),0.60-0.36(m,4H).

[0199] Step 10: Synthesis of 2-cyclopropyl-2-hydroxyacetic acid (intermediate 14-10-P1 / P2) Under a hydrogen atmosphere, intermediate 14-9-P1 (500 mg) was added to methanol (15 mL), and wet palladium carbon (10 mg, 10%) was added to the reaction solution, followed by stirring under a hydrogen atmosphere at 25° C. for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P1 (273 mg). 1 H NMR (400MHz, METHANOL-d4) δ3.63 (d, J=7.2 Hz, 1H), 1.21-1.09 (m, 1H), 0.61-0.40 (m, 4H). Under a hydrogen atmosphere, intermediate 14-9-P2 (500 mg) was added to methanol (15 mL), and wet palladium carbon (10 mg, 10%) was added to the reaction solution, followed by stirring under a hydrogen atmosphere at 25° C. for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give intermediate 14-10-P2 (279 mg). 1 H NMR (400MHz, METHANOL-d4) δ3.63 (d, J=7.2 Hz, 1H), 1.19-1.08 (m, 1H), 0.60-0.39 (m, 4H).

[0200] Step 11: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14-P1 / P2) Intermediate 14-8 (40.00 mg) and intermediate 14-10-P1 (28.11 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (46.02 mg) and N,N-diisopropylethylamine (31.28 mg) were added, and the reaction mixture was stirred at 25 °C for 1.5 h. After completion of the reaction, the reaction mixture was purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent (acetonitrile gradient ratio 16% to 46%, elution time 12 min)) to obtain compound 14-P1 (22.00 mg). MS m / z(ESI):520.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.62(t,J=5.7 Hz,1H),7.86(s,1H),7.52(s,1H),7.25(s,1H),6.51(s,1H),6.29(s,2H),5.47(s,2H),5.43(s,2H),4.73(d,J=5.9 Hz,2H),3.54(d,J=5.9 Hz,1H),1.93-1.78(m,2H),1.06-0.96(m,1H),0.87(t,J=7.3 Hz,3H),0.39-0.30(m,2H),0.29-0.21(m,2H).

[0201] Intermediate 14-8 (10.00 mg) and intermediate 14-10-P2 (8.27 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18.05 mg) and N,N-diisopropylethylamine (6.13 mg) were added, and the reaction mixture was stirred at 25 °C for 1.5 h. After completion of the reaction, the reaction mixture was directly purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent (acetonitrile gradient ratio 16% to 46%, elution time 12 min)) to obtain compound 14-P2 (8.00 mg). MS m / z(ESI):520.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.63(t,J=5.9 Hz,1H),7.86(s,1H),7.52(s,1H),7.24(s,1H),6.30(s,2H),5.46(s,2H),5.43(s,2H),4.72(d,J=6.0 Hz,2H),3.55(d,J=6.0 Hz,1H),1.92-1.81(m,2H),1.03-0.97(m,1H),0.88(t,J=7.3 Hz,3H),0.38-0.30(m,2H),0.28-0.22(m,2H).

[0202] The two isomers were each further analyzed by the following chiral supercritical fluid chromatography analytical method.

[0203] [Table 2-1] [Table 2-2] Compound 14-P1: Under the chiral supercritical fluid chromatography conditions described above, its retention time is 3.673 minutes. Compound 14-P2: Under the chiral supercritical fluid chromatography conditions described above, its retention time was 3.735 minutes.

[0204] Example 14-2: (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (compound 14-S) [ka] Step 1: Synthesis of (S)-4-benzyl-3-(2-cyclopropylacetyl)oxazolidin-2-one (Intermediate 3) Starting material 1 (150.0 g), 4-dimethylaminopyridine (160.15 g), and starting material 2 (221.2 g) were weighed and dissolved in 1500 mL of dichloromethane and stirred at room temperature for 15 min. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 359.0 g) was weighed and added to the reaction solution in several portions, followed by stirring at room temperature for approximately 5 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (1500 mL) and then washed sequentially with water (500 mL) twice, 2N HCl (500 mL) once, saturated sodium bicarbonate solution (500 mL) once, and saturated brine solution (500 mL) once. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to give the title compound (302 g). 1 H NMR(400 MHz, CDCl3)δ7.32-7.35(m,2H),7.26-7.29(m,1H),7.21-7.23(m,2H),4.68-4.71(m,1H),4.17-4.23(m,2H),3.3 0-3.33(dd,1H),2.91-2.95(dd,1H),2.78-2.82(m,2H),1.14-1.18(m,1H),0.59-0.63(m,2H),0.21-0.26(m,2H).

[0205] Step 2: Synthesis of (S)-4-benzyl-3-((S)-2-cyclopropyl-2-hydroxyacetyl)oxazolidin-2-one (Intermediate 5) Intermediate 3 (200 g) was weighed and dissolved in 2000 mL of anhydrous tetrahydrofuran. The mixture was stirred at -78°C for 15 min under nitrogen protection. Next, sodium bis(trimethylsilyl)amide (443.5 mL, 2 M tetrahydrofuran solution) was added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was stirred at -78°C for 30 min. Intermediate 4 (201.5 g) was dissolved in 700 mL of tetrahydrofuran to a clear solution and slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was stirred at -78°C for 2 h. 220 mL of glacial acetic acid was then added to the reaction mixture to quench the reaction. After the addition was complete, the mixture was gradually warmed to room temperature, 600 mL of 2N HCl was added to the reaction mixture, and the mixture was stirred at room temperature (20-25°C) for 10 h. The reaction mixture was then concentrated under reduced pressure, and ethyl acetate (1000 mL) and water (200 mL) were added to the residue and stirred for 20 min. The separated aqueous phase was extracted twice with ethyl acetate (500 mL × 2). The combined organic phases were washed twice with 400 mL of saturated NaHCO3 solution, 400 mL of saturated Na2S2O3 solution, and saturated brine. The resulting organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1 / 30) to give the title compound (128.7 g). 1 H NMR(400 MHz,CDCl3)δ7.37(dd,J=8.1,6.8 Hz,2H),7.33-7.29(m,1H),7.27-7.23(m,2H),4.81(dd,J=7.9,5.9 Hz,1H),4.72(ddt,J=10.0,7.5,2.9 Hz,1H),4.33(t,J=8.3Hz,1H),4.28(dd,J=9.1,2.5 Hz,1H),3.48(dd,J=8.2,3.9 Hz,1H),3.35(dd,J=13.5,3.4 Hz,1H),2.88(dd,J=13.5,9.4 Hz,1H),1.36-1.29(m,1H),0.62-0.44(m,4H).

[0206] Step 3: Synthesis of (S)-4-benzyl-3-((S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetyl)oxazolidin-2-one (Intermediate 6) Intermediate 5 (128.7 g) was weighed and dissolved in 1300 mL of dichloromethane. Imidazole (56.17 g) was added and the mixture was stirred in an ice bath for 15 min. Then, TBSCl (107.3 g) was added in several portions to the reaction mixture, and the mixture was stirred at room temperature for 3 h. 200 mL of 2N HCl was added to the reaction mixture, and the mixture was stirred for 20 min and then separated. The organic phase was washed twice each with 200 mL of saturated NaHCO3 solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=80:1) to obtain the title compound (160 g). 1 H NMR(400 MHz, CDCl3)δ7.35-7.37(m,2H),7.30-7.32(m,1H),7.26-7.29(m,2H),5.26-5.31(m,1H),4.67-4.71(m,1H),4.20-4.26(m,2H) ),3.41-3.44(dd,1H),2.72-2.76(dd,1H),1.25-1.31(m,1H),0.94(s,9H),0.54-0.56(m,2H),0.46-0.48(m,2H),0.12(s,6H).

[0207] Step 4: Synthesis of (S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylbenzyl acetate (Intermediate 7) Benzyl alcohol (62.18 g) was weighed out and dissolved in 500 mL of tetrahydrofuran, and the mixture was stirred at -25°C. n-Butyllithium (213.6 mL, 2.5 M tetrahydrofuran solution) was weighed out and slowly added dropwise to the reaction solution. After the addition was complete, the mixture was stirred at -25°C for 1 hour. Intermediate 6 (160 g) was weighed out and dissolved in 320 mL of tetrahydrofuran, and the mixture was slowly added dropwise to the reaction solution at -25°C. After the addition was complete, the mixture was stirred at -15°C for 3 hours. The reaction solution was quenched by adding saturated NH4Cl (200 mL) solution to the reaction solution. The mixture was then concentrated under reduced pressure, and 400 mL of methyl tert-butyl ether and 150 mL of water were added to the reaction mixture. The mixture was stirred for 30 minutes and then separated into layers. The aqueous phase was extracted twice with methyl tert-butyl ether (200 mL × 2). The combined organic phases were washed once with saturated brine (250 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=100:1) to obtain the title compound (125 g). 1 H NMR (400 MHz, CDCl3) δ7.28-7.40(m,5H),5.17-5.26(m,2H),3.86-3.89(m,1H),1.21-1.46(m,1H),0.90(s,9H),0.45-0.51(m,4H),0.05(s,6H).

[0208] Step 5: Synthesis of (S)-2-cyclopropyl-2-hydroxybenzyl acetate (Intermediate 8) Intermediate 7 (125 g) was weighed and dissolved in 1200 mL of tetrahydrofuran. Glacial acetic acid (35.1 g) was added and the mixture was stirred at room temperature for 5 min. Tetrabutylammonium fluoride (TBAF, 585 mL, 1 M tetrahydrofuran solution) was then added to the reaction mixture, and the mixture was allowed to react at 45 °C for 4 h. The reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran (600 mL). 300 mL of water and 400 mL of methyl tert-butyl ether were added to the residue, stirred for 20 min, and the mixture was separated. The aqueous phase was extracted twice with methyl tert-butyl ether (200 mL). The combined organic phase was washed twice with 200 mL of saturated NaHCO3 solution and then with saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 60:1) to give the title compound (68.9 g). 1 H NMR (400 MHz, CDCl3) δ7.27-7.39(m,5H),5.22-5.28(m,2H),3.82(d,1H),2.7(brs,1H),1.11-1.15(m,1H),0.41-0.56(m,4H). Intermediate 8 was further analyzed by the following chiral supercritical fluid chromatography conditions.

[0209] [Table 3] Under the chiral supercritical fluid chromatography conditions, the retention time of intermediate 8 was 3.013 minutes, which was essentially the same as the retention time (2.990 minutes) of intermediate 14-9-P1 in Example 14-1 under the same chromatographic analysis conditions. Intermediate 8 and intermediate 14-9-P1 had the same configuration, and were the same compound.

[0210] Step 6: Synthesis of (S)-2-cyclopropyl-2-hydroxyacetic acid (intermediate 9) Intermediate 8 (5 g) was dissolved in methanol (80 mL), and wet palladium-carbon (10% by mass, 0.7 g) was added to the reaction mixture, followed by stirring under a hydrogen atmosphere at 25° C. for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to give the title compound (2.4 g). 1 H NMR (400MHz, METHANOL-d4) δ = 3.63 (d, J = 7.3 Hz, 1H), 1.20-1.09 (m, 1H), 0.61-0.39 (m, 4H). Step 7: Synthesis of (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14-S) Intermediate 14-8 (90 mg) and Intermediate 9 (49.60 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphine salt (121.81 mg) and N,N-diisopropylethylamine (82.81 mg) were added. The reaction mixture was stirred at 25 °C for 16 h. After completion of the reaction, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Green ODS 150 × 30 mm × 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile], B%: 20% to 50%, 12 min) to obtain the title compound (21 mg). MS m / z(ESI):520.0[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=8.62(t,J=6.1 Hz,1H),7.87(s,1H),7.52(s,1H),7.24(s,1H),6.48(s,1H),6.30(s,2H),5.48(s,2H),5.43(s,2H),4.73(d,J=5.6 Hz,2H),3.54(d,J=5.5 Hz,1H),1.93-1.80(m,2H),1.05-0.97(m,1H),0.88(t,J=7.3 Hz,3H),0.39-0.30(m,2H),0.30-0.22(m,2H). Compound 14-S was further analyzed by the following chiral supercritical fluid chromatography conditions.

[0211] [Table 4] The retention time of compound 14-S under the chiral supercritical fluid chromatography conditions was 3.654 minutes, which was essentially identical to the retention time (3.673 minutes) of compound 14-P1 prepared in Example 14-1 under the same chromatographic analysis conditions. Therefore, it was determined that compound 14-S and compound 14-P1 prepared in Example 14-1 have the same configuration and are the same compound.

[0212] Example 15, (S)—N-((9-bromo-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b][1,7]naphthyridin-11-yl)methyl)-2-hydroxyacetamide (Compound 15) [ka] Step 1: Synthesis of 1-(5-amino-2-bromopyridin-4-yl)ethanone (Intermediate 15-2) Intermediate 15-1 (500 mg, 3.67 mmol) was dissolved in anhydrous tetrahydrofuran (90 mL), and sodium bicarbonate (617.04 mg, 7.34 mmol) and 2-pyrrolidone trihydrobromide (1.64 g, 5.03 mmol) were added thereto, and the reaction mixture was stirred at 25° C. for 8 hours. After completion of the reaction, the reaction mixture was filtered and purified by preparative thin-layer chromatography (silica, petroleum ether:ethyl acetate=20:1) to obtain the title compound (300 mg). MS m / z(ESI):214.9[M+H] + .

[0213] Step 2: Synthesis of N-(4-acetyl-6-bromopyridin-3-yl)acetamide (Intermediate 15-3) Intermediate 15-2 (150 mg, 697.52 mmol) was dissolved in dichloromethane (2 mL), the reaction mixture was cooled to 0 °C, and N,N-diisopropylethylamine (180.30 mg, 1.40 mmol) and acetyl chloride (109.51 mg, 1.40 mmol) were added thereto. The reaction mixture was stirred at 25 °C for 3 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure and purified by preparative thin-layer chromatography (silica, petroleum ether: ethyl acetate = 3:1) to obtain the title compound (100 mg). MS m / z (ESI): 257.0 [M + H] + .

[0214] Step 3: Synthesis of 1-(5-amino-2-bromopyridin-4-yl)-2-bromoethanone (Intermediate 15-4) Intermediate 15-3 (95.00 mg, 273.45 μmol) was dissolved in acetic acid (2 mL), and a solution of hydrogen bromide in acetic acid (100.57 g, 410.18 μmol, purity 33%) was added thereto. Liquid bromine (48.07 mg, 300.80 μmol) was then slowly added thereto, and the reaction mixture was stirred at 25° C. for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the title compound (45 mg) was obtained by preparative thin-layer chromatography (silica, petroleum ether:ethyl acetate=3:1). MS m / z(ESI):292.9[M+H] + .

[0215] Step 4: Synthesis of 1-(5-amino-2-bromopyridin-4-yl)-2-chloroethanone (Intermediate 15-5) Intermediate 15-4 (50.00 mg, 170.10 μmol) was dissolved in concentrated hydrochloric acid (1 mL), and the reaction mixture was stirred at 60°C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ice water (10 mL) and saturated sodium bicarbonate (10 mL) were added slowly, successively. Dichloromethane (30 mL) was then added, and the organic phase was washed with water (20 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to give the title compound (25 mg). MS m / z(ESI):248.9[M+H] + .

[0216] Step 5: Synthesis of (S)-9-bromo-11-(chloromethyl)-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b][1,7]naphthyridine-3,14(4H)-dione (Intermediate 15-6) Intermediate 15-5 (25 mg, 100.20 μmol) and intermediate 1-3 (26.38 mg, 100.20 μmol) were dissolved in toluene (0.5 mL), and pyridinium p-toluenesulfonate (2.52 mg, 10.02 μmol) was added. The reaction mixture was stirred at 90°C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ethanol (1 mL) was added. The reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL × 2) to obtain the title compound (30 mg). MS m / z(ESI):475.9[M+H] + .

[0217] Step 6: Synthesis of (S)-11-(aminomethyl)-9-bromo-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b][1,7]naphthyridine-3,14(4H)-dione (Intermediate 15-7) Intermediate 15-6 (30 mg, 62.93 μmol) was dissolved in ethanol (1 mL) and urotropine (17.64 mg, 125.86 μmol) was added. The reaction mixture was stirred at 80 °C for 2 h. After the reaction was completed, the reaction mixture was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.225% formic acid) and methanol with decreasing polarity as the eluent, with a gradient of 0% to 25% methanol, and an elution time of 12 min) to obtain the title compound (4 mg). MS m / z (ESI): 457.0 [M + H] + .

[0218] Step 7: Synthesis of (S)-N-((9-bromo-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b][1,7]naphthyridin-11-yl)methyl)-2-hydroxyacetamide (Compound 15) Intermediate 15-7 (4 mg, 8.75 μmol) and hydroxyacetic acid (3.33 mg, 43.74 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). HATU (4.99 mg, 13.12 μmol) and N,N-diisopropylethylamine (3.39 mg, 26.24 μmol) were added, and the reaction mixture was stirred at 25 °C for 2 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 8% to 28%, and an elution time of 12 min) to obtain the title compound (1.00 mg). MS m / z(ESI):515.0[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=9.38(s,1H),8.88(t,J=6.2 Hz,1H),8.76(s,1H),7.39(s,1H),6.57(s,1H),5.64-5.61(m,1H),5.60(s,2H),5.45(s,2H),4.80(d,J=6.0 Hz,2H),3.83(d,J=5.7 Hz,2H),1.92-1.80(m,2H),0.88(t,J=7.3 Hz,3H).

[0219] Example 16, (S)—N-((9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (Compound 16) [ka] Step 1: Synthesis of 1-(6-amino-3-chloro-2,4-difluorophenyl)-2-chloroethan-1-one (Intermediate 16-2) Boron trichloride (1M, 6.11 mL) was dissolved in 1,2-dichloroethane (12 mL). The reaction mixture was cooled to 0 °C, and reactant 16-1 (1 g, 6.11 mmol) and chloroacetonitrile (784.73 mg, 10.39 mmol) were added. The reaction mixture was stirred at 0 °C for 10 min, and aluminum trichloride (1.06 g, 7.95 mmol) was added. The reaction mixture was then heated to 25 °C under nitrogen protection and stirred for 10 min. The reaction mixture was stirred at 90 °C for 18 h under nitrogen protection. LC-MS confirmed the completion of the reaction. After cooling to room temperature, ice water (25 mL) and 5% hydrochloric acid (5 mL) were added slowly, successively, and the mixture was stirred at 25 °C for 30 min. Dichloromethane (50 mL) was then added, and the organic phase was washed with water (2 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. After concentration to dryness under reduced pressure, the crude product was subjected to preparative thin layer chromatography (silica, petroleum ether:ethyl acetate=9:1) to give the title compound (340 mg). MS m / z (ESI): 240.0 [M + H]+ .

[0220] Step 2: Synthesis of (S)-9-chloro-11-(chloromethyl)-4-ethyl-8,10-difluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Intermediate 16-3) Intermediate 16-2 (0.2 g, 833.22 μmol) and intermediate 1-3 (219.34 mg, 833.22 μmol) were dissolved in toluene (4 mL), and pyridinium p-toluenesulfonate (20.94 mg, 83.32 μmol) was added. The reaction mixture was stirred at 100° C. for 18 h. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, ethanol (1 mL) was added, and the reaction mixture was stirred at 25° C. for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL × 2) to obtain the title compound (190 mg) as a crude product. MS m / z(ESI):467.1[M+H] + .

[0221] Step 3: Synthesis of (S)-11-(aminomethyl)-9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (16-4) Intermediate 16-3 (50 mg, 107.01 μmol) was dissolved in ethanol (1 mL) and urotropine (45.00 mg, 321.03 μmol) was added. The reaction mixture was stirred at 80 °C for 1.5 h. Completion of the reaction was confirmed by LC-MS. After cooling to room temperature, the reaction mixture was concentrated to dryness under reduced pressure and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 2% to 32%, and an elution time of 12 min) to obtain the title compound (1.22 mg). MS m / z(ESI):448.0[M+H]+ . 1 H NMR(400MHz,DMSO-d6)δ=8.14(d,J=9.8 Hz,1H),7.36(s,1H),6.57(s,1H),5.55(s,2H),5.46(s,2H),4.35(d,J=3.0 Hz,2H),1.94-1.83(m,2H),0.88(t,J=7.3 Hz,3H).

[0222] Step 4: Synthesis of (S)-N-((9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (Compound 16) Compound 16-4 (5.75 mg, 12.84 μmol) and intermediate 9-2 (3.93 mg, 38.52 μmol) were dissolved in N,N-dimethylformamide (0.5 mL). HATU (7.32 mg, 19.26 μmol) and diisopropylethylamine (4.98 mg, 38.52 μmol) were added, and the reaction mixture was stirred at 30 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 14% to 34%, and an elution time of 12 min) to obtain the title compound (3 mg). MS m / z(ESI):532.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.46(t,J=5.9 Hz,1H),8.16(d,J=9.8 Hz,1H),7.36(s,1H),6.56(s,1H),6.33(s,1H),5.53(s,2H),5.45(s,2H),4.93(d,J=3.6 Hz,2H),1.92-1.82(m,2H),1.04-0.99(m,2H),0.90-0.87(m,2H),0.87-0.82(m,3H).

[0223] Example 17, (S)—N-((8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-2-methylpropanamide (Compound 17) [ka] Intermediate 6-4 (5 mg, 11.74 μmol) and intermediate 17-1 (2.44 mg, 23.48 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (8.95 mg, 23.48 μmol) and N,N-diisopropylethylamine (1.19 mg, 11.74 μmol) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm silica, 25 mm diameter, 100 mm length, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 22% to 42%, and an elution time of 12 min) to obtain the title compound (1 mg). MS m / z(ESI):512.1[M+H] + . 1 H NMR(400MHz,Methanol-d4)δ=8.32(s,1H),8.22(s,1H),7.67(s,1H),5.62(d,J=16.4 Hz,1H),5.52(s,2H),5.42(d,J=16.4 Hz,1H),5.01(s,2H),2.65(s,3H),2.05-1.94(m,2H),1.38(s,6H),1.03(t,J=7.5 Hz,3H).

[0224] Example 18, N-(((S)-8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-3-methylbutanamide (Compound 18) [ka] Intermediate 6-4 (5 mg, 11.74 μmol) and intermediate 18-1 (2.77 mg, 23.48 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (8.95 mg, 23.48 μmol) and N,N-diisopropylethylamine (1.19 mg, 11.74 μmol) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 25% to 45%, and an elution time of 12 min) to obtain the title compound (1.20 mg). MS m / z(ESI):526.1[M+H] + . 1 H NMR(400MHz,Methanol-d4)δ=8.30(s,1H),8.13(s,1H),7.61(s,1H),5.60(d,J=16.3 Hz,1H),5.56-5.45(m,2H),5.42-5.37(m,1H),5.00-4.90(m,2H),3.91(d,J=3.3 Hz,1H),3.13(d,J=6.5 Hz, 1H), 2.62 (s, 3H), 2.01-1.94 (m, 2H), 1.05-1.00 (m, 6H), 0.77-0.70 (m, 3H).

[0225] Example 19-1, 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19, Compound 19-P1 / P2) [ka] Step 1: Synthesis of 1-(benzo[d][1,3]dioxol-5-yl-2,2-d2)ethan-1-one (Intermediate 19-2) Intermediate 19-1 (3 g) was dissolved in anhydrous DMF (25 mL), deuterated dichloromethane (8.57 g) and potassium carbonate (8.18 g) were added, and the mixture was heated to 90 °C and stirred for 16 h. The reaction mixture was then added to water (100 mL) and extracted with ethyl acetate (200 mL × 2). The combined organic phase was washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (2.4 g). MS m / z (ESI): 167.1 [M + H] + .

[0226] Step 2: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl-2,2-d2)ethan-1-one (Intermediate 19-3) Intermediate 19-2 (2.4 g) was dissolved in acetic anhydride (10 mL), and concentrated nitric acid (32.50 g, 70% content) was added dropwise at 0 °C. After the addition was complete, the mixture was stirred at 0 °C for 10 min. The mixture was then warmed to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was added dropwise to ice water (200 mL), filtered, and the filter cake was dried to obtain the title compound (1.9 g). MS m / z (ESI): 212.0 [M + H] + . 1 H NMR(400 MHz,DMSO-d6)δ7.69(s,1H),7.30(s,1H),2.49(s,3H).

[0227] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (Intermediate 19-4) Intermediate 19-3 (1.8 g) was dissolved in acetic acid (25 mL), and acetic anhydride (1.84 g) and reduced iron powder (4.76 g) were added. The mixture was stirred at room temperature for 1 h. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (1.5 g). MS m / z(ESI):224.1[M+H] + .

[0228] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (Intermediate 19-5) A solution of HBr in acetic acid (2.39 g, 33% content) was added dropwise to a solution of intermediate 19-4 (1.45 g) in acetic anhydride (25 mL), followed by the dropwise addition of Br2 (1.07 g). After the addition was complete, the mixture was stirred at room temperature for 1 h. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. The residue was added to water (50 mL) and extracted with ethyl acetate (50 mL × 2). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (1.3 g). MS m / z(ESI):302.1[M+H] + .

[0229] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl-2,2-d2)-2-chloroethan-1-one (Intermediate 19-6) Intermediate 19-5 (1.2 g) and concentrated hydrochloric acid (144.82 mg) were dissolved in ethanol (15 mL), and the reaction mixture was stirred at 60 °C for 16 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a mixture of water (containing 0.05% NH4HCO3) and acetonitrile with decreasing polarity as the eluent (a gradient ratio of acetonitrile was 40% to 50%) to obtain the title compound (577 mg). MS m / z (ESI): 216.0 [M + H] + .

[0230] Step 6: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (Intermediate 19-7) Intermediate 19-6 (100.0 mg) and intermediate 1-3 (109.87 mg) were dissolved in toluene (1 mL) and acetic acid (1 mL), and pyridinium p-toluenesulfonate (5.24 mg) was added. The reaction mixture was stirred at 100°C for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature and then directly concentrated to dryness under reduced pressure. Ethanol (5 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (5 mL × 2) to obtain the title compound (100.0 mg). MS m / z (ESI): 443.0 [M + H] + .

[0231] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (Intermediate 19-8) Intermediate 19-7 (100.00 mg) was dissolved in absolute ethanol (1.5 mL) and anhydrous N,N-dimethylformamide (1.5 mL), and urotropine (94.97 mg) was added. The reaction mixture was stirred at 50°C for 6 hours. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high-performance liquid chromatography (column: Boston Green ODS 150 x 30 mm x 5 μm, mobile phase: [A: water (formic acid), B: acetonitrile], B%: 0% to 30%, 12 min) to obtain the title compound (25.0 mg). MS m / z(ESI):424.0[M+H] + .

[0232] Step 8: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19) Intermediate 19-8 (7 mg) and intermediate 11-1 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 20% to 50%, elution time 12 min)) to obtain the title compound (2.60 mg). MS m / z (ESI): 522.1 [M + H] + . 1H NMR(400MHz,DMSO-d6)δ=8.62(t,J=5.9 Hz,1H),7.84(s,1H),7.51(s,1H),7.24(s,1H),6.49(s,1H),5.48-5.41(m,5H),4.72(d,J=5.5 Hz,2H),3.59-3.52(m,1H),2.00-1.76(m,2H),1.05-0.96(m,1H),0.88(t,J=7.4 Hz,3H),0.37-0.30(m,2H),0.29-0.19(m,2H).

[0233] Step 9: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19-P1 / P2) [ka] Intermediate 19-8 (7 mg) and intermediate 14-10-P1 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 15% to 45%, and an elution time of 12 min) to obtain compound 19-P1 (3.30 mg). MS m / z (ESI): 522.1 [M + H] + . 1H NMR(400MHz,DMSO-d6)δ=8.62(t,J=6.0 Hz,1H),7.86(s,1H),7.52(s,1H),7.25(s,1H),6.51(s,1H),5.54-5.51(m,1H),5.47(s,2H),5.43(s,2H),4.72(d,J=6.0 Hz,2H),3.55-3.53(m,1H),1.94-1.78(m,2H),1.05-0.96(m,1H),0.88(t,J=7.3 Hz,3H),0.40-0.30(m,2H),0.29-0.19(m,2H).

[0234] Intermediate 19-8 (7 mg) and intermediate 14-10-P2 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent (acetonitrile gradient ratio 15% to 45%, elution time 12 min)) to obtain compound 19-P2 (4.0 mg). MS m / z (ESI): 522.1 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.62(t,J=6.1 Hz,1H),7.86(s,1H),7.52(s,1H),7.25(s,1H),6.50(s,1H),5.54-5.51(m,1H),5.46(s,2H),5.43(s,2H),4.72(d,J=5.8 Hz,2H),3.55-3.52(m,1H),1.94-1.80(m,2H),1.04-0.95(m,1H),0.88(t,J=7.4 Hz,3H),0.39-0.30(m,2H),0.29-0.21(m,2H).

[0235] The two isomers were each further analyzed by the following chiral supercritical fluid chromatography analytical method.

[0236] [Table 5]

[0237] Compound 19-P1: Under the above chiral high performance liquid chromatography conditions, its retention time is 2.877 minutes. Compound 19-P2: Under the above chiral high performance liquid chromatography conditions, its retention time was 2.690 minutes.

[0238] Confirmation of the configuration of compound 19-P1 (X-ray single crystal diffraction method) Single crystal cultivation method: Weigh 10 mg of compound 19-P1 sample and place it in a 1.5 mL centrifuge tube. Add 300 μL of pyridine and dissolve it with ultrasound until it becomes clear. Then seal it with a sealing film, poke three small holes in the sealing film with a needle, and slowly evaporate it at 20-30°C for 48 hours to obtain needle-shaped crystals.

[0239] The obtained single crystal samples were subjected to X-ray analysis, and the test results are shown in Table 1 and FIG.

[0240] [Table 6-1] [Table 6-2]

[0241] From the X-ray crystal diffraction experiments, the chemical structure of compound 19-P1 was determined to be: [ka] It was decided that:

[0242] Example 19-2 Synthesis of (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19-S) [ka] Intermediate 19-8 (2.4 g) and Intermediate 9 (1645.5 mg) were dissolved in anhydrous N,N-dimethylformamide (25 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphine salt (3.24 g) and N,N-diisopropylethylamine (1465.11 mg) were added. The reaction mixture was stirred at 25 °C for 3 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure. Ethyl acetate (100 mL) was added to the residue and stirred for 16 h. After filtration, methanol (50 mL) was added to the filter cake and stirred for 16 h. After filtration, the title compound (1.6 g) was obtained. MS m / z (ESI): 522.1 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.60(t,J=5.9 Hz,1H),7.84(s,1H),7.50(s,1H),7.23(s,1H),6.48(s,1H),5.49(d,J=5.1 Hz,1H),5.47-5.37(m,4H),4.71(d,J=5.8 Hz,2H),3.54(t,J=5.6 Hz,1H),1.97-1.75(m,2H),1.07-0.94(m,1H),0.87(t,J=7.3 Hz,3H),0.40-0.29(m,2H),0.29-0.20(m,2H).

[0243] Compound 19-S was further analyzed by the following chiral supercritical fluid chromatography analytical methods, respectively.

[0244] [Table 7]

[0245] The retention time of compound 19-S prepared in this example was 2.853 minutes under the above chiral supercritical fluid chromatography conditions, which was essentially identical to the retention time (2.877 minutes) of compound 19-P1 prepared in Example 19-1 under the same chromatographic conditions. Therefore, it was determined that compound 19-S and compound 19-P1 have the same configuration and are the same compound.

[0246] Example 20, (S)—N-((8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furo[3,2-f]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 20) [ka] Step 1: Synthesis of 1-(5-aminobenzofuran-4-yl)-2-chloroethanone (Intermediate 20-2) Boron trichloride (1M, 9.61 mL) was dissolved in dichloroethane (14 mL). The reaction mixture was cooled to 0 °C. Intermediate 20-1 (1.6 g, 12.02 mmol) and chloroacetonitrile (1.36 g, 18.03 mmol) were added to the mixture. The mixture was stirred at 0 °C for 10 min, and aluminum trichloride (1.92 g, 14.42 mmol) was then added. The reaction mixture was heated to 25 °C under nitrogen protection and stirred for 10 min. The reaction mixture was stirred at 90 °C for 18 h under nitrogen protection. After completion of the reaction, the reaction mixture was cooled to room temperature, and ice water (50 mL) and 5% HCl (10 mL) were added slowly, successively, and the mixture was stirred at 25 °C for 30 min. Dichloromethane (60 mL) was then added, and the organic phase was washed with water (30 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. Preparative thin layer chromatography (petroleum ether: (ethyl acetate + ethanol = 3:1) = 9:1) gave the title compound (300 mg). MS m / z(ESI):210.0[M+H] + . 1H NMR (400MHz, chloroform-d) δ=7.72(d,J=2.1 Hz,1H),7.53(d,J=9.0 Hz,1H),6.89(d,J=1.4 Hz,1H),6.66(d,J=9.0 Hz,1H),4.78(s,2H)

[0247] Step 2: Synthesis of (S)-15-(chloromethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (Intermediate 20-3) Intermediate 20-2 (200 mg, 954.07 μmol) and Intermediate 1-3 (251.15 mg, 954.07 μmol) were dissolved in anhydrous toluene (4 mL), and pyridinium p-toluenesulfonate (23.98 mg, 95.41 μmol) was added thereto. The reaction mixture was stirred at 90° C. for 16 h under nitrogen protection. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethanol (3 mL × 2) to obtain the title compound (300 mg). MS m / z(ESI):437.1[M+H] + .

[0248] Step 3: Synthesis of (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (Intermediate 20-4) Intermediate 20-3 (70 mg, 160.24 μmol) was dissolved in ethanol (0.5 mL) and anhydrous N,N-dimethylformamide (0.5 mL), and urotropine (89.85 mg, 640.96 μmol) was added. The reaction mixture was stirred at 25 °C for 3 h. After completion of the reaction, the mixture was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.225% formic acid) and methanol with decreasing polarity as the eluent, with a gradient of 5% to 25% methanol, and an elution time of 12 min) to obtain the title compound (17 mg). MS m / z(ESI):418.1[M+H] + .

[0249] Step 4: (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furo[3,2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (compound 20) Intermediate 20-4 (5.00 mg, 11.98 μmol) and hydroxyacetic acid (4.55 mg, 59.89 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). HATU (6.83 mg, 17.97 μmol) and N,N-diisopropylethylamine (4.64 mg, 35.94 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 15% to 35%, and an elution time of 12 min) to obtain the title compound (3 mg). MS m / z (ESI): 476.2 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.48-8.41(m,1H),8.35(d,J=1.8 Hz,1H),8.22(d,J=8.0 Hz,1H),8.14(d,J=8.0 Hz,1H),7.76(s,1H),7.35(s,1H),6.54(s,1H),5.58(t,J=5.6 Hz,1H),5.52(s,2H),5.45(s,2H),5.10(d,J=5.3 Hz,2H),3.88(d,J=5.6 Hz,2H),1.93-1.82(m,2H),0.89(t,J=7.2 Hz,3H).

[0250] Example 21, (S)—N-((9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 21) [ka] Compound 16-4 (7 mg, 15.63 μmol) and hydroxyacetic acid (1.78 mg, 25.48 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). HATU (11.89 mg, 31.26 μmol) and diisopropylethylamine (2.02 mg, 15.63 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 20% to 40%, and an elution time of 12 min) to obtain the title compound (1 mg). MS m / z(ESI):506.1[M+H] + . 1 H NMR(400MHz,Methanol-d4)δ=8.35(t,J=5.8 Hz,1H),8.15(dd,J=1.8,9.8 Hz,1H),7.36(s,1H),6.57(s,1H),5.59-5.50(m,3H),5.45(s,2H),4.91(d,J=3.2 Hz,2H),3.84(d,J=5.7 Hz,2H),1.90-1.80(m,2H),0.87(t,J=7.3 Hz,3H).

[0251] Example 22, (S)—N-((9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-2-methylpropanamide (Compound 22) [ka] Compound 16-4 (20 mg, 44.66 μmol) and intermediate 17-1 (13.95 mg, 133.98 μmol) were dissolved in N,N-dimethylformamide (0.5 mL). HATU (25.47 mg, 66.99 μmol) and N,N-diisopropylethylamine (17.32 mg, 133.98 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 20% to 40%, and an elution time of 12 min) to obtain the title compound (1.07 mg). MS m / z(ESI):534.2[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.30(t,J=5.9 Hz,1H),8.19-8.07(m,1H),7.36(s,1H),6.56(s,1H),5.55-5.45(m,3H),5.45(s,2H),4.90(d,J=3.7 Hz,2H),1.90-1.82(m,2H),1.24(d,J=4.3 Hz,6H),0.87(t,J=7.3 Hz,3H).

[0252] Example 23, N-(((S)-9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 23) [ka] Compound 16-4 (7.0 mg, 15.63 μmol) and intermediate 11-1 (9.08 mg, 78.16 μmol) were dissolved in N,N-dimethylformamide (0.5 mL), and HATU (8.92 mg, 23.45 μmol) and diisopropylethylamine (6.06 mg, 46.89 μmol) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 41% to 61%, and an elution time of 12 min) to obtain the title compound (7 mg). MS m / z (ESI): 546.2 [M + H] + .

[0253] Compound 23 (7 mg) was separated and purified by preparative supercritical fluid chromatography (column: DAICEL CHIRALCEL OD-H (250 mm × 30 mm, 5 μm), mobile phase: A: carbon dioxide, B: ethanol, B%: 50%, flow rate: 80 mL / min) to give compound 23-1 (2.1 mg, RT: 5.106 min) and compound 23-2 (2.09 mg, RT: 5.641 min).

[0254] Compound 23-1: 1 H NMR(400MHz,DMSO-d6)δ=8.32(t,J=5.5 Hz,1H),8.15(d,J=9.7 Hz,1H),7.36(s,1H),6.57(s,1H),5.53(s,2H),5.47(d,J=5.1 Hz,1H),5.45(s,2H),4.93-4.86(m,2H),2.02-1.96(m,1H),1.91-1.81(m,2H),1.04-0.96(m,1H),0.87(t,J=7.3 Hz,3H),0.37-0.31(m,2H),0.29-0.23(m,2H) MS m / z (ESI): 546.2 [M + H] + .

[0255] Compound 23-2: 1 H NMR(400MHz,DMSO-d6)δ=8.37-8.29(m,1H),8.15(d,J=9.9 Hz,1H),7.36(s,1H),6.57(s,1H),5.53(s,2H),5.50-5.46(m,1H),5.45(s,2H),4.96 -4.86(m,2H),2.10-1.95(m,1H),1.92-1.81(m,2H),1.04-0.98(m,1H),0.87(t,J=7.3 Hz,3H),0.40-0.31(m,2H),0.30-0.25(m,2H) MS m / z (ESI): 546.2 [M + H] + .

[0256] Example 25, (R)—N-(((S)-9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxypropanamide (Compound 25) [ka] Compound 16-4 (6 mg, 13.40 μmol) and intermediate 25-1 (2.41 mg, 26.80 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). HATU (10.09 mg, 26.80 μmol) and diisopropylethylamine (1.73 mg, 13.49 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 20% to 40%, and an elution time of 12 min) to obtain the title compound (1.80 mg). MS m / z(ESI):520.1[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=8.34(t,J=5.7 Hz,1H),8.15(d,J=9.9 Hz,1H),7.36(s,1H),6.57(s,1H),5.59(d,J=5.0 Hz,1H),5.51(s,2H),5.45(s,2H),4.90(s,2H),4.13-3.89(m,1H),1.95-1.77(m,2H),1.21(d,J=6.8 Hz,3H),0.87(t,J=7.3 Hz,3H).

[0257] Example 26, (S)—N-(((S)-9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxypropanamide (Compound 26) [ka] Compound 16-4 (6 mg, 13.40 μmol) and intermediate 26-1 (2.41 mg, 26.80 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). HATU (10.09 mg, 26.80 μmol) and N,N-diisopropylethylamine (1.73 mg, 13.49 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 20% to 40%, and an elution time of 12 min) to obtain the title compound (1.60 mg). MS m / z(ESI):520.1[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=8.34(t,J=5.7 Hz,1H),8.23-8.03(m,1H),7.36(s,1H),6.57(s,1H),5.59(d,J=5.0 Hz,1H),5.51(s,2H),5.45(s,2H),4.90(s,2H),4.07-3.96(m,1H),1.93-1.81(m,2H),1.21(d,J=6.8 Hz,3H),0.87(t,J=7.3 Hz,3H).

[0258] Example 27, (S)—N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furo[3,2-f]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 27) [ka] Step 1: Synthesis of (3-chloro-2-hydroxy-5-nitrophenyl)methanediol (Intermediate 27-2) Intermediate 27-1 (6 g) was dissolved in acetic acid (25 mL), the reaction mixture was cooled to 0°C, and nitric acid (9.64 g) was slowly added thereto. The reaction mixture was stirred at 25°C for 4 hours. After completion of the reaction, the reaction mixture was slowly added dropwise to ice water and then extracted three times with ethyl acetate (60 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated to dryness under reduced pressure to give the title compound (5.88 g). 1 H NMR(400MHz,METHANOL-d4)δ=8.16(d,J=1.4,2.4 Hz,1H),8.07-8.01(m,1H),5.68(s,1H)

[0259] Step 2: Synthesis of ethyl 7-chloro-5-nitrobenzofuran-2-carboxylate (intermediate 27-4) Intermediate 27-2 (5.88 g), Intermediate 27-3 (7.69 g), and potassium carbonate (7.41 g) were dissolved in acetone (60 mL), and the reaction mixture was stirred for 6 h at 70° C. After the reaction was completed, water (50 mL) was added thereto, followed by extraction with ethyl acetate (50 mL) three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated to dryness under reduced pressure to give the title compound (4.5 g). 1 H NMR (400 MHz, chloroform-d) δ = 8.55 (d, J = 2.1 Hz, 1H), 8.39 (d, J = 2.1 Hz, 1H), 7.68 (s, 1H), 4.49 (q, J = 7.1 Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H)

[0260] Step 3: Synthesis of 7-chloro-5-nitrobenzofuran-2-carboxylic acid (intermediate 27-5) Intermediate 27-4 (4.5 g) was dissolved in methanol (50 mL), and aqueous sodium hydroxide solution (2 g in 25 mL of HO) was slowly added dropwise thereto. The reaction mixture was stirred at 25 °C for 3 h. After the reaction was completed, water (120 mL) was added thereto and extracted twice with ethyl acetate (50 mL). The aqueous phase was adjusted to pH 1 with dilute hydrochloric acid and further extracted three times with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the title compound (4.0 g). 1 H NMR (400 MHz, chloroform-d) δ = 8.45 (d, J = 2.1 Hz, 1H), 8.25 (d, J = 2.1 Hz, 1H), 7.58 (s, 1H)

[0261] Step 4: Synthesis of 7-chloro-5-nitrobenzofuran (Intermediate 27-6) Intermediate 27-5 (4 g) and copper oxide (1.05 g) were dissolved in quinoline (28 mL). The reaction mixture was purged with nitrogen gas and stirred at 200 °C for 0.5 h. After completion of the reaction, the temperature was lowered to 0 °C, and dilute hydrochloric acid (80 mL) was slowly added dropwise thereto. Water (30 mL) was then added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the title compound (2.4 g). 1 H NMR (400 MHz, chloroform-d) δ = 8.49 (d, J = 2.1 Hz, 1H), 8.31 (d, J = 2.1 Hz, 1H), 7.88 (d, J = 2.3 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H)

[0262] Step 5: Synthesis of 7-chlorobenzofuran-5-amine (Intermediate 27-7) Intermediate 27-6 (2.4 g) and iron powder (1.55 g) were dissolved in methanol (5 mL), and aqueous ammonium chloride solution (148.91 mg, 5 mL) was added dropwise. Nitrogen gas was purged through the reaction solution, and the mixture was stirred at 80 °C for 0.5 h. After the reaction was completed, the temperature was lowered to 25 °C, water (10 mL) was added, and the mixture was extracted three times with ethyl acetate (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated to dryness under reduced pressure to give the title compound (1.2 g). MS m / z (ESI): 167.8 [M + H] + .

[0263] Step 6: Synthesis of 1-(5-amino-7-chlorobenzofuran-4-yl)-2-chloroethan-1-one (Intermediate 27-8) Boron trichloride (671.17 mg) was dissolved in 1,2-dichloroethane (8 mL), and the reaction mixture was cooled to 0 °C. Intermediate 27-7 (1.2 g) and chloroacetonitrile (702.75 mg) were added to the mixture. The mixture was stirred at 0 °C for 10 min, and then aluminum trichloride (1.24 g) was added. The mixture was then heated to 25 °C under nitrogen protection and stirred for 10 min. The mixture was stirred at 90 °C for 18 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, and ice water (5 mL) and 5% HCl (1 mL) were added slowly, successively, and the mixture was stirred at 25 °C for 30 min. Dichloromethane (4 mL) was then added, and the organic phase was washed with water (2 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. After filtration, the mixture was concentrated to dryness under reduced pressure, and the residue was subjected to preparative thin-layer chromatography (silica, petroleum ether: (ethyl acetate and ethanol 3:1 mixed solvent) = 9:1) to obtain the title compound (500 mg). MS m / z (ESI): 243.8 [M + H]+ .

[0264] Step 7: Synthesis of (S)-4-chloro-15-(chloromethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (Intermediate 27-9) Intermediate 27-8 (450 mg) and Intermediate 1-3 (480.35 mg) were dissolved in toluene (5 mL), and pyridinium p-toluenesulfonate (23.17 mg) was added. The reaction mixture was stirred at 90°C for 18 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ethanol (1 mL) was added. The reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered, and the filter cake was washed with petroleum ether (2 mL x 2) and dried to obtain the title compound (500 mg). MS m / z (ESI): 471.0 [M + H] + .

[0265] Step 8: Synthesis of (S)-15-(aminomethyl)-4-chloro-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (Intermediate 27-10) Intermediate 27-9 (450 mg) was dissolved in a mixture of methanol (1 mL) and N,N-dimethylformamide (1 mL), and urotropine (267.71 mg) was added. The reaction mixture was stirred at 50 °C for 4 h. After completion of the reaction, the reaction mixture was cooled to room temperature, concentrated hydrochloric acid (2 mL) was added, and the mixture was stirred. The mixture was then concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity as the eluent, with an acetonitrile gradient of 13% to 43%, and an elution time of 12 min) to obtain the title compound (60.0 mg). MS m / z(ESI):451.9[M+H] + .

[0266] Step 9: Synthesis of (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furo[3,2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 27) Intermediate 27-10 (10 mg) and 2-hydroxyacetic acid (5.05 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), HATU (16.83 mg) and diisopropylethylamine (2.86 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, with an acetonitrile gradient of 28% to 48%, and an elution time of 12 min) to obtain the title compound (6.0 mg). MS m / z(ESI):510.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.46(s,2H),8.36-8.20(m,1H),7.85(s,1H),7.35(s,1H),6.54(s,1H),5.58(t,J=5.6 Hz,1H),5.49(d,J=2.9 Hz,2H),5.45(s,2H),5.07(s,2H),3.88(d,J=5.6 Hz,2H),2.02-1.75(m,2H),0.89(t,J=7.3 Hz,3H).

[0267] Example 28, N-(((S)-4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furo[3,2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 28) [ka] Intermediate 27-10 (10 mg) and intermediate 11-1 (8.34 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), HATU (13.65 mg) and diisopropylethylamine (3.10 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 34% to 54%, and an elution time of 12 min) to obtain the title compound (6.2 mg). MS m / z(ESI):550.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.48-8.43(m,2H),8.29(s,1H),7.84(s,1H),7.35(s ,1H),6.56(s,1H),5.51(s,2H),5.45(s,2H),5.15-4.98(m,2H),3.57(d,J=6.0 Hz,1H),1.95-1.80(m,2H),1.10-1.00(m,1H),0.89(t,J=7.3 Hz,3H),0.41-0.32(m,2H),0.32-0.24(m,2H).

[0268] Example 29, 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-15-nitro-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 29) [ka] Step 1: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-15-nitro-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 29-1) Intermediate 14-7 (500.0 mg) was dissolved in sulfuric acid (15 mL), and the reaction mixture was cooled to 0 °C. Nitric acid (357.35 g, 70% purity) was then slowly added, and the reaction mixture was stirred at 25 °C for 1.5 h. After the reaction was complete, ice water (10 mL) was slowly added, followed by dichloromethane (30 mL). The organic phase was washed with 40 mL of water (20 mL × 2), and the washed organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure to give the title compound (280.0 mg) as a crude product. MS m / z (ESI): 486.0 [M + H] + .

[0269] Step 2: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-15-nitro-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 29-2) Intermediate 29-1 (270.0 mg) was dissolved in methanol (2 mL) and tetrahydrofuran (2 mL), and urotropine (233.73 mg) was added. The reaction mixture was stirred at 60 °C for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (YMC-Pack CN C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a mixture of water (containing 0.225% FA) and methanol with decreasing polarity as the eluent, with a methanol gradient of 9% to 29%, and an elution time of 12 min) to obtain the title compound (15.0 mg). MS m / z(ESI):467.1[M+H] + .

[0270] Step 3: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-15-nitro-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 29) Intermediate 29-2 (12.00 mg) and intermediate 11-1 (14.94 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and HATU (14.67 mg) and N,N-diisopropylethylamine (9.98 mg) were added. The reaction mixture was stirred at 25 °C for 1.5 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 15% to 45%, and an elution time of 12 min) to obtain the title compound (5.20 mg). MS m / z (ESI): 565.2 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.25(t,J=5.3 Hz,1H),7.81(s,1H),7.28(s,1H),6.52(d,J=2.6 Hz,3H),5.55(d,J=4.9 Hz,1H),5.43(s,2H),5.36(s,2H),4.48(d,J=5.1 Hz,2H),3.53(t,J=5.7 Hz,1H),1.91-1.83(m,2H),1.10-0.98(s,1H),0.87(t,J=7.3 Hz,3H),0.42-0.28(m,4H).

[0271] Example 30, N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 30) [ka] Step 1: Synthesis of 2-chloro-3,4-dihydroxybenzaldehyde (Intermediate 30-2) Intermediate 30-1 (20.0 g) was dissolved in anhydrous dichloromethane (100 mL), the reaction mixture was cooled to 0° C., and boron tribromide (87.41 g) was slowly added thereto. The reaction mixture was stirred at 25° C. for 4 h. After completion of the reaction, the reaction mixture was slowly poured into ice water, and ethyl acetate (200 mL) was added. The organic phase was washed with water (100 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was washed with a 1:4 solution of ethyl acetate and petroleum ether with stirring and filtered to give the title compound (14 g). MS m / z (ESI): 173.1 [M + H] + .

[0272] Step 2: Synthesis of 4-chlorobenzo[d][1,3]dioxole-5-carbaldehyde (Intermediate 30-3) Intermediate 30-2 (10.0 g) was dissolved in anhydrous N,N-dimethylformamide (100 mL), and cesium carbonate (28.32 g) and diiodomethane (23.28 g) were added. The reaction mixture was stirred at 100°C for 1 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and water (100 mL) was slowly added, followed by ethyl acetate (150 mL). The organic phase was washed with water (50 mL x 2). The washed organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to give the title compound (5.2 g). MS m / z(ESI):185.4[M+H] + .

[0273] Step 3: Synthesis of 1-(4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-ol (Intermediate 30-4) Intermediate 30-3 (5.0 g) was dissolved in anhydrous tetrahydrofuran (100 mL), and the reaction mixture was cooled to -78 °C. Methylmagnesium bromide (4.85 g, 3 M) was slowly added to the mixture. The reaction mixture was stirred at 25 °C for 4 h under nitrogen protection. After completion of the reaction, water (50 mL) was slowly added, followed by ethyl acetate (100 mL). The organic phase was washed with water (50 mL × 2). The washed organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative column chromatography (petroleum ether:ethyl acetate = 2:1) to give the title compound (5.3 g). 1 H NMR (400MHz, chloroform-d) δ=6.99(d,J=8.3 Hz,1H),6.69(d,J=8.1 Hz,1H),5.97(s,2H),5.13(q,J=6.4 Hz,1H),1.41(d,J=6.4 Hz,3H)

[0274] Step 4: Synthesis of 1-(4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-one (Intermediate 30-5) Intermediate 30-4 (5.2 g) was dissolved in anhydrous dichloromethane (100 mL). The reaction mixture was cooled to 0 °C and Dess-Martin reagent (DMP) (16.49 g) was slowly added. The reaction mixture was stirred at 25 °C for 2 h under nitrogen protection. After completion of the reaction, water (50 mL) was slowly added, followed by ethyl acetate (100 mL). The organic phase was washed with water (50 mL × 2) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative column chromatography (petroleum ether:ethyl acetate = 3:1) to give the title compound (3.0 g). MS m / z (ESI): 199.2 [M + H] + .

[0275] Step 5: Synthesis of 1-(4-chloro-6-nitrobenzo[d][1,3]dioxol-5-yl)ethan-1-one (Intermediate 30-6) Intermediate 30-5 (2.50 g) was dissolved in anhydrous dichloromethane (20 mL), and concentrated sulfuric acid (1.23 g) and nitric acid (5.67 g) were slowly added. The reaction mixture was stirred at 25 °C for 3 h. After completion of the reaction, the reaction mixture was slowly poured into ice water, and ethyl acetate (150 mL) was added. The organic phase was washed with water (50 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was subjected to preparative column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain the title compound (1.8 g). MS m / z(ESI):244.1[M+H] + .

[0276] Step 6: Synthesis of 1-(6-amino-4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-one (Intermediate 30-7) Intermediate 30-6 (0.80 g) was dissolved in anhydrous methanol (6 mL), and Raney Ni (400.0 mg) was added thereto. The reaction mixture was stirred under a hydrogen atmosphere at 25° C. for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to give the title compound (510.0 mg). MS m / z(ESI):214.2[M+H] + .

[0277] Step 7: Synthesis of N-(6-acetyl-7-chlorobenzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 30-8) Intermediate 30-7 (260.0 mg) was dissolved in anhydrous dichloromethane (5 mL), and the reaction mixture was cooled to 0° C. N,N-diisopropylethylamine (235.95 mg) and acetyl chloride (143.32 mg) were added thereto. The reaction mixture was stirred at 25° C. for 1.5 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative chromatography (petroleum ether:ethyl acetate=5:1) to obtain the title compound (140.0 mg). MS m / z (ESI): 256.0 [M + H] + .

[0278] Step 8: Synthesis of N-(6-(2-bromoacetyl)-7-chlorobenzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 30-9) Intermediate 30-8 (110.00 mg) was dissolved in acetic acid (2 mL), and a solution of hydrogen bromide in acetic acid (158.24 mg, 33% content) was added. Liquid bromine (72.20 mg) was then slowly added to the solution, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was slowly poured into ice water, and ethyl acetate (30 mL) was added. The organic phase was washed with water (20 mL × 2), and the washed organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to give the title compound (110.0 mg). MS m / z (ESI): 334.0 [M + H] + .

[0279] Step 9: Synthesis of 1-(6-amino-4-chlorobenzo[d][1,3]dioxol-5-yl)-2-chloroethan-1-one (Intermediate 30-10) Intermediate 30-9 (110.00 mg) was dissolved in absolute ethanol (1 mL) and concentrated hydrochloric acid (1 mL), and the reaction mixture was stirred at 60 °C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ice water (10 mL) and saturated sodium bicarbonate (10 mL) were added slowly, successively. Dichloromethane (30 mL) was then added, and the organic phase was washed with water (20 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 3:1) to obtain the title compound (75 mg). MS m / z (ESI): 248.0 [M + H] + .

[0280] Step 10: Synthesis of (S)-15-chloro-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 30-11) Intermediate 30-10 (75.00 mg) and intermediate 1-3 (83.57 mg) were dissolved in toluene (3 mL), and pyridinium p-toluenesulfonate (PPTS) (11.4 mg) was added. The reaction mixture was stirred at 90°C for 16 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and then ethanol (1 mL) was added. The reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL x 2) and dried to obtain the title compound (75.0 mg). MS m / z(ESI):475.1[M+H] + .

[0281] Step 11: Synthesis of (S)-14-(aminomethyl)-15-chloro-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 30-12) Intermediate 30-11 (70.00 mg) was dissolved in anhydrous methanol (2 mL) and anhydrous tetrahydrofuran (1 mL), and urotropine (61.94 mg) was added. The reaction mixture was stirred at 80 °C for 2 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity as the eluent, with an acetonitrile gradient of 5% to 25%, and an elution time of 12 min) to obtain the title compound (16.0 mg). MS m / z(ESI):456.1[M+H] + .

[0282] Step 12: Synthesis of N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 30) Intermediate 30-12 (5.00 mg) and intermediate 11-1 (6.37 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (6.26 mg) and N,N-diisopropylethylamine (4.25 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 31% to 51%, and an elution time of 12 min) to obtain the title compound (2.30 mg). MS m / z (ESI): 554.2 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=7.78(t,J=5.4 Hz,1H),7.35(s,1H),7.01(s,1H),6.30-6.25(m,1H),6.16(d,J=1.5 Hz,2H),5.27-5.21(m,2H),5.19(s,2H),4.93-4.78(m,2H),3.29(dd,J=2.4,6.1 Hz,1H),1.68-1.56(m,2H),0.81-0.72(m,1H),0.63(t,J=7.3 Hz,3H),0.14-0.06(m,2H),0.05-0.03(m,2H).

[0283] Example 31, (S)—N-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 31) [ka] Intermediate 30-12 (5 mg) and hydroxyacetic acid (4.17 mg) were dissolved in N,N-dimethylformamide (0.5 mL), and HATU (6.26 mg) and N,N-diisopropylethylamine (4.25 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 20% to 40%, and an elution time of 12 min) to obtain the title compound (2.50 mg). MS m / z(ESI):514.2[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.03(t,J=5.8 Hz,1H),7.60(s,1H),7.25(s,1H),6.54-6.48(m,1H),6.41(s,2H),5.48(s,2H),5.43(s,2H),5.12(d,J=6.0 Hz,2H),3.82(s,2H),1.93-1.78(m,2H),0.87(t,J=7.4 Hz,3H)

[0284] Example 33, (S)—N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopenta[f]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 33) [ka] Step 1: Synthesis of 7-chloro-2,3-dihydro-1H-inden-4-ol (Intermediate 33-2) Intermediate 33-1 (500 mg) was dissolved in anhydrous acetonitrile (5 mL), NCS (547 mg) was added, and the mixture was stirred at 25 °C for 2 hours. After the reaction was completed, water (10 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (30 mL) and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 100:1 to 10:1) to obtain the title compound (600 mg). MS m / z (ESI): 169.0 [M + H] + .

[0285] Step 2: Synthesis of 7-chloro-5-nitro-2,3-dihydro-1H-inden-4-ol (Intermediate 33-3) Intermediate 33-2 (10 g) was dissolved in AcOH (50 mL) and H2O (10 mL), and HNO3 (8.62 g, mass fraction 65%) was added thereto at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours. After completion of the reaction, the reaction mixture was slowly added to ice water (200 mL). After filtration, the mixture was concentrated under reduced pressure to remove the solvent, and the title compound (10 g) was obtained. MS m / z (ESI): 214.0 [M + H] + .

[0286] Step 3: Synthesis of 5-amino-7-chloro-2,3-dihydro-1H-inden-4-ol (Intermediate 33-4) Intermediate 33-3 (5 g) was dissolved in anhydrous DCM (50 mL), AcOH (14.04 g) and Zn (7.61 g) were added, and the reaction mixture was stirred at 25° C. for 12 hours. After the reaction was completed, water (200 mL) and ethyl acetate (200 mL) were added sequentially, and the organic phase was washed with saturated aqueous sodium bicarbonate (50 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to remove the solvent, yielding the title compound (4 g). MS m / z (ESI): 184.0 [M + H] + .

[0287] Step 4: Synthesis of 5-acetamido-7-chloro-2,3-dihydro-1H-inden-4-yl acetate (Intermediate 33-5) Intermediate 33-4 (4 g) was dissolved in anhydrous dichloromethane (40 mL), and acetic anhydride (AcO) (6.67 g) and triethylamine (TEA) (6.61 g) were added. The reaction mixture was stirred at 25 °C for 12 hours. After the reaction was completed, water (200 mL) and ethyl acetate (200 mL) were added sequentially. The organic phase was washed with saturated aqueous NaCl (50 mL × 2). The washed organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 5:1) to obtain the title compound (4 g). MS m / z (ESI): 268.0 [M + H] + .

[0288] Step 5: Synthesis of N-(7-chloro-4-hydroxy-2,3-dihydro-1H-inden-5-yl)acetamide (Intermediate 33-6) Intermediate 33-5 (4 g) was dissolved in anhydrous methanol (20 mL), KCO (6.19 g) was added, and the reaction mixture was stirred at 25 °C for 12 hours. After the reaction was complete, water (200 mL) and ethyl acetate (200 mL) were added sequentially, and the organic phase was washed with saturated aqueous NaCl (50 mL × 2). The washed organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 1:1) to obtain the title compound (2.8 g). MS m / z (ESI): 226.0 [M + H] + .

[0289] Step 6: Synthesis of 5-acetamido-7-chloro-2,3-dihydro-1H-inden-4-yl trifluoromethanesulfonate (Intermediate 33-7) Intermediate 33-6 (500 mg) was dissolved in dichloromethane (5 mL), trifluoromethanesulfonic anhydride (TfO) (749 mg) and triethylamine (TEA) (672 mg) were added, and the reaction mixture was stirred at 25 °C for 12 hours. After completion of the reaction, water (50 mL) and ethyl acetate (50 mL) were added sequentially, and the organic phase was washed with saturated aqueous NaCl (50 mL × 2). The washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 1:1) to obtain the title compound (580 mg). MS m / z(ESI):358.0[M+H] + .

[0290] Step 7: Synthesis of N-(4-(1-butoxyvinyl)-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (Intermediate 33-8) Intermediate 33-7 (430 mg) and vinyl n-butyl ether (360.60 mg) were dissolved in dioxane (20 mL). Diisopropylethylamine (DIEA) (466 mg), 1,1'-bis(diphenylphosphino)ferrocene (DPPF) (66 mg), and tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) (110 mg) were added. The reaction mixture was stirred at 80 °C for 16 h under nitrogen protection. After completion of the reaction, the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 1:1) to obtain the title compound (300 mg). MS m / z (ESI): 308.0 [M + H] + .

[0291] Step 8: Synthesis of N-(4-acetyl-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (Intermediate 33-9) Intermediate 33-8 (200 mg) was dissolved in dioxane (5 mL), 1N HCl (5 mL) was added, and the mixture was reacted with stirring for 2 hours at 25° C. After completion of the reaction, the organic phase was concentrated under reduced pressure to remove the solvent, and the title compound (150 mg) was obtained. MS m / z (ESI): 252.0 [M + H] + .

[0292] Step 9: Synthesis of N-(4-(2-bromoacetyl)-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (Intermediate 33-10) Intermediate 33-9 (300 mg) was dissolved in HBr / AcOH (4 mL, mass fraction 33%), NBS (318.20 mg) was added, and the reaction solution was reacted with stirring at 25° C. for 2 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the solvent, and the title compound (390 mg) was obtained. MS m / z(ESI):330.0[M+H] + .

[0293] Step 10: Synthesis of 1-(5-amino-7-chloro-2,3-dihydro-1H-inden-4-yl)-2-chloroethan-1-one (Intermediate 33-11) Intermediate 33-10 (300 mg) was dissolved in absolute ethanol, HCl (12 M, 8.00 mL) was added, and the reaction solution was stirred at 80° C. for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by preparative high-performance liquid chromatography (chromatography column: Gemini NX C18 5 μm × 10 × 150 mm, mobile phase: A: water (0.225% formic acid v / v), B: acetonitrile, B%: 30% to 70%) to obtain the title compound (64 mg). MS m / z (ESI): 244.0 [M + H] + .

[0294] Step 11: Synthesis of (S)-4-chloro-15-(chloromethyl)-8-ethyl-1,2,3,8,11,14-hexahydro-9H,12H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione (Intermediate 33-12) Intermediate 33-11 (45.00 mg) and Intermediate 1-3 (48.53 mg) were dissolved in toluene (1 mL), and pyridinium p-toluenesulfonate (4.63 mg) was added thereto. The reaction mixture was stirred at 90°C for 16 hours. After the reaction was completed, the mixture was cooled to room temperature, ethanol (1 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL x 2) and dried to obtain the title compound (80.0 mg). MS m / z (ESI): 471.1 [M + H] + .

[0295] Step 12: Synthesis of (S)-15-(aminomethyl)-4-chloro-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione (Intermediate 33-13) Intermediate 33-12 (40.00 mg) was dissolved in anhydrous methanol (1 mL) and anhydrous N,N-dimethylformamide (0.5 mL), and urotropine (35.69 mg) was added. The reaction mixture was stirred at 50 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.225% FA) and acetonitrile as the eluent, with an acetonitrile gradient of 35% to 55%, and an elution time of 12 min) to obtain the title compound (15.0 mg). MS m / z(ESI):452.1[M+H] + .

[0296] Step 13: Synthesis of (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (Compound 33) Intermediate 33-13 (5.00 mg) and hydroxyacetic acid (4.21 mg, 55.30 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), HATU (6.31 mg) and N,N-diisopropylethylamine (4.29 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.05% FA) and acetonitrile as the eluent, with an acetonitrile gradient of 32% to 52%, and an elution time of 12 min) to obtain the title compound (2.0 mg). MS m / z(ESI):510.3[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.36-8.30(m,1H),8.14(s,1H),7.31(s,1H),6.55(s,1H),5.44(s,2H),5.38(s,2H),4.96(d,J=5.3 Hz,2H),3.88(s,2H),3.74-3.66(m,2H),3.14(t,J=7.6 Hz,2H),2.27-2.19(m,2H),1.92-1.82(m,2H),0.88(t,J=7.3 Hz,3H).

[0297] Example 34, N-(((S)-4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 34) [ka] Intermediate 33-13 (5.00 mg) and intermediate 11-1 (6.42 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), HATU (6.31 mg) and diisopropylethylamine (4.29 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.05% FA) and acetonitrile as the eluent, with an acetonitrile gradient of 35% to 55%, and an elution time of 12 min) to obtain the title compound (2.0 mg). MS m / z(ESI):550.3[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.36-8.31(m,1H),8.14(s,1H),7.31(s,1H), 6.54(s,1H),5.47-5.41(m,3H),5.38(s,2H),4.96-4.90(m,2H),3.69(t,J=7.1 Hz,2H),3.56(t,J=5.8 Hz,1H),3.14(t,J=7.4 Hz,2H),2.27-2.18(m,2H),1.93-1.81(m,2H),1.09-1.03(m,1H),0.88(t,J=7.2 Hz,3H),0.41-0.26(m,4H).

[0298] Example 35, 2-cyclopropyl-N-(((S)-7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 35) [ka] Intermediate 12-11 (6 mg) and intermediate 11-1 (7.93 mg) were dissolved in N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.79 mg) and N,N-dimethyldiisopropylamine (5.29 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent (acetonitrile gradient ratio 15% to 45%, elution time 12 min)) to obtain the title compound (6.50 mg). MS m / z(ESI):538.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.21(t,J=6.0 Hz,1H),7.51(s,1H),7.26(s,1H),6.53(s,1H),6.40(s,2H),5.47(s,2H),5.43(s,2H),4.84(d,J=3.8 Hz,2H),3.53(d,J=6.3 Hz,1H),1.90-1.81(m,2H),1.01(d,J=5.5 Hz,1H),0.89-0.85(m,3H),0.33(d,J=6.0 Hz,2H),0.30-0.25(m,2H).

[0299] Synthesis of compounds 35-P1 and 35-P2 [ka] Intermediate 12-11 (6 mg) and intermediate 14-10-P1 (7.93 mg) were dissolved in N,N-dimethylformamide (1 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.79 mg) and N,N-dimethyldiisopropylamine (5.29 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 17% to 47%, and an elution time of 12 min) to obtain compound 35-P1 (2.87 mg). MS m / z(ESI):538.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.19(t,J=5.9 Hz,1H),7.51(s,1H),7.26(s,1H),6.51(s,1H),6.40(s,2H),5.47(s,2H),5.43(s,2H),4.84(d,J=4.6 Hz,2H),3.53(d,J=6.4 Hz,1H),1.91-1.80(m,2H),1.06-0.97(m,1H),0.87(t,J=7.3 Hz,3H),0.38-0.31(m,2H),0.31-0.24(m,2H).

[0300] Intermediate 12-11 (6 mg) and intermediate 14-10-P2 (4.76 mg) were dissolved in N,N-dimethylformamide (1 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (10.38 mg) and N,N-dimethyldiisopropylamine (1.76 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as the eluent, with an acetonitrile gradient of 17% to 47%, and an elution time of 12 min) to obtain compound 35-P2 (2.01 mg). MS m / z(ESI):538.1[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.26-8.16(m,1H),7.51(s,1H),7.26(s,1H),6.51(s ,1H),6.40(s,2H),5.46(s,2H),5.43(s,2H),4.87-4.82(m,2H),3.53(d,J=6.2 Hz,1H),1.94-1.80(m,2H),1.05-0.96(m,1H),0.87(t,J=7.3 Hz,3H),0.39-0.30(m,2H),0.31-0.20(m,2H).

[0301] The two isomers were each further analyzed by the following chiral supercritical fluid chromatography analytical method.

[0302] [Table 8]

[0303] Compound 35-P1: Under the above chiral high performance liquid chromatography conditions, its retention time is 3.519 minutes. Compound 35-P2: Under the above chiral high performance liquid chromatography conditions, its retention time was 3.573 minutes.

[0304] Example 36, (S)—N-((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 36) [ka] Intermediate 12-11 (6 mg) and hydroxyacetic acid (3.21 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (10.38 mg) and diisopropylethylamine (1.76 mg) were added, and the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, diameter 25 mm, length 100 mm, using a decreasing polarity mixture of water (containing 0.05% formic acid) and acetonitrile as the eluent (acetonitrile gradient ratio 18% to 48%, elution time 12 min)) to obtain the title compound (2.40 mg). MS m / z (ESI): 498.1 [M + H] + . 1 H NMR(400MHz,DMSO-d6)δ=8.20(t,J=5.9 Hz,1H),7.51(s,1H),7.26(s,1H),6.39(s,2H),5.46(s,2H),5.43(s,2H),4.85(d,J=4.3 Hz,2H),3.83(s,2H),1.92-1.80(m,2H),0.87(t,J=7.3 Hz,3H).

[0305] Example 37 Synthesis of N-((12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound L1-14), Isomers L1-14-P1 and L1-14-P2 [ka] Step 1: Synthesis of intermediate L1-14-2 The starting material L1-14-1 (25 g), lead acetate (43.79 g), and pyridine (6.98 g) were dissolved in a mixed solvent of tetrahydrofuran (600 mL) and toluene (200 mL), heated to 85 °C under a nitrogen atmosphere, and reacted with stirring for 18 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (chromatography column: ISCO®, 330 g SepaFlash® Silica Flash Column, mobile phase gradient 0% to 75% ethyl acetate / petroleum ether, flow rate 100 mL / min) to obtain the title compound (18 g). 1 H NMR(400MHz,METHANOL-d4)δ=7.82(d,J=7.5 Hz,2H),7.69(d,J=7.3 Hz,2H),7.44-7.38(m,2H),7.36-7.31(m,2H),5.22(s,2H),4.39(d,J=6.8 Hz, 2H), 4.28-4.22 (m, 1H), 3.81 (s, 2H), 2.03 (s, 3H). MS m / z (ESI): 391.1 [M + Na] + .

[0306] Step 2: Synthesis of intermediate L1-14-3 Intermediate L1-14-2 (5 g), 2-cyclopropyl-2-hydroxybenzyl acetate (8.40 g), and pyridinium p-toluenesulfonate (PPTS, 341.09 mg) were dissolved in dichloromethane (150 mL), and the reaction mixture was heated to 65°C under a nitrogen atmosphere and reacted with stirring for 48 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (chromatography column: ISCO®, 120 g SepaFlash® Silica Flash Column, mobile phase gradient: 0-45% ethyl acetate / petroleum ether, flow rate: 80 mL / min), and then further purified by high-performance liquid chromatography (chromatography column: Boston Prime C18 150 × 30 mm × 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile], B%: 42%-82%, 13 min) to obtain the title compound (1.4 g). MS m / z (ESI): 537.2 [M + Na] + .

[0307] Step 3: Synthesis of intermediates L1-14-4-P1 and L1-14-4-P2 Intermediate L1-14-3 (1.4 g) was dissolved in a mixed solvent of methanol (15 mL) and water (15 mL), and wet palladium on carbon (10% mass content, 0.15 g) was added. The reaction mixture was stirred under a hydrogen atmosphere at 25° C. for 16 h. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by high-performance liquid chromatography (chromatography column: Boston Prime C18 150 × 30 mm × 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile], B%: 24% to 64%, 13 min), and then further purified by supercritical fluid chromatography (chromatography column: DAICEL CHIRALPAK IC column, 10 μm silica, diameter 30 mm, length 250 mm, isopropanol (containing 0.1% aqueous ammonia) was used as the eluent), and intermediate L1-14-4-P1 (130 mg) and intermediate L1-14-4-P2 (130 mg) were obtained.

[0308] The two isomers were further analyzed by the following chiral high performance liquid chromatography analysis method.

[0309] The chiral high performance liquid chromatography conditions are as follows: [Table 9-1] [Table 9-2]

[0310] Intermediate L1-14-4-P1: Under the above chiral high performance liquid chromatography conditions, its retention time is 4.471 minutes. MS m / z (ESI): 447.5 [M + Na] + .

[0311] Intermediate L1-14-4-P2: Under the above chiral high performance liquid chromatography conditions, its retention time is 5.692 minutes. MS m / z (ESI): 447.3 [M + Na] + .

[0312] Step 4: Synthesis of intermediates L1-14-5-P1 and L1-14-5-P2 2-Chlorotrityl chloride resin (2-CTC-resin) (specification: approximately 1.19 mmol / g) (257 mg) was added to dichloromethane (3 mL), and intermediate L1-14-4-P1 (130 mg) and diisopropylethylamine (59.37 mg) were further added. The reaction mixture was shaken on a shaker at 25 °C under a nitrogen atmosphere for 16 h. After the reaction was completed, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) successively, this was repeated three times. The mixture was filtered, and the filter cake was dried to obtain intermediate L1-14-5-P1 (330 mg). Intermediate L1-14-4-P2 (130 mg) was used as a starting material according to the above method to prepare intermediate L1-14-5-P2 (350 mg).

[0313] Step 5: Synthesis of intermediates L1-14-6-P1 and L1-14-6-P2 Intermediate L1-14-5-P1 (330 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (1.08 g) was added. The reaction mixture was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was complete, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). The mixture was filtered and the filter cake was dried to obtain L1-14-6-P1 (220 mg). Intermediate L1-14-5-P2 (350 mg) was used as a starting material to prepare intermediate L1-14-6-P2 (220 mg) according to the above method.

[0314] Step 6: Synthesis of intermediates L1-14-7-P1 and L1-14-7-P2 Intermediate L1-14-6-P1 (220 mg) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (230.17 mg) were dissolved in N,N-dimethylformamide (5 mL), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (225.31 mg) and diisopropylethylamine (99.96 mg) were added to the reaction solution. The reaction solution was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was complete, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) successively, three times. Filtration and drying of the filter cake gave L1-14-7-P1 (377 mg). Intermediate L1-14-7-P2 (361 mg) was prepared from intermediate L1-14-6-P2 (220 mg) according to the method described above.

[0315] Step 7: Synthesis of intermediates L1-14-8-P1 and L1-14-8-P2 Intermediate L1-14-7-P1 (377 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (37.22 mg) was added. The reaction mixture was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was complete, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). The mixture was filtered and the filter cake was dried to obtain L1-14-8-P1 (270 mg). Intermediate L1-14-7-P2 (361 mg) was used as a starting material according to the above method to prepare intermediate L1-14-8-P2 (260 mg).

[0316] Step 8: Synthesis of intermediates L1-14-9-P1 and L1-14-9-P2 Intermediate L1-14-8-P1 (270 mg) was dissolved in N,N-dimethylformamide (5 mL), and N-((9H-fluoren-9-ylmethoxy)carbonyl)glycylglycine (209.58 mg), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (224.29 mg), and diisopropylethylamine (99.51 mg) were added sequentially. The reaction mixture was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was complete, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times. The mixture was filtered, and the filter cake was dried to obtain intermediate L1-14-9-P1 (403 mg). Intermediate L1-14-9-P2 (420 mg) was prepared from intermediate L1-14-8-P2 (260 mg) according to the above method.

[0317] Step 9: Synthesis of Intermediates L1-14-10-P1 and L1-14-10-P2 Intermediate L1-14-9-P1 (403 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (1.08 g) was added. The reaction mixture was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was complete, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). The mixture was filtered and the filter cake was dried to obtain L1-14-10-P1 (300 mg). Intermediate L1-14-9-P2 (420 mg) was used as a starting material according to the above method to prepare intermediate L1-14-10-P2 (320 mg).

[0318] Step 10: Synthesis of Intermediates L1-14-12-P1 and L1-14-12-P2 Intermediate L1-14-10-P1 (300 mg) was dissolved in N,N-dimethylformamide (5 mL), and compound L1-14-11 (182.13 mg) and diisopropylethylamine (99.41 mg) were added sequentially. The reaction mixture was placed on a shaker at 25 °C and shaken for 16 h. After the reaction was complete, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times. The mixture was filtered, and the filter cake was dried to obtain L1-14-12-P1 (374 mg). Intermediate L1-14-10-P2 (320 mg) and intermediate L1-14-11 (194.27 mg) were used as starting materials to prepare intermediate L1-14-12-P2 (387 mg) according to the method described above.

[0319] Step 11: Synthesis of Intermediates L1-14-13-P1 and L1-14-13-P2 Intermediate L1-14-12-P1 (374 mg) was added to a mixed solvent of dichloromethane (8 mL) and hexafluoroisopropanol (HFIP, 2 mL), and the reaction solution was shaken on a shaker at 25 °C for 0.5 h. After the reaction was completed, the reaction solution was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by high-performance liquid chromatography (chromatography column: Boston Prime C18 150 x 30 mm x 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile], B%: 15% to 35%, 9 min) to obtain intermediate L1-14-13-P1 (74 mg). 1H NMR(400MHz,METHANOL-d4)δ=7.33-7.20(m,5H),6.81(s,2H),4.77-4.70(m,2H),4.60-4.52(m,1H),3.96-3.70(m,6H),3.61(d,J=7.6 Hz,1H),3.55-3.47(m,2H),3.27-3.23(m,1H),3.05-2.98(m,1H),2.30(t,J=7.4 Hz,2H),1.72-1.55(m,4H),1.38-1.29(m,2H),1.16-1.07(m,1H),0.60-0.47(m,4H). MS m / z(ESI):679.7[M+Na] + . The raw material for intermediate L1-14-12-P2 (387 mg) is the same, the method mentioned above is the same, and the intermediate L1-14-13-P2 (86 mg) is manufactured. 1 H NMR(400MHz,METHANOL-d4)δ=7.40-7.21(m,5H),6.82(s,2H),4.81-4.67(m,2H),4.60-4.50(m,1H),3.97-3.70(m,6H),3.66-3.57(m,1H),3.56-3 .47(m,2H),3.27-3.22(m,1H),3.08-2.97(m,1H),2.35-2.26(m,2H),1.7 5-1.55(m,4H),1.41-1.32(m,2H),1.16-1.06(m,1H),0.60-0.45(m,4H). MS m / z(ESI):679.5[M+Na] + .

[0320] ステップ12: Synthesis of compounds L1-14-P1 and びL1-14-P2 Intermediate L1-14-13-P1 (31.17 mg), intermediate 14-8 (20 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (18.20 mg), pyridine (11.26 mg), and 1-hydroxybenzotriazole (12.83 mg) were dissolved in N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 2 h. After completion of the reaction, the mixture was purified by high-performance liquid chromatography (chromatography column: Boston Green ODS 150 x 30 mm x 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile], B%: 26% to 46%, 12 min) to obtain compound L1-14-P1 (12.3 mg). 1 H NMR(400MHz,DMSO-d6)δ=8.65(t,J=5.9 Hz,1H),8.58(t,J=6.6 Hz,1H),8.27(t,J=5.6 Hz,1H),8.17-8.02(m,2H),7.99(t,J=5.6 Hz,1H),7.79(s,1H),7.52(s,1H),7.28-7.19(m,5H),7.19-7.14(m,1H),6.98(s,2H),6.49(s,1H),6.29(d,J=3.7 Hz,2H),5.48-5.37(m,4H),4.85-4.70(m,2H),4.70-4.65(m,1H),4.52-4.45(m,1H),4.43-4.37(m,1H),3.79-3.54(m,7H),3.49(d,J=7.1 Hz,1H),3.08-3.01(m,1H),2.85-2.74(m,1H),2.14-2.06(m,2H),1.94-1.80( m,2H),1.52-1.42(m,4H),1.27-1.13(m,2H),1.01-0.92(m,1H),0.88(t,J=7.3 Hz,3H),0.41-0.28(m,4H). MS m / z(ESI):1060.3[M+H] + . Compound L1-14-P2 (11.4 mg) was prepared according to the above method using intermediate L1-14-13-P2 (31.17 mg) and intermediate 14-8 (20 mg) as starting materials. 1H NMR(400MHz,DMSO-d6)δ=8.72-8.52(m,2H),8.33-8.25(m,1H),8.17-7.94( m,3H),7.80(s,1H),7.51(s,1H),7.32-7.19(m,5H),7.18-7.12(m,1H),6.9 9(s,2H),6.49(s,1H),6.35-6.25(m,2H),5.48-5.36(m,4H),4.84-4.59(m, 3H),4.54-4.45(m,1H),4.44-4.35(m,1H),3.81-3.54(m,7H),3.49(d,J=6.7 Hz,1H),3.08-3.01(m,1H),2.87-2.73(m,1H),2.14-2.05(m,2H),1.95-1.77(m, 2H),1.53-1.39(m,4H),1.25-1.13(m,2H),1.03-0.82(m,4H),0.43-0.25(m,4H) MS m / z(ESI):1060.3[M+H] + .

[0321] The two isomers were further analyzed by chiral high performance liquid chromatography as follows.

[0322] The chiral high performance liquid chromatography conditions are as follows: [Table 10]

[0323] Compound L1-14-P1 had a retention time of 3.735 minutes under the above chiral high performance liquid chromatography conditions.

[0324] Compound L1-14-P2 had a retention time of 3.901 minutes under the above chiral high performance liquid chromatography conditions.

[0325] Example 38: N-((12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound L1-35-P1) [ka] Intermediate L1-14-13-P1 (8 mg), intermediate 12-11 (5.89 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.67 mg), pyridine (2.89 mg), and 1-hydroxybenzotriazole (3.29 mg) were dissolved in N,N-dimethylformamide (1 mL). The reaction mixture was stirred under a nitrogen atmosphere at 25 °C for 2 h. After completion of the reaction, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Prime C18 150 x 30 mm x 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile], B%: 23% to 45%, B%, 10 min) to obtain L1-35-P1 (2.3 mg). 1H NMR(400MHz,DMSO-d6)δ=8.59(t,J=6.1 Hz,1H),8.36-8.31(m,1H),8.28(t,J=5.6 Hz,1H),8.14-8.02(m,2H),8.00(t,J=5.8 Hz,1H),7.49(s,1H),7.27-7.16(m,6H),6.99(s,2H),6.52(s,1H),6.38(d,J=2.0 Hz,2H),5.49-5.41(m,4H),4.90-4.82(m,2H),4.69-4.62(m,1H),4.53-4.43(m,2H),3.79-3.54(m,7H),3.47(d,J=7.0 Hz,1H),3.06-3.00(m,1H),2.80-2.74(m,1H),2.10(t,J=7.3 Hz,2H),1.91-1.79(m,2H),1.51-1.41(m,4H),1.22-1.14(m,2H),1.02-0.94(m,1H),0.87(t,J=7.2 Hz,3H),0.40-0.27(m,4H). MS m / z (ESI): 1078.2 [M + H] + . Further analysis was carried out by supercritical fluid chromatography as follows.

[0326] The supercritical fluid chromatography conditions are as follows: [Table 11] Compound L1-35-P1 had a retention time of 3.732 minutes under the above supercritical fluid chromatography conditions.

[0327] Example 39-1: Synthesis of N-((12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (compound L1-19-P1), isomer L1-19-P2, and racemic L1-19 Intermediate L1-14-13-P1 (7.75 mg), intermediate 19-8 (5.0 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.53 mg), pyridine (2.80 mg), and 1-hydroxybenzotriazole (3.19 mg) were dissolved in N,N-dimethylformamide (1 mL). The reaction mixture was stirred under a nitrogen atmosphere at 25 °C for 2 h. After completion of the reaction, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Green ODS 150 x 30 mm x 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile], B%: 22% to 52%, B%, 12 min) to obtain L1-19-P1 (6.0 mg). MS m / z(ESI):1062.3[M+H] + . 1H NMR (400MHz, DMSO-d6) δ=8.74-8.69(m,1H),8.59(t,J=6.7 Hz,1H),8.32-8.26(t,J=5.6 Hz,1H),8.11(d,J=7.8 Hz,1H),8.06(t,J=5.3 Hz,1H),7.99-7.94(m,1H),7.80(s,1H),7.52(s,1H),7.27-7.20(m,5H),7.19-7.11(m,1H),6.99(s,2H),6.50(s,1H),5.49 -5.41(m,4H),4.84-4.72(m,2H),4.71-4.63(m,1H),4.53-4.48(m,1H),4.44-4.36(m,1H),3.77-3.56(m,6H),3.49(d,J=7.0 Hz,1H),3.06-3.02(m,1H),2.83-2.74(m,1H),2.10(t,J=7.6 Hz,2H),1.91-1.82(m,2H),1.50-1.42(m,4H),1.22-1.13(m,2H),1.02-0.90(s,1H),0.88(t,J=7.3 Hz,3H),0.38-0.29(m,4H).

[0328] The raw materials of intermediate L1-14-13-P2 (30.0 mg) and intermediate 19-8 (25.2 mg) were used, and the method mentioned above was used, and the compound L1-19-P2 (19.0 mg) was manufactured. MS m / z(ESI):1062.3[M+H] + . 1H NMR(400MHz,DMSO-d6)δ=8.73-8.63(m,1H),8.58(t,J=6.4 Hz,1H),8.30(t,J=5.7 Hz,1H),8.13(d,J=8.1 Hz,1H),8.07(t,J=5.8 Hz,1H),8.01(t,J=5.4 Hz,1H),7.81(s,1H),7.52(s,1H),7.28-7.19(m,5H),7.19-7.12(m,1H),7.00(s,2H),6.50(s,1H),5.48-5.39(m, 4H),4.84-4.71(m,2H),4.68-4.55(m,1H),4.53-4.43(m,1H),4.42-4.35(m,1H),3.77-3.54(m,6H),3.49(d,J=7.0 Hz,1H),3.08-3.02(m,1H),2.85-2.76(m,1H),2.09(t,J=7.5 Hz,2H),1.93-1.79(m,2H),1.52-1.39(m,4H),1.25-1.12(m,2H),1.02-0.94(m,1H),0.88(t,J=7.3 Hz,3H),0.44-0.24(m,4H). The two isomers were further analyzed by the following chiral high performance liquid chromatography analysis method.

[0329] The chiral high performance liquid chromatography conditions are as follows: [Table 12] L1-19-P1 has a retention time of 3.775 minutes under the above chiral supercritical fluid chromatography conditions; Under the above chiral supercritical fluid chromatography conditions, L1-19-P2 had a retention time of 3.973 minutes.

[0330] Synthesis of racemic compound L1-19 [ka] Intermediate L1-14-13 (30.00 mg, synthesized using 2-cyclopropyl-2-hydroxybenzyl acetate (racemic) as starting material according to the synthesis method for L1-14-13-P1), intermediate 19-8 (25.15 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17.52 mg), pyridine (10.84 mg), and 1-hydroxybenzotriazole (12.35 mg) were dissolved in N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 25 °C for 2 h under nitrogen protection. After completion of the reaction, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Green ODS 150 x 30 mm x 5 μm, mobile phase: [A: water (formic acid), B: acetonitrile], B%: 33% to 33%, 14 min) to obtain racemic compound L1-19 (15.4 mg). 1 H NMR(400MHz,DMSO-d6)δ=8.76-8.66(m,1H),8.59(t,J=6.8 Hz,1H),8.36-8.27(m,1H),8.12(d,J=6.6 Hz,1H),8.09-8.05(m,1H),8.04-8.00(m,1H),7.81(s,1H),7.52(s,1H),7.27-7.19(m,5H),7.17-7.12(m,1H),7.00(s,2H),6.50(s, 1H),5.49-5.42(m,4H),4.84-4.71(m,2H),4.71-4.63(m,1H),4.53-4.42(m,1H),4.44-4.35(m,1H),3.83-3.54(m,6H),3.49(d,J=7.1 Hz,1H),3.08-3.02(m,1H),2.86-2.73(m,1H),2.10(t,J=7.3 Hz,2H),1.93-1.79(m,2H),1.49-1.37(m,4H),1.25-1.13(m,2H),1.01-0.92(m,1H),0.88(t,J=7.2 Hz,3H),0.43-0.25(m,4H). MS m / z(ESI):1062.4[M+H] + .

[0331] Example 39-2: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (compound L1-19-S) [ka] Step 1: Synthesis of L1-14-3-S Intermediate L1-14-2 (28 g) and Intermediate 9 (23.5 g, prepared in Example 14-2) were dissolved in dichloromethane (25 mL). Silver trifluoromethanesulfonate (139.50 mg) was added to the reaction mixture, and the mixture was stirred at 25°C for 108 hours under nitrogen protection. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified using a flash silica gel column (mobile phase gradient: tetrahydrofuran / dichloromethanol: 0-7%, flow rate: 70 mL / min) to obtain the title compound (10.3 g). MS m / z (ESI): 537.3 [M + Na] + .

[0332] Step 2: Synthesis of L1-14-4-S Intermediate L1-14-3-S (10 g) was dissolved in tetrahydrofuran (100 mL), and wet palladium-carbon (10% mass content, 1 g) was added. The reaction mixture was stirred for 16 hours in a hydrogen atmosphere at 0° C. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. Petroleum ether / ethyl acetate (10 mL / 0.5 mL) was added to the residue, and the mixture was stirred for 2 hours. After filtration, the title compound (6 g) was obtained. MS m / z (ESI): 447.2 [M + Na] + . L1-14-4-S was further analyzed by the following chiral high performance liquid chromatography analytical method.

[0333] The chiral high performance liquid chromatography conditions are as follows: [Table 13]

[0334] Under the chiral supercritical fluid chromatography conditions, the retention time of L1-14-4-S was 4.385 min, which was essentially identical to the retention time (4.471 min) of compound L1-14-4-P1 under the same chromatographic conditions. Therefore, L1-14-4-S and L1-14-4-P1 have the same configuration and are the same compound.

[0335] Step 3: Synthesis of L1-19-S Intermediate L1-14-13-S (600 mg, prepared from L1-14-4-S using the synthesis method for L1-14-13-P1), intermediate 19-8 (502.93 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (350.31 mg), pyridine (216.82 mg), and 1-hydroxybenzotriazole (246.91 mg) were dissolved in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25 °C for 2 h under nitrogen protection. After completion of the reaction, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Green ODS 150 x 30 mm x 5 μm, mobile phase: [A: water (0.05% formic acid), B: acetonitrile], B%: 24% to 54%, B%, 12 min) to obtain the title compound (286 mg). 1H NMR(400MHz,DMSO-d6)δ=8.72-8.63(m,1H),8.62-8.52(m,1H),8.34-8.26(m,1H),8.1 6-7.94(m,3H),7.78(s,1H),7.51(s,1H),7.26-7.13(m,6H),6.99(s,1H),6.56-6.42(m ,1H),5.52-5.40(m,4H),4.85-4.60(m,3H),4.59-4.46(m,1H),4.44-4.38(m,1H),3.7 9-3.53(m,6H),3.52-3.44(m,2H),3.08-3.02(m,1H),2.85-2.75(m,1H),2.09(t,J=7.8 Hz,2H),1.95-1.75(m,2H),1.58-1.38(m,4H),1.26-1.09(m,2H),1.04-0.96(m,1H),0.88(t,J=7.1 Hz,3H),0.42-0.26(m,4H). MS m / z(ESI):1062.5[M+H] + . L1-19-S was further analyzed by the following chiral high performance liquid chromatography.

[0336] The chiral high performance liquid chromatography conditions are as follows: [Table 14]

[0337] Under the chiral supercritical fluid chromatography conditions, the retention time of L1-19-S was 3.748 minutes, which was essentially the same as the retention time (3.775 minutes) of compound L1-19-P1 prepared in Example 39-1 under the same chromatographic conditions. Therefore, it was determined that L1-19-S and L1-19-P1 have the same configuration and belong to the same compound.

[0338] For the synthesis of compounds LI-0 and L1-00, see patent documents WO2019195665A1 and WO2020063676A1, respectively. [ka]

[0339] Example 40: Preparation of antibody-drug conjugates 40.1 Antibodies: 40.1.1 Construction and Production of Anti-Human HER2 Monoclonal Antibodies The sequences of the anti-human HER2 monoclonal antibodies are shown in Table 2 below. The nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5, which contains a signal peptide and heavy chain constant region / light chain constant region sequences, to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL. After sequencing verification, the plasmid was extracted and transfected into host cells. Cell culture supernatant secreting the antibody was obtained by culturing.

[0340] [Table 15]

[0341] 40.1.2 Screening for anti-human p95HER2 monoclonal antibodies Anti-human p95HER2 monoclonal antibodies were produced by immunizing mice with hu p95HER2.ECD-Fc protein as the immunogen. Hybridoma cells were produced from splenic lymphocytes selected from mice with high and stable serum antibody titers. Positive hybridoma clones were obtained by screening using conventional methods in the art, such as ELISA and FACS. Antibodies were further produced using serum-free cell culture, purified, and sequenced. Mouse anti-human p95HER2 antibodies were obtained by sequencing.

[0342] Based on a comparison with the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, heavy and light chain variable region germline genes with high homology to mouse antibodies were selected as templates, respectively. The CDRs of the mouse antibody were then grafted onto the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on this, back mutations were performed as necessary to maintain the original affinity and / or hot spot mutations were performed to eliminate the risk of molecular modification. The final optimized humanized antibodies and their corresponding sequences are shown in Table 3.

[0343] [Table 16]

[0344] 40.1.3 Construction and Production of p95HER2 / HER2 Bispecific Antibodies The DVD-Ig format antibody consists of VH1-linker-VH2-CH1-Fc and VL1-linker-VL2-CL, where VH1 and VL1 target HER2, VH2 and VL2 target p95HER2, and the linker is optional. The specific sequences of the bispecific antibodies are shown in Table 4. According to the designed structure, the bispecific antibody light chain and heavy chain expression plasmids were constructed on the pTT5 vector, respectively, and expressed in HEK293 cells.

[0345] [Table 17]

[0346] 40.1.4 Construction and Production of Anti-Human CDH6 Antibodies The sequences of the anti-human CDH6 monoclonal antibodies CDH6-Ab and CDH6-Ab-1 are shown in Table 5 below (antibody sequences are derived from US20200171163A1).

[0347] [Table 18]

[0348] CDH6-Ab-2 and CDH6-Ab-3 were produced by immunizing mice with human CDH6-hFc protein and HEK293T cells overexpressing human CDH6 as the immunogen. Splenic lymphocytes from mice with high serum antibody titers were selected to produce hybridoma cells. Positive hybridoma clones were obtained by screening using conventional methods in the art, such as ELISA and FACS. The antibodies were further produced using serum-free cell culture, purified, and sequenced to obtain mouse-derived anti-human CDH6 antibodies and their variable region sequences. Based on a comparison with the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, heavy and light chain variable region germline genes with high homology to mouse antibodies were selected as templates, respectively. The CDRs of the mouse antibodies were grafted onto the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on this, back mutations were performed as necessary to maintain the original affinity and / or hot spot mutations were performed to eliminate the risk of molecular modification. The VH and VL corresponding to the final optimized humanized antibodies CDH6-Ab-2 and CDH6-Ab-3, as well as the CDR sequences classified by Kabat format, are shown in Table 6.

[0349] [Table 19-1] [Table 19-2]

[0350] The nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5, which contains a signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 70) and heavy chain constant region / light chain constant region sequences, to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL. The plasmid and transfection reagent PEI (Polysciences, product number 24765-1) were added to OPTI-MEM (Gibco, product number 11058021), mixed uniformly, and left to stand for 15 minutes. The mixture was then added to Expi293F cells (Thermofisher, product number A14527) and cultured in a shaker at 37°C, 120 rpm, and 5% CO2. The day after transfection, OPM-293 ProFeed (Shanghai OPM Biosciences, product number F081918-001) and 6 g / L glucose (Sigma, product number G7528) were added. Six days after transfection, antibody-secreting cell culture supernatant was obtained.

[0351] 40.1.5 Construction and Production of Anti-Human LIV-1 Monoclonal Antibodies The sequence of the anti-human LIV-1 monoclonal antibody LIV1-Ab-1 is shown in Table 7 below (the antibody sequence is derived from US20200165335A). The nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5, which contains a signal peptide and heavy chain constant region (sequence) / light chain constant region sequences, to obtain a recombinant plasmid expressing LIV1-Ab-1. After sequencing verification, the plasmid was extracted. The plasmid and transfection reagent PEI (Polysciences, product number: 24765-1) were added to OPTI-MEM (Gibco, product number: 11058021), mixed uniformly, and allowed to stand for 15 minutes. The mixture was then added to Expi293 cells (manufacturer: Thermofisher, product number: A14527) and cultured in a shaker at 5% CO2, 120 rpm, and 37°C. The day after transfection, OPM-293 ProFeed (Shanghai OPM Biosciences, product number: F081918-001) and 6 g / L glucose (manufacturer: Sigma, product number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0352] [Table 20]

[0353] 40.1.6 Construction and Production of Anti-Human ROR1 Monoclonal Antibodies The variable region sequence of the anti-human ROR1 monoclonal antibody ROR1-Ab-1 is derived from Patent WO2020198531A2, the variable region sequence of ROR1-Ab-2 is derived from Patent CN113521300A, and the variable region sequence of ROR1-Ab-3 is derived from Patent WO2020074724A1 (see Table 8). The nucleic acid sequences encoding the above antibody VH and VL were recombined into a pTT59 expression vector containing the CH and CL of human IgG1 to obtain recombinant plasmids expressing ROR1-Ab-1, ROR1-Ab-2, and ROR1-Ab-3, respectively.

[0354] Plasmids and transfection reagent PEI (Polysciences, 24765-1) were added to OPTI-MEM (Gibco, product number: 11058021), mixed uniformly, and left to stand for 15 minutes. Then, Expi293 cells (Thermofisher, A14527) were added and cultured in a shaker at 5% CO2, 120 rpm, and 37°C. The day after transfection, OPM-293 ProFeed (Shanghai OPM Biosciences, F081918-001) and 6 g / L glucose (Sigma, G7528) were added. On day 6 after transfection, the cell supernatant was collected.

[0355] [Table 21-1] [Table 21-2]

[0356] 40.2 Antibody Purification: The above antibodies were purified from cell culture supernatants using Protein A affinity chromatography. The Protein A affinity column was washed with 6 M guanidine hydrochloride (3-5 column volumes) followed by 3-5 column volumes of purified water. For example, 1x PBS (pH 7.4) was used as the equilibration buffer, and the chromatography column was equilibrated with 3-5 column volumes. The cell supernatant was loaded at a slow flow rate and allowed to bind. The flow rate was controlled to achieve a retention time of approximately 1 min or longer. After binding was complete, the column was washed with 1x PBS (pH 7.4) for 3-5 column volumes until the UV absorbance returned to baseline. The sample was eluted with 0.1 M acetic acid / sodium acetate (pH 3.0-3.5) buffer, and the elution peak was collected by UV monitoring. The eluted product was quickly adjusted to pH 5-6 with 1 M Tris-HCl (pH 8.0) and temporarily stored. The eluted product can be subjected to solution exchange using methods well known to those skilled in the art, such as ultrafiltration using an ultrafiltration tube to concentrate the product and then exchange the solution with a desired buffer system, or using molecular exclusion such as G-25 desalting to exchange the solution with a desired buffer system, or using a high-resolution molecular exclusion column such as Superdex 200 to remove multimeric components from the eluted product, thereby improving the purity of the sample. After purification, proteins that meet the purity requirements can be dialyzed for solution exchange and then subjected to subsequent coupling and detection.

[0357] 40.3 Coupling: The above antibody was added to an Amicon-Ultra-30kD ultrafiltration tube and concentrated and exchanged into a 50 mM phosphate, 150 mM NaCl, 1 mM EDTA, pH 6.5 buffer solution. A 7-8x 10 mM tris(2-carboxyethyl)phosphine solution (TCEP) was added to the antibody solution, and the mixture was reduced for 2-3 hours on a thermostatic metal shaker at 25°C. A 15-20x DMSO solution of the corresponding drug-linker compound (prepared according to Examples 37-39 or with reference to the methods in Examples 37-39) was added to the reaction system, and the reaction solution was allowed to couple for 2-16 hours at 25°C. The reaction product was concentrated and exchanged into a phosphate (PBS) buffer by ultrafiltration to remove unreacted free small molecule toxins. Purity and DAR values ​​of the ADC product were analyzed using SEC and LC-MS.

[0358] 40.4 SEC Purity Analysis: Test protein samples were analyzed using SEC-HPLC to characterize the molecular size uniformity of the recombinant protein and measure its purity. The HPLC used in this method was an Agilent 1260, the chromatography column was a TSKgel G3000SWXL (purchased from Tosoh Bioscience), the mobile phase was 200 mM phosphate buffer, pH 7.0 / isopropanol (v / v 9:1), the detection temperature was 25°C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading amount was 50 μg, and the analysis time was 40 min.

[0359] 40.5 DAR Value Measurement: The DAR values ​​of the ADC molecules were measured using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) combined method. First, the test ADC molecules were treated with PNGase F to remove N-glycosylation, then further treated with dithiothreitol (DTT) and incubated at 37°C for 1 hour to reduce them to light and heavy chains. Then, they were analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected onto a Waters ACQUITY Protein BEH size-exclusion chromatography column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min, and the analysis time was 30 min. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometric parameters were spray voltage 3.8 kV, capillary heating temperature 300°C, sheath gas flow rate 35 arb, and parent ion scan range 800–3000. Finally, the mass spectrometry data analysis software Biopharma Finder was used. 4.1, deconvolution was performed using the Respect algorithm, and the molecular weight information of the mass spectrometry peaks of the light chain and heavy chain and the mass spectrometry response signals of each component were calculated, thereby calculating the DAR value of the test ADC sample.

[0360] An isotype control antibody Ab-ISO (anti-FITC-hIgG1 antibody) was prepared in the same manner as above and coupled with a Linker+Payload compound to obtain an isotype control for the corresponding ADC.

[0361] Using the same method as above, antibodies ROR1-Ab-1, ROR1-Ab-2, and ROR1-Ab-3 were each coupled with compound L1-0 to produce ADC-L1-0-8, ADC-L1-0-9, and ADC-L1-0-10, with DAR values ​​of 6.6, 5.5, and 7.5, respectively. Antibody LIV1-Ab-1 was coupled with compound L1-0 to produce ADC-L1-0-7, with a DAR value of 6.7. Antibodies trastuzumab and pertuzumab were each coupled with compound L1-00 to produce ADC-L1-00-2. and ADC-L1-00-11, with DAR values ​​of 6.8 and 7.3, respectively. Antibodies trastuzumab and pertuzumab were coupled to compound L1-0 to obtain ADC-L1-0-2 and ADC-L1-0-11, with DAR values ​​of 7.3 in each case. Antibodies CDH6-Ab, CDH6-Ab-2, and CDH6-Ab-3 were coupled to compound L1-0 to obtain ADC-L1-0-3, ADC-L1-0-5, and ADC-L1-0-6, with DAR values ​​of 7.4, 7.4, and 7.2, respectively.

[0362] [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4] [Table 22-5]

[0363] Example 41: Biological activity and related properties testing All compounds in the following test examples were prepared according to the methods of the above examples of this disclosure.

[0364] Test Example 1: Antitumor cell proliferation activity test of the compound of formula (DH) Cells and materials: Human colorectal cancer cell line HCT116 was purchased from KYINNO Biotechnology, human breast cancer cell line SKBR3 was purchased from ATCC, human ovarian cancer cell line OVCAR3 was purchased from ATCC, bovine serum (Gibco #10099-141C), McCoy's 5A medium (Gibco #16600-082), 1640 medium (Gibco #A10491-01), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA), bovine insulin (Solarbio #I8040) was purchased from Solarbio, and 96-well plates (Greiner Bio-one #655098) was purchased from Corning (USA), and Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0365] Cell culture: HCT116 and SKBR3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0366] Cell proliferation activity was assayed using Cell-Titer Glo reagent to detect the inhibitory activity of compounds on the proliferation of three cell lines: HCT116, SKBR3, and OVCAR3. HCT116 cells (1500 cells per well), SKBR3 cells (3000 cells per well), and OVCAR3 cells (5000 cells per well) were seeded into a 96-well plate and incubated at 37°C and 5% CO2 for 24 hours. After adding the test compound solution (the compound was dissolved in DMSO to a 1 mM compound concentration, then diluted to 3 μM with DMSO, followed by 3-fold dilutions for a total of nine concentrations), 10 μL of the prepared compound solution was transferred to the 96-well plate to achieve final concentrations ranging from 0 to 300 nM. The plates were then incubated at 37°C and 5% CO2. HCT116 cells were cultured for 3 days, and SKBR3 and OVCAR3 cells were cultured for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0367] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0368] Data analysis: % Inhibition was calculated and compound IC 50 was fitted.

[0369] Percent inhibition (% Inhibition) = 1-100% × (Signal-Bottom) / (Top-Bottom) Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0370] Test Results: Under these experimental conditions, the compounds of the present disclosure exhibited relatively strong growth inhibitory activity against HCT116 cells, SKBR3 cells, and OVCAR3 cells. The corresponding anti-cell proliferation activities of the compounds of the present disclosure are shown in Table 10.

[0371] [Table 23]

[0372] In the above table, the symbols used to indicate binding activity have the following meanings:

[0373] "++++" indicates the IC value of the test compound's cell growth inhibitory activity against the cells. 50 The range is shown to be <10 nM.

[0374] "++" indicates the IC50 inhibitory activity of the test compound against the cells 50 The range is indicated as 10 to 100 nM.

[0375] "N / A" indicates not tested.

[0376] Test Example 2: Biacore measurement of anti-p95HER2 / HER2 dual antibody BsAb02-P This experiment was performed using a Biacore 8K (GE) instrument, employing multi-cycle kinetics to compare the test antibody BsAb02-P with human p95HER2 (hu p95HER2.ECD-Fc, SEQ ID NO: 104, the italics are the extracellular domain of human p95HER2, and the underlined Fc tag: MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT). EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The affinity of the antibody to HER2 (purchased from Acro, HE2-H5225) was measured.

[0377] The experimental running buffer was 1x HBS-EP+ buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (Cat. #BR-1006-69, GE). The flow cell temperature was set to 25°C, and the sample vial temperature was set to 16°C. Both were pretreated with the running buffer. A fixed amount of test antibody was affinity-captured onto a Protein A biosensor chip (Cat. #29127556, GE). A fixed concentration of human p95HER2 antigen or human HER2 antigen was then applied to the chip surface. The reaction signal was detected in real time using a Biacore 8K instrument (GE) to obtain binding and dissociation curves. After each dissociation cycle, the antigen-antibody complex was washed and regenerated with a pH 1.5 glycine-HCl regeneration solution (Cat. #BR-1003-54, GE). Specifically, the binding process was detected by injecting different concentrations of human p95HER2 and human HER2 antigen in solution for 240 seconds at a flow rate of 30 μL / min. Starting from 50 nM, the solution was diluted 1:1 to set up a series of concentration gradients, and the dissociation time reached 900 seconds. Finally, the chip surface was regenerated by washing with 10 mM glycine-HCl solution (pH 1.5) at a flow rate of 30 μL / min for 30 seconds.

[0378] The data obtained from the experiment was fitted with a (1:1) Langmuir model using GE Biacore 8K Evaluation version 2.0 software to obtain the association rate (Ka), dissociation rate (Kd), and affinity values ​​(KD), which are specifically shown in Tables 11 and 12. The experimental results demonstrate that the p95HER2 / HER2 dual antibody of the present disclosure can bind to human p95HER2 and HER2 with high affinity.

[0379] [Table 24]

[0380] [Table 25]

[0381] Test Example 3-1: Detection of binding activity of anti-HER2 ADC and antibody to SKBR3 tumor cells by flow cytometry (FACS) SKBR3 cells (from ATCC) were harvested and counted, and 2 × 10 5 Cells were seeded at 1000 cells / well in a 96-well plate (Corning, 3795). Gradient-diluted test samples were added and incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 goat anti-human Fc secondary antibody (JacksonImmuno, 109-605-098) was added and incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and analyzed using a flow cytometer (BD FACSCanto TM The mean fluorescence intensity (MFI) was analyzed using Graphpad Prism software. The data were subjected to a four-parameter curve fitting analysis to obtain the EC 50 The values ​​were obtained and the results are shown in Table 13.

[0382] [Table 26]

[0383] Test Example 3-2: Binding activity test of anti-ROR1 ADC and antibody to stable transformed cell line MCF7-hROR1 clone 2G2 Construction of stable transformant cell lines A nucleotide sequence encoding the human ROR1 amino acid sequence (SEQ ID NO: 105) was cloned into the pLVX lentiviral vector, and viral particles were produced in HEK293T cells. MCF7 cells (purchased from the Chinese Academy of Sciences) were infected with lentivirus and selectively cultured in DMEM (Gibco, product number 11995073) containing 10% (w / w) fetal bovine serum (ExCell Bio, product number FND500) containing 5 μg / mL puromycin (Gibco, product number A1113803) for one week. Using ROR1-Ab-1 and goat anti-mouse IgG (H+L) antibodies (Jackson, product number 115605006), positive cells with different expression levels were sorted into 96-well plates using a FACS Aria III flow cytometer (BD Biosciences). The cells were then cultured at 37°C with 5% (v / v) CO2 for approximately two weeks. Some of the cells were then selected for proliferation. Cells with relatively good proliferation, high fluorescence intensity, and uniformity were selected and subsequently expanded and cryopreserved in liquid nitrogen. The identity of the monoclonal stable transformant cells is shown in Table 14.

[0384] [Table 27] Full length human ROR1 amino acid sequence (SEQ ID NO: 105): QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSD KCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILY Flow cytometry (FACS) detection of binding activity of anti-ROR1 ADCs and antibodies to the stably transformed MCF7-hROR1 clone 2G2 cell line The stable MCF7-hROR1 clone 2G2 transformants constructed in the above steps were expanded to logarithmic growth phase in T-75 cell culture flasks, centrifuged to discard the supernatant, and the cell pellet was washed twice with PBS. The test ADC and antibody were added at an initial concentration of 100 nM, diluted 5-fold to eight points, and incubated at 4°C for 1 h. After washing twice with PBS, the secondary antibody, Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (H+L) (purchased from Jackson Immuno, product number: 109-605-088), was added and incubated at 4°C for 1 h. After washing twice with PBS, the cells were resuspended and analyzed by FACS (FACS Canto™, purchased from BD). A four-parameter curve fitting analysis was performed on the data using Graphpad Prism software to determine the EC. 50 The values ​​were obtained and the results are shown in Table 15.

[0385] [Table 28-1] [Table 28-2]

[0386] Test Example 3-3: Detection of binding activity of anti-LIV-1 ADC and antibody to OVCAR3 tumor cells by flow cytometry (FACS) OVCAR3 cells (derived from ATCC) belong to a tumor model with high LIV-1 expression. After harvesting, OVCAR3 cells were counted and found to be 2 × 10 5 Cells were seeded at 1000 cells / well in a 96-well plate (Corning, 3795). Gradient-diluted test samples were added and incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 Goat anti-human Fc secondary antibody (JacksonImmuno, 109-605-098) was added and incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and analyzed using a flow cytometer (BD FACSCanto TM The mean fluorescence intensity (MFI) was analyzed using Graphpad Prism software. The data were subjected to a four-parameter curve fitting analysis to obtain the EC 50 The values ​​were obtained and the results are shown in Table 16.

[0387] [Table 29]

[0388] Test Example 4-1, Anti-HER2-ADC Tumor Cell Proliferation Activity Test 1 Cells and materials: Human breast cancer cell line SKBR3 was purchased from ATCC, human breast cancer cell line SKBR3-p95HER2 was constructed by Hainan Xiansheng Ziming Pharmaceutical Co., Ltd. (for the construction method, refer to patent application CN202210094806.6), human ovarian cancer cell line SKOV3 was purchased from ATCC, bovine serum, McCoy's 5a medium, penicillin-streptomycin and 0.25% Trypsin-EDTA were purchased from Gibco (USA, product number is the same as in Test Example 1), 96-well plates were purchased from Corning (USA, product number is the same as in Test Example 1), and Cell-Titer Glo reagent was purchased from Promega (USA, product number is the same as in Test Example 1).

[0389] Cell culture: SKBR3 cells, SKBR3-p95HER2 cells, and SKOV3 cells are all cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO2. Cells in the logarithmic growth phase can be used for experiments.

[0390] Detection of cell proliferation activity: Cell-Titer Glo reagent was used to detect the inhibitory activity of ADCs on the proliferation of three cell lines: SKBR3, SKBR3-p95HER2, and SKOV3. SKBR3 cells (3,000 cells per well), SKBR3-p95HER2 cells (3,000 cells per well), and SKOV3 cells (600 cells per well) were seeded into 96-well plates and cultured at 37°C and 5% CO for 24 hours. After adding the test ADC solution (the ADC was diluted with the corresponding cell culture medium to adjust the ADC concentration to 20 nM or 200 nM, and then diluted three-fold with culture medium to a total of eight concentrations), 10 μL of the prepared ADC solution was transferred to the 96-well plate to achieve an initial final concentration of 2 nM or 20 nM. The cells were then cultured at 37°C and 5% CO. SKBR3 cells, SKBR3-p95HER2 cells, and SKOV3 cells were cultured for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0391] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0392] Data analysis: % Inhibition was calculated and compound IC 50 was fitted.

[0393] Percent inhibition (% Inhibition) = 1-100% × (Signal-Bottom) / (Top-Bottom) Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0394] Experimental results: Under the experimental conditions, the ADC of the present disclosure exhibited relatively strong growth inhibitory activity against SKBR3 cells, SKBR3-p95HER2 cells, and SKOV3 cells, as shown in Table 17 in detail.

[0395] [Table 30]

[0396] Test Example 4-2, Anti-HER2-ADC Tumor Cell Proliferation Activity Test 2 Cells and Materials: Human breast ductal carcinoma cell line T47D (medium-expressing cell line) was purchased from ATCC, human breast cancer cell line SKBR3 was purchased from ATCC, McCoy's 5a medium (Gibco #16600-082), 1640 medium (Gibco #A10491-01), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA), 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0397] Cell culture: SKBR3 cells are cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and T47D cells are cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Both cell lines are cultured at 37°C in 5% CO2, and cells in the logarithmic growth phase can be used for experiments.

[0398] Detection of cell proliferation activity: Cell-Titer Glo reagent was used to detect the inhibitory activity of ADCs on the proliferation of two cell lines, SKBR3 and T47D. SKBR3 and T47D cells were digested and dispersed from cell culture flasks, resuspended in the corresponding fresh medium, and the cell density was adjusted. T47D cells were seeded at 2000 cells / 90 μL per well into a 96-well plate and cultured overnight at 37°C and 5% CO2. The ADC was diluted to 1000 nM with complete medium and diluted three-fold for a total of eight concentration gradients. 10 μL of the diluted ADC solution was then transferred to the 96-well plate, resulting in an initial final ADC concentration of 100 nM. The 96-well plate was cultured at 37°C and 5% CO2 for 7 days. Cell-Titer Glo reagent was added to detect cell activity.

[0399] SKBR3 cells were seeded into a 96-well plate at 5000 cells / 90 μL per well and cultured overnight at 37°C in 5% CO2. The ADC was diluted to 1000 nM with complete medium and diluted five-fold to a total of nine gradient concentrations. 10 μL of the diluted ADC solution was then transferred to the 96-well plate, resulting in an initial final ADC concentration of 100 nM. The 96-well plate was cultured at 37°C in 5% CO2 for 3 days. Cell-Titer Glo reagent was added to detect cell activity.

[0400] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0401] Data Analysis: The percent inhibition (% Inhibition) was calculated and the compound IC 50 was fitted.

[0402] Percent inhibition (% Inhibition) = 1 - 100% x (Signal-Bottom) / (Top-Bottom).

[0403] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0404] Test Results: Under these experimental conditions, the anti-HER2-ADCs of the present disclosure exhibited relatively strong growth inhibitory activity against the human ductal carcinoma cell line T47D and the human breast cancer cell line SKBR3, as shown in Table 18.

[0405] [Table 31]

[0406] Test Example 5: Cell-binding activity of anti-CDH6 antibodies to their corresponding ADCs OVCAR3 cells were harvested and counted, and 2 x 10 5 Cells were seeded at 1000 cells / well in a 96-well plate (Corning, 3795). Gradient-diluted test samples were added and incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 Goat anti-human Fc secondary antibody (JacksonImmuno, 109-605-098) was added and incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and analyzed using a flow cytometer (BD FACSCanto TMThe mean fluorescence intensity (MFI) was analyzed using Graphpad Prism software. The data were subjected to a four-parameter curve fitting analysis to obtain the EC 50 The values ​​were obtained and the results are shown in Table 19.

[0407] [Table 32]

[0408] Test Example 6-1: Anti-CDH6-ADC anti-tumor cell proliferation activity test Cells and Materials: Human ovarian cancer cell line OVCAR3 (a CDH6-high expressing cell line) was purchased from ATCC. Human ovarian teratoma cell line PA-1 was purchased from ATCC. Bovine serum, 1640 medium (Gibco #A10491-01), MEM medium (Gibco #11095-080), MEM NEAA (Gibco #11140-050), sodium pyruvate (Gibco #11360-070), penicillin-streptomycin, and 0.25% trypsin-EDTA (Gibco #25200-056) were purchased from Gibco. Bovine insulin was purchased from Solarbio. 96-well plates were purchased from Corning (USA). Cell-Titer Glo reagent was purchased from Promega (USA).

[0409] Cell culture: OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin at 37°C in 5% CO2. PA-1 cells were cultured in MEM medium containing 10% fetal bovine serum, 1% MEM NEAA, 1% sodium pyruvate, and 1% penicillin-streptomycin at 37°C in 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0410] Detection of cell proliferation activity: Cell-Titer Glo reagent was used to detect the inhibitory activity of ADCs on the proliferation of two cell lines, OVCAR3 and PA-1. OVCAR3 and PA-1 cells were digested and dispersed from cell culture flasks, resuspended in the corresponding fresh medium, and the cell density was adjusted. OVCAR3 cells (5,000 cells per well) and PA-1 cells (800 cells per well) were seeded into 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. After adding the test ADC solution (the ADC was diluted with the corresponding cell culture medium to a concentration of 100 nM, then diluted three-fold with medium for a total of eight concentrations), 10 μL of the prepared ADC solution was transferred to the 96-well plate and cultured at 37°C and 5% CO2 for a final initial concentration of 0–10 nM. OVCAR3 and PA-1 cells were cultured for 5 days. Cell-Titer Glo reagent was added to detect cell proliferation.

[0411] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0412] Data analysis: % Inhibition was calculated and compound IC 50 was fitted.

[0413] Percent inhibition (% Inhibition) = 1-100% × (Signal-Bottom) / (Top-Bottom) Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0414] Experimental Results: Under the experimental conditions, the anti-CDH6-ADC of the present disclosure exhibited relatively strong growth inhibitory activity against both the human ovarian cancer cell line OVCAR3 and the human ovarian teratoma cell line PA-1 cells, as shown in Table 20.

[0415] [Table 33-1] [Table 33-2]

[0416] Test Example 6-2: Anti-LIV-1-ADC antitumor cell proliferation activity test Cells and Materials: Human ovarian cancer cell line OVCAR3 and human non-small cell lung cancer cell line H838 were purchased from ATCC. 1640 medium (Gibco #A10491-01), penicillin-streptomycin (Gibco #15140-122), and 0.25% trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA). Bovine insulin (Solarbio #I8040) was purchased from Solarbio (USA). 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA). Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0417] Cell culture: OVCAR3 cells are cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin at 37°C and 5% CO2. H838 cells are cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase can be used for experiments.

[0418] Detection of cell proliferation activity: Cell-Titer Glo reagent was used to detect the inhibitory activity of ADCs on the proliferation of OVCAR3 and H838 cell lines. OVCAR3 or H838 cells were digested and dispersed from cell culture flasks, resuspended in the corresponding fresh medium, and seeded into 96-well plates at 1500 cells / 90 μL per well for OVCAR3 cells and 450 cells / 90 μL per well for H838 cells. The cells were then cultured overnight at 37°C and 5% CO2. The ADC was diluted to 5000 nM with complete medium and diluted three-fold for a total of eight gradients. 10 μL of the diluted ADC solution was then transferred to the 96-well plate, resulting in an initial final ADC concentration of 500 nM. The 96-well plate was then cultured at 37°C and 5% CO2 for 5 days. Cell-Titer Glo reagent was added to detect cell proliferation.

[0419] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0420] Data Analysis: The percent inhibition (% Inhibition) was calculated and the compound IC 50 was fitted.

[0421] Percent inhibition (% Inhibition) = 1 - 100% x (Signal-Bottom) / (Top-Bottom).

[0422] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0423] Test Results: Under these experimental conditions, the anti-LIV-1 ADCs of the present disclosure exhibited relatively strong growth inhibitory activity against the human ovarian cancer cell line OVCAR3 and the human non-small cell lung cancer cell line H838, as shown in Table 21 in detail.

[0424] [Table 34]

[0425] Test Example 6-3: Anti-ROR1-ADC anti-tumor cell proliferation activity test Cells and Materials: Human breast cancer cell line hROR1-MCF7 was constructed at Hainan Xiansheng Ziming Pharmaceutical Co., Ltd. DMEM medium (Gibco #11995-065), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA). 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA). Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0426] Cell culture: hROR1-MCF7 cells are cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO2. Cells in the logarithmic growth phase can be used for experiments.

[0427] Detection of cell proliferation activity: Cell-Titer Glo reagent was used to detect the inhibitory activity of ADCs on the proliferation of the hROR1-MCF7 cell line. hROR1-MCF7 cells were digested and dispersed from cell culture flasks, resuspended in the corresponding fresh medium, and the cell density was adjusted. The hROR1-MCF7 cells were seeded into a 96-well plate at 1700 cells / 90 μL per well and cultured overnight at 37°C and 5% CO2. The ADC was diluted to 500 nM with complete medium and diluted four-fold to a total of nine concentration gradients. 10 μL of the diluted ADC solution was then transferred to the 96-well plate, resulting in an initial final ADC concentration of 50 nM. The 96-well plate was cultured at 37°C and 5% CO2 for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0428] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0429] Data Analysis: The percent inhibition (% Inhibition) was calculated and the compound IC 50 was fitted.

[0430] Percent inhibition (% Inhibition) = 1 - 100% x (Signal-Bottom) / (Top-Bottom).

[0431] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0432] Test Results: Under these experimental conditions, the anti-ROR1-ADCs of the present disclosure exhibited relatively strong growth inhibitory activity against the human ovarian cancer cell line hROR1-MCF7, as shown in Table 22 in detail.

[0433] [Table 35] Test Example 7, OVCAR3 subcutaneous tumor model drug efficacy evaluation-1 Experimental Reagents: Human ovarian carcinoma OVCAR3 cells were purchased from ATCC, RPMI-1640 medium was purchased from Gibco (product number A104910), fetal bovine serum was purchased from Excell (product number FND500), penicillin-streptomycin was purchased from Gibco (product number 15140122), bovine insulin was purchased from Yeasen (product number 40107ES60), 0.25% trypsin-EDTA was purchased from Gibco (product number 25200-072), D-PBS (phosphate buffered saline without calcium and magnesium ions) was purchased from Hyclone (product number SH30256.01), and Matrigel was purchased from Corning (product number 356237).

[0434] Experimental Method: Animal information: Balb / c nude mice, female, 5-6 weeks old, weighing approximately 14-20 grams. The animals were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment, with each cage individually ventilated and ventilated. All animals were fed standard certified commercial laboratory diets and had free access to water.

[0435] Cell Culture: Human ovarian cancer OVCAR3 cell line was cultured in vitro in RPMI-1640 supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin, and 10 μg / mL bovine insulin at 37°C in a 5% CO2 incubator. Cells were passaged weekly by routine digestion with 0.25% trypsin-EDTA digestion solution. Cells were harvested and counted when 80%-90% cell saturation and the required number were reached.

[0436] Cell seeding: 0.1 mL of OVCAR3 cell suspension (1 × 10 7 Mice were subcutaneously inoculated with tumor cells (containing 1000 cells and RPMI-1640:Matrigel at a volume ratio of 1:1) into the armpits. On day 26 after cell inoculation, the mice were randomly divided into groups based on tumor volume and administered the treatment. The day of grouping was designated Day 0.

[0437] Tumor Measurements and Laboratory Indicators: Tumor diameters were measured twice a week using a vernier caliper. Tumor volume was calculated using the formula V = 0.5a × b 2 where a and b indicate the long and short diameters of the tumor, respectively. Mice were weighed twice a week.

[0438] The tumor growth inhibition rate (TGI) (%) was used to evaluate the tumor-inhibiting effect of the test drug: TGI (%) = [(1 - (mean tumor volume at the end of treatment in a treatment group - mean tumor volume at the start of treatment in that treatment group) / (mean tumor volume at the end of treatment in the vehicle control group - mean tumor volume at the start of treatment in the vehicle control group)] × 100%.

[0439] Test Results: In the mouse subcutaneously implanted tumor OVCAR3 model, ADC-L1-14-P1-5 had a significant inhibitory effect on tumor growth after a single intravenous administration at a dose of 3 mg / kg (P<0.0001). The results are shown in Table 23 and Figure 2.

[0440] [Table 36]

[0441] In the mouse subcutaneously implanted tumor OVCAR3 model, ADC-L1-19-P1-5 had a significant inhibitory effect on tumor growth after a single intravenous administration at a dose of 3 mg / kg (P<0.0001). The results are shown in Table 24 and Figure 3.

[0442] [Table 37-1] [Table 37-2]

[0443] Test Example 8, ADC plasma stability test The ADC molecules (final concentration 100 μg / mL) were incubated with human plasma (Oribiotech, PB021-C) and monkey plasma (Shinuoda Biological Technology Co., Ltd., SND-X0107) in an incubator at 37°C. The day of incubation was designated as day 0, and samples were subsequently taken on days 7, 14, and 28, respectively, for detection of free small molecules.

[0444] A 20 μL sample was taken, 300 μL of the internal standard working solution (prepared in acetonitrile) was added, and the mixture was vortexed for 5 minutes to mix evenly, centrifuged (14,000 rpm) for 5 minutes, and 4 μL of the supernatant was injected into an LC-MS / MS (API 6500+) for analysis, and the results are shown in Table 25. The results indicate that the tested ADC molecules are relatively stable in both human and monkey plasma.

[0445] [Table 38]

[0446] Test Example 9: OVCAR3 subcutaneous tumor model drug efficacy evaluation-2 Experimental Reagents: Human ovarian cancer OVCAR3 cells: ATCC RPMI-1640 culture solution: Gbico, Cat No.: A104910 Fetal bovine serum: Excell, FND500 Bovine insulin: Yeasen, 40107ES60 0.25% Trypsin-EDTA: Gibco, Cat No: 25200-072 D-PBS (phosphate buffer solution without calcium and magnesium ions): Hyclone, Cat. No.: SH30256.01 Matrigel:Corning, Cat.No.:356237 Experimental Method: Animal information: Balb / c nude mice, female, 5-6 weeks old, weighing approximately 14-20 grams. The animals were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment, with each cage individually ventilated and ventilated. All animals were fed standard certified commercial laboratory diets and had free access to water.

[0447] Cell Culture: Human ovarian cancer OVCAR3 cell line was cultured in vitro in RPMI-1640 (cell culture medium) supplemented with 20% fetal bovine serum, 1% Pen-Strep, and 10 μg / mL bovine insulin at 37°C in a 5% CO2 incubator. Cells were passaged weekly by routine digestion with 0.25% trypsin-EDTA digestion solution. When cell saturation reached 80%-90% and the required number of cells was reached, cells were harvested and counted.

[0448] Cell seeding: 0.1mL / (1×10 7 OVCAR3 cell suspension (containing 1000 cells) in RPMI-1640:Matrigel (volume ratio: 1:1) was inoculated subcutaneously into the armpits of each mouse. On day 23 after cell inoculation, mice were randomly assigned to groups based on tumor volume and administered the treatment. The day of grouping was designated Day 0.

[0449] Administration: ADC-L1-19-P1-7 and the isotype control ADC-L1-19-P1-ISO were both administered intraperitoneally (Q4D) at a dose of 3 mg / kg, with 6 mice per group.

[0450] Tumor Measurements and Laboratory Indicators: Tumor diameters were measured twice a week using a vernier caliper. Tumor volume was calculated using the formula V = 0.5a × b 2 where a and b indicate the long and short diameters of the tumor, respectively. Mice were weighed twice a week.

[0451] The tumor growth inhibition rate (TGI) (%) was used to evaluate the tumor inhibitory effect of the compound: TGI (%) = [(1 - (mean tumor volume at the end of treatment for a treatment group - mean tumor volume at the start of treatment for that treatment group) / (mean tumor volume at the end of treatment for the vehicle control group - mean tumor volume at the start of treatment for the vehicle control group)] × 100%.

[0452] Test Results: See Table 26, Figures 4 and 5.

[0453] [Table 39]

[0454] Testing conclusion: In a mouse subcutaneous tumor xenograft OVCAR3 model, the compound ADC-L1-19-P1-7 of the present disclosure and the isotype control ADC-L1-19-P1-ISO had a significant inhibitory effect on tumor growth when administered intraperitoneally at 3 mg / kg once every four days (P<0.0001), but the tumor inhibition of the isotype control ADC-L1-19-P1-ISO was significantly weaker than that of ADC-L1-19-P1-7 (P<0.001). In this example, the doses tested were not found to affect mouse body weight, did not cause any mouse deaths, and were well tolerated by the mice.

[0455] Test Example 10: Efficacy evaluation of NCI-H838 subcutaneous tumor model Experimental Reagents: Human lung cancer NCI-H838 cells: Cobioer RPMI-1640 culture solution: Gbico, Cat No.: 61870-036 Fetal bovine serum: Gibco, Cat. No.: 10099-141C 0.25% Trypsin-EDTA: Gibco, Cat No: 25200-072 D-PBS (phosphate buffer solution without calcium and magnesium ions): Hyclone, Cat. No.: SH30256.01 Matrigel:Corning, Cat.No.:356237 Experimental Method: Animal information: B-NDG mice, female, 5-6 weeks old, weighing approximately 14-20 grams. The animals were purchased from Baiaosaitu. The mice were housed in an SPF-grade environment, with each cage individually ventilated and ventilated. All animals were fed standard certified commercial laboratory diets and had free access to water.

[0456] Cell Culture: Human lung cancer NCI-H838 cell line was cultured in vitro in RPMI-1640 (cell culture medium) supplemented with 10% fetal bovine serum and 1% Pen-Strep at 37°C in a 5% CO2 incubator. Cells were passaged twice weekly by routine digestion with 0.25% trypsin-EDTA digestion solution. When cell saturation reached 80%-90% and the required number of cells was reached, cells were harvested and counted.

[0457] Cell seeding: 0.1mL / (1×10 7 A suspension of NCI-H838 cells (containing 100 cells) in RPMI-1640:Matrigel (volume ratio: 1:1) was inoculated subcutaneously into the armpits of each mouse. On day 23 after cell inoculation, mice were randomly assigned to groups based on tumor volume and administered the treatment. The day of grouping was designated Day 0.

[0458] Administration: ADC-L1-19-P1-7 and the isotype control ADC-L1-19-P1-ISO were both administered intraperitoneally (Q4D) at a dose of 3 mg / kg, with 6 mice per group.

[0459] Tumor Measurements and Laboratory Indicators: Tumor diameters were measured twice a week using a vernier caliper. Tumor volume was calculated using the formula V = 0.5a × b 2 where a and b indicate the long and short diameters of the tumor, respectively. Mice were weighed twice a week.

[0460] The tumor growth inhibition rate (TGI) (%) was used to evaluate the tumor inhibitory effect of the compound: TGI (%) = [(1 - (mean tumor volume at the end of treatment for a treatment group - mean tumor volume at the start of treatment for that treatment group) / (mean tumor volume at the end of treatment for the vehicle control group - mean tumor volume at the start of treatment for the vehicle control group)] × 100%.

[0461] Test Results: See Table 27, Figures 6 and 7.

[0462] [Table 40] Testing conclusion: In the mouse subcutaneous tumor NCI-H838 model, the compound ADC-L1-19-P1-7 of the present disclosure and the isotype control ADC-L1-19-P1-ISO had a significant inhibitory effect on tumor growth when administered intraperitoneally at 3 mg / kg once every four days (P<0.0001), but the tumor inhibition of the isotype control ADC-L1-19-P1-ISO was significantly weaker than that of ADC-L1-19-P1-7 (P<0.001). In this example, the doses tested were not found to affect mouse body weight, did not cause any mouse deaths, and were well tolerated by the mice.

[0463] Test Example 11: Bystander effect test of anti-LIV-1 ADC Cells and Materials: Human ovarian cancer cell line OVCAR3 was purchased from ATCC, human non-small cell lung cancer cell line NCI-H838-hLIV1-KO was constructed in-house, bovine serum (Gibco #10099-141C), 1640 medium (Gibco #A10491-01), penicillin-streptomycin (Gibco #15140-122), and 0.25% trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA), bovine insulin (Solarbio #I8040) was purchased from Solarbio, 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0464] Construction of NCI-H838-hLIV1-KO: Six sgRNAs were designed against the human LIV1 gene. The sgRNAs were then cloned into the pLVX lentiviral vector and viral particles were produced in HEK293T cells (purchased from the Chinese Academy of Sciences). NCI-H838 (purchased from ATCC) cells were infected with lentivirus and selectively cultured in RPMI 1640 medium containing 10% (w / w) fetal bovine serum and 1.5 μg / mL puromycin (purchased from Gibco, product number A1113802) for two weeks to obtain the H838-LIV1 KO pool cell line. H838-LIV1 KO monoclonal cells were labeled with human anti-LIV1 antibody (Ladiratuzumab, self-produced) and goat anti-human IgG (H+L) antibody (Jackson, product number: 109605088) and sorted into 96-well plates using a FACSAria II flow cytometer (purchased from BD Biosciences). They were then cultured at 37°C and 5% (v / v) CO2 for approximately two weeks. Some monoclonal wells were selected for expansion. The expanded clones were screened by flow cytometry. Monoclonal cell lines with relatively high proliferation and low fluorescence intensity were selected and further expanded and cryopreserved in liquid nitrogen.

[0465] Cell culture: OVCAR3 cells are cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin. NCI-H838-hLIV1-KO cells are cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Both cell lines are cultured at 37°C in 5% CO2. Cells in the logarithmic growth phase can be used for experiments.

[0466] Detection of bystander effects: After incubation of the test ADC with LIV-1-positive OVCAR3 cells, the supernatant was transferred to LIV-1-negative NCI-H838-hLIV1-KO cells and continued incubation. The effect of the supernatant on the proliferation activity of NCI-H838-hLIV1-KO cells was detected using Cell-Titer Glo reagent to reflect the bystander effect of the ADC. OVCAR3 cells were digested and dispersed from the cell culture flask, resuspended in the corresponding fresh medium, adjusted to a cell density of 10,000 cells / 180 μL / well, seeded into a 96-well plate, and cultured overnight at 37°C and 5% CO2. The ADC was diluted to 5,000 nM with complete medium and diluted five-fold for a total of eight concentration gradients. 20 μL of the diluted ADC solution was then transferred to the 96-well plate, i.e., the initial ADC concentration was 500 nM. The 96-well plate was cultured at 37°C and 5% CO2 for 5 days. 150 μL of the supernatant from the OVCAR3 cell culture plate was aspirated and transferred to a plate of NCI-H838-hLIV1-KO cells (450 cells / 50 μL / well) seeded one day earlier, and cultured at 37°C and 5% CO2 for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0467] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0468] Data Analysis: The percent inhibition (% Inhibition) was calculated and the compound IC 50 was fitted.

[0469] Percent inhibition (% Inhibition) = 1 - 100% x (Signal-Bottom) / (Top-Bottom).

[0470] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0471] Test Results: Under these experimental conditions, the supernatant obtained after incubation of the test ADC with LIV-1-positive cells OVCAR3 was subsequently incubated with LIV-1-negative cells NCI-H838-hLIV1-KO, and the killing of the negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 28, indicating that the anti-LIV1-ADC of the present disclosure has a relatively good bystander effect.

[0472] [Table 41]

[0473] Test Example 12: Bystander effect test of anti-ROR1-ADC Cells and Materials: Human breast cancer cell line hROR1-MCF7 was constructed at Hainan Xiansheng Ziming Pharmaceutical Co., Ltd. Human breast cancer cell line MCF7 was purchased from ATCC. Bovine serum (Gibco #10099-141C), DMEM medium (Gibco #11995-065), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA). 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA). Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0474] Cell culture: hROR1-MCF7 cells and MCF7 cells are cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO2. Cells in the logarithmic growth phase can be used for experiments.

[0475] Detection of bystander effect: After incubation of the test ADC with ROR1-positive hROR1-MCF7 cells, the supernatant was transferred to ROR1-negative MCF7 cells and continued incubation. The effect of the supernatant on MCF7 cell proliferation activity was detected using Cell-Titer Glo reagent to reflect the bystander effect of the ADC. hROR1-MCF7 cells were digested and dispersed from the cell culture flask, resuspended in the corresponding fresh medium, and the cell density was adjusted to 20,000 cells / 180 μL / well. They were then seeded into a 96-well plate and cultured overnight at 37°C and 5% CO2. The ADC was diluted to 5,000 nM with complete medium and diluted three-fold for a total of eight concentration gradients. 20 μL of the diluted ADC solution was then transferred to the 96-well plate, i.e., the initial ADC concentration was 500 nM. The 96-well plate was cultured at 37°C and 5% CO2 for 5 days. 150 μL of the supernatant from the hROR1-MCF7 cell culture plate was aspirated and transferred to a plate of MCF7 cells seeded one day earlier (1,500 cells / 50 μL / well), and cultured at 37°C in 5% CO for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0476] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0477] Data Analysis: The percent inhibition (% Inhibition) was calculated and the compound IC 50 was fitted.

[0478] Percent inhibition (% Inhibition) = 1 - 100% x (Signal-Bottom) / (Top-Bottom).

[0479] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0480] Test Results: Under these experimental conditions, the supernatant obtained after incubation of the test ADC with ROR1-positive cells hROR1-MCF7 was subsequently incubated with ROR1-negative cells MCF7, and the killing of the negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 29, indicating that the anti-ROR1-ADC of the present disclosure has a relatively good bystander effect.

[0481] [Table 42]

[0482] Test Example 13: Bystander effect test of anti-HER2-ADC Cells and Materials: Human breast cancer cell line SKBR3 was purchased from ATCC, human small cell lung cancer cell line NCI-H2171 was purchased from ATCC, bovine serum (Gibco #10099-141C), McCoy's 5a medium (Gibco #16600-082), 1640 medium (Gibco #A10491-01), penicillin-streptomycin (Gibco #15140-122), and 0.25% trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA), 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0483] Cell culture: SKBR3 cells are cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and NCI-H2171 cells are cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Both cell lines are cultured at 37°C in 5% CO2, and cells in the logarithmic growth phase can be used for experiments.

[0484] Detection of bystander effect: After incubation of the ADC with HER2-positive SKBR3 cells, the supernatant was transferred to HER2-negative NCI-H2171 cells and continued incubation. The effect of the supernatant on the proliferation activity of NCI-H2171 cells was detected using Cell-Titer Glo reagent to reflect the bystander effect of the ADC. SKBR3 cells were digested and dispersed from the cell culture flask, resuspended in the corresponding fresh medium, and the cell density was adjusted to 10,000 cells / 180 μL / well. They were seeded into 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC was diluted to 5,000 nM with complete medium and diluted three-fold for a total of eight concentration gradients. 20 μL of the diluted ADC solution was then transferred to the 96-well plate, i.e., the initial ADC concentration was 500 nM. The 96-well plate was cultured at 37°C and 5% CO2 for 3 days. 150 μL of the supernatant from the SKBR3 cell culture plate was aspirated and transferred to a plate of NCI-H2171 cells seeded one day earlier (6,000 cells / 50 μL / well), and cultured at 37°C in 5% CO for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0485] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0486] Data Analysis: The percent inhibition (% Inhibition) was calculated and the compound IC 50 was fitted.

[0487] Percent inhibition (% Inhibition) = 1 - 100% x (Signal-Bottom) / (Top-Bottom).

[0488] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0489] Test Results: Under these experimental conditions, the supernatant obtained after incubation of the test ADC with HER2-positive cells SKBR3 was subsequently incubated with HER2-negative cells NCI-H2171, and the killing of the negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 30, indicating that the anti-HER2-ADC of the present disclosure has a relatively good bystander effect.

[0490] [Table 43]

[0491] Test Example 14: Bystander effect test of anti-CDH6-ADC Cells and materials: Human ovarian cancer cell line OVCAR3 was purchased from ATCC, human ovarian cancer cell line SKOV3 was purchased from ATCC, bovine serum (Gibco #10099-141C), 1640 medium (Gibco #A10491-01), McCoy's 5a medium (Gibco #16600-082), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA), bovine insulin (Solarbio #I8040) was purchased from Solarbio (USA), and 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA). Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega Corporation (USA).

[0492] Cell culture: OVCAR3 cells are cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin. SKOV3 cells are cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Both cell lines are cultured at 37°C in 5% CO2. Cells in the logarithmic growth phase can be used for experiments.

[0493] Detection of bystander effect: After incubation of the ADC with CDH6-positive OVCAR3 cells, the supernatant was transferred to CDH6-negative SKOV3 cells and incubated. The effect of the supernatant on SKOV3 cell proliferation was detected using Cell-Titer Glo reagent to reflect the bystander effect of the ADC. OVCAR3 cells were digested and dispersed from cell culture flasks, resuspended in the corresponding fresh medium, and the cell density was adjusted to 20,000 cells / 180 μL / well. Then, the cells were seeded into 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC was diluted to 4500 nM with complete medium and diluted three-fold for a total of eight concentration gradients. 20 μL of the diluted ADC solution was then transferred to the 96-well plate, resulting in an initial ADC concentration of 450 nM. The 96-well plate was cultured at 37°C and 5% CO2 for 5 days. 150 μL of the supernatant from the OVCAR3 cell culture plate was aspirated and transferred to a plate of SKOV3 cells seeded one day earlier (700 cells / 50 μL / well), and cultured at 37°C in 5% CO for 7 days. Cell-Titer Glo reagent was added to detect cell activity.

[0494] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group was treated with the same volume of medium without adding cells, and all other operations were the same as those of the experimental group. The positive control group was treated with no test drug, and all other operations were the same as those of the experimental group.

[0495] Data Analysis: The percent inhibition (% Inhibition) was calculated and the compound IC 50was fitted.

[0496] Percent inhibition (% Inhibition) = 1 - 100% x (Signal-Bottom) / (Top-Bottom).

[0497] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0498] Test Results: Under these experimental conditions, the supernatant obtained after incubation of the test ADC with CDH6-positive OVCAR3 cells was subsequently incubated with CDH6-negative SKOV3 cells, and the killing of the negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 31, indicating that the anti-CDH6-ADC of the present disclosure has a relatively good bystander effect.

[0499] [Table 44]

Claims

1. The general structural formula is Pc-(L-D) ｎ A ligand-drug complex or a pharmaceutically acceptable salt thereof, wherein Pc is a ligand unit, L is a linker unit, D is a drug unit represented by the following formula (D-I), 【Chemical 1】 wherein, X is selected from NH or O, R １ is selected from halogen, CN, C １ -C ６ alkyl group, C ３ -C ６ cycloalkyl group or C ２ -C ６ alkynyl group, and the C １ -C ６ alkyl group, C ３ -C ６ cycloalkyl group or C ２ -C ６ alkynyl group is optionally substituted with one or more R ａ１ s, X １ is selected from CR ２ or N, and R ２ is selected from H, halogen, CN, or R １ , R ２ forms, together with the atoms connecting thereto, a 5- to 6-membered heterocyclyl group, and the 5- to 6-membered heterocyclyl group is optionally substituted with one or more R ａ２ s, R ４ is selected from H, C １ -C ３ alkyl group, C ３ -C ６ cycloalkyl group or 4- to 7-membered heterocyclyl group, and the C １ -C ３ alkyl group, C ３ -C ６ cycloalkyl group or 4- to 7-membered heterocyclyl group is optionally substituted with one or more R ａ４ and R ５ is selected from H, halogen, CN, NH ２ or NO ２ or R １ R ５ , together with the atoms to which they are attached, forms a 5- to 6-membered heterocyclyl group, a 5- to 6-membered heteroaryl group or C ５ -C ７ cycloalkenyl group, and the 5- to 6-membered heterocyclyl group, 5- to 6-membered heteroaryl group or C ５ -C ７ cycloalkenyl group is optionally substituted with one or more R ａ５ s, R ６ is selected from H or C １ -C ３ and an alkyl group, R ７ is selected from H, C １ -C ３ alkyl group or C ３ -C ６ cycloalkyl group, or R ６ , R ７ , together with the C atom to which they are attached, forms a C ３ -C ６ cycloalkyl group, and the said C ３ -C ６ cycloalkyl group is optionally substituted with one or more R ａ７ ; Each R ａ１ , R ａ２ , R ａ４ , R ａ５ , R ａ７ is independently selected from D, halogen, CN, =O, OH, NH ２ , C １ -C ３ alkyl group, C ３ -C ６ cycloalkyl group or 4- to 7-membered heterocyclyl group, and the OH, NH ２ , C １ -C ３ alkyl group, C ３ -C ６ cycloalkyl group or 4- to 7-membered heterocyclyl group is optionally substituted with one or more R ｂ ; Each R ｂ is independently halogen, CN, =O, C １ -C ３ alkyl group, OH, O(C １ -C ３ alkyl group), NH ２ , NH(C １ -C ３ alkyl group) or N(C １ -C ３ alkyl group) ２ selected from As conditions, i) when R １ is selected from methyl groups and R ２ is selected from F, R ６ , R ７ form a cyclopropyl group together with the C atom to which they are attached, ii) when X is selected from NH, R ５ is not selected from H, and iii) the compound represented by formula (D-I) is 【Chemical 2】 does not contain, and n is a real number from 1 to 16, a ligand-drug complex or a pharmaceutically acceptable salt thereof.

2. R １ is selected from halogen, C １ -C ３ alkyl group, C ３ -C ６ cycloalkyl group or C ２ -C ３ alkynyl group, The ligand-drug complex or a pharmaceutically acceptable salt thereof according to Claim 1.

3. R ２ is selected from H, halogen, CN, or R １ , R ２ forms a 5- or 6-membered heterocyclyl group together with the atoms connecting thereto, the 5- or 6-membered heterocyclyl group contains 1 or 2 oxygen atoms as ring atoms, and the 5- or 6-membered heterocyclyl group is optionally substituted with one or more D atoms The ligand-drug complex or a pharmaceutically acceptable salt thereof according to Claim 1 or 2.

4. R ２ is selected from H, F or Cl, or R １ , R ２ together with the atoms connected thereto [Chemical Formula 3] forming, The ligand-drug complex or a pharmaceutically acceptable salt thereof according to Claim 1 or 2.

5. R ５ is selected from H, Cl, F, NH ２ or NO ２ or R １ R ５ together with the atoms connected thereto 【Chemical Formula 4】 forming, The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 4.

6. R ４ is selected from H or C １ -C ３ alkyl group The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 5.

7. R ７ is selected from H, C １ -C ３ alkyl group or C optionally substituted with one or more D ３ -C ６ cycloalkyl group, or R ６ , R ７ , together with the C atom to which they are attached, forms a C ３ -C ６ cycloalkyl group The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 6.

8. The structural unit 【Chemical Formula 5】 is 【Chemical Formula 6】 selected from The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 7.

9. The drug unit represented by formula (D-I) is selected from the drug units represented by formula (D-Ia), [Chemical Formula 7] Among them, R １ , R ２ , R ４ , R ５ , R ６ , R ７ is as defined in any one of claims 1 to 8. The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 8.

10. The compound represented by formula (D-I) is a compound shown below: 【Chemical Formula 8】 【Chem.】 [Chemical] selected from The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 9.

11. The linker unit L is 【Chemical Formula 9】 selected from, the a-terminus of which is covalently bonded to the ligand unit Pc, and the b-terminus of which is covalently bonded to the drug unit D, wherein m1 and m2 are each independently selected from integers of 2 to 8, m3 is selected from integers of 1 to 16, L １ , L ２ is each independently selected from peptide residues consisting of 1 to 8 amino acids, and the peptide residue is further optionally halogen, CN, =O, C １ -C ６ alkyl group, OH, O(C １ -C ６ alkyl group), NH ２ , NH(C １ -C ６ alkyl group), N(C １ -C ６ alkyl group) ２ , C ３ -C ６ substituted with one or more substituents of a cycloalkyl group and a 4- to 7-membered heterocyclyl group The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 10.

12. Said L １ , L ２ is independently selected from peptide residues consisting of 2, 3 or 4 amino acids, and said peptide residue is further optionally substituted with one or more substituents selected from halogen, CN, =O, C １ -C ６ alkyl group, OH, O(C １ -C ６ alkyl group), NH ２ , NH(C １ -C ６ alkyl group), N(C １ -C ６ alkyl group) ２ , C ３ -C ６ substituted with one or more substituents selected from cycloalkyl groups and 4- to 7-membered heterocyclyl groups The ligand-drug complex or a pharmaceutically acceptable salt thereof according to Claim 11.

13. The aforementioned L １ is a Gly-Gly-Phe-Gly tetrapeptide residue or an Ala-Ala-Ala tripeptide residue, and the aforementioned L ２ is a Gly-Gly-Phe-Gly tetrapeptide residue or a Val-Lys dipeptide residue. The ligand-drug complex or a pharmaceutically acceptable salt thereof according to Claim 11 or 12.

14. m1 is selected from 5, m2 is selected from 2, m3 is selected from 8, The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 11 to 13.

15. The linker unit L is 【Chemical 10】 selected from, the a-terminal of which is covalently bonded to the ligand unit Pc, and the b-terminal is covalently bonded to the drug unit D, The ligand-drug complex or a pharmaceutically acceptable salt thereof according to any one of Claims 11 to 14.

16. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is the following ligand-drug conjugate or a pharmaceutically acceptable salt thereof: 【Chemical 11】 【Chem.】 【Chem.】 [Chemical] 【Chem.】 selected from wherein Pc and n are as defined in claim 1, The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15.

17. The ligand unit Pc is selected from a polypeptide, an antibody or an antigen-binding fragment thereof, The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16.

18. The ligand unit Pc can specifically bind to one or more antigens selected from the group consisting of HER2, p95HER2, HER3, CD3, CD16, ROR1, DLL3, CDH6, CD70, CD5, CD20, BCMA, EGFR, VEGFR and LIV-1, The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 17.

19. The Pc is an antibody or an antigen-binding fragment thereof that specifically binds to HER2, p95HER2, CDH6, ROR1 or LIV-1. The antibody or an antigen-binding fragment thereof includes a heavy chain variable region (VH) or / and a light chain variable region (VL). Optionally, among them, (1) the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 included in the VH shown in SEQ ID NO: 1, 3, 19, 21, 37, 46, 54, 56, 71, 80, 82 or 84, or / and the light chain variable region includes LCDR1, LCDR2 and LCDR3 included in the VL shown in SEQ ID NO: 2, 4, 20, 22, 38, 47, 55, 57, 72, 81, 83 or 85, or (2) the heavy chain variable region or / and the light chain variable region includes an amino acid sequence having at least 80% identity compared to each CDR in HCDR1-3 or / and LCDR1-3 described in group (1), or an amino acid sequence in which at most three insertion, deletion or substitution mutations occur. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 17 or 18.

20. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein, among them, the HCDR1-3 and / or the LCDR1-3 are (1) The HCDR1-3 are SEQ ID NO: 7-9, and / or the LCDR1-3 are SEQ ID NO: 10-12, (2) The HCDR1-3 are SEQ ID NO: 13-15, and / or the LCDR1-3 are SEQ ID NO: 16-18, (3) The HCDR1-3 are SEQ ID NO: 23-25, and / or the LCDR1-3 are SEQ ID NO: 26-28, (4) The HCDR1-3 are SEQ ID NO: 29-31, and / or the LCDR1-3 are SEQ ID NO: 32-34, (5) The HCDR1-3 are SEQ ID NO: 40-42, and / or the LCDR1-3 are SEQ ID NO: 43-45, (6) The HCDR1-3 are SEQ ID NO: 48-50, and / or the LCDR1-3 are SEQ ID NO: 51-53, (7) The HCDR1-3 are SEQ ID NO: 58-60, and / or the LCDR1-3 are SEQ ID NO: 61-63, (8) The HCDR1-3 are SEQ ID NO: 64-66, and / or the LCDR1-3 are SEQ ID NO: 67-69, (9) The HCDR1-3 are SEQ ID NO: 74-76, and / or the LCDR1-3 are SEQ ID NO: 77-79, (10) The HCDR1-3 are SEQ ID NO: 86-88, and / or the LCDR1-3 are SEQ ID NO: 89-91, (11) The HCDR1-3 are SEQ ID NO: 92-94, and / or the LCDR1-3 are SEQ ID NO: 95-97, (12) The HCDR1-3 are SEQ ID NO: 98-100, and / or the LCDR1-3 are SEQ ID NO: 101-103, or (13) The HCDR1-3 and / or the LCDR1-3 have an amino acid sequence with at least 80% identity or at most three insertion, deletion or substitution mutations compared to each CDR in the HCDR1-3 or / and LCDR1-3 described in any one of groups (1)-(12), selected from The ligand-drug conjugate according to claim 19 or a pharmaceutically acceptable salt thereof.

21. The antibody or antigen-binding fragment thereof comprises a heavy-chain variable region (VH) or / and a light-chain variable region (VL), wherein the heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, 3, 19, 21, 37, 46, 54, 56, 71, 80, 82 or 84, or / and the light-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 20, 22, 38, 47, 55, 57, 72, 81, 83 or 85, or the heavy-chain variable region or / and the light-chain variable region each comprises an amino acid sequence having at least 80% identity compared to any one of the above heavy-chain variable regions or / and light-chain variable regions. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 17 or 18.

22. The antibody or antigen-binding fragment comprises a heavy-chain constant region sequence and / or a light-chain constant region sequence. Optionally, the heavy-chain constant region and / or the light-chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc. Optionally, the heavy-chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant regions, and the light-chain constant region is selected from human or mouse kappa constant region or lambda constant region. Optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain consists of the VH and the heavy-chain constant region, the heavy-chain constant region has the amino acid sequence shown in SEQ ID NO: 5, 39 or 73, the light chain consists of the VL and the light-chain constant region, and the light-chain constant region has the amino acid sequence shown in SEQ ID NO:

6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 17 to 21.

23. The antibody is selected from Trastuzumab, Pertuzumab or Rituximab. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 17 to 22.

24. A drug-linker compound having the general structural formula L'-D or a pharmaceutically acceptable salt thereof, wherein the drug unit D is as defined in any one of claims 1 to 10, the linker unit L' is 【Chemical 12】 selected from, the b-terminus of which is covalently bonded to the drug unit D, L １ L ２ L, m1, m2, m3 are as defined in any one of claims 11 to 15 the drug-linker compound or a pharmaceutically acceptable salt thereof.

25. L' is 【Chemical 13】 selected from, the b-terminus of which is covalently bonded to the drug unit D, m1 is selected from 5, and L １ is selected from Gly-Gly-Phe-Gly tetrapeptide residues or Ala-Ala-Ala tripeptide residues, the drug-linker compound or a pharmaceutically acceptable salt thereof according to claim 24. Claim 26 L' is 【Chemical Formula 14】 selected from, the b-terminus of which is covalently bonded to the drug unit D, m2 is selected from 2, m3 is selected from 8, and L ２ is selected from a Gly-Gly-Phe-Gly tetrapeptide residue or a Val-Lys dipeptide residue, the drug-linker compound according to claim 24 or a pharmaceutically acceptable salt thereof. Claim 27 L' is the following chemical structure: 【Chemical Formula 15】 selected from, the b terminus of which is covalently bonded to the drug unit D, the drug-linker compound according to any one of claims 24 to 26 or a pharmaceutically acceptable salt thereof. Claim 28 the following compound or a pharmaceutically acceptable salt thereof: 【Chemical 16】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 selected from, the drug-linker compound according to any one of claims 24 to 27 or a pharmaceutically acceptable salt thereof. Claim 29 A pharmaceutical composition comprising the ligand-drug complex according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive. Claim 30 Use of the ligand-drug complex according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 29, in the manufacture of a drug for treating tumors. Claim 31 A method for producing the ligand-drug complex according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, comprising the step of coupling the drug-linker compound according to any one of claims 24 to 28 with a ligand, optionally, the ligand is an antibody or an antigen-binding fragment thereof. A method for producing the ligand-drug complex according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof. Claim 32 A method for treating a tumor in a subject in need thereof, comprising administering to the subject the ligand-drug complex according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 29. Claim 33 Use of the ligand-drug complex according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 29, for treating a tumor in a subject.

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