Camptothecin derivatives and ligand-drug conjugates

Abstract

This disclosure relates to a class of novel ligand-drug conjugates or pharmaceutically acceptable salts thereof, specifically providing structures with the general formula Pc-(L-D). n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof, methods for its preparation, pharmaceutical compositions containing said conjugate, and its use in the treatment of tumors.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese patent application No. 202210515898.0 filed on May 12, 2022, Chinese patent application No. 202210999585.7 filed on August 19, 2022, and Chinese patent application No. 202310406469.4 filed on April 14, 2023, which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This disclosure pertains to the field of biomedicine and relates to a class of novel ligand-drug conjugates, their preparation methods, pharmaceutical compositions containing the conjugates, and their use as antitumor drugs. Background Technology

[0004] Antibody-drug conjugates (ADCs), as novel targeted therapies, combine the tumor-targeting properties of antibodies with the highly effective killing effects of toxins by linking monoclonal antibodies that specifically bind to tumor cell surface antigens to biologically active toxin molecules. This avoids the drawbacks of lower efficacy of the former and excessive toxicity and poor drug-likeness of the latter. Compared to traditional chemotherapy drugs, ADCs can precisely target tumor cells and reduce the impact on normal cells, achieving a safer and more effective anti-tumor effect.

[0005] In 2000, the first antibody-drug conjugate (ADC), Mylotarg (gemtuzumab ozogamicin), was approved by the FDA for the treatment of acute myeloid leukemia (AML) in adults. In 2011, the FDA approved the novel targeted ADC drug Adcetris (bretuximab vedotin) for the treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma. Both Mylotarg and Adcetris are treatments for hematologic malignancies. In 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the FDA for the treatment of HER2-positive advanced or metastatic breast cancer that is resistant to trastuzumab and paclitaxel, becoming the first approved ADC for the treatment of solid tumors.

[0006] Antibody-adjuvant drugs (ADCs) generally consist of three parts: antibody, linker, and toxin. Camptothecin derivatives are one type of toxin used in ADC development; they exert their anti-tumor effect by inhibiting topoisomerase I. Daiichi Sankyo developed the ADC drug Enhertu (Trastuzumab deruxtecan, DS-8201) targeting HER2, using the camptothecin derivative ezetezine as the toxin. It was approved by the FDA in 2019. Clinical studies have shown that Enhertu has good therapeutic effects on HER2-positive breast cancer, gastric cancer, and non-small cell lung cancer.

[0007] Although several ADC drugs are currently on the market, safety and drug resistance issues remain, indicating a significant unmet clinical need. Therefore, there is an urgent need in this field to further develop more effective and safer ADC drugs. Summary of the Invention

[0008] This disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD). n ,

[0009] in,

[0010] Pc is a ligand unit;

[0011] L represents the connecting subunit;

[0012] D is the drug unit represented by the following formula (DI):

[0013]

[0014] in,

[0015] X is selected from NH or O;

[0016] R 1 Selected from halogens, CN, C1-C6 alkyl, C3-C6 cycloalkyl, or C2-C6 ynyl, wherein the C1-C6 alkyl, C3-C6 cycloalkyl, or C2-C6 ynyl group is optionally surrounded by one or more R groups. a1 replace;

[0017] X1 is selected from CR 2 Or N;

[0018] R 2 Selected from H, halogen, CN, or R 1 R 2 The atoms bonded to them together form a 5-6 membered heterocyclic group, which is optionally bonded by one or more R atoms. a2 replace;

[0019] R 4 Selected from H, C1-C3 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic groups, wherein the C1-C3 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group is optionally surrounded by one or more R groups. a4 replace;

[0020] R 5 Selected from H, halogens, CN, NH2 or NO2, or R 1 R 5 The atoms bonded to them together form a 5-6 membered heterocyclic group, a 5-6 membered heteroaryl group, or a C5-C7 cycloalkenyl group, wherein the 5-6 membered heterocyclic group, 5-6 membered heteroaryl group, or C5-C7 cycloalkenyl group is optionally surrounded by one or more R a5 replace;

[0021] R 6 Selected from H or C1-C3 alkyl groups;

[0022] R 7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, or R 6 R 7 The C atoms attached thereto form a C3-C6 cycloalkyl group, which is optionally bonded by one or more R atoms. a7 replace;

[0023] Each R a1 R a2 R a4 R a5 R a7 Independently selected from D, halogen, CN, =O, OH, NH2, C1-C3 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group, wherein the OH, NH2, C1-C3 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group is optionally surrounded by one or more R b replace;

[0024] Each R b It is independently selected from halogen, CN, =O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2;

[0025] The condition is: i) when R 1 Selected from methyl, R 2 When selected from F, R 6 R 7 Together with the C atoms attached to it, they form a cyclopropyl group;

[0026] ii) When X is selected from NH, R 5 Not selected from H; iii) The compound shown in formula (DI) does not contain

[0027] Furthermore, n is a real number from 1 to 16.

[0028] In some implementation schemes, each R a1 R a2 R a4 R a5 R a7 It is independently selected from D, halogen, CN, =O, OH, NH2, C1-C3 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic groups.

[0029] In some implementation schemes, each R a2 and R a7 Selected independently from D.

[0030] In some implementation schemes, R 1 It is selected from halogens, C1-C3 alkyl groups, C3-C6 cycloalkyl groups, or C2-C3 alkynyl groups.

[0031] In some implementation schemes, R 1 Selected from Cl, Br, methyl, cyclopropyl or ethynyl.

[0032] In some implementation schemes, R 1 Selected from Cl, Br, or methyl.

[0033] In some implementation schemes, R 2 Selected from H, halogen, CN, or R 1 R 2 The atoms connected to them together form a 5-6 membered heterocyclic group, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocyclic group is optionally replaced by one or more D atoms.

[0034] In some implementation schemes, R 2 Selected from H, halogen, CN, or R 1 R 2 The atoms connected to them together form a 5-6 membered heterocyclic group, which contains one or two oxygen atoms as ring atoms.

[0035] In some implementation schemes, R 2 Selected from H, F, or Cl, or R 1 R 2 The atoms connected to them together form

[0036] In some implementation schemes, R 2 Selected from H, F, or Cl, or R 1 R 2 The atoms connected to them together form

[0037] In some implementation schemes, R 5 Selected from H, halogens, NH2 or NO2, or R 1 R 5 The atoms bonded to them together form a 5-6 membered heteroaryl or a C5-C6 cycloalkenyl group, wherein the 5-6 membered heteroaryl or C5-C6 cycloalkenyl group is optionally surrounded by one or more R groups. a5 replace.

[0038] In some implementation schemes, R 5 Selected from H, Cl, F, NH2 or NO2, or R 1 R 5 The atoms connected to them together form

[0039] In some implementation schemes, R 4 Selected from H or C1-C3 alkyl groups.

[0040] In some implementation schemes, R 4 Selected from H.

[0041] In some implementation schemes, R 6 Selected from H or methyl.

[0042] In some implementation schemes, R 7 Selected from H, C1-C3 alkyl, or optional C3-C6 cycloalkyl substituted with one or more D, or R 6 R 7 Together with the C atoms attached thereto, they form C3-C6 cycloalkyl groups.

[0043] In some implementation schemes, R 7 Selected from H, methyl, isopropyl, or cyclopropyl, optionally substituted with one or more D, or R 6 R 7 The C atoms attached to it together form a cyclopropyl group.

[0044] In some implementation schemes, R 1 R 2 Together with the atoms they are connected to, they form R 6 Selected from H or methyl, R 7 Selected from H, methyl, isopropyl, or cyclopropyl, optionally substituted with one or more D, or R 6 R 7 The C atoms attached to it together form a cyclopropyl group.

[0045] In some implementation schemes, structural units Selected from

[0046] In some implementation schemes, R 1 Selected from methyl, R 2 Selected from F, R 6 R 7 The C atoms attached to it together form a cyclopropyl group.

[0047] In some implementations, X is selected from NH, R 5 Selected from Cl, F, NH2 or NO2.

[0048] In some embodiments, the pharmaceutical unit represented by formula (DI) is selected from the pharmaceutical unit represented by formula (D-Ia):

[0049]

[0050] Among them, R 1 R 2 R 4 R 5 R 6 R 7 As defined above.

[0051] In some embodiments, the compound represented by formula (DI) is selected from the following compounds:

[0052]

[0053]

[0054]

[0055]

[0056] In some embodiments, this disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD). n ,

[0057] in,

[0058] Pc, L, and n are defined as above;

[0059] D is selected from one of the following compounds:

[0060]

[0061]

[0062] In some implementations, the connecting subunit L is selected from... Its a-terminus is covalently connected to the ligand unit Pc, and its b-terminus is covalently connected to the drug unit D. Here, m1 and m2 are each independently selected from integers 2 to 8, m3 is selected from integers 1 to 16, and L... 1 L 2 Each peptide residue is independently selected from 1 to 8 amino acids, and the peptide residue is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic groups.

[0063] In some implementations, the L 1 L 2 Each peptide residue is independently selected from 2, 3, or 4 amino acids, and the peptide residue is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, and 4-7 membered heterocyclic groups.

[0064] In some implementations, the L 1 It consists of Gly-Gly-Phe-Gly tetrapeptide residues or Ala-Ala-Ala tripeptide residues.

[0065] In some implementations, the L 2 It consists of Gly-Gly-Phe-Gly tetrapeptide residues or Val-Lys dipeptide residues.

[0066] In some implementations, m1 is selected from 5.

[0067] In some implementations, m2 is selected from 2 and m3 is selected from 8.

[0068] In some implementations, the connecting subunit L is selected from the following chemical structures:

[0069]

[0070] Its a-end is covalently connected to the ligand unit Pc, and its b-end is covalently connected to the drug unit D.

[0071] In some implementations, the general formula of this disclosure is Pc-(LD). n The ligand-drug conjugate or its pharmaceutically acceptable salt is selected from the following compounds or their pharmaceutically acceptable salts:

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] Pc and n are defined as described above.

[0079] In some implementations, the aforementioned general formula is Pc-(LD). n The ligand-drug conjugate or its pharmaceutically acceptable salt thereof, wherein the ligand unit Pc is selected from a polypeptide, an antibody or its antigen-binding fragment.

[0080] In some implementations, 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, VEGF, and LIV-1.

[0081] In some embodiments, the antibody or its antigen-binding fragment is monospecific, bispecific, trispecific, or tetraspecific.

[0082] In some implementations, the antibody is selected from Trastuzumab, Pertuzumab, or Rituximab.

[0083] In some embodiments, the Pc is an antibody or antigen-binding fragment thereof that specifically binds to HER2, p95HER2, CDH6, ROR1, or LIV-1; the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL);

[0084] In some embodiments, (1) the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 contained 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 SEQ ID NO: 1, 3, 19, 21, 37, 46, 54, 56, 71, 80, 82, or 84. NO: 2, 4, 20, 22, 38, 47, 55, 57, 72, 81, 83 or 85, which contain LCDR1, LCDR2 and LCDR3; or (2) the heavy chain variable region and / or the light chain variable region comprises an amino acid sequence having at least 80% identity with each CDR in HCDR1-3 or / and LCDR1-3 of group (1), or an amino acid sequence having at most 3 insertion, deletion or substitution mutations; 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 or / and the LCDR1-3 are determined according to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system.

[0085] In some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, or / and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein HCDR1-3 and / or LCDR1-3 are selected from the following:

[0086] (1) The HCDR1-3 is SEQ ID NO:7-9; or / and the LCDR1-3 is SEQ ID NO:10-12;

[0087] (2) The HCDR1-3 is SEQ ID NO:13-15; or / and the LCDR1-3 is SEQ ID NO:16-18;

[0088] (3) The HCDR1-3 is SEQ ID NO:23-25; or / and the LCDR1-3 is SEQ ID NO:26-28;

[0089] (4) The HCDR1-3 is SEQ ID NO:29-31; or / and the LCDR1-3 is SEQ ID NO:32-34;

[0090] (5) The HCDR1-3 is SEQ ID NO:40-42; or / and the LCDR1-3 is SEQ ID NO:43-45;

[0091] (6) The HCDR1-3 is SEQ ID NO:48-50; or / and the LCDR1-3 is SEQ ID NO:51-53;

[0092] (7) The HCDR1-3 is SEQ ID NO:58-60; or / and the LCDR1-3 is SEQ ID NO:61-63;

[0093] (8) The HCDR1-3 is SEQ ID NO:64-66; or / and the LCDR1-3 is SEQ ID NO:67-69;

[0094] (9) The HCDR1-3 is SEQ ID NO:74-76; or / and the LCDR1-3 is SEQ ID NO:77-79;

[0095] (10) The HCDR1-3 is SEQ ID NO: 86-88; or / and the LCDR1-3 is SEQ ID NO: 89-91;

[0096] (11) The HCDR1-3 is SEQ ID NO: 92-94; or / and the LCDR1-3 is SEQ ID NO: 95-97;

[0097] (12) The HCDR1-3 is SEQ ID NO: 98-100; or / and the LCDR1-3 is SEQ ID NO: 101-103; or,

[0098] (13) The HCDR1-3 or / and the LCDR1-3 have an amino acid sequence that is at least 80% identical to each CDR in any group of HCDR1-3 or / and LCDR1-3 in groups (1)-(12), or has at most 3 insertion, deletion or substitution mutations; preferably, the HCDR1-3 or / and the LCDR1-3 have an amino acid sequence that is at least 80% identical to each CDR in any group of HCDR1-3 or / and LCDR1-3 in groups (1)-(12); further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0099] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or 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, and / or 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 and / or the light chain variable region each comprise an amino acid sequence having at least 80% identity with any of the aforementioned heavy chain variable regions and / or light chain variable regions; further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0100] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence, wherein the heavy chain constant region and / or light chain constant region are selected from a complete constant region sequence or a fragment thereof, the constant region fragment comprising CH1, a 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 regions or lambda constant regions; optionally, the antibody or its antigen-binding fragment comprises a complete heavy chain and a light chain, wherein the heavy chain is composed of the VH and the heavy chain constant region, the heavy chain constant region having an amino acid sequence as shown in SEQ ID NO:5, 39, or 73, and the light chain is composed of the VL and the light chain constant region, the light chain constant region having an amino acid sequence as shown in SEQ ID NO:6.

[0101] In some implementations, the aforementioned general formula is Pc-(LD). n The ligand-drug conjugate or its pharmaceutically acceptable salt, wherein n is selected from a real number from 1 to 16, for example n is selected from a real number from 2 to 12, for example n is selected from a real number from 4 to 10, for example n is selected from a real number from 5 to 9, for example n is selected from a real number from 6 to 8.

[0102] In some implementations, n is selected from real numbers from 5 to 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.

[0103] This disclosure also provides a drug-linker compound or a pharmaceutically acceptable salt thereof, having the general structural formula L'-D, wherein:

[0104] Drug unit D is as defined above;

[0105] L' is selected from Its b-end is covalently connected to drug unit D, L 1 L 2 m1, m2, and m3 are defined as described above.

[0106] In some implementations, L' is selected from Its b-end is covalently connected to the drug unit D, and m1 is selected from 5, L 1 Selected from Gly-Gly-Phe-Gly tetrapeptide residues or Ala-Ala-Ala tripeptide residues.

[0107] In some implementations, L' is selected from Its b-end is covalently connected to drug unit D, m2 is selected from 2, m3 is selected from 8, and L 2 Selected from Gly-Gly-Phe-Gly tetrapeptide residues or Val-Lys dipeptide residues.

[0108] In some implementations, L' is selected from one of the following chemical structures:

[0109]

[0110] Its b-end is covalently connected to the drug unit D.

[0111] In some embodiments, the drug-linker compound of the general formula L'-D disclosed herein, or a pharmaceutically acceptable salt thereof, is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] This disclosure also provides compounds represented by the formula (DH) or pharmaceutically acceptable salts thereof:

[0118]

[0119] in,

[0120] R 1 X1, X, R 4 R 5 R 6 R 7 As defined above.

[0121] In some embodiments, the compound represented by formula (DH) or a pharmaceutically acceptable salt thereof is selected from the compounds shown below or pharmaceutically acceptable salts thereof:

[0122]

[0123]

[0124]

[0125]

[0126] On the other hand, this disclosure provides a pharmaceutical composition comprising the aforementioned general formula Pc-(LD). n The ligand-drug conjugate or its pharmaceutically acceptable salt and pharmaceutically acceptable excipient.

[0127] On the other hand, this disclosure provides a method for treating mammalian tumors, comprising administering to a mammal in need of treatment, preferably a human, a therapeutically effective amount of the aforementioned general formula Pc-(LD). n The ligand-drug conjugate or its pharmaceutically acceptable salt, or a pharmaceutical composition thereof.

[0128] On the other hand, this disclosure provides the aforementioned general formula as Pc-(LD). n The use of ligand-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for treating tumors.

[0129] On the other hand, this disclosure provides the aforementioned general formula as Pc-(LD). n The use of ligand-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the treatment of tumors.

[0130] On the other hand, this disclosure provides a treatment for tumors using the aforementioned general formula Pc-(LD). n The ligand-drug conjugate or its pharmaceutically acceptable salt, or a pharmaceutical composition thereof.

[0131] On the other hand, this disclosure provides a pharmaceutical composition comprising a compound represented by the formula (DH) above the present disclosure or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0132] On the other hand, this disclosure provides a method for treating mammalian tumors, comprising administering to a mammal, preferably a human, a therapeutically effective amount of a compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0133] On the other hand, this disclosure provides the use of the compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating tumors.

[0134] On the other hand, this disclosure provides the use of the compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the treatment of tumors.

[0135] On the other hand, this disclosure provides compounds of the aforementioned formula (DH) for treating tumors, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof.

[0136] On the other hand, this disclosure provides a general formula of Pc-(LD). n A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, comprising the step of conjugating a drug-linker compound of the formula L'-D disclosed herein with the aforementioned ligand, wherein optionally, the ligand is an antibody or an antigen-binding fragment thereof.

[0137] On the other hand, this disclosure provides a general formula of Pc-(LD). n A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, comprising the step of linking the aforementioned drug unit D of this disclosure to the aforementioned ligand unit Pc; optionally, the linking is via the aforementioned linker unit L; optionally, the ligand is an antibody or an antigen-binding fragment thereof.

[0138] On the other hand, this disclosure provides the preparation of compounds represented by the aforementioned formula (DH) or pharmaceutically acceptable salts thereof in the preparation of the aforementioned general formula Pc-(LD). n The use of the ligand-drug conjugate or its pharmaceutically acceptable salt in the preparation of the aforementioned drug-linker compound of general formula L'-D or its pharmaceutically acceptable salt.

[0139] The ligand-drug conjugates disclosed herein have significant anti-inflammatory activity and / or reduced toxic side effects.

[0140] Brief description of the attached figures

[0141] Figure 1 The results of X-ray single-crystal diffraction analysis of compound 19-P1 are shown.

[0142] Figure 2The tumor growth curve of the OVCAR3 subcutaneous tumor model is shown.

[0143] Figure 3 The tumor growth curve of the OVCAR3 subcutaneous tumor model is shown.

[0144] Figure 4 The tumor growth curve of the OVCAR3 subcutaneous tumor model is shown.

[0145] Figure 5 The curves showing the weight change of mice in the OVCAR3 subcutaneous tumor model are displayed.

[0146] Figure 6 The tumor growth curve of the H838 subcutaneous tumor model is shown.

[0147] Figure 7 The curve of weight change in H838 subcutaneous tumor model mice is shown.

[0148] Terminology Definitions and Explanations

[0149] Unless otherwise stated, the terms used in this disclosure have the following meanings: the definitions of groups and terms recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0150] The term "ligand" refers to a macromolecular compound that recognizes and binds to antigens or receptors associated with target cells. The role of ligands is to deliver drugs to the target cell population that has bound to them. 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 this disclosure, a ligand or ligand unit is designated as Pc, and the ligand can form a linker bond with a linker unit via heteroatoms on the ligand. In some embodiments of this disclosure, the ligand is selected from antibodies or antigen-binding fragments, and the antibody is selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; in some embodiments of this disclosure, the antibody is a monoclonal antibody.

[0151] The term "linker" or "linker unit" refers to a chemical structural segment or chemical bond that is connected to a ligand at one end and to a drug at the other end.

[0152] The term "drug" refers to a small molecule compound that is biologically active in a living organism. In some embodiments of this disclosure, the drug is a glucocorticoid receptor agonist or its corresponding phosphate ester molecule that has anti-inflammatory function.

[0153] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In some embodiments of this disclosure, "ligand-drug conjugate" is an antibody-drug conjugate (ADC), where an ADC refers to a monoclonal antibody or antibody fragment linked to a biologically active drug via a stable linker unit.

[0154] 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 disclosed herein, DAR is defined by the variable "n," which can be either an integer or a decimal.

[0155] The term “antibody” is used in its broadest sense to refer to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or from the variable regions of the immunoglobulin light chain, thereby enabling it to specifically bind to an antigen. The term “antibody” as used herein encompasses a variety of forms and structures, provided they exhibit the desired antigen-binding activity. The term “antibody” as used herein includes alternative protein scaffolds or artificial scaffolds having transplanted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, 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 also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for use in transplanting CDRs, including but not limited to tendinins, fibronectins, peptide aptamers, etc.

[0156] The term "antibody" in this article includes typical "quadruple-chain antibodies," which belong to immunoglobulins composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of 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 in the N-to-C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to-C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0157] The term "antibody" in this article also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by camels such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lamaguanicoe), and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0158] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0159] The term "antibody" in this article includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0160] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variants (e.g., containing naturally occurring mutations or generated during the manufacturing process of the formulation, such variants are typically present in small amounts), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. The modifier "monoclonal" herein should not be construed as requiring the production of the antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be prepared using a variety of techniques, including (but not limited to) hybridoma techniques, recombinant DNA methods, phage library display techniques, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0161] The term "natural antibody" refers to antibodies produced and paired by the immune system of multicellular organisms. The term "engineered antibody" used in this article refers to non-natural antibodies obtained through techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), and bispecific antibodies.

[0162] The term "monospecific" refers to having one or more binding sites, where each binding site binds to the same epitope of the same antigen.

[0163] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as "bispecific," "trispecific," and "quadrispecific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0164] The term "valence" indicates the number of binding sites present in an antibody / antigen binding molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" indicate the presence of one, two, four, and six binding sites in an antibody / antigen binding molecule, respectively.

[0165] In this article, "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably and refer to antibodies with structures that are substantially similar to those of natural antibodies.

[0166] In this document, "antigen-binding fragment" and "antibody fragment" are used interchangeably. They do not possess the complete structure of a full antibody, but only contain a portion or a local variant of the full antibody, which has the ability to bind antigens. "Antigen-binding fragment" or "antibody fragment" in this document includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0167] Papain digestion of the intact antibody produces two identical antigen-binding fragments, called “Fab” fragments, each containing variable domains for both the heavy and light chains, as well as a constant domain for the light chain and a first constant domain (CH1) for the heavy chain. Thus, the term “Fab fragment” as used herein refers to the antibody fragment containing the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. The Fab’ fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab’-SH is the Fab’ fragment in which the cysteine ​​residues in the constant domain carry a free thiol group. Pepsin treatment produces the F(ab’)2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0168] The “Fv fragment” is the smallest fragment produced by IgG and IgM, containing a complete antigen binding site. The Fv fragment has the same binding properties as Fab and similar three-dimensional binding properties. The VH and VL chains of the Fv fragment are linked together through non-covalent interactions.

[0169] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).

[0170] The term "diabody" refers to a single polypeptide chain in which the VH and VL domains are expressed, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0171] The term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a specific species or belong to a specific antibody class or subclass), and another portion of the light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the 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" can include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0172] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the FR and / or constant regions within the variable region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to strengthen the immune response.

[0173] The term "fully human antibody" refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. "Fully human antibody" as used herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, "fully human antibody" as used herein does not include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0174] The term "variable region" refers to the region within the heavy or light chain of an antibody that enables the antibody to bind to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity. In this article, the terms "complementarity-determining region" and "CDR" are used interchangeably. They generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is called the complementarity-determining region because it can form precise complementarity with the antigenic epitope in its spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "frame region" and "FR region" are used interchangeably in this article. They refer to the amino acid residues in the antibody heavy chain variable region or light chain variable region other than the CDR. Typically, an antibody variable region consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0175] The term "CDR" in this paper can be labeled and defined in a manner known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. The tools and websites 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). The CDR in this paper includes overlaps and subsets of amino acid residues defined in different ways.

[0176] The term "heavy chain constant region" in this document refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with the Fc receptor, and has a more conserved amino acid sequence relative to the variable domains of the antibody. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes "full-length heavy chain constant region" and "heavy chain constant region fragments," the former having a structure substantially similar to the natural antibody constant region, while the latter comprises only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domains.

[0177] The term "light chain constant region" in this article refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0178] The term "Fc" in this document refers to the carboxyl-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody, typically containing 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 the immunoglobulin heavy chain can vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include Lys447.

[0179] The term "identity" used herein can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences 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 or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.

[0180] Taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percentage of identity between the two sequences varies with the common positions shared by the sequences.

[0181] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at www.gcg.com) in the GAP program integrated into the GCG software package can be used to determine the percentage of identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. As another example, the GAP program in the GCG software package (available at www.gcg.com) can be used to determine the percentage of identity between two nucleotide sequences using an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0182] In this article, "n is a real number from 1 to 16" means that n is any real number greater than or equal to 1 and less than or equal to 16.

[0183] In this article Indicates the connection site.

[0184] The diagrammatic representation of racemic or enantiomerically pure compounds in this article is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, wedge bonds and virtual wedge bonds are used. The absolute configuration of a solid center is represented by black solid bonds and imaginary bonds. It indicates the relative configuration of a stereocenter (such as the cis-trans configuration of alicyclic compounds).

[0185] The term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom between two positions within a molecule. The compounds disclosed herein can exhibit tautomerism. Tautomers can exist in two or more interconvertible forms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer typically yield a mixture whose physicochemical properties are consistent with those of the mixture of compounds. The equilibrium position depends on the intramolecular chemical characteristics. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form is dominant; while in phenols, the enol form is dominant. This disclosure encompasses all tautomeric forms of the compounds.

[0186] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0187] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds, and therefore may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E- and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Alkyl groups or other substituents may contain additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms. All such isomers involved in all substituents, and mixtures thereof, are also included within the scope of the definition of the compounds disclosed herein. The compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0188] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0189] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “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, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0190] When any variable (e.g., R) a R b When a group appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is surrounded by two R... bReplaced, then each R b Each has its own independent options.

[0191] When the number of a linking group is 0, such as -(CH2)0-, it indicates that the linking group is a bond.

[0192] When one of the variables is selected as a chemical bond or does not exist, it means that the two groups it is connected to are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0193] If the linking group mentioned in this article does not specify its linking direction, then its linking direction is arbitrary. For example, when the structural unit... When X is selected from "C1-C3 alkylene-O", X can either connect ring A and ring B in a left-to-right direction to form "ring A-C1-C3 alkylene-O-ring B", or connect ring A and ring B in a right-to-left direction to form "ring A-C1-C3 alkylene-ring B".

[0194] C in this article m -C n , refers to having an integer number of carbon atoms in the range mn.

[0195] The term "alkyl" refers to a compound with the general formula C10. n H 2n+1 The alkyl group can be straight-chain or branched. The term "C1-C6 alkyl" should be understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. 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, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0196] The “C1-C6 alkyl” mentioned in this article may further include “C1-C3 alkyl”.

[0197] The term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one triple bond. For example, the term "C2-C6 alkynyl" should be understood to preferably represent a straight or branched hydrocarbon group containing one or more triple bonds and having 2, 3, 4, 5, or 6 carbon atoms. Examples of "C2-C6 alkynyl" include, but are not limited to, ethynyl (-C≡CH), propynyl-1-alkynyl (1-propynyl, -C≡CCH3), propynyl-2-alkynyl (-CH2C≡CH), butynyl-1-alkynyl, butynyl-2-alkynyl, or butynyl-3-alkynyl. "C2-C6 alkynyl" may include "C2-C3 alkynyl," and examples of "C2-C3 alkynyl" include ethynyl (-C≡CH), propynyl-1-alkynyl (1-propynyl, -C≡CCH3), and propynyl-2-alkynyl (-CH2C≡CH).

[0198] The term "cycloalkyl" refers to a fully saturated carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0199] The term "cycloalkenyl" refers to a non-aromatic carbocyclic group that is not fully saturated and exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is typically a 5- to 8-membered ring. The term "C5-C7 cycloalkenyl" refers to a cycloalkenyl group with 5, 6, or 7 ring atoms, including but not limited to cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. The term "C5-C7 cycloalkenyl" can include the range "C5-C6 cycloalkenyl". The term "C5-C6 cycloalkenyl" refers to a cycloalkenyl group with 5 or 6 ring atoms, including but not limited to cyclopentenyl, cyclopentadienyl, cyclohexenyl, and cyclohexadienyl.

[0200] The term "heterocyclic group" refers to a fully saturated or partially saturated monocyclic, fused, spirocyclic, or bridged ring group containing 1-5 heteroatoms or heterogroups (i.e., groups containing heteroatoms). These "heteroatoms or heterogroups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, etc. The term "4-7 membered heterocyclic group" refers to a heterocyclic group with 4, 5, 6, or 7 ring atoms, containing 1-3 independently selected heteroatoms or heterogroups as described above. Examples of 4-membered heterocyclic groups include, but are not limited to, azirrobutane and oxobutane; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, 4,5-dihydrooxazole or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, trithiaalkyl, tetrahydropyridinyl or 4H-[1,3,4]thiadiazinyl; examples of 7-membered heterocyclic groups include, but are not limited to, diazacycloheptane. Preferably, "4-7-membered heterocyclic group" can include the ranges of "4-7-membered heterocyclic alkyl", "5-6-membered heterocyclic group", "5-6-membered heterocyclic alkyl", etc.

[0201] The term "5-6 membered heteroaryl" refers to an aromatic cyclic group having 5 or 6 ring atoms, and comprising 1-3, preferably 1-2, heteroatoms independently selected from N, O, and S. Specifically, the 5-6 membered heteroaryl is selected from thiophene, furanyl, pyrroleyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrazinyl, or triazinyl, etc.

[0202] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0203] The term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated individual, such as the progression of cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or remission are used, they also imply elimination, disappearance, or non-occurrence.

[0204] The term "effective dose" refers to the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously.

[0205] The term "subject" refers to an organism that receives treatment for a specific disease or condition as described in this disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals that receive treatment for a disease or condition.

[0206] The amount of the disclosed compound constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0207] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0208] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0209] 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 the administration of the disclosed compounds to an organism.

[0210] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients 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.

[0211] The word “comprise” or “include” and its English variants such as comprises or comprising can be understood as having an open, non-exclusive meaning, that is, “including but not limited to”.

[0212] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 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.

[0213] Certain isotope-labeled compounds of this disclosure (e.g., using...) 3 H and 14 C-labeling can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e., 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this disclosure can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0214] The pharmaceutical compositions disclosed herein are suitable for parenteral administration, such as in suitable unit dosage forms as sterile solutions, suspensions, or lyophilized products. For example, the pharmaceutical compositions disclosed herein may be in the form of sterile aqueous solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions disclosed herein may accept other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution, during use.

[0215] In all methods of administration of the compounds described herein, the daily dose is from 0.001 mg / kg to 600 mg / kg body weight, preferably from 0.05 mg / kg to 200 mg / kg body weight, more preferably from 0.1 mg / kg to 100 mg / kg body weight, in the form of single or separate doses.

[0216] The compounds disclosed herein can be prepared by a variety of synthetic methods 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 substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0217] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0218] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see 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 by reference in their entirety. Detailed Implementation

[0219] The present disclosure is described in detail below with reference to embodiments, but this does not imply any adverse limitation thereof. The present disclosure has been described in detail herein, including specific embodiments thereof. It will be apparent to those skilled in the art that various changes can be made to the specific embodiments of the present disclosure without departing from the spirit and scope thereof. All reagents used in this disclosure are commercially available and can be used without further purification.

[0220] Unless otherwise stated, the proportions expressed for mixed solvents are volume-based.

[0221] Unless otherwise stated, % refers to wt%.

[0222] Compounds are processed manually or Software naming conventions are used; commercially available compounds use supplier catalog names.

[0223] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are measured in units of 10⁻⁶. -6 (ppm). The solvents used for NMR determination were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., with tetramethylsilane (TMS) as the internal standard; "IC 50 "Hardest effective concentration (HIC)" refers to the concentration at which half of the maximum inhibitory effect is achieved. 50 "Refers to the concentration that produces half of the maximum effect concentration, i.e., the concentration that causes 50% of the maximum effect."

[0224] Example 1: (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-11H-[1,4]dioxacyclo[2,3-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 1)

[0225]

[0226] Step 1: Synthesis of 1-(7-amino-2,3-dihydrobenzo[b][1,4]dioxhexane-6-yl)-2-chloroethane-1-one (intermediates 1-2)

[0227] Reactant 1-1 (500 mg, 3.31 mmol) was dissolved in 1,2-dichloroethane (3 mL). The reaction solution was cooled to 0 °C, and boron trichloride (1 M, 2.65 mL) and aluminum trichloride (573.36 mg, 4.30 mmol) were added. Under nitrogen protection, chloroacetonitrile (299.67 mg, 3.97 mmol) was added, and the reaction solution was stirred at 90 °C for 16 h under nitrogen protection. The reaction was detected by LC-MS to indicate completion. After the reaction was cooled to room temperature, ice water (30 mL) and 1N HCl (10 mL) were added sequentially, and the mixture was stirred for 30 min. Dichloromethane (30 mL * 3) was added to the reaction solution for extraction three times. The combined organic phases were washed with saturated brine (30 mL), and the washed organic phase was dried over anhydrous sodium sulfate. The crude product was concentrated to dryness under reduced pressure, and the crude product was subjected to preparative thin-layer chromatography (silica, petroleum ether: ethyl acetate = 9:1) to obtain the title compound (210 mg).

[0228] MS m / z(ESI): 228.0 [M+H] + .

[0229] Step 2: Synthesis of (S)-15-(chloromethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxanecyclo[2,3-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(8H)-dione (intermediates 1-4)

[0230] Intermediates 1-2 (100 mg, 439.28 μmol) and 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. The reaction was confirmed by LC-MS. After the reaction mixture cooled to room temperature, it was filtered, and the filter cake was washed with ethanol (5 mL * 2) to obtain the crude title compound (130 mg).

[0231] MS m / z (ESI): 455.1 [M+H] + .

[0232] Step 3: Synthesis of (S)-15-(azidomethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxanecyclo[2,3-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(8H)-dione (intermediates 1-5)

[0233] Intermediate 1-4 (120 mg, 263.82 μmol) was dissolved in dimethyl sulfoxide (1 mL), and sodium azide (25.73 mg, 395.73 μmol) was added. The reaction mixture was stirred at 25 °C for 3 h under nitrogen protection. The reaction was confirmed by LC-MS. Ice water (2 mL) was added and the mixture was stirred for 0.5 h. The mixture was then filtered to obtain the crude title compound (90 mg).

[0234] MS m / z (ESI): 462.1 [M+H] + .

[0235] Step 4: Synthesis of (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxanecyclo[2,3-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(8H)-dione (intermediates 1-6)

[0236] Intermediate 1-5 (90 mg, 195.05 μmol) was dissolved in anhydrous toluene (1 mL), and triethyl phosphite (81.02 mg, 487.62 μmol) was added. The reaction mixture was stirred at 100 °C for 3 h under nitrogen protection. The reaction mixture was then cooled to 25 °C, and hydrochloric acid-methanol solution (0.5 mL) was added. The reaction mixture was stirred at 85 °C for 16 h under nitrogen protection. The reaction was confirmed to be complete by LC-MS. After the reaction mixture cooled to room temperature, it was filtered to obtain the title compound (18 mg).

[0237] MS m / z (ESI): 436.1 [M+H] + .

[0238] Step 5: Synthesis of (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-11H-[1,4]dioxanecyclo[2,3-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 1)

[0239] 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). Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea 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. The reaction was completed by LC-MS. The reaction solution was filtered and purified by preparative high performance liquid chromatography (YMC-ActusTriart C18 column, 5 μm silica, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 10%-30%, elution time 12 min) to give the title compound (7 mg).

[0240] MS m / z(ESI): 494.1 [M+H] + .

[0241] 1 H NMR (400MHz, DMSO-d6) δ = 8.69 (t, J = 6.0Hz, 1H), 7.92 (s, 1H), 7.56 (s, 1H), 7.26 (s, 1H), 6.49 (s, 1H), 5.57 (t, J = 5.7Hz, 1 H), 5.46 (s, 2H), 5.43 (s, 2H), 4.74 (d, J = 6.0Hz, 2H), 4.44 (s, 4H), 3.82 (d, J = 5.6Hz, 2H), 1.94-1.80 (m, 2H), 0.88 (m, 3H).

[0242] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyacetamide (Compound 3)

[0243]

[0244] Step 1: Synthesis of 1-(2-amino-5-chloro-4-fluorophenyl)-2-chloroethane-1-one (intermediate 3-2)

[0245] Boron trichloride (1M, 13.74 mL) was dissolved in 1,2-dichloroethane (24 mL). The reaction solution 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 was stirred at 0 °C for 10 min, and then aluminum trichloride (2.38 g, 17.86 mmol) was added. The reaction solution was then heated to 25 °C under nitrogen protection and stirred for 10 min. The reaction solution was then stirred at 90 °C under nitrogen protection for 18 h. The reaction was detected as complete by LC-MS. After the reaction was cooled to room temperature, ice water (50 mL) and 5% HCl (10 mL) were slowly added sequentially, and the mixture was stirred at 25 °C for 30 min. Then, dichloromethane (50 mL) was added, and the organic phase was washed with water (2 mL x 2). The washed organic phase was dried over 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; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 40%-60%, elution time 10 min) to give the title compound (320 mg).

[0246] MS m / z(ESI): 222.0 [M+H] + .

[0247] Step 2: Synthesis of (S)-9-chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (intermediate 3-3)

[0248] 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-toluenesulfonic acid salt (24.90 mg, 99.08 μmol) was added. The reaction mixture was stirred at 100 °C for 18 h. The reaction was confirmed by LC-MS. After the reaction mixture was cooled to room temperature, ethanol (1 mL) was added, and the 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 crude title compound (270 mg).

[0249] MS m / z(ESI): 449.0 [M+H] + .

[0250] Step 3: Synthesis of (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (3-4)

[0251] Intermediate 3-3 (50 mg, 111.29 μmol) was dissolved in ethanol (1 mL), and hexamethylenetetramine (23.40 mg, 166.94 μmol) was added. The reaction mixture was stirred at 90 °C for 1.5 h. The reaction was detected by LC-MS. After the reaction was cooled to room temperature, it 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; eluent: a mixture of water (containing 0.225% formic acid) and methanol with decreasing polarity; methanol gradient ratio 0%-30%, elution time 12 min) to obtain the title compound (22 mg).

[0252] MS m / z (ESI): 430.1 [M+H] + .

[0253] 1 H NMR (400MHz, DMSO-d6) δ = 8.71 (d, J = 8.0Hz, 1H), 8.23 ​​(d, J = 10.3Hz, 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).

[0254] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyacetamide (compound 3)

[0255] 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 to the solution, and the reaction mixture was stirred at 25 °C for 1.5 h. The reaction was confirmed by LC-MS. The reaction mixture was filtered, concentrated to dryness under reduced pressure, and 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; eluent: a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 10%-40%, elution time 12 min) to obtain the title compound (2.20 mg).

[0256] MS m / z(ESI): 488.1 [M+H] + .

[0257] 1 H NMR (400MHz, DMSO-d6) δ=8.90-8.85(m,2H),8.20(d,J=10.3Hz,1H),7.35(s,1H),6.55(s,1H),5.60(t,J=5.7H z,1H),5.56(s,2H),5.45(s,2H),4.83(d,J=6.0Hz,2H),3.83(d,J=5.8Hz,2H),1.93-1.81(m,2H),0.88(m,3H).

[0258] Example 5: (S)-N-((9-bromo-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxane-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyacetamide (Compound 5)

[0259]

[0260] Step 1: Synthesis of 1-(2-amino-5-bromo-4-fluorophenyl)-2-chloroethane-1-one (intermediate 5-2)

[0261] Boron trichloride (1M, 10.53 mL) was dissolved in 1,2-dichloroethane (24 mL). The reaction solution 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 was stirred at 0 °C for 10 min, and then aluminum trichloride (1.82 g, 13.68 mmol) was added. The reaction solution was stirred at 25 °C for 10 min under nitrogen protection. Subsequently, the temperature was raised to 90 °C and stirred for 18 h. The reaction was detected as complete by LC-MS. After the reaction was cooled to room temperature, ice water (50 mL) and 5% HCl (10 mL) were slowly added sequentially, and the mixture was stirred at 25 °C for 30 min. Then, dichloromethane (50 mL) was added, and the organic phase was washed with water (2 mL x 2). The washed organic phase was dried over anhydrous sodium sulfate. The title compound (380 mg) was obtained by preparative high performance liquid chromatography purification (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 39%-49%, elution time 12 min).

[0262] MS m / z(ESI): 265.9 [M+H] + .

[0263] Step 2: Synthesis of (S)-9-bromo-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (intermediate 5-3)

[0264] 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-toluenesulfonic acid salt (22.63 mg, 90.06 μmol) was added. The reaction mixture was stirred at 100 °C for 18 h. The reaction was confirmed by LC-MS. After the reaction mixture cooled to room temperature, ethanol (1 mL) was added, and the 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 crude title compound (200 mg).

[0265] MS m / z(ESI): 493.0 [M+H] + .

[0266] Step 3: Synthesis of (S)-11-(aminomethyl)-9-bromo-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (intermediate 5-4)

[0267] Intermediate 5-3 (200 mg, 405.10 μmol) was dissolved in ethanol (4 mL), and hexamethylenetetramine (113.58 mg, 810.19 μmol) was added. The reaction mixture was stirred at 90 °C for 1.5 h. The reaction was detected by LC-MS. After the reaction was cooled to room temperature, it 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, 25 mm diameter, 100 mm length; eluent of a mixture of water (containing 0.225% formic acid) and acetonitrile in decreasing polarity; acetonitrile gradient ratio 6%-26%, elution time 12 min) to obtain the title compound (30 mg).

[0268] MS m / z(ESI): 476.0 [M+H] + .

[0269] 1H NMR (400MHz, DMSO-d6) δ = 8.83 (d, J = 7.4Hz, 1H), 8.18 (d, J = 9.8Hz, 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=7.3Hz,3H).

[0270] Step 4: Synthesis of (S)-N-((9-bromo-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxane-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyacetamide (compound 5)

[0271] 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. The reaction was confirmed by LC-MS. The reaction mixture was filtered, concentrated to dryness under reduced pressure, and 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 mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, acetonitrile gradient ratio 6%-36%, elution time 12 min) to give the title compound (2.09 mg).

[0272] MS m / z(ESI): 532.0 [M+H] + .

[0273] 1 H NMR (400MHz, DMSO-d6) δ = 9.00 (d, J = 7.5Hz, 1H), 8.86 (t, J = 5.9Hz, 1H), 8.15 (d, J = 9.8Hz, 1H), 7.35 (s, 1H), 6.54 (s, 1H), 5.60 (t, J=5.7Hz,1H),5.56(s,2H),5.45(s,2H),4.83(d,J=5.8Hz,2H),3.83(d,J=5.9Hz,2H),1.94-1.82(m,2H),0.88(t,J=7.4Hz,3H).

[0274] Example 6: (S)-N-((8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxane-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyacetamide (Compound 6)

[0275]

[0276] Step 1: Synthesis of 1-(2-amino-4-chloro-5-methylphenyl)-2-chloroethane-1-one (intermediate 6-2)

[0277] Boron trichloride (1M, 2.82 mL) was dissolved in 1,2-dichloroethane (8 mL). The reaction solution 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 was stirred at 0 °C for 10 min, and then aluminum trichloride (612.09 mg, 4.59 mmol) was added. The reaction solution was stirred at 25 °C for 10 min under nitrogen protection. Then the reaction solution was heated to 90 °C and stirred for 18 h. The reaction was detected by LC-MS to indicate completion. After the reaction was cooled to room temperature, ice water (25 mL) and 5% HCl (5 mL) were slowly added sequentially, and the mixture was stirred at 25 °C for 30 min. Then dichloromethane (20 mL) was added, and the organic phase was washed with water (20 mL * 2). The washed organic phase was dried with an appropriate amount of anhydrous sodium sulfate. The title compound (500 mg) was obtained by preparative thin-layer chromatography (silica, petroleum ether: ethyl acetate = 9:1).

[0278] MS m / z(ESI): 218.0 [M+H] +

[0279] Step 2: Synthesis of (S)-8-chloro-11-(chloromethyl)-4-ethyl-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (intermediate 6-3)

[0280] 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-toluenesulfonic acid salt (34.57 mg, 137.56 μmol) was added. The reaction mixture was stirred at 100 °C for 18 h. The reaction was confirmed by LC-MS. After the reaction mixture was cooled to room temperature, ethanol (1 mL) was added, and the 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 crude title compound (230 mg).

[0281] MS m / z (ESI): 445.1 [M+H] + .

[0282] Step 3: Synthesis of (S)-11-(aminomethyl)-8-chloro-4-ethyl-4-hydroxy-9-methyl-1,12-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (intermediate 6-4)

[0283] Intermediate 6-3 (49.56 mg, 111.29 μmol) was dissolved in ethanol (0.5 mL), and hexamethylenetetramine (23.40 mg, 166.94 μmol) was added. The reaction mixture was stirred at 90 °C for 1.5 h. The reaction was detected by LC-MS. After the reaction was cooled to room temperature, it was concentrated to dryness under reduced pressure and purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length; eluent of a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 2%-32%, elution time 12 min) to give the title compound (11.0 mg).

[0284] MS m / z(ESI): 426.2 [M+H] + .

[0285] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyacetamide (compound 6)

[0286] 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. The reaction was confirmed by LC-MS. The reaction mixture was filtered, concentrated to dryness under reduced pressure, and 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 mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity as the eluent, acetonitrile gradient ratio 10%-40%, elution time 12 min) to obtain the title compound (3.00 mg).

[0287] MS m / z (ESI): 484.1 [M+H] + .

[0288] 1 H NMR (400MHz, DMSO-d6) δ = 8.77 (t, J = 6.1Hz, 1H), 8.52 (s, 1H), 8.26 (s, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.59 (t, J = 5.8Hz, 1H), 5 .52(s,2H),5.44(s,2H),4.85(d,J=6.0Hz,2H),3.84(d,J=5.6Hz,2H),2.60(s,3H),1.91-1.82(m,2H),0.88(t,J=7.3Hz,3H)

[0289] Example 7: (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 7)

[0290]

[0291] Step 1: Synthesis of 4,6-dibromo-2,3-dihydro-1H-inden-5-amine (intermediate 7-2)

[0292] 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, followed by stirring overnight at room temperature. The reaction solution 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 * 2). The resulting organic phase was dried over anhydrous sodium sulfate, mixed with silica gel, and then placed on diatomaceous earth. It was washed with 500 mL of petroleum ether, and the filtrate was concentrated to give the title compound (17.0 g).

[0293] MS m / z(ESI): 289.9 [M+H] + .

[0294] Step 2: Synthesis of 4-bromo-2,3-dihydro-1H-inden-5-amine (intermediate 7-3)

[0295] 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 reaction mixture was reacted at 90 °C for 3 h. The reaction mixture was cooled to room temperature, concentrated to remove acetic acid, and the residue was dissolved in 100 mL of ethyl acetate. The pH was adjusted to approximately 8 with a saturated sodium carbonate aqueous solution, filtered, and the organic phase was separated from the filtrate. The aqueous phase was then extracted with ethyl acetate (50 mL * 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the title compound (10 g).

[0296] MS m / z(ESI): 212.0 [M+H] + .

[0297] Step 3: Synthesis of N-(4-bromo-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 7-4)

[0298] Intermediate 7-3 (10.0 g) was dissolved in 100 mL of anhydrous dichloromethane, and triethylamine (10.6 g) was added. Acetyl chloride (5.5 g) was then slowly added dropwise at 0 °C, and the reaction mixture was stirred overnight at room temperature. The reaction solution 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).

[0299] MS m / z(ESI): 254.0 [M+H] + .

[0300] Step 4: Synthesis of N-(4-acetyl-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 7-5)

[0301] 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, followed by the addition of bis(triphenylphosphine)palladium dichloride (1.8 g). The reaction mixture was stirred overnight at 100 °C. The reaction solution 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 solution was filtered through diatomaceous earth, and the filtrate was diluted with 100 mL of ethyl acetate and washed with water (100 mL x 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).

[0302] MS m / z(ESI): 218.1 [M+H] + .

[0303] Step 5: Synthesis of N-(4-(2-bromoacetyl)-2,3-dihydro-1H-inden-5-yl)acetamide (intermediates 7-6)

[0304] Intermediate 7-5 (4.7 g) was dissolved in 50 mL of acetic acid, and 7.3 g of 33% hydrobromic acid-acetic acid solution was added. Bromine (2.85 g) was slowly added dropwise at room temperature, and the reaction was continued to be stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was poured into ice water and stirred until a large amount of solid precipitated. The mixture was filtered, the filter cake was washed with petroleum ether, and the resulting solid was dried to give the title compound (5.0 g).

[0305] MS m / z(ESI): 296.0 [M+H] + .

[0306] Step 6: Synthesis of 1-(5-amino-2,3-dihydro-1H-inden-4-yl)-2-chloroethyl-1-one (intermediate 7-7)

[0307] 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 system was stirred at 80 °C for 2 h. The reaction system was cooled to room temperature, and the solvent was removed by concentration under reduced pressure. The residue was dissolved in 100 mL of dichloromethane, and the pH was adjusted to approximately 7 with saturated sodium bicarbonate aqueous solution. The organic phase was separated, and the aqueous phase was extracted again with dichloromethane (50 mL * 2). The organic phases were combined and 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 give the title compound (840 mg).

[0308] MS m / z(ESI): 210.0 [M+H] + .

[0309] Step 7: Synthesis of (S)-15-(chloromethyl)-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12-dione (intermediates 7-8)

[0310] Intermediate 7-7 (100 mg) and intermediate 1-3 (125.55 mg) were dissolved in toluene (5 mL), and pyridinium p-toluenesulfonic acid salt (5.99 mg) was added. The reaction mixture was stirred at 90 °C for 18 h. After the reaction mixture cooled 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 petroleum ether (2 mL * 2) to obtain the title compound (180 mg).

[0311] MS m / z(ESI): 437.0 [M+H] + .

[0312] Step 8: Synthesis of (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12-dione (intermediate 7-9)

[0313] Intermediate 7-8 (50 mg) was dissolved in a mixed solution of methanol (1 mL) and N,N-dimethylformamide (1 mL), and hexamethylenetetramine (483.13 mg) was added. The reaction mixture was stirred at 50 °C for 4 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and concentrated hydrochloric acid (0.5 mL) was added and 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, 25 mm diameter, 100 mm length; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 20%-40%, elution time 12 min) to give the title compound (22.0 mg).

[0314] MS m / z (ESI): 418.2 [M+H] + .

[0315] Step 9: Synthesis of (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (compound 7)

[0316] Intermediate 7-9 (15 mg) and glycolic 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 the reaction was completed, 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, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 30%-50%, elution time 12 min) to give the title compound (9.0 mg).

[0317] MS m / z (ESI): 476.2 [M+H] + .

[0318] 1H NMR (400MHz, DMSO-d6) δ = 8.28 (t, J = 5.1Hz, 1H), 8.02 (d, J = 8.5Hz, 1H), 7.78 (d, J=8.3Hz,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=5.0Hz,2H),3.88(d,J=5.5Hz,2H),3.57(t,J=7.2Hz,2H),3.0 9(t,J=7.4Hz,2H),2.23-2.15(m,2H),1.95-1.80(m,2H),0.88(t,J=7.3Hz,3H).

[0319] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (Compound 9)

[0320]

[0321] Intermediate 9-1 (6.00 mg, 14.66 μmol, 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 to the solution, and the reaction mixture was stirred at 25 °C for 1 h. The reaction was detected by LC-MS upon completion. The reaction solution was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 25 mm diameter, 100 mm length; eluent: a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 6%-36%, elution time 12 min) to obtain the title compound (3.00 mg).

[0322] MS m / z(ESI): 494.2 [M+H] + .

[0323] 1H NMR (400MHz, DMSO-d6) δ = 8.96 (t, J = 6.0Hz, 1H), 8.50 (d, J = 8.3Hz, 1H), 7.90 (d, J = 10.9Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 6.30 (s, 1H), 5.5 2(s,2H),5.44(s,2H),4.85(d,J=5.9Hz,2H),2.53(s,3H),1.92-1.83(m,2H),1.05-1.00(m,2H),0.88(t,J=7.3Hz,3H),0.85-0.81(m,2H).

[0324] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (Compound 10)

[0325]

[0326] 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. The reaction was confirmed by LC-MS. 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; eluent: a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 16%-46%, elution time 12 min) to give the title compound (2.20 mg).

[0327] MS m / z (ESI): 510.1 [M+H] + .

[0328] 1H NMR (400MHz, DMSO-d6) δ = 8.96 (t, J = 5.9Hz, 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 = 6.0Hz, 2H), 2.59 (s, 3H), 1.94-1.80 (m, 2H), 1.01 (d, J = 3.0Hz, 2H), 0.88 (t, J = 7.3Hz, 3H), 0.83 (d, J = 3.0Hz, 2H).

[0329] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 11)

[0330]

[0331] 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. The reaction was confirmed by LC-MS. The reaction mixture was filtered, concentrated to dryness under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 μm, 25 mm diameter, 100 mm length; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 16%-46%, elution time 12 min) to give the title compound (3.0 mg).

[0332] MS m / z (ESI): 524.1 [M+H] + .

[0333] 1H NMR (400MHz, DMSO-d6) δ = 8.66 (t, J = 5.9Hz, 1H), 8.47 (s, 1H), 8.26 (s, 1H), 7.32(s,1H),6.54(s,1H),5.53(d,J=5.0Hz,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=7.3Hz,3H),0.34-0.31(m,2H),0.29-0.20(m,2H).

[0334] Example 12: (S)-2-amino-N-((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)acetamide (compound 12)

[0335]

[0336] Step 1: Synthesis of 1-(2-fluoro-3,4-dimethoxyphenyl)ethane-1-one (intermediate 12-2)

[0337] Intermediate 12-1 (25.0 g) was dissolved in 1,2-dichloroethane (DCE, 250 mL). The reaction solution was cooled to 0 °C, and aluminum trichloride (64.04 g) was slowly added. Acetyl chloride (64.04 g) was then added dropwise. The reaction solution was stirred at 0 °C for 2 h under a nitrogen atmosphere. After the reaction was complete, water (300 mL) was added, and the mixture was extracted with ethyl acetate (150 mL x 3 times). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography. 120g Rapid silica column chromatography with a gradient of 0–50% petroleum ether / ethyl acetate at a flow rate of 70 mL / min yielded the title compound (21.0 g).

[0338] MS m / z(ESI): 199.0 [M+H] + .

[0339] Step 2: Synthesis of 1-(2-fluoro-3,4-dihydroxyphenyl)ethane-1-one (intermediate 12-3)

[0340] Intermediate 12-2 (15.00 g) was dissolved in anhydrous dichloromethane (DCM, 150 mL). The reaction solution was cooled to -78 °C, and boron tribromide (56.88 g) was slowly added dropwise. The reaction solution was stirred at -78 °C for 2 h under a nitrogen atmosphere, and then heated to 0 °C for 4 h. After the reaction was completed, the reaction solution was slowly quenched by adding ice water. After quenching, the solution was extracted with ethyl acetate (150 mL * 3 times). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography. 120g Rapid silica column chromatography with a gradient of 0–50% petroleum ether / ethyl acetate at a flow rate of 70 mL / min yielded the title compound (8.50 g).

[0341] MS m / z(ESI): 171.0 [M+H] + .

[0342] Step 3: Synthesis of 1-(4-fluorobenzo[d][1,3]dioxacyclopenten-5-yl)ethane-1-one (intermediate 12-4)

[0343] 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 the reaction was complete, the reaction mixture was slowly poured into water and extracted with ethyl acetate (50 mL * 3 times). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography. 24g The title compound (2.0 g) was obtained by rapid silica gel column chromatography with a gradient of 0–15% petroleum ether / ethyl acetate at a flow rate of 60 mL / min.

[0344] MS m / z(ESI): 183.0 [M+H] + .

[0345] Step 4: Synthesis of 1-(4-fluoro-6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)ethane-1-one (intermediate 12-5)

[0346] Intermediate 12-4 (2.0 g) was dissolved in anhydrous dichloromethane (15 mL), and concentrated sulfuric acid (5.38 g, 98% by mass) was added. The reaction solution was cooled to 0 °C. Then, concentrated nitric acid (3.46 g, 68% by mass) was slowly added dropwise to the reaction solution. The reaction solution was stirred at 25 °C for 2 h. After the reaction was complete, the reaction solution was slowly added dropwise to ice water (50 mL), followed by the addition of ethyl acetate (50 mL). The organic phase was washed with water (50 mL x 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. The residue was purified by silica gel column chromatography. 24g The title compound (1.8 g) was obtained by rapid silica column chromatography with a gradient of 0–40% petroleum ether / ethyl acetate at a flow rate of 60 mL / min.

[0347] 1 ¹H NMR (400MHz, deuterated chloroform) δ = 7.51 (s, 1H), 6.26 (s, 2H), 2.63 (s, 3H).

[0348] Step 5: Synthesis of 1-(6-amino-4-fluorobenzo[d][1,3]dioxacyclopenten-5-yl)ethane-1-one (intermediate 12-6)

[0349] 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 the reaction was completed, the mixture was allowed to cool 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), 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 (1.5 g).

[0350] MS m / z(ESI): 198.0 [M+H] + .

[0351] Step 6: Synthesis of N-(6-acetyl-7-fluorobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide (intermediate 12-7)

[0352] Intermediate 12-6 (500.0 mg) was dissolved in anhydrous dichloromethane (5 mL), and pyridine (601.79 mg) was added. Acetyl chloride (398.14 mg) was then 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 the reaction was complete, the organic phase was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography. 12g The title compound (380.0 mg) was obtained by rapid silica column chromatography with a gradient of 0–40% petroleum ether / ethyl acetate at a flow rate of 60 mL / min.

[0353] 1 ¹H 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).

[0354] Step 7: Synthesis of N-(6-(2-bromoacetyl)-7-fluorobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide (intermediate 12-8)

[0355] 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% purity) was added. Liquid bromine (256.42 mg) was then slowly added dropwise to the reaction mixture. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, the mixture was slowly poured into ice water and stirred for 0.5 h. The mixture was filtered, and the filter cake was washed with water (20 mL x 2). The filter cake was dried to give the title compound (400.0 mg).

[0356] MS m / z (ESI): 317.8 [M+H] + .

[0357] Step 8: Synthesis of 1-(6-amino-4-fluorobenzo[d][1,3]dioxacyclopenten-5-yl)-2-chloroethane-1-one (intermediate 12-9)

[0358] Intermediate 12-8 (400.0 mg) was dissolved in anhydrous 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 completed, the mixture was cooled to room temperature, and ice water (20 mL) was slowly added sequentially. The pH was adjusted to 8 with saturated sodium bicarbonate, and then 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).

[0359] MS m / z(ESI): 232.0 [M+H] + .

[0360] Step 9: Synthesis of (S)-14-(chloromethyl)-7-ethyl-15-fluoro-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediates 12-10)

[0361] Intermediate 12-9 (200.0 mg) and intermediate 1-3 (227.32 mg) were dissolved in toluene (3 mL), and pyridinium p-toluenesulfonic acid salt (21.70 mg) was added. The reaction mixture was stirred at 90 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature, and 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 (5 mL * 2) to give the title compound (320.0 mg).

[0362] MS m / z(ESI): 459.0 [M+H] + .

[0363] Step 10: Synthesis of (S)-14-(aminomethyl)-7-ethyl-15-fluoro-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediate 12-11)

[0364] Intermediate 12-10 (260.00 mg) was dissolved in anhydrous methanol (1 mL) and anhydrous N,N-dicarboxamide (1 mL), and hexamethylenetetramine (238.32 mg) was added. The reaction mixture was stirred at 50 °C for 3 h. After the reaction was completed, the 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 in decreasing polarity as the eluent (acetonitrile gradient ratio 0%-30%, elution time 14 min) to give the title compound (85.0 mg).

[0365] MS m / z(ESI): 440.0 [M+H] + .

[0366] 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 = 7.3Hz, 3H).

[0367] Step 11: Synthesis of (S)-(2-(((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)amino)-2-oxoethyl)carbamate tert-butyl ester (intermediate 12-12)

[0368] Intermediate 12-11 (7 mg) and 2-((tert-butoxycarbonyl)amino)acetic acid (5.58 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (12.11 mg) and diisopropylethylamine (2.06 mg) were added to the solution, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, the reaction mixture was concentrated to dryness to give the title compound (8.00 mg).

[0369] MS m / z (ESI): 597.3 [M+H] + .

[0370] 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]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)acetamide (compound 12)

[0371] Intermediate 12-12 (6 mg) was dissolved in dichloromethane (0.5 mL), and trifluoroacetic acid (902.27 mg) was added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, 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, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 2%-32%, elution time 12 min) to give the title compound (1.3 mg).

[0372] MS m / z(ESI): 497.1 [M+H] + .

[0373] 1H 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=7.1Hz,3H).

[0374] Example 13: 2-Cyclopropyl-N-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 13)

[0375]

[0376] Intermediates 7-9 (10 mg) and 11-1 (8.34 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (13.66 mg) and diisopropylethylamine (3.10 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, 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, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 34%-54%, elution time 12 min) to give the title compound (0.8 mg).

[0377] MS m / z (ESI): 516.2 [M+H] + .

[0378] 1 H NMR (400MHz, DMSO-d6) δ = 8.25 (t, J = 5.0Hz, 1H), 8.01 (d, J = 8.5Hz, 1H), 7.78 (d, J =8.5Hz,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=6.3Hz,2H),3.08(t,J=7.7Hz,2H),2.23-2.16(m,2H ),1.92-1.72(m,2H),1.15-1.01(m,1H),0.88(t,J=7.4Hz,3H),0.46-0.19(m,4H)

[0379] Example 14 - 1,2-Cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (Compound 14)

[0380]

[0381] Step 1: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)acetone (intermediate 14-2)

[0382] 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 complete, saturated sodium bicarbonate solution was slowly added to the reaction mixture to adjust the pH to 7-8, and then dichloromethane (100 mL) was added. The organic phase was washed with water (50 mL x 2), and the washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The title compound (5 g) was obtained by preparative thin-layer chromatography (petroleum ether: ethyl acetate = 1:2).

[0383] MS m / z(ESI): 210.0 [M+H] + .

[0384] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxacyclopenten-5-yl)acetone (intermediate 14-3)

[0385] Intermediate 14-2 (2.37 g, 11.33 mmol) was dissolved in anhydrous ethanol (25 mL), and palladium on carbon (0.2 g, 10% purity) was added. The reaction mixture was stirred at 25 °C for 16 h under hydrogen protection. After the reaction was completed, the reaction mixture was filtered, and the filter cake was washed twice with ethyl acetate. The filtrate was concentrated to dryness under reduced pressure to obtain the title compound (1.6 g).

[0386] MS m / z (ESI): 180.1 [M+H] + .

[0387] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxacyclopenten-5-yl)acetamide (intermediate 14-4)

[0388] Intermediate 14-3 (1.0 g, 5.58 mmol) was dissolved in dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and N,N-diisopropylethylamine (DIEA) (1.08 g, 8.37 mmol) and acetyl chloride (569.55 mg, 7.26 mmol) were added. The reaction solution was stirred at 25 °C for 1.5 h. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure to give the title compound (1.23 g).

[0389] MS m / z(ESI): 222.1 [M+H] + .

[0390] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxacyclopenten-5-yl)acetamide (intermediate 14-5)

[0391] 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, 33% purity) was added. Then, liquid bromine (959.69 mg, 6.01 mmol) was slowly added, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, the mixture was poured into ice water and stirred for 10 min. The mixture was filtered, the filter cake was washed twice with water, concentrated to dryness under reduced pressure, and ethyl acetate (2 mL) and petroleum ether (10 mL) were added to the residue. The mixture was stirred at 25 °C for 0.5 h. The mixture was filtered, and the filter cake was dried to give the title compound (500 mg).

[0392] MS m / z(ESI): 300.0 [M+H] + .

[0393] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxacyclopenten-5-yl)-2-chloroethyl ketone (intermediate 14-6)

[0394] Intermediate 14-5 (0.2 g, 666.43 μmol) was dissolved in anhydrous 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 mixture was cooled to room temperature, and ice water (10 mL) and saturated sodium bicarbonate (10 mL) were slowly added sequentially, followed by dichloromethane (50 mL). The organic phase was washed with water (20 mL x 2), and the washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The title compound (160 mg) was obtained by preparative thin-layer chromatography (petroleum ether: ethyl acetate = 6:1).

[0395] MS m / z(ESI): 214.0 [M+H] + .

[0396] Step 6: Synthesis of (S)-14-(bromomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediate 14-7)

[0397] 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-toluenesulfonic acid salt (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 mixture was cooled to room temperature, and 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 dried to give the title compound (60 mg).

[0398] MS m / z(ESI): 441.1 [M+H] + .

[0399] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediate 14-8)

[0400] Intermediate 14-7 (55.00 mg, 124.76 μmol) was dissolved in ethanol (1 mL), and hexamethylenetetramine (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 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, 25 mm diameter, 100 mm length; eluent: a mixture of water (containing 0.225% formic acid) and methanol in decreasing polarity; methanol gradient ratio 0%-27%, elution time 12 min) to obtain the title compound (10 mg).

[0401] MS m / z(ESI): 422.1 [M+H] + .

[0402] Step 8: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14)

[0403] 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). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (11.50 mg, 30.26 μmol) and N,N-diisopropylethylamine (7.82 mg, 60.51 μmol) were added to the solution. The reaction mixture was stirred at 25 °C for 1.5 h. After the reaction was completed, the reaction solution was filtered and purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5μm, 30mm diameter, 150mm length; water (containing 0.225% formic acid) and acetonitrile in decreasing polarity mixture as eluent; acetonitrile gradient ratio 4%-44%, elution time 9 minutes) to obtain the title compound (3mg).

[0404] MS m / z (ESI): 520.1 [M+H] + .

[0405] 1 H NMR(400MHz, DMSO-d6)δ=8.37(t,J=5.8Hz,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=5.0Hz,1H),5.23( s,2H),5.18(s,2H),4.48(d,J=5.5Hz,2H),1.66-1.55(m,2H),0.76-0. 40(m,1H),0.63(t,J=7.3Hz,3H),0.14-0.06(m,2H),0.05-0.04(m,2H).

[0406]

[0407] Step 9: Preparation of 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (intermediate 14-9-P1 / P2)

[0408] Intermediate 14-9 was resolved to prepare isomers 14-9-P1 and 14-9-P2. Intermediate 14-9 (1.3 g) was subjected to supercritical fluid chromatography (DAICEL CHIRALPAK AD column, 10 μm silica, 30 mm diameter, 250 mm length; using ethanol (containing 0.1% ammonia) as eluent) to obtain intermediates 14-9-P1 (600 mg) and 14-9-P2 (600 mg).

[0409] The two isomers were further analyzed under the following chiral supercritical fluid chromatography conditions.

[0410]

[0411] Intermediate 14-9-P1:

[0412] Under the above chiral supercritical fluid chromatography conditions, its retention time was 2.990 minutes;

[0413] 1 H NMR (400MHz, METHANOL-d4) δ7.43-7.29(m,5H),5.29-5.16(m,2H),3.67(d,J=7.6Hz,1H),1.19-1.07(m,1H),0.58-0.38(m,4H).

[0414] Intermediate 14-9-P2:

[0415] Under the above chiral supercritical fluid chromatography conditions, its retention time was 2.661 minutes;

[0416] 1 H NMR (400MHz, METHANOL-d4) δ7.46-7.28 (m, 5H), 5.30-5.16 (m, 2H), 3.67 (d, J = 7.6Hz, 1H), 1.21-1.03 (m, 1H), 0.60-0.36 (m, 4H).

[0417] Step 10: Synthesis of 2-cyclopropyl-2-hydroxyacetic acid (intermediate 14-10-P1 / P2)

[0418] Under a hydrogen atmosphere, intermediate 14-9-P1 (500 mg) was added to methanol (15 mL), and wet palladium on carbon (10 mg, 10%) was added to the reaction solution. The reaction solution was stirred at 25 °C for 16 hours under a hydrogen atmosphere. After the reaction was completed, the reactants were filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P1 (273 mg).

[0419] 1 H NMR (400MHz, METHANOL-d4) δ3.63 (d, J = 7.2Hz, 1H), 1.21-1.09 (m, 1H), 0.61-0.40 (m, 4H).

[0420] Under a hydrogen atmosphere, intermediate 14-9-P2 (500 mg) was added to methanol (15 mL), and wet palladium on carbon (10 mg, 10%) was added to the reaction solution. The reaction solution was stirred at 25 °C for 16 hours under a hydrogen atmosphere. After the reaction was completed, the reactants were filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P2 (279 mg).

[0421] 1 H NMR (400MHz, METHANOL-d4) δ3.63 (d, J = 7.2Hz, 1H), 1.19-1.08 (m, 1H), 0.60-0.39 (m, 4H).

[0422] Step 11: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14-P1 / P2)

[0423] 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'-tetramethylurea hexafluorophosphate (HATU) (46.02 mg) and N,N-diisopropylethylamine (31.28 mg) were added to the solution, and the reaction mixture was stirred at 25 °C for 1.5 h. After the reaction was complete, the reaction solution was purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent was a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity (acetonitrile gradient ratio 16%-46%, elution time 12 min) to obtain compound 14-P1 (22.00 mg).

[0424] MS m / z (ESI): 520.1 [M+H] + .

[0425] 1H NMR (400MHz, DMSO-d6) δ = 8.62 (t, J = 5.7Hz, 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=5.9Hz,2H),3.54(d,J=5.9Hz,1H),1.93-1.78(m,2H),1.06-0. 96(m,1H),0.87(t,J=7.3Hz,3H),0.39-0.30(m,2H),0.29-0.21(m,2H).

[0426] Intermediate 14-8 (10.00 mg) and intermediate 14-10-P2 (8.27 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (18.05 mg) and N,N-diisopropylethylamine (6.13 mg) were added. The reaction mixture was stirred at 25 °C for 1.5 h. After the reaction was completed, 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; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 16%-46%, elution time 12 min) to obtain compound 14-P2 (8.00 mg).

[0427] MS m / z (ESI): 520.1 [M+H] + .

[0428] 1 H NMR (400MHz, DMSO-d6) δ = 8.63 (t, J = 5.9Hz, 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=6.0Hz,2H),3.55(d,J=6.0Hz,1H),1.92-1.81(m,2H),1.03-0.97(m,1H),0.88(t,J=7.3Hz,3H),0.38-0.30(m,2H),0.28-0.22(m,2H).

[0429] The two isomers were further analyzed using the following chiral supercritical fluid chromatography method.

[0430]

[0431]

[0432] Compound 14-P1:

[0433] Under the above chiral supercritical fluid chromatography conditions, its retention time was 3.673 minutes;

[0434] Compound 14-P2:

[0435] Under the above chiral supercritical fluid chromatography conditions, its retention time was 3.735 minutes.

[0436] Example 14-2: (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14-S)

[0437]

[0438] Step 1: Synthesis of (S)-4-benzyl-3-(2-cyclopropylacetyl)oxazolidin-2-one (intermediate 3)

[0439] 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 portions. After the addition was complete, the mixture was stirred at room temperature for approximately 5 h. After the reaction was complete, the reaction solution was diluted with 1500 mL of dichloromethane, and then washed twice with 500 mL of water, once with 500 mL of 2N HCl, once with 500 mL of saturated sodium bicarbonate solution, and once with 500 mL of saturated saline solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (302 g).

[0440] 1 H NMR (400MHz, 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), 3.30-3.33(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).

[0441] Step 2: Synthesis of (S)-4-benzyl-3-((S)-2-cyclopropyl-2-hydroxyacetyl)oxazolidin-2-one (intermediate 5)

[0442] Weigh 200 g of intermediate 3 and dissolve it in 2000 mL of anhydrous tetrahydrofuran. Stir at -78 °C for 15 min under nitrogen protection. Then, add sodium bis(trimethylsilyl)amino (443.5 mL, 2 M tetrahydrofuran solution) dropwise to the reaction solution. After the addition is complete, stir the reaction solution at -78 °C for 30 min. Dissolve 201.5 g of intermediate 4 in 700 mL of tetrahydrofuran until dissolved, and slowly add this solution dropwise to the reaction solution. After the addition is complete, stir at -78 °C for 2 h. Then, add 220 mL of glacial acetic acid to the reaction solution to quench the reaction. After the addition is complete, gradually raise the temperature to room temperature and add 600 mL of 2N HCl. Stir at room temperature (20-25 °C) for 10 h. Then, concentrate the reaction solution under reduced pressure, and add 1000 mL of ethyl acetate and 200 mL of water to the residue, stirring for 20 min. The aqueous phase was extracted twice with ethyl acetate (500 mL x 2), the organic phases were combined, and washed twice each with 400 mL of saturated NaHCO3 solution, 400 mL of saturated Na2S2O3 solution, and saturated brine. The obtained organic phase was dried over anhydrous sodium sulfate, filtered, and 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).

[0443] 1 H NMR(400MHz, CDCl3)δ7.37(dd,J=8.1,6.8Hz,2H),7.33-7.29(m,1H),7.27-7.23(m ,2H),4.81(dd,J=7.9,5.9Hz,1H),4.72(ddt,J=10.0,7.5,2.9Hz,1H),4.33(t,J=8 .3Hz,1H),4.28(dd,J=9.1,2.5Hz,1H),3.48(dd,J=8.2,3.9Hz,1H),3.35(dd,J=13 .5,3.4Hz,1H),2.88(dd,J=13.5,9.4Hz,1H),1.36-1.29(m,1H),0.62-0.44(m,4H).

[0444] Step 3: Synthesis of (S)-4-benzyl-3-((S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetyl)oxazolidin-2-one (intermediate 6)

[0445] 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. TBSCl (107.3 g) was then added to the reaction mixture in portions, 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. The mixture was 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, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 80:1) to give the title compound (160 g).

[0446] 1 H NMR (400MHz, 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).

[0447] Step 4: Synthesis of (S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylbenzyl acetate (intermediate 7)

[0448] Weigh 62.18 g of benzyl alcohol and dissolve it in 500 mL of tetrahydrofuran, stirring at -25 °C. Measure 213.6 mL of n-butyllithium (in 2.5 M tetrahydrofuran solution) and slowly add it dropwise to the reaction solution. After the addition is complete, stir at -25 °C for 1 h. Weigh 160 g of intermediate 6 and dissolve it in 320 mL of tetrahydrofuran, slowly adding it dropwise to the reaction solution at -25 °C. After the addition is complete, stir at -15 °C for 3 h. Quench the reaction by adding 200 mL of saturated NH4Cl 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 solution. The mixture was stirred for 30 min, separated, and the aqueous phase was extracted twice with methyl tert-butyl ether (200 mL x 2). The organic phases were combined, washed once with saturated brine (250 mL), 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 = 100:1) to give the title compound (125 g).

[0449] 1H NMR (400MHz, 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).

[0450] Step 5: Synthesis of (S)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester (intermediate 8)

[0451] 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 solution, and the reaction mixture was placed at 45 °C for 4 h. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran (600 mL), and 300 mL of water and 400 mL of methyl tert-butyl ether were added to the residue. The mixture was stirred for 20 min, separated, and the aqueous phase was extracted twice with methyl tert-butyl ether (200 mL). The combined organic phases were washed twice each with 200 mL of saturated NaHCO3 solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and 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).

[0452] 1 H NMR (400MHz, 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).

[0453] Intermediate 8 was further analyzed under the following chiral supercritical fluid chromatography conditions.

[0454]

[0455] Intermediate 8 had a retention time of 3.013 minutes under the chiral supercritical fluid chromatography conditions described above; this 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 have the same configuration and are the same compound.

[0456] Step 6: Synthesis of (S)-2-cyclopropyl-2-hydroxyacetic acid (intermediate 9)

[0457] Intermediate 8 (5 g) was dissolved in methanol (80 mL), and wet palladium on carbon (10% by mass, 0.7 g) was added to the reaction solution. The mixture was stirred at 25 °C for 16 h under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (2.4 g).

[0458] 1 H NMR (400MHz, METHANOL-d4) δ = 3.63 (d, J = 7.3Hz, 1H), 1.20-1.09 (m, 1H), 0.61-0.39 (m, 4H).

[0459] Step 7: Synthesis of (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14-S)

[0460] 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-tetramethylurea 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 the reaction was completed, the reaction mixture was purified by high performance liquid chromatography (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 20%-50%, 12 min) to obtain the title compound (21 mg).

[0461] MS m / z(ESI): 520.0 [M+H] + .

[0462] 1 H NMR (400MHz, DMSO-d6) δ = 8.62 (t, J = 6.1Hz, 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=5.6Hz,2H),3.54(d,J=5.5Hz,1H),1.93-1.80(m,2H),1.05-0. 97(m,1H),0.88(t,J=7.3Hz,3H),0.39-0.30(m,2H),0.30-0.22(m,2H).

[0463] Compound 14-S was further analyzed under the following chiral supercritical fluid chromatography conditions.

[0464]

[0465] Compound 14-S had a retention time of 3.654 minutes under the chiral supercritical fluid chromatography conditions described above; this is essentially consistent with the retention time (3.673 minutes) of compound 14-P1 prepared in Example 14-1 under the same chromatographic conditions. Therefore, it is determined that compound 14-S and compound 14-P1 prepared in Example 14-1 have the same configuration and are the same compound.

[0466] Example 15: (S)-N-((9-bromo-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b][1,7]naphthidin-11-yl)methyl)-2-hydroxyacetamide (Compound 15)

[0467]

[0468] Step 1: Synthesis of 1-(5-amino-2-bromopyridin-4-yl)acetone (intermediate 15-2)

[0469] 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 tribromide (1.64 g, 5.03 mmol) were added. The reaction mixture was stirred at 25 °C for 8 h. After the reaction was completed, the reaction mixture was filtered, and the title compound (300 mg) was obtained by preparative thin-layer chromatography (silica, petroleum ether: ethyl acetate = 20:1).

[0470] MS m / z(ESI): 214.9 [M+H] + .

[0471] Step 2: Synthesis of N-(4-acetyl-6-bromopyridin-3-yl)acetamide (intermediate 15-3)

[0472] Intermediate 15-2 (150 mg, 697.52 mmol) was dissolved in dichloromethane (2 mL). The reaction solution 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. The reaction solution was stirred at 25 °C for 3 h. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure, and the title compound (100 mg) was obtained by preparative thin-layer chromatography (silica, petroleum ether: ethyl acetate = 3:1).

[0473] MS m / z(ESI): 257.0 [M+H] + .

[0474] Step 3: Synthesis of 1-(5-amino-2-bromopyridin-4-yl)-2-bromoethylone (intermediate 15-4)

[0475] 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, 33% purity) was added. Then, liquid bromine (48.07 mg, 300.80 μmol) was slowly added, and the mixture was stirred at 25 °C for 1 h. After the reaction was completed, the reaction solution 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).

[0476] MS m / z(ESI): 292.9 [M+H] + .

[0477] Step 4: Synthesis of 1-(5-amino-2-bromopyridin-4-yl)-2-chloroethyl ketone (intermediate 15-5)

[0478] 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 mixture was cooled to room temperature, and ice water (10 mL) and saturated sodium bicarbonate (10 mL) were slowly added sequentially, followed by dichloromethane (30 mL). The organic phase was washed with water (20 mL x 2), and then dried over an appropriate amount of anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to obtain the title compound (25 mg).

[0479] MS m / z(ESI): 248.9 [M+H] + .

[0480] Step 5: Synthesis of (S)-9-bromo-11-(chloromethyl)-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b][1,7]naphthidine-3,14(4H)-dione (intermediate 15-6)

[0481] 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-toluenesulfonic acid salt (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 mixture was cooled to room temperature, and 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).

[0482] MS m / z (ESI): 475.9 [M+H] + .

[0483] Step 6: Synthesis of (S)-11-(aminomethyl)-9-bromo-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b][1,7]naphthidine-3,14(4H)-dione (intermediate 15-7)

[0484] Intermediate 15-6 (30 mg, 62.93 μmol) was dissolved in ethanol (1 mL), and hexamethylenetetramine (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 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, 25 mm diameter, 100 mm length; eluent: a mixture of water (containing 0.225% formic acid) and methanol in decreasing polarity; methanol gradient ratio 0%-25%, elution time 12 min) to obtain the title compound (4 mg).

[0485] MS m / z(ESI): 457.0 [M+H] + .

[0486] 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]indolazino[1,2-b][1,7]naphthid-11-yl)methyl)-2-hydroxyacetamide (compound 15)

[0487] Intermediate 15-7 (4 mg, 8.75 μmol) and glycolic 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 the reaction was complete, 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; eluent: a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 8%-28%, elution time 12 min) to give the title compound (1.00 mg).

[0488] MS m / z(ESI): 515.0 [M+H] + .

[0489] 1 H NMR (400MHz, DMSO-d6) δ = 9.38 (s, 1H), 8.88 (t, J = 6.2Hz, 1H), 8.76 (s, 1H), 7.39 (s, 1H), 6.57 (s, 1H), 5.64-5.61 (m, 1 H), 5.60 (s, 2H), 5.45 (s, 2H), 4.80 (d, J = 6.0Hz, 2H), 3.83 (d, J = 5.7Hz, 2H), 1.92-1.80 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

[0490] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (Compound 16)

[0491]

[0492] Step 1: Synthesis of 1-(6-amino-3-chloro-2,4-difluorophenyl)-2-chloroethane-1-one (intermediate 16-2)

[0493] Boron trichloride (1M, 6.11 mL) was dissolved in 1,2-dichloroethane (12 mL). The reaction solution 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 was stirred at 0 °C for 10 min, and then aluminum trichloride (1.06 g, 7.95 mmol) was added. The reaction solution was then heated to 25 °C under nitrogen protection and stirred for 10 min. The reaction solution was then stirred at 90 °C under nitrogen protection for 18 h. The reaction was confirmed by LC-MS. After the reaction was cooled to room temperature, ice water (25 mL) and 5% hydrochloric acid (5 mL) were slowly added sequentially, and the mixture was stirred at 25 °C for 30 min. Then, dichloromethane (50 mL) was added, and the organic phase was washed with water (2 mL x 2). The washed organic phase was dried over anhydrous sodium sulfate. The crude product was concentrated 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 (340 mg).

[0494] MS m / z(ESI): 240.0 [M+H] + .

[0495] 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]indolazino[1,2-b]quinoline-3,14(4H)-dione (intermediate 16-3)

[0496] 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-toluenesulfonic acid salt (20.94 mg, 83.32 μmol) was added. The reaction mixture was stirred at 100 °C for 18 h. The reaction was confirmed by LC-MS. After the reaction mixture was cooled to room temperature, ethanol (1 mL) was added, and the 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 crude title compound (190 mg).

[0497] MS m / z (ESI): 467.1 [M+H] + .

[0498] 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]indolazino[1,2-b]quinoline-3,14(4H)-dione (16-4)

[0499] Intermediate 16-3 (50 mg, 107.01 μmol) was dissolved in ethanol (1 mL), and hexamethylenetetramine (45.00 mg, 321.03 μmol) was added. The reaction mixture was stirred at 80 °C for 1.5 h. The reaction was detected by LC-MS. After the reaction was cooled to room temperature, it 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; eluent of a mixture of water (containing 0.225% formic acid) and acetonitrile in decreasing polarity; acetonitrile gradient ratio 2%-32%, elution time 12 min) to give the title compound (1.22 mg).

[0500] MS m / z(ESI): 448.0 [M+H] + .

[0501] 1 H NMR (400MHz, DMSO-d6) δ = 8.14 (d, J = 9.8Hz, 1H), 7.36 (s, 1H), 6.57 (s, 1H), 5.55 (s, 2H), 5.46 (s, 2H), 4.35 (d, J = 3.0Hz, 2H), 1.94-1.83 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

[0502] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (compound 16)

[0503] 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), and HATU (7.32 mg, 19.26 μmol) and diisopropylethylamine (4.98 mg, 38.52 μmol) were added. The reaction mixture was stirred at 30 °C for 1 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, 25 mm diameter, 100 mm length; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 14%-34%, elution time 12 min) to obtain the title compound (3 mg).

[0504] MS m / z(ESI): 532.1 [M+H] + .

[0505] 1 H NMR (400MHz, DMSO-d6) δ = 8.46 (t, J = 5.9Hz, 1H), 8.16 (d, J = 9.8Hz, 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=3.6Hz,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).

[0506] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxy-2-methylpropionamide (Compound 17)

[0507]

[0508] 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). HATU (8.95 mg, 23.48 μmol) and N,N-diisopropylethylamine (1.19 mg, 11.74 μmol) were added to the solution, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, the reaction solution was filtered and purified by preparative high performance liquid chromatography (Waters Xbridge C18 column, 5 μm silica, 25 mm diameter, 100 mm length; eluent: a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 22%-42%, elution time 12 min) to obtain the title compound (1 mg).

[0509] MS m / z (ESI): 512.1 [M+H] + .

[0510] 1 H NMR (400MHz, Methanol-d4) δ = 8.32 (s, 1H), 8.22 (s, 1H), 7.67 (s, 1H), 5.62 (d, J = 16.4Hz, 1H), 5.52 (s, 2H) ,5.42(d,J=16.4Hz,1H),5.01(s,2H),2.65(s,3H),2.05-1.94(m,2H),1.38(s,6H),1.03(t,J=7.5Hz,3H).

[0511] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxy-3-methylbutyramide (Compound 18)

[0512]

[0513] 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). HATU (8.95 mg, 23.48 μmol) and N,N-diisopropylethylamine (1.19 mg, 11.74 μmol) were added to the solution, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, the reaction solution was filtered and purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 μm, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 25%-45%, elution time 12 min) to give the title compound (1.20 mg).

[0514] MS m / z(ESI): 526.1 [M+H] + .

[0515] 1 H NMR (400MHz, Methanol-d4) δ = 8.30 (s, 1H), 8.13 (s, 1H), 7.61 (s, 1H), 5.60 (d, J = 16.3Hz, 1H), 5.56-5.45 (m, 2H), 5.42-5.37 (m, 1H), 5 .00-4.90(m,2H),3.91(d,J=3.3Hz,1H),3.13(d,J=6.5Hz,1H),2.62(s,3H),2.01-1.94(m,2H),1.05-1.00(m,6H),0.77-0.70(m,3H).

[0516] Example 19-1, 2-Cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19, Compound 19-P1 / P2)

[0517]

[0518] Step 1: Synthesis of 1-(benzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)ethane-1-one (intermediate 19-2)

[0519] Intermediate 19-1 (3 g) was dissolved in anhydrous DMF solution (25 mL), and deuterated dichloromethane (8.57 g) and potassium carbonate (8.18 g) were added. After the addition was complete, the mixture was heated to 90 °C and stirred for 16 h. The reaction solution was then added to water (100 mL) and extracted with ethyl acetate (200 mL * 2). The combined organic phases were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, 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 (2.4 g).

[0520] MS m / z (ESI): 167.1 [M+H] + .

[0521] Step 2: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)ethane-1-one (intermediate 19-3)

[0522] Intermediate 19-2 (2.4 g) was dissolved in anhydrous acetic acid (10 mL), and concentrated nitric acid (32.50 g, 70% purity) was added dropwise at 0 °C. After the addition was complete, the mixture was stirred at 0 °C for 10 min. The temperature was then raised to room temperature and stirred for 1 h. After the reaction was complete, the reaction mixture was added dropwise to ice water (200 mL), filtered, and the filter cake was dried to give the title compound (1.9 g).

[0523] MS m / z(ESI): 212.0 [M+H] + .

[0524] 1 H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.30(s,1H),2.49(s,3H).

[0525] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)acetamide (intermediate 19-4)

[0526] 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 complete, 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).

[0527] MS m / z(ESI): 224.1 [M+H] + .

[0528] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)acetamide (intermediate 19-5)

[0529] A solution of HBr in acetic acid (2.39 g, 33% purity) was added dropwise to an anhydrous acetic acid solution (25 mL) containing 1.45 g of intermediate 19-4, followed by the 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 solution 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, filtered, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (1.3 g).

[0530] MS m / z (ESI): 302.1 [M+H] + .

[0531] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)-2-chloroethane-1-one (intermediate 19-6)

[0532] 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 the reaction was completed, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high performance liquid chromatography (YMC-ActusTriart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; water (containing 0.05% NH4HCO3) and a mixture of acetonitrile with decreasing polarity as eluent (acetonitrile gradient ratio 40%-50%) to give the title compound (577 mg).

[0533] MS m / z(ESI): 216.0 [M+H] + .

[0534] Step 6: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 19-7)

[0535] 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-toluenesulfonic acid (5.24 mg) was added. The reaction mixture was stirred at 100 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated to dryness under reduced pressure. Ethanol (5 mL) was added, and the mixture was stirred at 25 °C for 0.5 h. The mixture was filtered, and the filter cake was washed with ethanol (5 mL * 2) to obtain the title compound (100.0 mg).

[0536] MS m / z(ESI): 443.0 [M+H] + .

[0537] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 19-8)

[0538] Intermediate 19-7 (100.00 mg) was dissolved in anhydrous ethanol (1.5 mL) and anhydrous N,N-dicarboxamide (1.5 mL), and hexamethylenetetramine (94.97 mg) was added. The reaction mixture was stirred at 50 °C for 6 h. After the reaction was completed, 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*30 mm*5 μm; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 0%-30%, 12 min) to give the title compound (25.0 mg).

[0539] MS m / z(ESI): 424.0 [M+H] + .

[0540] Step 8: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (compound 19)

[0541] 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'-tetramethylurea hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, the reaction mixture was filtered and purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 μm, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 20%-50%, elution time 12 min) to give the title compound (2.60 mg).

[0542] MS m / z(ESI): 522.1 [M+H] + .

[0543] 1 H NMR (400MHz, DMSO-d6) δ=8.62(t,J=5.9Hz,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= 5.5Hz,2H),3.59-3.52(m,1H),2.00-1.76(m,2H),1.05-0.96(m,1H),0.88(t,J=7.4Hz,3H),0.37-0.30(m,2H),0.29-0.19(m,2H).

[0544] Step 9: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (compound 19-P1 / P2)

[0545]

[0546] Intermediate 19-8 (7 mg) and intermediate 14-10-P1 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, 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, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 15%-45%, elution time 12 min) to obtain compound 19-P1 (3.30 mg).

[0547] MS m / z(ESI): 522.1 [M+H] + .

[0548] 1 H 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=6.0Hz,2H),3.55-3.53(m,1H),1.94-1.78(m,2H),1.05-0. 96(m,1H),0.88(t,J=7.3Hz,3H),0.40-0.30(m,2H),0.29-0.19(m,2H).

[0549] Intermediate 19-8 (7 mg) and intermediate 14-10-P2 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, 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, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 15%-45%, elution time 12 min) to give compound 19-P2 (4.0 mg).

[0550] MS m / z(ESI): 522.1 [M+H] + .

[0551] 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=5.8Hz,2H),3.55-3.52(m,1H),1.94-1.80(m,2H),1.04-0. 95(m,1H),0.88(t,J=7.4Hz,3H),0.39-0.30(m,2H),0.29-0.21(m,2H).

[0552] The two isomers were further analyzed using the following chiral supercritical fluid chromatography method.

[0553]

[0554]

[0555] Compound 19-P1:

[0556] Under the above chiral high-performance liquid chromatography conditions, its retention time was 2.877 minutes;

[0557] Compound 19-P2:

[0558] Under the above chiral high-performance liquid chromatography conditions, its retention time was 2.690 minutes.

[0559] Configuration confirmation of compound 19-P1 (X-ray single crystal diffraction method)

[0560] 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, sonicate to dissolve, seal with sealing film, poke three small holes in the sealing film with a needle, and slowly volatilize at 20-30℃ for 48 h to obtain needle-shaped crystals.

[0561] The obtained single-crystal samples were subjected to X-ray analysis, and the test results are shown in Table 1 and 2. Figure 1 .

[0562] Table 1 Single crystal samples and crystal data of compound 19-P1

[0563]

[0564] The chemical structure of compound 19-P1 was determined to be as follows through the X-ray crystal diffraction experiments described above:

[0565]

[0566] 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]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (compound 19-S)

[0567]

[0568] Intermediate 19-8 (2.4 g) and intermediate 9 (1645.5 mg) were dissolved in anhydrous N,N-dimethylformamide (25 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphine salt (3.24 g) and N,N-diisopropylethylamine (1465.11 mg) were added to the solution, and the reaction mixture was stirred at 25 °C for 3 h. After the reaction was complete, the solution was concentrated to dryness under reduced pressure. Ethyl acetate (100 mL) was added to the residue and the mixture was stirred for 16 h. After filtration, methanol (50 mL) was added to the filter cake and the mixture was stirred for 16 h. After filtration, the title compound (1.6 g) was obtained.

[0569] MS m / z(ESI): 522.1 [M+H] + .

[0570] 1 H NMR (400MHz, DMSO-d6) δ = 8.60 (t, J = 5.9Hz, 1H), 7.84 (s, 1H), 7.50 (s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 5.49 (d, J = 5.1Hz, 1H), 5.47-5.37 (m, 4H), 4 .71(d,J=5.8Hz,2H),3.54(t,J=5.6Hz,1H),1.97-1.75(m,2H),1.07-0. 94(m,1H),0.87(t,J=7.3Hz,3H),0.40-0.29(m,2H),0.29-0.20(m,2H).

[0571] Compound 19-S was further analyzed using the following chiral supercritical fluid chromatography method.

[0572]

[0573] The retention time of compound 19-S obtained in this embodiment was 2.853 minutes under the chiral supercritical fluid chromatography conditions described above; this is essentially consistent with the retention time (2.877 minutes) of compound 19-P1 obtained in Example 19-1 under the same chromatographic analysis conditions. Therefore, it is determined that compounds 19-S and 19-P1 have the same configuration and are the same compound.

[0574] Example 20: (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 20)

[0575]

[0576] Step 1: Synthesis of 1-(5-aminobenzofuran-4-yl)-2-chloroethyl ketone (intermediate 20-2)

[0577] Boron trichloride (1M, 9.61 mL) was dissolved in dichloroethane (14 mL). The reaction solution was cooled to 0 °C, and intermediate 20-1 (1.6 g, 12.02 mmol) and chloroacetonitrile (1.36 g, 18.03 mmol) were added. The reaction was stirred at 0 °C for 10 min, and then aluminum trichloride (1.92 g, 14.42 mmol) was added. The reaction solution was heated to 25 °C under nitrogen protection and stirred for 10 min. The reaction solution was then stirred at 90 °C under nitrogen protection for 18 h. After the reaction was completed, the reaction solution was cooled to room temperature, and ice water (50 mL) and 5% HCl (10 mL) were slowly added sequentially. The mixture was stirred at 25 °C for 30 min, and then dichloromethane (60 mL) was added. The organic phase was washed with water (30 mL * 2), and the washed organic phase was dried with an appropriate amount of anhydrous sodium sulfate. The title compound (300 mg) was obtained by preparative thin-layer chromatography (petroleum ether: (ethyl acetate + ethanol = 3:1) = 9:1).

[0578] MS m / z(ESI): 210.0 [M+H] + .

[0579] 1 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)

[0580] Step 2: Synthesis of (S)-15-(chloromethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(8H)-dione (intermediate 20-3)

[0581] 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-toluenesulfonic acid salt (23.98 mg, 95.41 μmol) was added. The reaction mixture was stirred at 90 °C for 16 h under nitrogen protection. After the reaction was completed, the reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethanol (3 mL * 2) to give the title compound (300 mg).

[0582] MS m / z (ESI): 437.1 [M+H] + .

[0583] Step 3: Synthesis of (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(8H)-dione (intermediate 20-4)

[0584] Intermediate 20-3 (70 mg, 160.24 μmol) was dissolved in ethanol (0.5 mL) and anhydrous N,N-dimethylformamide (0.5 mL), and hexamethylenetetramine (89.85 mg, 640.96 μmol) was added. The reaction mixture was stirred at 25 °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 (YMC-Actus Triart C18 column 5 μm, 25 mm diameter, 100 mm length; eluent of a mixture of water (containing 0.225% formic acid) and methanol in decreasing polarity; methanol gradient ratio 5%-25%, elution time 12 min) to give the title compound (17 mg).

[0585] MS m / z (ESI): 418.1 [M+H] + .

[0586] Step 4: (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 20)

[0587] Intermediate 20-4 (5.00 mg, 11.98 μmol) and glycolic 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 the reaction was complete, 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; eluent: a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 15%-35%, elution time 12 min) to obtain the title compound (3 mg).

[0588] MS m / z (ESI): 476.2 [M+H] + .

[0589] 1 H NMR (400MHz, DMSO-d6) δ=8.48-8.41(m,1H),8.35(d,J=1.8Hz,1H),8.22(d,J=8.0Hz,1H),8.14(d,J=8.0Hz,1H),7.76(s,1H),7.35(s,1H),6.54 (s,1H),5.58(t,J=5.6Hz,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).

[0590] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxyacetamide (Compound 21)

[0591]

[0592] Compound 16-4 (7 mg, 15.63 μmol) and glycolic 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 the reaction was complete, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; eluent: a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 20%-40%, elution time 12 min) to obtain the title compound (1 mg).

[0593] MS m / z (ESI): 506.1 [M+H] + .

[0594] 1 H NMR (400MHz, Methanol-d4) δ = 8.35 (t, J = 5.8Hz, 1H), 8.15 (dd, J = 1.8, 9.8Hz, 1H), 7.36 (s, 1H), 6.57 (s, 1H), 5.59- 5.50(m,3H),5.45(s,2H),4.91(d,J=3.2Hz,2H),3.84(d,J=5.7Hz,2H),1.90-1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0595] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxy-2-methylpropionamide (Compound 22)

[0596]

[0597] 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 to the solution, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, the reaction solution was filtered and purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 20%-40%, elution time 12 min) to give the title compound (1.07 mg).

[0598] MS m / z (ESI): 534.2 [M+H] + .

[0599] 1 H NMR (400MHz, DMSO-d6) δ = 8.30 (t, J = 5.9Hz, 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=3.7Hz,2H),1.90-1.82(m,2H),1.24(d,J=4.3Hz,6H),0.87(t,J=7.3Hz,3H).

[0600] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 23)

[0601]

[0602] 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 the reaction was completed, 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; eluent of a mixture of water (containing 0.225% formic acid) and acetonitrile in decreasing polarity; acetonitrile gradient ratio 41%-61%, elution time 12 min) to obtain the title compound (7 mg).

[0603] MS m / z (ESI): 546.2 [M+H] + .

[0604] Compound 23 (7 mg) was 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 obtain compound 23-1 (2.1 mg, RT: 5.106 min) and compound 23-2 (2.09 mg, RT: 5.641 min).

[0605] Compound 23-1:

[0606] 1 H NMR (400MHz, DMSO-d6) δ = 8.32 (t, J = 5.5Hz, 1H), 8.15 (d, J = 9.7Hz, 1H), 7.36 (s, 1H), 6.57 (s, 1H), 5.53 (s, 2H), 5.47 (d, J = 5.1Hz, 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=7.3Hz,3H),0.37-0.31(m,2H),0.29-0.23(m,2H)

[0607] MS m / z (ESI): 546.2 [M+H] + .

[0608] Compound 23-2:

[0609] 1H NMR (400MHz, DMSO-d6) δ=8.37-8.29(m,1H),8.15(d,J=9.9Hz,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=7.3Hz,3H),0.40-0.31(m,2H),0.30-0.25(m,2H)

[0610] MS m / z (ESI): 546.2 [M+H] + .

[0611] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxypropionamide (Compound 25)

[0612]

[0613] 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 to the solution, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, the reaction solution was filtered and purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 μm, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 20%-40%, elution time 12 min) to give the title compound (1.80 mg).

[0614] MS m / z (ESI): 520.1 [M+H] + .

[0615] 1H NMR (400MHz, DMSO-d6) δ = 8.34 (t, J = 5.7Hz, 1H), 8.15 (d, J = 9.9Hz, 1H), 7.36 (s, 1H), 6.57 (s, 1H), 5.59 (d, J = 5.0Hz, 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=6.8Hz,3H),0.87(t,J=7.3Hz,3H).

[0616] 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]indolazino[1,2-b]quinoline-11-yl)methyl)-2-hydroxypropionamide (Compound 26)

[0617]

[0618] 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 the reaction was complete, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; eluent: a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 20%-40%, elution time 12 min) to give the title compound (1.60 mg).

[0619] MS m / z (ESI): 520.1 [M+H] + .

[0620] 1 H NMR (400MHz, DMSO-d6) δ = 8.34 (t, J = 5.7Hz, 1H), 8.23-8.03 (m, 1H), 7.36 (s, 1H), 6.57 (s, 1H), 5.59 (d, J = 5.0Hz, 1H), 5.5 1(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=6.8Hz,3H),0.87(t,J=7.3Hz,3H).

[0621] Example 27: (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 27)

[0622]

[0623] Step 1: Synthesis of (3-chloro-2-hydroxy-5-nitrophenyl)methylene glycol (intermediate 27-2)

[0624] Intermediate 27-1 (6 g) was dissolved in acetic acid (25 mL), and the reaction solution was cooled to 0 °C. Nitric acid (9.64 g) was slowly added to the solution. The reaction solution was stirred at 25 °C for 4 h. After the reaction was completed, the reaction solution 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 concentrated to dryness under reduced pressure to give the title compound (5.88 g).

[0625] 1 H NMR (400MHz, METHANOL-d4) δ = 8.16 (d, J = 1.4, 2.4Hz, 1H), 8.07-8.01 (m, 1H), 5.68 (s, 1H)

[0626] Step 2: Synthesis of ethyl 7-chloro-5-nitrobenzuran-2-carboxylate (intermediate 27-4)

[0627] 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 at 70 °C for 6 h. After the reaction was complete, water (50 mL) was added, and the mixture was 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 (4.5 g).

[0628] 1 ¹H NMR (400MHz, chloroform-d) δ=8.55(d,J=2.1Hz, 1H), 8.39(d,J=2.1Hz, 1H), 7.68(s, 1H), 4.49(q,J=7.1Hz, 2H), 1.46(t,J=7.1Hz, 3H)

[0629] Step 3: Synthesis of 7-chloro-5-nitrobenzofuran-2-carboxylic acid (intermediate 27-5)

[0630] Intermediate 27-4 (4.5 g) was dissolved in methanol (50 mL), and sodium hydroxide aqueous solution (2 g in 25 mL H₂O) was slowly added dropwise. The reaction mixture was stirred at 25 °C for 3 h. After the reaction was complete, water (120 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL). The pH of the aqueous phase was adjusted to 1 with dilute hydrochloric acid, and the mixture was extracted three more 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).

[0631] 1 ¹H NMR (400MHz, chloroform-d) δ = 8.45 (d, J = 2.1 Hz, 1H), 8.25 (d, J = 2.1 Hz, 1H), 7.58 (s, 1H)

[0632] Step 4: Synthesis of 7-chloro-5-nitrobenzofuran (intermediate 27-6)

[0633] Intermediate 27-5 (4 g) and copper oxide (1.05 g) were dissolved in quinoline (28 mL). The reaction mixture was purged with nitrogen and stirred at 200 °C for 0.5 h. After the reaction was complete, the temperature was lowered to 0 °C, and dilute hydrochloric acid (80 mL) was slowly added dropwise. Then, water (30 mL) was 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).

[0634] 1 ¹H NMR (400MHz, chloroform-d) δ = 8.49 (d, J = 2.1Hz, 1H), 8.31 (d, J = 2.1Hz, 1H), 7.88 (d, J = 2.3Hz, 1H), 7.02 (d, J = 2.3Hz, 1H)

[0635] Step 5: Synthesis of 7-chlorobenzofuran-5-amine (intermediate 27-7)

[0636] Intermediate 27-6 (2.4 g) and iron powder (1.55 g) were dissolved in methanol (5 mL), and ammonium chloride aqueous solution (148.91 mg, 5 mL) was added dropwise. The reaction mixture was purged with nitrogen and stirred at 80 °C for 0.5 h. After the reaction was complete, 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 concentrated to dryness under reduced pressure to give the title compound (1.2 g).

[0637] MS m / z (ESI): 167.8 [M+H] + .

[0638] Step 6: Synthesis of 1-(5-amino-7-chlorobenzofuran-4-yl)-2-chloroethyl-1-one (intermediate 27-8)

[0639] Boron trichloride (671.17 mg) was dissolved in 1,2-dichloroethane (8 mL). The reaction solution was cooled to 0 °C, and intermediate 27-7 (1.2 g) and chloroacetonitrile (702.75 mg) were added. The reaction was stirred at 0 °C for 10 min, and then aluminum trichloride (1.24 g) was added. The reaction solution was then heated to 25 °C under nitrogen protection and stirred for 10 min. The reaction solution was then stirred at 90 °C under nitrogen protection for 18 h. After the reaction was completed, the reaction solution was cooled to room temperature, and ice water (5 mL) and 5% HCl (1 mL) were slowly added sequentially. The mixture was stirred at 25 °C for 30 min, and then dichloromethane (4 mL) was added. The organic phase was washed with water (2 mL * 2), and the washed organic phase was dried with an appropriate amount of anhydrous sodium sulfate. After filtration, the solution was concentrated to dryness under reduced pressure. The residue was subjected to preparative thin-layer chromatography (silica, petroleum ether: (3 / 1 mixture of ethyl acetate and ethanol) = 9:1) to obtain the title compound (500 mg).

[0640] MS m / z(ESI): 243.8 [M+H] + .

[0641] Step 7: Synthesis of (S)-4-chloro-15-(chloromethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(8H)-dione (intermediate 27-9)

[0642] Intermediate 27-8 (450 mg) and intermediate 1-3 (480.35 mg) were dissolved in toluene (5 mL), and pyridinium p-toluenesulfonic acid salt (23.17 mg) was added. The reaction mixture was stirred at 90 °C for 18 h. After the reaction was completed, the mixture was cooled to room temperature, and 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 petroleum ether (2 mL * 2) and dried to give the title compound (500 mg).

[0643] MS m / z(ESI): 471.0 [M+H] + .

[0644] Step 8: Synthesis of (S)-15-(aminomethyl)-4-chloro-8-ethyl-8-hydroxy-11,14-dihydro-12H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12(8H)-dione (intermediate 27-10)

[0645] Intermediate 27-9 (450 mg) was dissolved in a mixed solution of methanol (1 mL) and N,N-dimethylformamide (1 mL), and hexamethylenetetramine (267.71 mg) was added. The reaction mixture was stirred at 50 °C for 4 h. After the reaction was completed, the reaction mixture was cooled to room temperature, concentrated hydrochloric acid (2 mL) was added and stirred, and then concentrated to dryness under reduced pressure. The residue was purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 μm, 25 mm diameter, 100 mm length; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 13%-43%, elution time 12 min) to give the title compound (60.0 mg).

[0646] MS m / z (ESI): 451.9 [M+H] + .

[0647] Step 9: Synthesis of (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (compound 27)

[0648] Intermediate 27-10 (10 mg) and 2-hydroxyacetic acid (5.05 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (16.83 mg) and diisopropylethylamine (2.86 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, the reaction mixture was filtered, and the residue was purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 μm, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 28%-48%, elution time 12 min) to give the title compound (6.0 mg).

[0649] MS m / z (ESI): 510.1 [M+H] + .

[0650] 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 = 5.6Hz, 1 H), 5.49 (d, J = 2.9Hz, 2H), 5.45 (s, 2H), 5.07 (s, 2H), 3.88 (d, J = 5.6Hz, 2H), 2.02-1.75 (m, 2H), 0.89 (t, J = 7.3Hz, 3H).

[0651] Example 28: N-(((S)-4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furano[3,2-f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 28)

[0652]

[0653] Intermediate 27-10 (10 mg) and intermediate 11-1 (8.34 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (13.65 mg) and diisopropylethylamine (3.10 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, 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, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 34%-54%, elution time 12 min) to give the title compound (6.2 mg).

[0654] MS m / z (ESI): 550.1 [M+H] + .

[0655] 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=6.0Hz,1H),1.95-1.80(m,2H),1.10-1.00(m,1H),0.89(t,J=7.3Hz,3H),0.41-0.32(m,2H),0.32-0.24(m,2H).

[0656] Example 29: 2-Cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-15-nitro-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (Compound 29)

[0657]

[0658] Step 1: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-15-nitro-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediate 29-1)

[0659] Intermediate 14-7 (500.0 mg) was dissolved in sulfuric acid (15 mL), and the reaction solution was cooled to 0°C. Nitric acid (357.35 g, 70% purity) was then slowly added, and the reaction solution was stirred at 25°C for 1.5 h. After the reaction was complete, ice water (10 mL) was slowly added sequentially, followed by dichloromethane (30 mL). The organic phase was washed with 40 mL of water (20 mL * 2), and then dried over an appropriate amount of anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure to obtain the crude title compound (280.0 mg).

[0660] MS m / z(ESI): 486.0 [M+H] + .

[0661] Step 2: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-15-nitro-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediate 29-2)

[0662] Intermediate 29-1 (270.0 mg) was dissolved in methanol (2 mL) and tetrahydrofuran (2 mL), and hexamethylenetetramine (233.73 mg) was added. The reaction mixture was stirred at 60 °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 (YMC-Pack CN C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent: a mixture of water (containing 0.225% FA) and methanol in decreasing polarity; methanol gradient ratio 9%-29%, elution time 12 min) to give the title compound (15.0 mg).

[0663] MS m / z (ESI): 467.1 [M+H] + .

[0664] 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]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 29)

[0665] 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 the reaction was complete, 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; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 15%-45%, elution time 12 min) to give the title compound (5.20 mg).

[0666] MS m / z (ESI): 565.2 [M+H] + .

[0667] 1 H NMR (400MHz, DMSO-d6) δ = 8.25 (t, J = 5.3Hz, 1H), 7.81 (s, 1H), 7.28 (s, 1H), 6.52 (d, J = 2.6Hz, 3H), 5.55 (d, J = 4.9Hz, 1H), 5.43 (s, 2H), 5. 36 (s, 2H), 4.48 (d, J = 5.1Hz, 2H), 3.53 (t, J = 5.7Hz, 1H), 1.91-1.83 (m, 2H), 1.10-0.98 (s, 1H), 0.87 (t, J = 7.3Hz, 3H), 0.42-0.28 (m, 4H).

[0668] Example 30: N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 30)

[0669]

[0670] Step 1: Synthesis of 2-chloro-3,4-dihydroxybenzaldehyde (intermediate 30-2)

[0671] Intermediate 30-1 (20.0 g) was dissolved in anhydrous dichloromethane (100 mL). The reaction solution was cooled to 0 °C, and boron tribromide (87.41 g) was slowly added. The reaction solution was stirred at 25 °C for 4 h. After the reaction was completed, the reaction solution was slowly poured into ice water, and then ethyl acetate (200 mL) was added. The organic phase was washed with water (100 mL * 2). The washed organic phase was dried with an appropriate amount of anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was washed with an ethyl acetate:petroleum ether = 1:4 solution and filtered to obtain the title compound (14 g).

[0672] MS m / z (ESI): 173.1 [M+H] + .

[0673] Step 2: Synthesis of 4-chlorobenzo[d][1,3]dioxacyclopentene-5-carboxaldehyde (intermediate 30-3)

[0674] 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 complete, the mixture was cooled to room temperature, and water (100 mL) was slowly added sequentially, followed by ethyl acetate (150 mL). The organic phase was washed with water (50 mL x 2), and 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 to obtain the title compound (5.2 g).

[0675] MS m / z (ESI): 185.4 [M+H] + .

[0676] Step 3: Synthesis of 1-(4-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)ethanol-1-ol (intermediate 30-4)

[0677] Intermediate 30-3 (5.0 g) was dissolved in anhydrous tetrahydrofuran (100 mL). The reaction solution was cooled to -78 °C, and methyl magnesium bromide (4.85 g, 3 M) was slowly added. The reaction solution was stirred at 25 °C for 4 h under nitrogen protection. After the reaction was completed, water (50 mL) was slowly added in sequence, followed by ethyl acetate (100 mL). The organic phase was washed with water (50 mL * 2), and the washed organic phase was dried with an appropriate amount of 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 = 2:1) to give the title compound (5.3 g).

[0678] 1¹H NMR (400MHz, chloroform-d) δ=6.99(d,J=8.3Hz, 1H), 6.69(d,J=8.1Hz, 1H), 5.97(s, 2H), 5.13(q,J=6.4Hz, 1H), 1.41(d,J=6.4Hz, 3H)

[0679] Step 4: Synthesis of 1-(4-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)ethyl-1-one (intermediate 30-5)

[0680] Intermediate 30-4 (5.2 g) was dissolved in anhydrous dichloromethane (100 mL). The reaction solution was cooled to 0 °C, and Dysmart reagent (DMP) (16.49 g) was slowly added. The reaction solution was stirred at 25 °C for 2 h under nitrogen protection. After the reaction was complete, water (50 mL) was slowly added sequentially, followed by ethyl acetate (100 mL). The organic phase was washed with water (50 mL x 2) and dried over an appropriate amount of 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).

[0681] MS m / z (ESI): 199.2 [M+H] + .

[0682] Step 5: Synthesis of 1-(4-chloro-6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)ethyl-1-one (intermediate 30-6)

[0683] 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 the reaction was complete, the reaction mixture was slowly poured into ice water, and then ethyl acetate (150 mL) was added. The organic phase was washed with water (50 mL * 2), and the washed organic phase was dried with an appropriate amount of anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was subjected to preparative column chromatography (petroleum ether: ethyl acetate = 2:1) to give the title compound (1.8 g).

[0684] MS m / z(ESI): 244.1 [M+H] + .

[0685] Step 6: Synthesis of 1-(6-amino-4-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)ethyl-1-one (intermediate 30-7)

[0686] Intermediate 30-6 (0.80 g) was dissolved in anhydrous methanol (6 mL), and Raney Ni (400.0 mg) was added. The reaction mixture was stirred at 25 °C for 16 h under a hydrogen atmosphere. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (510.0 mg).

[0687] MS m / z(ESI): 214.2 [M+H] + .

[0688] Step 7: Synthesis of N-(6-acetyl-7-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide (intermediate 30-8)

[0689] Intermediate 30-7 (260.0 mg) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and N,N-diisopropylethylamine (235.95 mg) and chloroacetyl (143.32 mg) were added. The reaction solution was stirred at 25 °C for 1.5 h. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative chromatography (petroleum ether: ethyl acetate = 5:1) to give the title compound (140.0 mg).

[0690] MS m / z(ESI): 256.0 [M+H] + .

[0691] Step 8: Synthesis of N-(6-(2-bromoacetyl)-7-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide (intermediate 30-9)

[0692] 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% purity) was added. Then, liquid bromine (72.20 mg) was slowly added to the reaction mixture, which was stirred at 25 °C for 1 h. After the reaction was complete, the reaction mixture was slowly poured into ice water, and then ethyl acetate (30 mL) was added. The organic phase was washed with water (20 mL x 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 obtain the title compound (110.0 mg).

[0693] MS m / z(ESI): 334.0 [M+H] + .

[0694] Step 9: Synthesis of 1-(6-amino-4-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)-2-chloroethyl-1-one (intermediate 30-10)

[0695] Intermediate 30-9 (110.00 mg) was dissolved in anhydrous 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 mixture was cooled to room temperature, and ice water (10 mL) and saturated sodium bicarbonate (10 mL) were slowly added sequentially, followed by dichloromethane (30 mL). 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. 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 give the title compound (75 mg).

[0696] MS m / z(ESI): 248.0 [M+H] + .

[0697] Step 10: Synthesis of (S)-15-chloro-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediate 30-11)

[0698] 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 h. After the reaction was completed, the mixture was cooled to room temperature, and 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 dried to give the title compound (75.0 mg).

[0699] MS m / z (ESI): 475.1 [M+H] + .

[0700] Step 11: Synthesis of (S)-14-(aminomethyl)-15-chloro-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (intermediate 30-12)

[0701] Intermediate 30-11 (70.00 mg) was dissolved in anhydrous methanol (2 mL) and anhydrous tetrahydrofuran (1 mL), and hexamethylenetetramine (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; eluent: a mixture of water (containing 0.225% formic acid) and acetonitrile in decreasing polarity; acetonitrile gradient ratio 5%-25%, elution time 12 min) to give the title compound (16.0 mg).

[0702] MS m / z (ESI): 456.1 [M+H] + .

[0703] Step 12: Synthesis of N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (compound 30)

[0704] 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 the reaction was completed, 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; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 31%-51%, elution time 12 min) to give the title compound (2.30 mg).

[0705] MS m / z (ESI): 554.2 [M+H] + .

[0706] 1H NMR (400MHz, DMSO-d6)δ=7.78(t,J=5.4Hz,1H),7.35(s,1H),7.01(s,1H),6.30-6.25(m,1H),6.16(d,J=1.5Hz,2H),5.27-5.21(m,2H),5.19(s,2H) ,4.93-4.78(m,2H),3.29(dd,J=2.4,6.1Hz,1H),1.68-1.56(m,2H),0.81- 0.72(m,1H),0.63(t,J=7.3Hz,3H),0.14-0.06(m,2H),0.05-0.03(m,2H).

[0707] Example 31: (S)-N-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (Compound 31)

[0708]

[0709] Intermediate 30-12 (5 mg) and glycolic 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 the reaction was completed, 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; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 20%-40%, elution time 12 min) to give the title compound (2.50 mg).

[0710] MS m / z (ESI): 514.2 [M+H] + .

[0711] 1 H NMR (400MHz, DMSO-d6) δ = 8.03 (t, J = 5.8Hz, 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=6.0Hz,2H),3.82(s,2H),1.93-1.78(m,2H),0.87(t,J=7.4Hz,3H)

[0712] Example 33: (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (Compound 33)

[0713]

[0714] Step 1: Synthesis of 7-chloro-2,3-dihydro-1H-inden-4-ol (Intermediate 33-2)

[0715] Intermediate 33-1 (500 mg) was dissolved in anhydrous acetonitrile (5 mL), and NCS (547 mg) was added. After the addition was complete, the mixture was stirred at 25 °C for 2 hours. After the reaction was complete, water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL * 3 times). The organic layer 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 ~ 10:1) to give the title compound (600 mg).

[0716] MS m / z(ESI): 169.0 [M+H] + .

[0717] Step 2: Synthesis of 7-chloro-5-nitro-2,3-dihydro-1H-inden-4-ol (intermediate 33-3)

[0718] Intermediate 33-2 (10 g) was dissolved in AcOH (50 mL) and H₂O (10 mL), and HNO₃ (8.62 g, 65% by mass) was added at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours. After the reaction was complete, the reaction mixture was slowly added to ice water (200 mL). After filtration, the solvent was removed by concentration under reduced pressure to obtain the title compound (10 g).

[0719] MS m / z(ESI): 214.0 [M+H] + .

[0720] Step 3: Synthesis of 5-amino-7-chloro-2,3-dihydro-1H-inden-4-ol (intermediate 33-4)

[0721] Intermediate 33-3 (5 g) was dissolved in anhydrous DCM (50 mL), and AcOH (14.04 g) and Zn (7.61 g) were added. 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. The organic phase was washed with saturated sodium bicarbonate aqueous solution (50 mL * 2), and 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).

[0722] MS m / z(ESI): 184.0 [M+H] + .

[0723] Step 4: Synthesis of 5-acetamido-7-chloro-2,3-dihydro-1H-inden-4-ylacetate (intermediate 33-5)

[0724] Intermediate 33-4 (4 g) was dissolved in anhydrous dichloromethane (40 mL), and acetic anhydride (Ac₂O) (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 complete, water (200 mL) and ethyl acetate (200 mL) were added sequentially. The organic phase was washed with saturated NaCl aqueous solution (50 mL * 2), and 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 5:1) to give the title compound (4 g).

[0725] MS m / z(ESI): 268.0 [M+H] + .

[0726] Step 5: Synthesis of N-(7-chloro-4-hydroxy-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 33-6)

[0727] Intermediate 33-5 (4 g) was dissolved in anhydrous methanol (20 mL), and K2CO3 (6.19 g) was added. 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. The organic phase was washed with saturated NaCl aqueous solution (50 mL * 2), and 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 give the title compound (2.8 g).

[0728] MS m / z(ESI): 226.0 [M+H] + .

[0729] Step 6: Synthesis of 5-acetamido-7-chloro-2,3-dihydro-1H-inden-4-yltrifluoromethanesulfonate (intermediate 33-7)

[0730] Intermediate 33-6 (500 mg) was dissolved in dichloromethane (5 mL), and trifluoromethanesulfonic anhydride (Tf₂O) (749 mg) and triethylamine (TEA) (672 mg) were added. The reaction mixture was stirred at 25 °C for 12 hours. After the reaction was complete, water (50 mL) and ethyl acetate (50 mL) were added sequentially. The organic phase was washed with saturated NaCl aqueous solution (50 mL * 2), and 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 give the title compound (580 mg).

[0731] MS m / z(ESI): 358.0 [M+H] + .

[0732] Step 7: Synthesis of N-(4-(1-butoxyvinyl)-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 33-8)

[0733] Intermediate 33-7 (430 mg) and vinyl n-butyl ether (360.60 mg) were dissolved in dioxane (20 mL), and diisopropylethylamine (DIEA) (466 mg), 1,1'-bis(diphenylphosphine)ferrocene (DPPF) (66 mg), and tris(dibenzylacetone)dipalladium (Pd2(dba)3) (110 mg) were added. The reaction mixture was stirred at 80 °C under nitrogen protection for 16 hours. After the reaction was completed, the reaction mixture was diluted with water (50 mL), extracted with ethyl acetate (50 mL * 3 times), and the organic phases were combined 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 = 20:1 to 1:1) to give the title compound (300 mg).

[0734] MS m / z(ESI): 308.0 [M+H] + .

[0735] Step 8: Synthesis of N-(4-acetyl-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 33-9)

[0736] Intermediate 33-8 (200 mg) was dissolved in dioxane (5 mL), and 1 N HCl (5 mL) was added. The mixture was stirred at 25 °C for 2 hours. After the reaction was completed, the organic phase was concentrated under reduced pressure to remove the solvent, yielding the title compound (150 mg).

[0737] MS m / z(ESI): 252.0 [M+H] + .

[0738] Step 9: Synthesis of N-(4-(2-bromoacetyl)-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 33-10)

[0739] Intermediate 33-9 (300 mg) was dissolved in HBr / AcOH (4 mL, 33% by mass), and NBS (318.20 mg) was added. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain the title compound (390 mg).

[0740] MS m / z(ESI): 330.0 [M+H] + .

[0741] Step 10: Synthesis of 1-(5-amino-7-chloro-2,3-dihydro-1H-inden-4-yl)-2-chloroethane-1-one (intermediate 33-11)

[0742] Intermediate 33-10 (300 mg) was dissolved in anhydrous ethanol and HCl (12 M, 8.00 mL) was added. The reaction mixture was stirred at 80 °C for 2 hours. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified by preparative high performance liquid chromatography (HPLC) (column: Gemini NX C18 5 μm*10*150 mm; mobile phase: A: water (0.225% formic acid v / v), B: acetonitrile; B%: 30%-70%) to obtain the title compound (64 mg).

[0743] MS m / z(ESI): 244.0 [M+H] + .

[0744] Step 11: Synthesis of (S)-4-chloro-15-(chloromethyl)-8-ethyl-1,2,3,8,11,14-hexahydro-9H,12H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12-dione (intermediate 33-12)

[0745] Intermediate 33-11 (45.00 mg) and intermediate 1-3 (48.53 mg) were dissolved in toluene (1 mL), and pyridinium p-toluenesulfonic acid salt (4.63 mg) was added. The reaction mixture was stirred at 90 °C for 16 h. After the reaction was complete, the mixture was cooled to room temperature, and 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 dried to give the title compound (80.0 mg).

[0746] MS m / z(ESI): 471.1 [M+H] + .

[0747] Step 12: Synthesis of (S)-15-(aminomethyl)-4-chloro-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9,12-dione (intermediates 33-13)

[0748] Intermediate 33-12 (40.00 mg) was dissolved in anhydrous methanol (1 mL) and anhydrous N,N-dimethylformamide (0.5 mL), and hexamethylenetetramine (35.69 mg) was added. The reaction mixture was stirred at 50 °C for 16 h. After the reaction was complete, 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; eluent: a mixture of water (containing 0.225% FA) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 35%-55%, elution time 12 min) to give the title compound (15.0 mg).

[0749] MS m / z (ESI): 452.1 [M+H] + .

[0750] Step 13: Synthesis of (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-hydroxyacetamide (compound 33)

[0751] Intermediate 33-13 (5.00 mg) and glycolic acid (4.21 mg, 55.30 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (6.31 mg) and N,N-diisopropylethylamine (4.29 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, 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; eluent: a mixture of water (containing 0.05% FA) and acetonitrile with decreasing polarity; acetonitrile gradient ratio 32%-52%, elution time 12 min) to give the title compound (2.0 mg).

[0752] MS m / z (ESI): 510.3 [M+H] + .

[0753] 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=7.6Hz,2H),2.27-2.19(m,2H),1.92-1.82(m,2H),0.88(t,J=7.3Hz,3H).

[0754] Example 34: N-(((S)-4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopentadieno[f]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-15-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (Compound 34)

[0755]

[0756] Intermediate 33-13 (5.00 mg) and intermediate 11-1 (6.42 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and HATU (6.31 mg) and diisopropylethylamine (4.29 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was complete, 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; water (containing 0.05% FA) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 35%-55%, elution time 12 min) to obtain the title compound (2.0 mg).

[0757] MS m / z (ESI): 550.3 [M+H] + .

[0758] 1H 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=7.1H z,2H),3.56(t,J=5.8Hz,1H),3.14(t,J=7.4Hz,2H),2.27-2.18(m,2H),1.9 3-1.81(m,2H),1.09-1.03(m,1H),0.88(t,J=7.2Hz,3H),0.41-0.26(m,4H).

[0759] Example 35: 2-Cyclopropyl-N-(((S)-7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (Compound 35)

[0760]

[0761] 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'-tetramethylurea 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 the reaction was completed, 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; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 15%-45%, elution time 12 min) to give the title compound (6.50 mg).

[0762] MS m / z (ESI): 538.1 [M+H] + .

[0763] 1H NMR (400MHz, DMSO-d6) δ=8.21(t,J=6.0Hz,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= 3.8Hz, 2H), 3.53 (d, J = 6.3Hz, 1H), 1.90-1.81 (m, 2H), 1.01 (d, J = 5.5Hz, 1H), 0.89-0.85 (m, 3H), 0.33 (d, J = 6.0Hz, 2H), 0.30-0.25 (m, 2H).

[0764] Synthesis of compounds 35-P1 and 35-P2

[0765]

[0766] 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'-tetramethylurea 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 the reaction was completed, 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; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 17%-47%, elution time 12 min) to obtain compound 35-P1 (2.87 mg).

[0767] MS m / z (ESI): 538.1 [M+H] + .

[0768] 1 H NMR (400MHz, DMSO-d6) δ = 8.19 (t, J = 5.9Hz, 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=4.6Hz,2H),3.53(d,J=6.4Hz,1H),1.91-1.80(m,2H),1.06-0.97(m,1H),0.87(t,J=7.3Hz,3H),0.38-0.31(m,2H),0.31-0.24(m,2H).

[0769] 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'-tetramethylurea 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 the reaction was completed, 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; water (containing 0.225% formic acid) and a mixture of acetonitrile with decreasing polarity as eluent; acetonitrile gradient ratio 17%-47%, elution time 12 min) to obtain compound 35-P2 (2.01 mg).

[0770] MS m / z (ESI): 538.1 [M+H] + .

[0771] 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=6.2Hz,1H),1.94-1.80(m,2H),1.05-0.96(m,1H),0.87(t,J=7.3Hz,3H),0.39-0.30(m,2H),0.31-0.20(m,2H).

[0772] The two isomers were further analyzed using the following chiral supercritical fluid chromatography method.

[0773]

[0774]

[0775] Compound 35-P1:

[0776] Under the above chiral high-performance liquid chromatography conditions, its retention time was 3.519 minutes;

[0777] Compound 35-P2:

[0778] Under the above chiral high-performance liquid chromatography conditions, its retention time was 3.573 minutes.

[0779] Example 36: (S)-N-((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (Compound 36)

[0780]

[0781] Intermediate 12-11 (6 mg) and glycolic acid (3.21 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (10.38 mg) and diisopropylethylamine (1.76 mg) were added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, 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, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 18%-48%, elution time 12 min) to give the title compound (2.40 mg).

[0782] MS m / z (ESI): 498.1 [M+H] + .

[0783] 1 H NMR (400MHz, DMSO-d6) δ = 8.20 (t, J = 5.9Hz, 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 = 4.3Hz, 2H), 3.83 (s, 2H), 1.92-1.80 (m, 2H), 0.87 (t, J = 7.3Hz, 3H).

[0784] 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]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)-3,8,11,14,17-pentoxo-5-oxy-2,7,10,13,16-pentazaoctadecane-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamamide (compound L1-14), isomers L1-14-P1 and L1-14-P2

[0785]

[0786] Step 1: Synthesis of intermediate L1-14-2

[0787] 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). The mixture was heated to 85 °C under a nitrogen atmosphere and stirred 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 (column: [column number missing]). 330g The title compound (18 g) was obtained by using a Silica Flash Column with a mobile phase gradient of 0–75% ethyl acetate / petroleum ether at a flow rate of 100 mL / min.

[0788] 1 H NMR (400MHz, METHANOL-d4) δ = 7.82 (d, J = 7.5Hz, 2H), 7.69 (d, J = 7.3Hz, 2H), 7.44-7.38 (m, 2H), 7. 36-7.31(m,2H),5.22(s,2H),4.39(d,J=6.8Hz,2H),4.28-4.22(m,1H),3.81(s,2H),2.03(s,3H).

[0789] MS m / z (ESI): 391.1 [M+Na] + .

[0790] Step 2: Synthesis of intermediate L1-14-3

[0791] Intermediate L1-14-2 (5 g), 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (8.40 g), and pyridinium p-toluenesulfonate (PPTS, 341.09 mg) were dissolved in dichloromethane (150 mL). The reaction mixture was heated to 65 °C under a nitrogen atmosphere and stirred for 48 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 (column: [column number missing]). 120g The sample was purified by high-performance liquid chromatography (HPLC) using a Silica Flash Column with a mobile phase gradient of 0–45% ethyl acetate / petroleum ether at a flow rate of 80 mL / min. The chromatographic column was a Boston Prime C18 150*30 mm*5 μm column. The mobile phase consisted of [A: water (0.225% formic acid), B: acetonitrile]; B%: 42%–82%, 13 min, yielding the title compound (1.4 g).

[0792] MS m / z (ESI): 537.2 [M+Na] + .

[0793] Step 3: Synthesis of intermediates L1-14-4-P1 and L1-14-4-P2

[0794] 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% by mass, 0.15 g) was added. The reaction solution was stirred at 25 °C for 16 h under a hydrogen atmosphere. 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 (column: Boston Prime C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 24%-64%, 13min). Subsequently, it was further purified by supercritical fluid chromatography (column: DAICEL CHIRALPAK IC column, 10μm silica, 30mm diameter, 250mm length; isopropanol (containing 0.1% ammonia) as eluent). The purification yielded intermediates L1-14-4-P1 (130mg) and L1-14-4-P2 (130mg).

[0795] The two isomers were further analyzed using the following chiral high-performance liquid chromatography method.

[0796] The chiral high-performance liquid chromatography conditions are as follows:

[0797]

[0798] Intermediate L1-14-4-P1:

[0799] Under the above chiral high-performance liquid chromatography conditions, its retention time was 4.471 minutes;

[0800] MS m / z (ESI): 447.5 [M+Na] + .

[0801] Intermediate L1-14-4-P2:

[0802] Under the above chiral high-performance liquid chromatography conditions, its retention time was 5.692 minutes;

[0803] MS m / z (ESI): 447.3 [M+Na] + .

[0804] Step 4: Synthesis of intermediates L1-14-5-P1 and L1-14-5-P2

[0805] 257 mg of 2-chlorotriphenylmethyl chloride resin (2-CTC-resin) (specification: approx. 1.19 mmol / g) was added to 3 mL of dichloromethane, followed by intermediate L1-14-4-P1 (130 mg) and diisopropylethylamine (59.37 mg). The reaction mixture was reacted in a shaker at 25 °C under a nitrogen atmosphere for 16 h. After the reaction was complete, the resin was washed sequentially with 10 mL of methanol and 10 mL of dichloromethane, repeated three times. After filtration and drying of the filter cake, intermediate L1-14-5-P1 (330 mg) was obtained.

[0806] Using intermediate L1-14-4-P2 (130 mg) as raw material, intermediate L1-14-5-P2 (350 mg) was prepared according to the above method.

[0807] Step 5: Synthesis of intermediates L1-14-6-P1 and L1-14-6-P2

[0808] Intermediate L1-14-5-P1 (330 mg) was dissolved in N,N-dimethylformamide (5 mL), and piperidine (1.08 g) was added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was completed, the resin was washed successively with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. After filtration, the filter cake was dried to obtain L1-14-6-P1 (220 mg).

[0809] Using intermediate L1-14-5-P2 (350 mg) as raw material, intermediate L1-14-6-P2 (220 mg) was prepared according to the above method.

[0810] Step 6: Synthesis of intermediates L1-14-7-P1 and L1-14-7-P2

[0811] Intermediate L1-14-6-P1 (220 mg) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-phenylalanine (230.17 mg) were dissolved in N,N-dimethylformamide (5 mL). Benzotriazole-N,N,N',N'-tetramethylurea 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 sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated three times. After filtration and drying of the filter cake, L1-14-7-P1 (377 mg) was obtained.

[0812] Using intermediate L1-14-6-P2 (220 mg) as raw material, intermediate L1-14-7-P2 (361 mg) was prepared according to the above method.

[0813] Step 7: Synthesis of intermediates L1-14-8-P1 and L1-14-8-P2

[0814] Intermediate L1-14-7-P1 (377 mg) was dissolved in N,N-dimethylformamide (5 mL), and piperidine (37.22 mg) was added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was completed, the resin was washed successively with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. After filtration, the filter cake was dried to obtain L1-14-8-P1 (270 mg).

[0815] Using intermediate L1-14-7-P2 (361 mg) as raw material, intermediate L1-14-8-P2 (260 mg) was prepared according to the above method.

[0816] Step 8: Synthesis of intermediates L1-14-9-P1 and L1-14-9-P2

[0817] Intermediate L1-14-8-P1 (270 mg) was dissolved in N,N-dimethylformamide (5 mL), followed by the sequential addition of N-((9H-fluoren-9-ylmethoxy)carbonyl)glycylglycine (209.58 mg), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (224.29 mg), and diisopropylethylamine (99.51 mg). 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 sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated three times. After filtration and drying of the filter cake, intermediate L1-14-9-P1 (403 mg) was obtained.

[0818] Using intermediate L1-14-8-P2 (260 mg) as raw material, intermediate L1-14-9-P2 (420 mg) was prepared according to the above method.

[0819] Step 9: Synthesis of intermediates L1-14-10-P1 and L1-14-10-P2

[0820] Intermediate L1-14-9-P1 (403 mg) was dissolved in N,N-dimethylformamide (5 mL), and piperidine (1.08 g) was added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was completed, the resin was washed successively with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. After filtration, the filter cake was dried to obtain L1-14-10-P1 (300 mg).

[0821] Using intermediate L1-14-9-P2 (420 mg) as raw material, intermediate L1-14-10-P2 (320 mg) was prepared according to the above method.

[0822] Step 10: Synthesis of intermediates L1-14-12-P1 and L1-14-12-P2

[0823] Intermediate L1-14-10-P1 (300 mg) was dissolved in N,N-dimethylformamide (5 mL), followed by the sequential addition of compound L1-14-11 (182.13 mg) and diisopropylethylamine (99.41 mg). 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 sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated three times. After filtration and drying of the filter cake, L1-14-12-P1 (374 mg) was obtained.

[0824] Using intermediates L1-14-10-P2 (320 mg) and L1-14-11 (194.27 mg) as raw materials, intermediate L1-14-12-P2 (387 mg) was prepared according to the above method.

[0825] Step 11: Synthesis of intermediates L1-14-13-P1 and L1-14-13-P2

[0826] 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 (HPLC) (column: Boston Prime C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 15%-35%, 9 min) to obtain intermediate L1-14-13-P1 (74 mg).

[0827] 1 H 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=7.6Hz,1H),3.55-3 .47(m,2H),3.27-3.23(m,1H),3.05-2.98(m,1H),2.30(t,J=7.4Hz,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).

[0828] MS m / z (ESI): 679.7 [M+Na] + .

[0829] Using intermediate L1-14-12-P2 (387 mg) as raw material, intermediate L1-14-13-P2 (86 mg) was prepared according to the above method.

[0830] 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).

[0831] MS m / z (ESI): 679.5 [M+Na] + .

[0832] Step 12: Synthesis of compounds L1-14-P1 and L1-14-P2

[0833] 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 solution was stirred at 25 °C for 2 h under a nitrogen atmosphere. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Boston Green ODS 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 26%-46%, 12 min), yielding compound L1-14-P1 (12.3 mg).

[0834] 1H NMR (400MHz, DMSO-d6) δ=8.65(t,J=5.9Hz,1H),8.58(t,J=6.6Hz,1H),8.27(t,J=5.6Hz,1H),8.17-8.02(m,2H),7.99(t,J=5.6Hz,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=3.7Hz,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=7.1Hz,1H),3.08-3.01(m,1H),2.85-2.74(m,1 H),2.14-2.06(m,2H),1.94-1.80(m,2H),1.52-1.42(m,4H),1.27-1.1 3(m,2H),1.01-0.92(m,1H),0.88(t,J=7.3Hz,3H),0.41-0.28(m,4H).

[0835] MS m / z (ESI): 1060.3 [M+H] + .

[0836] Using intermediates L1-14-13-P2 (31.17 mg) and 14-8 (20 mg) as raw materials, compound L1-14-P2 (11.4 mg) was prepared according to the above method.

[0837] 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),6.99(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=6.7Hz,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)

[0838] MS m / z (ESI): 1060.3 [M+H] + .

[0839] The two isomers were further analyzed using the following chiral high-performance liquid chromatography method.

[0840] The chiral high-performance liquid chromatography conditions are as follows:

[0841]

[0842] The retention time of compound L1-14-P1 under the above-mentioned chiral high-performance liquid chromatography conditions is 3.735 minutes.

[0843] The retention time of compound L1-14-P2 under the above-mentioned chiral high-performance liquid chromatography conditions was 3.901 minutes.

[0844] 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]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)-3,8,11,14,17-pentoxo-5-oxy-2,7,10,13,16-pentazaoctadecane-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexamamide (compound L1-35-P1)

[0845]

[0846] 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 solution was stirred at 25 °C for 2 h under a nitrogen atmosphere. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 23%-45%, 10 min) to obtain L1-35-P1 (2.3 mg).

[0847] 1 H NMR (400MHz, DMSO-d6) δ=8.59(t,J=6.1Hz,1H),8.36-8.31(m,1H),8.28(t,J=5.6Hz,1H),8.14-8.02(m,2H),8.00(t,J=5.8Hz, 1H),7.49(s,1H),7.27-7.16(m,6H),6.99(s,2H),6.52(s,1H),6.38(d,J=2.0Hz,2H),5.49-5.41(m,4H),4.90-4.82(m,2H),4.6 9-4.62(m,1H),4.53-4.43(m,2H),3.79-3.54(m,7H),3.47(d,J=7.0Hz,1H),3.06-3.00(m,1H),2.80-2.74(m,1H),2.10(t,J=7. 3Hz,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=7.2Hz,3H),0.40-0.27(m,4H).

[0848] MS m / z (ESI): 1078.2 [M+H] + .

[0849] Further analysis was performed using the following supercritical fluid chromatography method.

[0850] The supercritical fluid chromatography conditions are as follows:

[0851]

[0852] The retention time of compound L1-35-P1 under the above supercritical fluid chromatography conditions was 3.732 minutes.

[0853] 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]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentoxo-5-oxy-2,7,10,13,16-pentazaoctadecane-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamamide (compound L1-19-P1), isomer L1-19-P2 and racemic L1-19

[0854] 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 solution was stirred at 25 °C for 2 h under a nitrogen atmosphere. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 22%-52%, 12 min) to obtain L1-19-P1 (6.0 mg).

[0855] MS m / z (ESI): 1062.3 [M+H] + .

[0856] 1H NMR (400MHz, DMSO-d6) δ = 8.74-8.69 (m, 1H), 8.59 (t, J = 6.7Hz, 1H), 8.32-8.26 (t, J = 5.6Hz, 1H), 8.11 (d, J = 7.8Hz, 1H), 8.06 (t, J = 5.3Hz, 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=7.0Hz,1H),3.06-3.02(m,1H),2.83-2.74(m,1H) ,2.10(t,J=7.6Hz,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=7.3Hz,3H),0.38-0.29(m,4H).

[0857] Using intermediates L1-14-13-P2 (30.0 mg) and 19-8 (25.2 mg) as raw materials, compound L1-19-P2 (19.0 mg) was prepared according to the above method.

[0858] MS m / z (ESI): 1062.3 [M+H] + .

[0859] 1H NMR (400MHz, DMSO-d6) δ=8.73-8.63(m,1H),8.58(t,J=6.4Hz,1H),8.30(t,J=5.7Hz,1H),8.13(d,J=8.1Hz,1H),8.07(t,J=5.8Hz,1H),8.01( t,J=5.4Hz,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=7.0Hz,1H),3.08-3.02(m,1H),2.85-2.76(m,1H) ,2.09(t,J=7.5Hz,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=7.3Hz,3H),0.44-0.24(m,4H).

[0860] The two isomers were further analyzed using the following chiral high-performance liquid chromatography method.

[0861] The chiral high-performance liquid chromatography conditions are as follows:

[0862]

[0863] Under the above chiral supercritical fluid chromatography conditions, the retention time of L1-19-P1 is 3.775 minutes;

[0864] Under the chiral supercritical fluid chromatography conditions described above, the retention time of L1-19-P2 is 3.973 minutes.

[0865] Synthesis of racemic compound L1-19

[0866]

[0867] Intermediate L1-14-13 (30.00 mg, synthesized from 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (racemate) according to the L1-14-13-P1 synthesis method), 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 solution was stirred for 2 h at 25 °C under nitrogen protection. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 33%-33%, 14 min) to obtain racemate compound L1-19 (15.4 mg).

[0868] 1 H NMR (400MHz, DMSO-d6) δ=8.76-8.66(m,1H),8.59(t,J=6.8Hz,1H),8.36-8.27(m,1H),8.12(d,J=6.6Hz,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=7.1Hz,1H),3.08-3.02(m,1H),2.86-2.73(m,1H),2. 10(t,J=7.3Hz,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=7.2Hz,3H),0.43-0.25(m,4H).

[0869] MS m / z (ESI): 1062.4 [M+H] + .

[0870] 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]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentoxo-5-oxy-2,7,10,13,16-pentazaoctadecane-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexamamide (compound L1-19-S)

[0871]

[0872] Step 1: Synthesis of L1-14-3-S

[0873] Intermediate L1-14-2 (28 g) and intermediate 9 (23.5 g, prepared from Example 14-2) were dissolved in dichloromethane (25 mL). Silver trifluoromethanesulfonate (139.50 mg) was added to the reaction solution. The reaction solution was stirred at 25 °C for 108 h under nitrogen protection. 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 rapid silica gel column chromatography (mobile phase gradient tetrahydrofuran / dichloromethanol: 0–7%, flow rate 70 mL / min) to give the title compound (10.3 g).

[0874] MS m / z (ESI): 537.3 [M+Na] + .

[0875] Step 2: Synthesis of L1-14-4-S

[0876] Intermediate L1-14-3-S (10 g) was dissolved in tetrahydrofuran (100 mL), and wet palladium on carbon (10% by mass, 1 g) was added. The reaction mixture was stirred at 0 °C under a hydrogen atmosphere for 16 h. After the reaction was completed, 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 h. After filtration, the title compound (6 g) was obtained.

[0877] MS m / z (ESI): 447.2 [M+Na] + .

[0878] L1-14-4-S was further analyzed using the following chiral high-performance liquid chromatography methods.

[0879] The chiral high-performance liquid chromatography conditions are as follows:

[0880]

[0881]

[0882] Under the chiral supercritical fluid chromatography conditions described above, the retention time of L1-14-4-S is 4.385 minutes, which is essentially the same as the retention time (4.471 minutes) 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.

[0883] Step 3: Synthesis of L1-19-S

[0884] Intermediate L1-14-13-S (600 mg, prepared from L1-14-4-S using the synthetic method of 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 under nitrogen protection for 2 h. After the reaction was completed, the reaction mixture was purified by high performance liquid chromatography (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (0.05% formic acid), B: acetonitrile]; B%: 24%-54%, 12 min) to obtain the title compound (286 mg).

[0885] 1 H NMR (400MHz, DMSO-d6)δ=8.72-8.63(m,1H),8.62-8.52(m,1H),8.34-8.26(m,1H),8.16-7.94(m,3H),7.78(s,1H),7.5 1(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),3.79-3.53(m,6H),3.52-3.44(m,2H),3.08-3.02(m,1H),2.85-2.75(m,1H),2.09(t,J=7.8Hz,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=7.1Hz,3H),0.42-0.26(m,4H).

[0886] MS m / z (ESI): 1062.5 [M+H] + .

[0887] L1-19-S was further analyzed using the following chiral high-performance liquid chromatography method.

[0888] The chiral high-performance liquid chromatography conditions are as follows:

[0889]

[0890] Under the chiral supercritical fluid chromatography conditions described above, the retention time of L1-19-S was 3.748 minutes, which is essentially consistent with the retention time (3.775 minutes) of compound L1-19-P1 obtained in Example 39-1 under the same chromatographic analysis conditions. Therefore, it is determined that L1-19-S and L1-19-P1 have the same configuration and belong to the same compound.

[0891] The synthesis of compounds LI-0 and L1-00 was performed with reference to patent documents WO2019195665A1 and WO2020063676A1, respectively.

[0892]

[0893] Example 40: Preparation of antibody-drug conjugates

[0894] 40.1 Antibody:

[0895] 40.1.1 Construction and Production of Anti-human HER2 Monoclonal Antibodies

[0896] The sequence of the anti-human HER2 monoclonal antibody is shown in Table 2 below. The nucleic acid sequences encoding the antibodies 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. After culturing, the cell culture supernatant secreting the antibody was obtained.

[0897] Table 2. Sequence information and CDR analysis of anti-human HER2 antibodies (based on Kabat partitioning)

[0898]

[0899]

[0900] 40.1.2 Screening for anti-human p95HER2 monoclonal antibodies

[0901] Anti-human p95HER2 monoclonal antibodies were generated by immunizing mice. The immunogen was the hu p95HER2.ECD-Fc protein. Hybridoma cells were prepared from splenic lymphocytes of mice with high and plateauing antibody titers in their serum. Positive hybridoma clones were screened using standard methods in the field, such as ELISA and FACS. Antibodies were further prepared using serum-free cell culture, purified, and sequenced. Sequencing yielded mouse-derived anti-human p95HER2 antibodies.

[0902] By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, germline genes of heavy and light chain variable regions with high homology to murine antibodies were selected as templates. The CDRs of murine antibodies were transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on this, reversion mutations were performed as needed to ensure the original affinity and / or hotspot mutations were performed to eliminate the risk of molecular modification. The final optimized humanized antibodies and corresponding sequences are shown in Table 3.

[0903] Table 3. Optimized humanized antibody sequence information and CDR analysis (based on Kabat partitioning)

[0904]

[0905] 40.1.3 Construction and Production of p95HER2 / HER2 Bispecific Antibody

[0906] An antibody in DVD-Ig form was constructed from VH1-linker-VH2-CH1-Fc and VL1-linker-VL2-CL, where VH1 and VL1 target HER2, and VH2 and VL2 target p95HER2; the linker can be omitted. The specific sequences of the bispecific antibody are shown in Table 4. Bispecific antibody light and heavy chain expression plasmids were constructed on the pTT5 vector according to the designed structure and expressed in HEK293 cells.

[0907] Table 4. Sequence information of p95HER2 / HER2 bispecific antibody

[0908]

[0909]

[0910] 40.1.4 Construction and Production of Anti-Human CDH6 Antibody

[0911] The sequences of the anti-human CDH6 monoclonal antibodies CDH6-Ab and CDH6-Ab-1 are shown in Table 5 below (antibody sequences are from US20200171163A1).

[0912] Table 5. Sequence information and CDR analysis of anti-human CDH6 antibodies CDH6-Ab and CDH6-Ab-1 (based on Kabat classification)

[0913]

[0914]

[0915] CDH6-Ab-2 and CDH6-Ab-3 were generated by immunizing mice. The immunogens were human CDH6-hFc protein and HEK293T cells overexpressing human CDH6. Hybridoma cells were prepared from splenic lymphocytes of mice with high antibody titers in their serum. Positive hybridoma clones were obtained by screening using conventional methods in the field, such as ELISA and FACS. Antibodies were further prepared using serum-free cell culture, and the antibodies were purified and sequenced to obtain mouse anti-human CDH6 antibodies and their variable region sequences. By comparing the germline gene database of human antibody heavy and light chain variable regions (IMGT, http: / / imgt.cines.fr) and the MOE (Molecular Operating Environment) software, germline genes of heavy and light chain variable regions with high homology to murine antibodies were selected as templates. The CDRs of murine antibodies were transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on this, reversion mutations were performed as needed to ensure the original affinity and / or hotspot mutations were performed to eliminate the risk of molecular modification. The VH and VL sequences corresponding to the finally optimized humanized antibodies CDH6-Ab-2 and CDH6-Ab-3, as well as the CDR sequences divided according to the Kabat format, are shown in Table 6.

[0916] Table 6. Sequence information and CDR analysis of anti-human CDH6 antibodies CDH6-Ab-2 and CDH6-Ab-3 (based on Kabat partitioning)

[0917]

[0918]

[0919] The nucleic acid sequences encoding the antibodies VH and VL were recombined into the expression vector pTT5 containing 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, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed, and incubated for 15 min. This mixture was then added to Expi293F cells (Thermofisher, catalog number: A14527) and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. On the second day after transfection, OPM-293ProFeed (Shanghai Aopomai, catalog number: F081918-001) and 6 g / L glucose (Sigma, catalog number: G7528) were added. On the sixth day after transfection, the cell culture supernatant that secretes antibodies was obtained.

[0920] 40.1.5 Construction and Production of Anti-Human LIV-1 Monoclonal Antibodies

[0921] The sequence of the anti-human LIV-1 monoclonal antibody LIV1-Ab-1 is shown in Table 7 below (antibody sequence source: US20200165335A). The nucleic acid sequences encoding the antibodies VH and VL were recombined into the expression vector pTT5, which contains a signal peptide and a heavy chain constant region (sequence) / light chain constant region sequence, to obtain a recombinant plasmid expressing LIV1-Ab-1. After sequencing verification, the plasmid was extracted. The plasmid and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed, and incubated for 15 min. The mixture was then added to Expi293 cells (Thermofisher, catalog number: A14527) and cultured in a shaker at 37°C and 120 rpm with 5% CO2. On the second day after transfection, OPM-293ProFeed (Shanghai Optima, catalog number: F081918-001) and 6 g / L glucose (manufacturer: Sigma, catalog number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0922] Table 7. Sequence information and CDR analysis of anti-human LIV-1 antibodies (based on Kabat partitioning)

[0923]

[0924]

[0925] 40.1.6 Construction and Production of Anti-Human ROR1 Monoclonal Antibodies

[0926] 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 antibodies VH and VL were recombined into pTT59 expression vectors containing human IgG1 at CH and CL, respectively, to obtain recombinant plasmids expressing ROR1-Ab-1, ROR1-Ab-2, and ROR1-Ab-3.

[0927] The plasmid and transfection reagent PEI (Polysciences, 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed well, and incubated for 15 min. This mixture was then added to Expi293 cells (Thermofisher, A14527) and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. On the second day after transfection, OPM-293ProFeed (Shanghai OPMI, F081918-001) and 6 g / L glucose (Sigma, G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0928] Table 8. Sequence information and CDR analysis of anti-human ROR1 antibodies (based on Kabat partitioning)

[0929]

[0930]

[0931] 40.2 Antibody Purification: The antibodies were purified from cell culture supernatant using Protein A affinity chromatography. The Protein A affinity column was washed with 3-5 column volumes of 6M guanidine hydrochloride, followed by 3-5 column volumes of pure water. The column was equilibrated with 3-5 column volumes of 1×PBS (pH 7.4) buffer. Cell supernatant was loaded at a low flow rate to bind, maintaining a retention time of approximately 1 min or longer. After binding, the column was washed with 3-5 column volumes of 1×PBS (pH 7.4) until the UV absorbance returned to baseline. Sample elution was performed using 0.1M acetate / sodium acetate (pH 3.0-3.5) buffer. The elution peak was collected based on UV monitoring. The elution product was temporarily stored by rapidly adjusting the pH to 5-6 with 1M Tris-HCl (pH 8.0). The elution products can be replaced using methods well-known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube followed by replacement with the desired buffer system, or size exclusion chromatography (e.g., G-25 desalting column) to replace the buffer system, or high-resolution size exclusion columns (e.g., Superdex 200) to remove aggregate components from the elution products to improve sample purity. Proteins that meet the purity requirements after purification are dialyzed to change the buffer and then subjected to subsequent coupling and detection.

[0932] 40.3 Coupling: The antibody was added to an Amicon-Ultra-30kD ultrafiltration tube and concentrated to a buffer solution of 50mM phosphate, 150mM NaCl, 1mM EDTA, pH 6.5. 7-8 times the volume of 10mM tris(2-carboxyethyl)phosphine solution (TCEP) was added to the antibody solution, and the mixture was incubated on a constant-temperature metal shaker at 25°C for 2-3 hours to reduce the antibody. 15-20 times the volume of the corresponding drug-linker compound (prepared according to Examples 37-39, or prepared according to the methods of Examples 37-39) was added to the reaction system, and the reaction solution was coupled at 25°C for 2-16 hours. The reaction product was ultrafiltered and concentrated to a phosphate-buffered saline (PBS) buffer to remove unreacted free small molecule toxins. The purity and DAR value of the ADC product were analyzed using SEC and LC-MS methods.

[0933] 40.4 SEC Purity Analysis: The SEC-HPLC method was used to analyze the protein samples, characterize the molecular size uniformity of the recombinant protein, and determine the purity of the recombinant protein. The HPLC used in this method was an Agilent 1260, the column was a TSK gel 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℃, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 min.

[0934] 40.5 DAR Value Determination: The DAR value of ADC molecules was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecules were treated with PNGase F to remove N-sugar modification, then treated with dithiothreitol (DTT) and incubated at 37°C for 1 h to reduce them to light and heavy chains. Analysis was then performed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected into a Waters ACQUITY Protein BEH size-exclusion 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 spectrometry parameters were: spray voltage 3.8 kV, capillary heating temperature 300°C, sheath gas flow rate 35 arb, and precursor ion scan range 800-3000. Finally, the mass spectrometry data was analyzed using the Biopharma Finder software. 4.1 The Respect algorithm is used for deconvolution processing to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component, thereby calculating the DAR value of the ADC sample to be tested.

[0935] The same method was used to prepare the isotype control antibody Ab-ISO (anti-FITC-hIgG1 antibody), and it was conjugated with a Linker+Payload compound to obtain the isotype control of the corresponding ADC.

[0936] Using the same method described above, antibodies ROR1-Ab-1, ROR1-Ab-2, and ROR1-Ab-3 were conjugated with compound L1-0 to prepare 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 conjugated with compound L1-0 to obtain ADC-L1-0-7, with a DAR value of 6.7; antibodies trastuzumab and pertuzumab were conjugated with compound L1-00 to obtain ADC-L The DAR values ​​of ADC-L1-0-2 and ADC-L1-0-11 were 6.8 and 7.3, respectively. ADC-L1-0-2 and ADC-L1-0-11 were obtained by conjugating antibodies trastuzumab and pertuzumab with compound L1-0, respectively, with a DAR value of 7.3. ADC-L1-0-3, ADC-L1-0-5, and ADC-L1-0-6 were obtained by conjugating antibodies CDH6-Ab, CDH6-Ab-2, and CDH6-Ab-3 with compound L1-0, respectively, with DAR values ​​of 7.4, 7.4, and 7.2, respectively.

[0937] Table 9 Antibody-drug conjugates, their DAR values ​​and SEC purity

[0938]

[0939]

[0940]

[0941]

[0942]

[0943]

[0944] Example 41: Biological activity and related property testing

[0945] The compounds in the following test examples were all prepared according to the methods described in the embodiments of this disclosure.

[0946] Test Example 1: Test of the antitumor cell proliferation activity of compound (DH)

[0947] Cells and Materials: Human colorectal cancer cell line HCT116 was purchased from Kangyuan Bochuang; 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; 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA); Cell-Titer... Glo reagent (Promega#G7568) was purchased from Promega (USA).

[0948] 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. Only cells in the logarithmic growth phase were used for experiments.

[0949] Cell proliferation activity assay: The inhibitory activity of the compound on the proliferation of three cell lines, HCT116, SKBR3, and OVCAR3, was detected using Cell-Titer Glo reagent. HCT116 cells (1500 cells per well), SKBR3 cells (3000 cells per well), and OVCAR3 cells (5000 cells per well) were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. After adding the test compound solution (the compound was dissolved in DMSO to a concentration of 1 mM, then diluted to 3 μM using DMSO, resulting in 9 three-fold dilutions, and 10 μL of the prepared compound solution was transferred to each well to a final concentration of 0-300 nM), and the plates were cultured at 37°C and 5% CO2 for further incubation. HCT116 cells were cultured for 3 days, and SKBR3 and OVCAR3 cells for 5 days. Cell-Titer Glo reagent was then added to assess cell viability.

[0950] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[0951] Data analysis: Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[0952] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom)

[0953] 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.

[0954] Experimental results:

[0955] Under the experimental conditions described herein, the disclosed compounds exhibited strong inhibitory activity against the proliferation of HCT116, SKBR3, and OVCAR3 cells. The specific anti-proliferative activities of the disclosed compounds are shown in Table 10.

[0956] Table 10 shows the anti-tumor cell proliferation activity of compounds of formula (DH).

[0957]

[0958]

[0959] In the table above, the symbols used to indicate binding activity have the following meanings:

[0960] "+++" indicates the IC50 value of the test compound on cell proliferation inhibition. 50 The range is <10nM.

[0961] "++" indicates the IC50 value of the test compound on cell proliferation inhibition. 50 The range is 10–100 nM.

[0962] “N / A” indicates that it was not tested.

[0963] Test Example 2: Biacore determination of anti-p95HER2 / HER2 bispecific antibody BsAb02-P

[0964] This experiment was performed using a Biacore 8K (GE) instrument. Multi-cycle kinetics were employed to determine the interaction between the test antibody BsAb02-P and human p95HER2 (hu p95HER2.ECD-Fc, SEQ ID NO:104, where italics represent the extracellular region of human p95HER2 and the underline represents Fc). Tags: MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKThe affinity of the protein (prepared by self-expression) and HER2 (purchased from Acro, HE2-H5225) was measured.

[0965] The experimental run buffer was 1×HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P2O) (Cat.#BR-1006-69, GE), with the flow cell temperature set at 25℃ and the sample chamber temperature at 16℃. Both were pretreated with the run buffer. A certain amount of the test antibody was affinity-captured using a Protein A biosensor chip (Cat.#29127556, GE), and then a certain concentration of human p95HER2 antigen or human HER2 antigen flowed onto the chip surface. The reaction signal was detected in real time using a Biacore 8K instrument (GE) to obtain binding and dissociation curves. After dissociation in each cycle, the antigen-antibody complex was regenerated by washing with a glycine-hydrochloric acid regeneration solution (Cat.#BR-1003-54, GE) at pH 1.5. Specifically, the binding process was detected by injecting different concentrations of human p95HER2 and human HER2 antigen into the solution for 240 seconds at a flow rate of 30 μL / min, starting from 50 nM and diluted 1:1 to set a series of concentration gradients; the dissociation time was as long as 900 seconds, and finally the chip surface was regenerated by washing with 10 mM glycine-hydrochloric acid solution (pH 1.5) at a flow rate of 30 μL / min for 30 seconds.

[0966] The experimental data were fitted using the GE Biacore 8K Evaluation version 2.0 software with a (1:1) Langmuir model to obtain the binding rate (Ka), dissociation rate (Kd), and affinity (KD), as shown in Tables 11-12. The experimental results demonstrate that the p95HER2 / HER2 bispecific antibody of this disclosure can bind to human p95HER2 and HER2 with high affinity.

[0967] Table 11. Reactivity affinity of p95HER2 / HER2 bispecific antibody with human p95HER2 protein.

[0968]

[0969] Table 12. Reactivity affinity of p95HER2 / HER2 bispecific antibody with human HER2 protein.

[0970]

[0971] Test Example 3-1: Flow Cytometry (FACS) Detection of the Binding Activity of Anti-HER2ADC and Antibody to SKBR3 Tumor Cells

[0972] SKBR3 cells (derived from ATCC) were collected and counted at a ratio of 2 × 10⁻⁶. 5 Cells were seeded per well in a 96-well plate (Corning, 3795). Serially diluted test samples were added, and the cells were incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 goat anti-human Fc secondary antibody (Jackson Immuno, 109-605-098) was added, and the cells were incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and analyzed by flow cytometry (BD FACSCanto). TM II) Analyze the mean fluorescence intensity (MFI). Use Graphpad Prism software to perform four-parameter curve fitting analysis on the data to obtain EC. 50 The values ​​are shown in Table 13.

[0973] Table 13. FACS detection of binding reaction between anti-HER2ADC and antibody and SKBR3 tumor cells

[0974]

[0975] Test Example 3-2: Assay of the binding activity of anti-ROR1 ADC and antibody with stable transgenic cells MCF7-hROR1 clone 2G2

[0976] Construction of stable cell lines

[0977] The nucleotide sequence encoding the human ROR1 amino acid sequence (SEQ ID NO: 105) was cloned into the pLVX lentiviral vector, and viral particles were prepared in HEK293T cells. After lentiviral infection, the MCF7 cell line (purchased from the Chinese Academy of Sciences) was selectively cultured for one week in DMEM (Gibco, catalog number 11995073) containing 5 μg / ml puromycin (Gibco, catalog number A1113803) and 10% (w / w) fetal bovine serum (ExCell Bio, catalog number FND500). Positive cell populations with different expression levels were sorted into 96-well plates using a FACS Aria III flow cytometer (purchased from BDBiosciences) using ROR1-Ab-1 and goat anti-mouse IgG (H+L) antibody (Jackson, catalog number: 115605006). The plates were incubated at 37°C with 5% (v / v) CO2. After approximately two weeks, a portion of the cells were selected for expansion. Positive cell populations with good growth, high fluorescence intensity, and good homogeneity were further expanded and cryopreserved in liquid nitrogen. The identification of the monoclonal stable transgenic cell line is shown in Table 14.

[0978] Table 14. FACS detection results of human ROR1 protein in MCF7 stable cell lines.

[0979]

[0980] Full-length human ROR1 amino acid sequence (SEQ ID NO: 105):

[0981]

[0982] Flow cytometry (FACS) was used to detect the binding activity of anti-ROR1 ADC and antibody to stable transgenic cells MCF7-hROR1 clone2G2.

[0983] The stable cell line MCF7-hROR1 clone 2G2 constructed in the above steps was cultured in a T-75 cell culture flask until the logarithmic growth phase. After centrifugation and discarding the culture supernatant, the cell pellet was washed twice with PBS. The ADC and antibody to be tested were added at an initial concentration of 100 nM, serially diluted 5-fold at 8 spots, and incubated at 4°C for 1 hour. After washing twice with PBS, secondary antibody (Alexa) was added. 647 AffiniPure Goat Anti-Human IgG (H+L) (purchased from Jackson Immuno, catalog number: 109-605-088) were incubated at 4°C for 1 hour. After washing twice with PBS, the cells were resuspended and analyzed using FACS (FACS Canto™, purchased from BD). Four-parameter curve fitting analysis was performed using Graphpad Prism software to obtain EC... 50 The values ​​are shown in Table 15.

[0984] Table 15. FACS detection of the binding activity of anti-ROR1 ADC and antibody to MCF7-hROR1 clone 2G2 cells

[0985]

[0986] Test Example 3-3: Flow Cytometry (FACS) Detection of the Binding Activity of Anti-LIV-1 ADC and Antibody to OVCAR3 Tumor Cells

[0987] OVCAR3 cells (derived from ATCC) belong to a tumor model with high LIV-1 expression. OVCAR3 cells were collected and counted at a ratio of 2 × 10⁻⁶. 5 Cells were seeded per well in a 96-well plate (Corning, 3795). Serially diluted test samples were added, and the cells were incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 Goat anti-human Fc secondary antibody (Jackson Immuno, 109-605-098) was added, and the cells were incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and analyzed by flow cytometry (BD FACSCanto). TMII) Analyze the mean fluorescence intensity (MFI). Use Graphpad Prism software to perform four-parameter curve fitting analysis on the data to obtain EC. 50 The values ​​are shown in Table 16.

[0988] Table 16. FACS detection of the binding reaction between anti-LIV-1 ADC and antibody and OVCAR3 tumor cells

[0989]

[0990] Test Example 4-1, Anti-HER2-ADC Tumor Cell Proliferation Activity Test 1

[0991] Cells and materials: The human breast cancer cell line SKBR3 was purchased from ATCC; the human breast cancer cell line SKBR3-p95HER2 was constructed by Simcere Pharmaceutical (construction method is described in patent application CN202210094806.6); the 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, catalog number same as test example 1); 96-well plates were purchased from Corning (USA, catalog number same as test example 1); and Cell-Titer Glo reagent was purchased from Prometheus (USA, catalog number same as test example 1).

[0992] Cell culture: SKBR3 cells, SKBR3-p95HER2 cells and SKOV3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C and 5% CO2. Only cells in the logarithmic growth phase were used for experiments.

[0993] Cell proliferation activity assay: The inhibitory activity of ADCs on the proliferation of three cell lines (SKBR3, SKBR3-p95HER2, and SKOV3) was detected using Cell-Titer Glo reagent. SKBR3 cells (3000 cells per well), SKBR3-p95HER2 cells (3000 cells per well), and SKOV3 cells (600 cells per well) were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. The ADC solution was then added (ADC was diluted with the corresponding cell culture medium to adjust the concentration to 20 nM or 200 nM, and then serially diluted 3-fold with culture medium to obtain 8 concentrations; 10 μL of the prepared ADC solution was transferred to each 96-well plate to maintain a final concentration of 2 nM or 20 nM), and the plates were cultured at 37°C and 5% CO2. SKBR3, SKBR3-p95HER2, and SKOV3 cells were cultured for 5 days. Cell-Titer Glo reagent was then added to assess cell viability.

[0994] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[0995] Data analysis: Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[0996] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom)

[0997] 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.

[0998] Experimental results: Under the experimental conditions, the disclosed ADC showed strong inhibitory activity against SKBR3 cells, SKBR3-p95HER2 cells and SKOV3 cells, as detailed in Table 17.

[0999] Table 17 Anti-tumor cell proliferation activity of ADCs

[1000]

[1001] “N / A” indicates that it was not tested.

[1002] Test Example 4-2, Anti-HER2-ADC Tumor Cell Proliferation Activity Test 2

[1003] Cells and materials: Human breast ductal carcinoma cell line T47D (medium expression 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); Cell-TiterGlo reagent (Promega#G7568) was purchased from Promega (USA).

[1004] Cell culture: SKBR3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum + 1% penicillin-streptomycin, and T47D cells were cultured in 1640 medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. Both cell lines were cultured at 37°C and 5% CO2. Only cells in the logarithmic growth phase were used for experiments.

[1005] Cell proliferation activity assay: The inhibitory activity of ADC on the proliferation of SKBR3 and T47D cell lines was detected using Cell-Titer Glo reagent. SKBR3 and T47D cells were digested and dispersed from the cell culture flasks, resuspended in the corresponding fresh culture medium, and the cell density was adjusted. T47D cells were seeded at 2000 cells / 90 μL / well in 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 1000 nM with complete culture medium, and then serially diluted 3-fold (8 concentration gradients) was performed. 10 μL of the diluted ADC solution was then transferred to each 96-well plate, resulting in an initial ADC concentration of 100 nM. The 96-well plates were cultured at 37°C and 5% CO2 for 7 days. Cell-Titer Glo reagent was added to assess cell viability.

[1006] SKBR3 cells were seeded at 5000 cells / 90 μL / well in 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 1000 nM with complete culture medium and serially diluted 5-fold (9 concentration gradients in total). Then, 10 μL of the diluted ADC solution was transferred to each 96-well plate, resulting in an initial ADC concentration of 100 nM. The 96-well plates were incubated at 37°C and 5% CO2 for 3 days. Cell-Titer Glo reagent was added to assess cell viability.

[1007] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1008] Data Analysis:

[1009] Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1010] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom).

[1011] 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.

[1012] Experimental results:

[1013] Under the experimental conditions described herein, the anti-HER2-ADC exhibited strong inhibitory activity against the human breast ductal carcinoma cell line T47D and the human breast cancer cell line SKBR3, as shown in Table 18.

[1014] Table 18 Anti-tumor cell proliferation activity of ADCs

[1015]

[1016] Test Example 5: Cell-binding activity of anti-CDH6 antibody and corresponding ADC

[1017] OVCAR3 cells were collected and counted at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in a 96-well plate (Corning, 3795). Serially diluted test samples were added, and the cells were incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 Goat anti-human Fc secondary antibody (Jackson Immuno, 109-605-098) was added, and the cells were incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and analyzed by flow cytometry (BD FACSCanto). TM II) Analyze the mean fluorescence intensity (MFI). Use Graphpad Prism software to perform four-parameter curve fitting analysis on the data to obtain EC. 50 The values ​​are shown in Table 19.

[1018] Table 19 Binding activities of CDH6 antibody and CDH6-ADC with OVCAR3 cells

[1019]

[1020] Test Example 6-1: Anti-CDH6-ADC Anti-tumor Cell Proliferation Activity Test

[1021] Cells and Materials: Human ovarian cancer cell line OVCAR3 (CDH6-overexpressing cell line) and human ovarian teratoma cell line PA-1 were 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 Prometheus (USA).

[1022] 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 and 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 and 5% CO2. Only cells in the logarithmic growth phase were used for experiments.

[1023] Cell proliferation activity assay: The inhibitory activity of ADCs on the proliferation of OVCAR3 and PA-1 cell lines was detected using Cell-Titer Glo reagent. OVCAR3 and PA-1 cells were digested and dispersed from the cell culture flasks, resuspended in their corresponding fresh culture media, and the cell density was adjusted. OVCAR3 cells (5000 cells per well) and PA-1 cells (800 cells per well) were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. The ADC solution was then added (ADC was diluted with the corresponding cell culture medium to a concentration of 100 nM, and then serially diluted 3-fold to obtain 8 concentrations; 10 μL of the prepared ADC solution was transferred to each well to a final concentration of 0-10 nM), and the plates were cultured at 37°C and 5% CO2 for 5 days. Cell viability was then assessed using Cell-Titer Glo reagent.

[1024] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1025] Data analysis: Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1026] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom)

[1027] 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.

[1028] Experimental results: Under the experimental conditions, the disclosed anti-CDH6-ADC showed strong inhibitory activity against the proliferation of human ovarian cancer cell line OVCAR3 and human ovarian teratoma cell line PA-1, as detailed in Table 20.

[1029] Table 20 Anti-tumor cell proliferation activity of ADCs

[1030]

[1031] Test Example 6-2: Anti-LIV-1-ADC Anti-tumor Cell Proliferation Activity Test

[1032] 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. 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA). Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[1033] Cell culture: OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum + 2 μg / mL bovine insulin + 1% penicillin-streptomycin at 37°C and 5% CO2. H838 cells were cultured in 1640 medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C and 5% CO2. Only cells in the logarithmic growth phase could be used for experiments.

[1034] Cell proliferation activity assay: The inhibitory activity of ADC on the proliferation of OVCAR3 and H838 cell lines was detected using Cell-Titer Glo reagent. OVCAR3 or H838 cells were digested and dispersed from the cell culture flasks, resuspended in the corresponding fresh medium, and the cell density was adjusted to 1500 cells / 90 μL / well for OVCAR3 and 450 cells / 90 μL / well for H838. The cells were seeded in 96-well plates and incubated overnight at 37°C and 5% CO2. The ADC concentration was diluted to 5000 nM with complete medium and serially diluted 3-fold (8 concentration gradients). Then, 10 μL of the diluted ADC solution was transferred to each 96-well plate, resulting in an initial ADC concentration of 500 nM. The 96-well plates were incubated at 37°C and 5% CO2 for 5 days. Cell-Titer Glo reagent was then added to assess cell viability.

[1035] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1036] Data Analysis:

[1037] Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1038] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom).

[1039] 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.

[1040] Experimental results:

[1041] Under the experimental conditions described herein, the anti-LIV-1-ADC exhibited strong inhibitory activity against the human ovarian cancer cell line OVCAR3 and the human non-small cell lung cancer cell line H838, as detailed in Table 21.

[1042] Table 21 Anti-tumor cell proliferation activity of ADC

[1043]

[1044] Test Example 6-3: Anti-ROR1-ADC Anti-tumor Cell Proliferation Activity Test

[1045] Cells and Materials: The human breast cancer cell line hROR1-MCF7 was constructed by Simcere. 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).

[1046] Cell culture: hROR1-MCF7 cells were cultured in DMEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C and 5% CO2. Only cells in the logarithmic growth phase could be used for experiments.

[1047] Cell proliferation activity assay: The inhibitory activity of ADC on the proliferation of hROR1-MCF7 cells was detected using Cell-Titer Glo reagent. 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 1700 cells / 90 μL / well. Cells were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 500 nM with complete medium and then serially diluted 4-fold (9 concentration gradients). 10 μL of each diluted ADC solution was then transferred to each 96-well plate, resulting in an initial ADC concentration of 50 nM. The 96-well plates were cultured at 37°C and 5% CO2 for 5 days. Cell-Titer Glo reagent was then added to assess cell viability.

[1048] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1049] Data Analysis:

[1050] Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1051] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom).

[1052] 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.

[1053] Experimental results:

[1054] Under the experimental conditions described herein, the anti-ROR1-ADC exhibited strong inhibitory activity against the human ovarian cancer cell line hROR1-MCF7, as detailed in Table 22.

[1055] Table 22 Anti-tumor cell proliferation activity of ADCs

[1056]

[1057]

[1058] Test Case 7: Efficacy Evaluation of OVCAR3 Subcutaneous Tumor Model - 1

[1059] Experimental reagents: Human ovarian cancer OVCAR3 cells were purchased from ATCC; RPMI-1640 medium was purchased from Gibco (catalog number A104910); fetal bovine serum was purchased from Excel (catalog number FND500); penicillin-streptomycin was purchased from Gibco (catalog number 15140122); bovine insulin was purchased from Yeasen (catalog number 40107ES60); 0.25% trypsin-EDTA was purchased from Gibco (catalog number 25200-072); D-PBS (calcium and magnesium phosphate buffer) was purchased from Hyclone (catalog number SH30256.01); and Matrigel was purchased from Corning (catalog number 356237).

[1060] Experimental methods:

[1061] Animal Information: Balb / c nude mice, female, 5-6 weeks old, weighing approximately 14-20 grams. The animals were purchased from Beijing Vital River Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[1062] Cell culture: Human ovarian cancer OVCAR3 cell line was cultured in vitro under the following conditions: RPMI-1640 medium supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin, and 10 μg / ml bovine insulin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged once a week using a routine digestion process with 0.25% trypsin-EDTA digestion solution. Cells were harvested and counted when the cell saturation reached 80%-90% and the desired number was achieved.

[1063] Cell seeding: 0.1 ml of OVCAR3 cell suspension (containing 1×10⁻⁶ cells) was added. 7100 cells (RPMI-1640:Matrigel volume ratio 1:1) were subcutaneously injected into the axilla of each mouse. On day 26 post-inoculation, mice were randomly assigned to receive the drug based on tumor volume, with the grouping day being Day 0.

[1064] Tumor measurements and experimental indicators:

[1065] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5axb 2 , where a and b represent the long and short diameters of the tumor, respectively. Mouse body weight was measured twice weekly.

[1066] The tumor-suppressive efficacy of the test drug was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[1067] Experimental results:

[1068] In the mouse subcutaneous xenograft OVCAR3 model, ADC-L1-14-P1-5, at a dose of 3 mg / kg, significantly inhibited tumor growth after a single intravenous administration (P<0.0001). Results are shown in Table 23 and... Figure 2 .

[1069] Table 23 Tumor volume in the OVCAR3 subcutaneous tumor model

[1070]

[1071] In the mouse subcutaneous xenograft OVCAR3 model, ADC-L1-19-P1-5, at a dose of 3 mg / kg, significantly inhibited tumor growth after a single intravenous administration (P<0.0001). Results are shown in Table 24 and... Figure 3 .

[1072] Table 24 Tumor Volume in OVCAR3 Subcutaneous Tumor Model

[1073]

[1074]

[1075] Test Example 8: ADC Plasma Stability Test

[1076] The ADC molecules (final concentration of 100 μg / ml) were incubated with human plasma (Aoneng Biotechnology, PB021-C) and monkey plasma (Sinuoda Biotechnology, SND-X0107) in an incubator at 37°C. The day of incubation was marked as day 0. Samples were then collected on days 7, 14, and 28 for the detection of free small molecules.

[1077] Take 20 μL of sample, add 300 μL of internal standard working solution (prepared with acetonitrile), vortex mix for 5 minutes, centrifuge for 5 minutes (14000 rpm), and inject 4 μL of supernatant into LC-MS / MS (API 6500+) for analysis. The results are shown in Table 25. The results indicate that the tested ADC molecule is relatively stable in both human and monkey plasma.

[1078] Table 25 Plasma stability of CDH6-ADC

[1079]

[1080] N / A indicates that no free small molecules were detected and therefore free small molecules could not be calculated.

[1081] Test Example 9: Efficacy Evaluation of OVCAR3 Subcutaneous Tumor Model - 2

[1082] Experimental reagents:

[1083] Human ovarian cancer OVCAR3 cells: ATCC

[1084] RPMI-1640 culture medium: Gbico; Cat No.: A104910

[1085] Fetal bovine serum: Excell, FND500

[1086] Bovine insulin: Yeasen, 40107ES60

[1087] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[1088] D-PBS (Calcium and Magnesium-Free Phosphate Buffer): Hyclone, Cat. No.: SH30256.01

[1089] Matrigel:Corning,Cat.No.:356237

[1090] Experimental methods:

[1091] Animal Information: Balb / c nude mice, female, 5-6 weeks old, weighing approximately 14-20 grams. The animals were purchased from Beijing Vital River Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[1092] Cell culture: Human ovarian cancer OVCAR3 cell line was cultured in vitro under the following conditions: RPMI-1640 (cell culture medium) with 20% fetal bovine serum, 1% Pen Strep, and 10 μg / ml bovine insulin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged once a week using a routine digestion process with 0.25% trypsin-EDTA digestion solution. When cell saturation reached 80%-90% and the required number was achieved, cells were harvested and counted.

[1093] Cell inoculation: 0.1 ml of OVCAR3 cell suspension (RPMI-1640:Matrigel, volume ratio 1:1) was subcutaneously inoculated into the axilla of each mouse. On day 23 post-inoculation, mice were randomly assigned to groups based on tumor volume, with the grouping day designated as Day 0.

[1094] Administration: The dosage of ADC-L1-19-P1-7 and the isotype control ADC-L1-19-P1-ISO was 3 mg / kg, administered intraperitoneally (Q4D). Six mice were used in each group.

[1095] Tumor measurements and experimental indicators:

[1096] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5axb 2 , where a and b represent the long and short diameters of the tumor, respectively. Mouse body weight was measured twice weekly.

[1097] The antitumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[1098] Experimental results:

[1099] See Table 26. Figure 4 and Figure 5 .

[1100] Table 26 Tumor Volume in OVCAR3 Subcutaneous Tumor Model

[1101]

[1102] Experimental conclusion:

[1103] In a mouse subcutaneous xenograft OVCAR3 model, the disclosed compound ADC-L1-19-P1-7 and its isotype control ADC-L1-19-P1-ISO, administered intraperitoneally at 3 mg / kg every four days, significantly inhibited tumor growth (P<0.0001); however, the isotype control ADC-L1-19-P1-ISO showed significantly weaker tumor inhibition than ADC-L1-19-P1-7 (P<0.001). In this embodiment, no effect on mouse body weight or death was observed at the tested doses, and the mice tolerated the treatment well.

[1104] Test Case 10: Efficacy Evaluation of NCI-H838 Subcutaneous Tumor Model

[1105] Experimental reagents:

[1106] Human lung cancer NCI-H838 cells: Kebai

[1107] RPMI-1640 culture medium: Gbico; Cat No.: 61870-036

[1108] Fetal bovine serum: Gibco; Cat No.: 10099-141C

[1109] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[1110] D-PBS (Calcium and Magnesium-Free Phosphate Buffer): Hyclone, Cat. No.: SH30256.01

[1111] Matrigel:Corning,Cat.No.:356237

[1112] Experimental methods:

[1113] Animal Information: B-NDG mice, female, 5-6 weeks old, weighing approximately 14-20 grams. The animals were purchased from Biocytogen and housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[1114] Cell culture: Human lung cancer NCI-H838 cell line was cultured in vitro under the following conditions: RPMI-1640 (cell culture medium) with 10% fetal bovine serum and 1% Pen Strep, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using a routine digestion process with 0.25% trypsin-EDTA digestion solution. When cell saturation reached 80%-90% and the required number was achieved, cells were harvested and counted.

[1115] Cell inoculation: 0.1 ml (containing 1 × 10⁷ NCI-H838 cell suspension) (RPMI-1640: Matrigel, volume ratio 1:1) was subcutaneously inoculated into the axilla of each mouse. On day 23 post-inoculation, mice were randomly assigned to groups based on tumor volume, with the grouping day designated as Day 0.

[1116] Administration: The dosage of ADC-L1-19-P1-7 and the isotype control ADC-L1-19-P1-ISO was 3 mg / kg, administered intraperitoneally (Q4D). Six mice were used in each group.

[1117] Tumor measurements and experimental indicators:

[1118] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5axb 2 , where a and b represent the long and short diameters of the tumor, respectively. Mouse body weight was measured twice weekly.

[1119] The antitumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[1120] Experimental results:

[1121] See Table 27. Figure 6 and Figure 7 .

[1122] Table 27 Tumor Volume in H838 Subcutaneous Tumor Model

[1123]

[1124] Experimental conclusion:

[1125] In the mouse subcutaneous xenograft tumor NCI-H838 model, the disclosed compound ADC-L1-19-P1-7 and the isotype control ADC-L1-19-P1-ISO, administered intraperitoneally at 3 mg / kg once every four days, significantly inhibited tumor growth (P<0.0001); however, 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 embodiment, no effect on mouse body weight or death was observed at the tested doses, and the mice tolerated the treatment well.

[1126] Test Example 11: Bystander Effect Test of Anti-LIV-1-ADC

[1127] Cells and Materials: The human ovarian cancer cell line OVCAR3 was purchased from ATCC; the 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).

[1128] Construction method of NCI-H838-hLIV1-KO: Six sgRNAs targeting the human LIV1 gene were designed, and then the sgRNAs were cloned into the pLVX lentiviral vector. Viral particles were prepared in HEK293T cells (purchased from the Chinese Academy of Sciences). After lentiviral infection of the NCI-H838 (purchased from ATCC) cell line, the cells were selectively cultured for 2 weeks in RPMI 1640 medium containing 10% (w / w) fetal bovine serum with 1.5 μg / ml puromycin (purchased from Gibco, catalog number: A1113802) to obtain the H838-LIV1 KO pool cell line. H838-LIV1KO monoclonal cells were labeled with human anti-LIV1 antibody (Ladiratuzumab, self-produced) and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088) and sorted into 96-well plates using a FACSAriaII flow cytometer (purchased from BD Biosciences). The cells were incubated at 37°C with 5% (v / v) CO2 for approximately two weeks. A subset of wells were selected for amplification. The amplified clones were screened by flow cytometry. Cell lines with good growth, low fluorescence intensity, and monoclonal cell lines were selected for further expansion and cryopreservation in liquid nitrogen.

[1129] Cell culture: OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum + 2 μg / mL bovine insulin + 1% penicillin-streptomycin, and NCI-H838-hLIV1-KO cells were cultured in 1640 medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. Both cell lines were cultured at 37°C and 5% CO2. Only cells in the logarithmic growth phase were used for experiments.

[1130] Bystander effect detection: The supernatant from incubation of the test ADC with LIV-1 positive OVCAR3 cells was transferred to LIV-1 negative NCI-H838-hLIV1-KO cells for further incubation. The effect of this supernatant on the proliferation activity of NCI-H838-hLIV1-KO cells was detected using Cell-Titer Glo reagent, thus reflecting the bystander effect of ADC. OVCAR3 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. The cells were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 5000 nM with complete medium, and a five-fold serial dilution was performed for a total of eight concentration gradients. Then, 20 μL of the diluted ADC solution was transferred to each 96-well plate, resulting in an initial ADC concentration of 500 nM. The 96-well plates were cultured at 37°C and 5% CO2 for 5 days. Transfer 150 μL of supernatant from an OVCAR3 cell culture plate to an NCI-H838-hLIV1-KO cell plate seeded one day prior (450 cells / 50 μL / well) and incubate at 37°C and 5% CO2 for 5 days. Add Cell-Titer Glo reagent to assess cell viability.

[1131] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1132] Data Analysis:

[1133] Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1134] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom).

[1135] 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.

[1136] Experimental results:

[1137] Under the experimental conditions, the supernatant of the ADC to be tested after incubation with LIV-1 positive cells OVCAR3 was further incubated with LIV-1 negative cells NCI-H838-hLIV1-KO. The killing effect on negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 28, indicating that the anti-LIV1-ADC disclosed in this paper has a good bystander effect.

[1138] Table 28 Bystander Effect Against LIV1-ADC

[1139]

[1140] Test Example 12: Bystander Effect Test of ROR1-ADC

[1141] Cells and Materials: The human breast cancer cell line hROR1-MCF7 was constructed by Simcere and Sinconel. The 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).

[1142] Cell culture: hROR1-MCF7 cells and MCF7 cells were cultured in DMEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C and 5% CO2. Only cells in the logarithmic growth phase could be used for experiments.

[1143] Bystander effect detection: The supernatant from incubating the test ADC with ROR1-positive hROR1-MCF7 cells was transferred to ROR1-negative MCF7 cells for further incubation. The effect of this supernatant on the proliferation activity of MCF7 cells was detected using Cell-Titer Glo reagent, thus reflecting the bystander effect of 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. The cells were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 5000 nM with complete medium, and a three-fold serial dilution was performed for a total of 8 concentration gradients. Then, 20 μL of the diluted ADC solution was transferred to each 96-well plate, resulting in an initial ADC concentration of 500 nM. The 96-well plates were cultured at 37°C and 5% CO2 for 5 days. Transfer 150 μL of supernatant from the hROR1-MCF7 cell culture plate to a plate of MCF7 cells seeded one day in advance (1500 cells / 50 μL / well), and incubate at 37°C and 5% CO2 for 5 days. Add Cell-Titer Glo reagent to detect cell viability.

[1144] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1145] Data Analysis:

[1146] Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1147] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom).

[1148] 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.

[1149] Experimental results:

[1150] Under the experimental conditions, the supernatant of the ADC to be tested after incubation with ROR1 positive cells hROR1-MCF7 was further incubated with ROR1 negative cells MCF7. The killing effect on negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 29, indicating that the anti-ROR1-ADC disclosed in this paper has a good bystander effect.

[1151] Table 29 Bystander Effect Against ROR1-ADC

[1152]

[1153] Test Example 13: Bystander Effect Test of Anti-HER2-ADC

[1154] Cells and materials: Human breast cancer cell line SKBR3 and human small cell lung cancer cell line NCI-H2171 were 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). Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[1155] Cell culture: SKBR3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum + 1% penicillin-streptomycin, and NCI-H2171 cells were cultured in 1640 medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. Both cell lines were cultured at 37°C and 5% CO2. Only cells in the logarithmic growth phase were used for experiments.

[1156] Bystander effect detection: The supernatant from incubation of ADC with HER2-positive SKBR3 cells was transferred to HER2-negative NCI-H2171 cells for further incubation. The effect of this supernatant on the proliferation activity of NCI-H2171 cells was detected using Cell-Titer Glo reagent, thus reflecting the bystander effect of 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. Cells were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 5000 nM with complete medium and serially diluted 3-fold (8 concentration gradients). 20 μL of the diluted ADC solution was then transferred to each 96-well plate, resulting in an initial ADC concentration of 500 nM. The 96-well plates were cultured at 37°C and 5% CO2 for 3 days. Transfer 150 μL of supernatant from an SKBR3 cell culture plate to an NCI-H2171 cell plate seeded one day prior (6000 cells / 50 μL / well) and incubate at 37°C and 5% CO2 for 5 days. Add Cell-Titer Glo reagent to assess cell viability.

[1157] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1158] Data Analysis:

[1159] Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1160] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom).

[1161] 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.

[1162] Experimental results:

[1163] Under the experimental conditions, the supernatant of the ADC to be tested after incubation with HER2-positive cells SKBR3 was further incubated with HER2-negative cells NCI-H2171. The killing effect on negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 30, indicating that the anti-HER2-ADC disclosed in this paper has a good bystander effect.

[1164] Table 30 Bystander Effect in Anti-HER2-ADC

[1165]

[1166] Test Example 14: Bystander Effect Test for Anti-CDH6-ADC

[1167] Cells and materials: Human ovarian cancer cell line OVCAR3 and SKOV3 were 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. 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA). Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[1168] Cell culture: OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum + 2 μg / mL bovine insulin + 1% penicillin-streptomycin, and SKOV3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. Both cell lines were cultured at 37°C and 5% CO2. Only cells in the logarithmic growth phase were used for experiments.

[1169] Bystander effect detection: The supernatant from incubation of ADC with CDH6-positive OVCAR3 cells was transferred to CDH6-negative SKOV3 cells for further incubation. The effect of this supernatant on the proliferation activity of SKOV3 cells was detected using Cell-Titer Glo reagent, thus reflecting the bystander effect of ADC. OVCAR3 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. The cells were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 4500 nM with complete medium, and then serially diluted 3-fold (8 concentration gradients) was performed. 20 μL of the diluted ADC solution was then transferred to each 96-well plate, resulting in an initial ADC concentration of 450 nM. The 96-well plates were cultured at 37°C and 5% CO2 for 5 days. Transfer 150 μL of supernatant from an OVCAR3 cell culture plate to a pre-seeded SKOV3 cell plate (700 cells / 50 μL / well) and incubate at 37°C and 5% CO2 for 7 days. Add Cell-Titer Glo reagent to assess cell viability.

[1170] A negative control group and a positive control group were set up as the bottom and top, respectively. The negative control group was treated with no cells but with the same volume of culture medium, and all other procedures were the same as the experimental group. The positive control group was treated with no test drug, and all other procedures were the same as the experimental group.

[1171] Data Analysis:

[1172] Calculate the percentage inhibition (%Inhibition) and fit the IC50 of the compound. 50 .

[1173] Inhibition percentage (%Inhibition) = 1 - 100% * (Signal-Bottom) / (Top-Bottom).

[1174] 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.

[1175] Experimental results:

[1176] Under the experimental conditions, the supernatant of the ADC to be tested after incubation with CDH6 positive cells OVCAR3 was further incubated with CDH6 negative cells SKOV3. The killing effect on negative cells was detected by adding Cell-Titer Glo reagent. The results are shown in Table 31, indicating that the anti-CDH6-ADC disclosed in this paper has a good bystander effect.

[1177] Table 31 Bystander Effect in Anti-CDH6-ADC

[1178]

[1179]

Claims

1. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structure of Pc-(L-D) n wherein: Pc is a ligand unit; L represents the connecting subunit; D is the drug unit represented by the following formula (DI): (DI) in, X is O; X1is selected from CR 2 ; R 1 , R 2 , together with the atom to which they are attached, form a 5-6 membered heterocyclyl group which is unsubstituted or substituted by one or more R a2 ; R 4 It is H; R 5 Selected from H, halogens, or CN; R 6 Selected from H; R 7 Selected from unsubstituted or D-substituted cyclopropyl groups; R a2 It is D; Furthermore, n is a real number from 1 to 16, and The connecting subunit L is selected from or Its a-terminus is covalently connected to the ligand unit Pc, and its b-terminus is covalently connected to the drug unit D. Here, m1 and m2 are each independently selected from integers 2 to 8, m3 is selected from integers 1 to 16, and L... 1 L 2 Each peptide residue is independently selected from 1 to 8 amino acids.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 R 2 The atoms connected to them together form a 5-6 membered heterocyclic group, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocyclic group is unsubstituted or substituted by one or more D atoms.

3. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 R 2 The atoms connected to them together form , or .

4. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein R 5 Selected from H, Cl, or F.

5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the structural unit Selected from , , or .

6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the pharmaceutical unit represented by formula (DI) is selected from the pharmaceutical unit represented by formula (D-Ia): (D-Ia) in, R 1 R 2 R 4 R 5 R 6 R 7 As defined in any one of claims 1 to 3.

7. The ligand-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (DI) is selected from the following compounds: or .

8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The L 1 L 2 Each peptide residue is independently selected from 2, 3, or 4 amino acid residues.

9. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The L 1 The L is a Gly-Gly-Phe-Gly tetrapeptide residue or an Ala-Ala-Ala tripeptide residue. 2 It consists of Gly-Gly-Phe-Gly tetrapeptide residues or Val-Lys dipeptide residues.

10. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, m1 is selected from 5, m2 is selected from 2, and m3 is selected from 8.

11. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1, 7 to 10, wherein, Connecting subunit L is selected from , 、 or Its a-end is covalently connected to the ligand unit Pc, and its b-end is covalently connected to the drug unit D.

12. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The ligand-drug conjugate or its pharmaceutically acceptable salt is selected from the following ligand-drug conjugates or their pharmaceutically acceptable salts: or Wherein Pc and n are as defined in claim 1.

13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The ligand unit Pc is selected from polypeptides, antibodies, or their antigen-binding fragments.

14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, 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, VEGF, and LIV-1.

15. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein, The Pc is an antibody or antigen-binding fragment thereof that specifically binds to HER2, p95HER2, CDH6, ROR1, or LIV-1.

16. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein, The antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein HCDR1-3 and LCDR1-3 are selected from the following: (1) The HCDR1-3 is SEQ ID NO:7-9; and the LCDR1-3 is SEQ ID NO:10-12; (2) The HCDR1-3 is SEQ ID NO:13-15; and the LCDR1-3 is SEQ ID NO:16-18; (3) The HCDR1-3 is SEQ ID NO:23-25; and the LCDR1-3 is SEQ ID NO:26-28; (4) The HCDR1-3 is SEQ ID NO:29-31; and the LCDR1-3 is SEQ ID NO:32-34; (5) The HCDR1-3 is SEQ ID NO:40-42; and the LCDR1-3 is SEQ ID NO:43-45; (6) The HCDR1-3 is SEQ ID NO:48-50; and the LCDR1-3 is SEQ ID NO:51-53; (7) The HCDR1-3 is SEQ ID NO:58-60; and the LCDR1-3 is SEQ ID NO:61-63; (8) The HCDR1-3 is SEQ ID NO:64-66; and the LCDR1-3 is SEQ ID NO:67-69; (9) The HCDR1-3 is SEQ ID NO:74-76; and the LCDR1-3 is SEQ ID NO:77-79; (10) The HCDR1-3 is SEQ ID NO:86-88; and the LCDR1-3 is SEQ ID NO:89-91; (11) The HCDR1-3 is SEQ ID NO:92-94; and the LCDR1-3 is SEQ ID NO:95-97; or (12) The HCDR1-3 is SEQ ID NO:98-100; and the LCDR1-3 is SEQ ID NO:101-103.

17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein, The antibody is selected from Trastuzumab, Pertuzumab, or Rituximab.

18. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, n is a real number selected from 4 to 10.

19. A drug-linker compound or a pharmaceutically acceptable salt thereof, having the general structural formula L'-D, wherein: Drug unit D is defined as in any one of claims 1-7; Connecting subunit L' is selected from or Its b-end is covalently connected to drug unit D, L 1 L 2 m1, m2, and m3 are as defined in any one of claims 1, 8 to 10.

20. The drug-linker compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein, L' is selected from Its b-end is covalently connected to the drug unit D, m1 is selected from 5, and L 1 Selected from Gly-Gly-Phe-Gly tetrapeptide residues or Ala-Ala-Ala tripeptide residues.

21. The drug-linker compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein, L' is selected from Its b-end is covalently connected to drug unit D, m2 is selected from 2, m3 is selected from 8, and L 2 Selected from Gly-Gly-Phe-Gly tetrapeptide residues or Val-Lys dipeptide residues.

22. The drug-linker compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein, L' is selected from the following chemical structures: or Its b-end is covalently connected to the drug unit D.

23. The drug-linker compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following compounds or pharmaceutically acceptable salts thereof: or 。 24. A pharmaceutical composition comprising any one of claims 1 to 18, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

25. Use of any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 24, in the preparation of a medicament for treating tumors.

26. A method for preparing the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, comprising the step of conjugating the drug-linker compound according to any one of claims 19 to 23 to the ligand.

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