WO2023217227A1 - 喜树碱类衍生物及配体-药物偶联物 - Google Patents

喜树碱类衍生物及配体-药物偶联物 Download PDF

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WO2023217227A1
WO2023217227A1 PCT/CN2023/093503 CN2023093503W WO2023217227A1 WO 2023217227 A1 WO2023217227 A1 WO 2023217227A1 CN 2023093503 W CN2023093503 W CN 2023093503W WO 2023217227 A1 WO2023217227 A1 WO 2023217227A1
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ligand
pharmaceutically acceptable
acceptable salt
seq
reaction solution
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PCT/CN2023/093503
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French (fr)
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付雅媛
李桢
陈常艳
柴晓鹃
于智勇
唐锋
曹卓晓
唐任宏
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先声再明医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure belongs to the field of biomedicine and relates to a class of ligand-drug conjugates with novel structures, their preparation methods, pharmaceutical compositions containing the conjugates and their use as anti-tumor drugs.
  • Antibody-drug conjugate as a new type of targeted drug, combines the tumor targeting properties of antibodies by linking monoclonal antibodies that specifically bind tumor cell surface antigens to biologically active toxin molecules. Combined with the high-efficiency killing effect of toxin molecules, it also avoids the shortcomings of the former's low efficacy and the latter's excessive side effects and poor drugability. Compared with traditional chemotherapy drugs in the past, ADC drugs can accurately target tumor cells and reduce the impact on normal cells to achieve safer and more effective anti-tumor effects.
  • the first antibody drug conjugate Mylotarg (gemtuzumab ozogamicin) was approved by the US FDA for the treatment of adult acute myeloid leukemia (AML).
  • AML adult acute myeloid leukemia
  • the U.S. FDA approved the new targeted ADC drug Adcetris (bretuximab vedotin) for the treatment of Hodgkin lymphoma and systemic anaplastic large cell lymphoma.
  • Mylotarg and Adcetris are both treatments for blood tumors.
  • Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA for the treatment of HER2-positive advanced or metastatic breast cancer that is resistant to trastuzumab (Trastuzumab) and paclitaxel. It is the first An approved ADC drug for the treatment of solid tumors.
  • ADC generally consists of three parts: antibody, linker and toxin.
  • Camptothecin derivatives are one type of toxin used in the development of ADCs, which achieve anti-tumor effects by inhibiting topoisomerase I.
  • Daiichi Sankyo developed the ADC drug Enhertu (Trastuzumab deruxtecan, DS-8201) using the camptothecin derivative ixotecan as a toxin and targeting the HER2 target. It was approved for marketing by the US FDA in 2019. . Clinical studies have shown that Enhertu has a good therapeutic effect on HER2-positive breast cancer, gastric cancer, and non-small cell lung cancer.
  • the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n ,
  • Pc is the ligand unit
  • L is the connecting subunit
  • D is a drug unit represented by the following formula (DI):
  • X is selected from NH or O
  • R 1 is selected from halogen, CN, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or C 2 -C 6 alkynyl, the C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl
  • the base or C 2 -C 6 alkynyl group is optionally substituted by one or more R a1 ;
  • X 1 is selected from CR 2 or N;
  • R 2 is selected from H, halogen, CN, or R 1 , R 2 and the atoms to which they are connected together form a 5-6 membered heterocyclic group, and the 5-6 membered heterocyclic group is optionally substituted by one or more R a2 ;
  • R 4 is selected from H, C 1 -C 3 alkyl, C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl, said C 1 -C 3 alkyl, C 3 -C 6 cycloalkyl or The 4-7 membered heterocyclyl group is optionally substituted by one or more R a4 ;
  • R 5 is selected from H, halogen, CN, NH 2 or NO 2 , or R 1 and R 5 and the atoms to which they are connected together form a 5-6 membered heterocyclyl group, a 5-6 membered heteroaryl group or C 5 -C 7 Cycloalkenyl, the 5-6-membered heterocyclyl, 5-6-membered heteroaryl or C 5 -C 7 cycloalkenyl is optionally substituted by one or more R a5 ;
  • R 6 is selected from H or C 1 -C 3 alkyl
  • R 7 is selected from H, C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl, or R 6 , R 7 and the C atoms to which they are connected together form a C 3 -C 6 cycloalkyl group, and the C 3 - C 6 cycloalkyl is optionally substituted by one or more R a7 ;
  • the conditions are: i) when R 1 is selected from methyl and R 2 is selected from F, R 6 , R 7 and the C atom to which they are connected together form a cyclopropyl group;
  • n is a real number from 1 to 16.
  • each R a2 and R a7 are independently selected from D.
  • R 1 is selected from halogen, C 1 -C 3 alkyl, C 3 -C 6 cycloalkyl, or C 2 -C 3 alkynyl.
  • R1 is selected from Cl, Br, methyl, cyclopropyl, or ethynyl.
  • R1 is selected from Cl, Br, or methyl.
  • R 2 is selected from H, halogen, CN, or R 1 , R 2 and the atoms to which they are connected together form a 5-6 membered heterocyclyl group containing 1 or 2 oxygen atoms as ring atoms, and the 5-6-membered heterocyclic group is optionally substituted by one or more D atoms.
  • R 2 is selected from H, halogen, CN, or R 1 , R 2 and the atoms to which they are connected together form a 5-6 membered heterocyclyl group containing 1 or 2 oxygen atoms as ring atoms.
  • R 2 is selected from H, F, or Cl, or R 1 , R 2 and the atoms to which they are attached together form
  • R 2 is selected from H, F, or Cl, or R 1 , R 2 and the atoms to which they are attached together form
  • R 5 is selected from H, halogen, NH 2 or NO 2 , or R 1 , R 5 and the atoms to which they are attached together form a 5-6 membered heteroaryl or C 5 -C 6 cycloalkenyl group,
  • the 5-6 membered heteroaryl or C 5 -C 6 cycloalkenyl group is optionally substituted by one or more Ra5 .
  • R 5 is selected from H, Cl, F, NH 2 or NO 2 , or R 1 , R 5 and the atoms to which they are attached together form
  • R4 is selected from H or Ci - C3 alkyl.
  • R4 is selected from H.
  • R6 is selected from H or methyl.
  • R 7 is selected from H, C 1 -C 3 alkyl, or C 3 -C 6 cycloalkyl optionally substituted with one or more D, or the C atom to which R 6 , R 7 is attached Together they form C 3 -C 6 cycloalkyl.
  • R 7 is selected from H, methyl, isopropyl, or cyclopropyl optionally substituted by one or more D, or R 6 , R 7 and the C atom to which they are attached together form cyclopropyl.
  • R 1 , R 2 and the atoms to which they are respectively attached together form R 6 is selected from H or methyl
  • R 7 is selected from H, methyl, isopropyl or cyclopropyl optionally substituted by one or more D
  • R 6 and R 7 and the C atom to which they are connected together form a ring.
  • R 1 is selected from methyl
  • R 2 is selected from F
  • R 6 , R 7 and the C atom to which they are connected together form a cyclopropyl group.
  • X is selected from NH and R5 is selected from Cl, F, NH2 , or NO2 .
  • the pharmaceutical unit represented by formula (DI) is selected from the group consisting of pharmaceutical units represented by formula (D-Ia):
  • R 1 , R 2 , R 4 , R 5 , R 6 and R 7 are as defined above.
  • the compound represented by formula (DI) is selected from the following compounds:
  • the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n ,
  • Pc, L, n are as defined above;
  • D is selected from one of the following compounds:
  • linker unit L is selected from Its a-end is covalently connected to the ligand unit Pc, and its b-end is covalently connected to the drug unit D, wherein m1 and m2 are each independently selected from integers 2 to 8, m3 is selected from integers 1 to 16, L 1 and L 2
  • m1 and m2 are each independently selected from integers 2 to 8
  • m3 is selected from integers 1 to 16, L 1 and L 2
  • each of L 1 and L 2 is independently selected from a peptide residue consisting of 2, 3 or 4 amino acids,
  • the L1 is a Gly-Gly-Phe-Gly tetrapeptide residue or an Ala-Ala-Ala tripeptide residue.
  • the L is a Gly-Gly-Phe-Gly tetrapeptide residue or a Val-Lys dipeptide residue.
  • m1 is selected from 5.
  • m2 is selected from 2 and m3 is selected from 8.
  • linker unit L is selected from the following chemical structures:
  • a-end is covalently connected to the ligand unit Pc, and its b-end is covalently connected to the drug unit D.
  • the ligand-drug conjugate of the present disclosure having the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • 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.
  • the antibody or antigen-binding fragment thereof is monospecific, bispecific, trispecific, or tetraspecific.
  • the antibody is selected from Trastuzumab, Pertuzumab, or Rituximab.
  • the Pc is an antibody or antigen-binding fragment thereof that specifically binds HER2, p95HER2, CDH6, ROR1 or LIV-1; the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and light chain variable region (VL);
  • the heavy chain variable region includes the VH shown in SEQ ID NO: 1, 3, 19, 21, 37, 46, 54, 56, 71, 80, 82 or 84. Containing HCDR1, HCDR2 and HCDR3; or/and the light chain variable region includes the VL shown in SEQ ID NO: 2, 4, 20, 22, 38, 47, 55, 57, 72, 81, 83 or 85 LCDR1, LCDR2 and LCDR3 contained in; or (2) the heavy chain variable region or/and the light chain variable region include HCDR1-3 or/and LCDR1-3 as described in group (1) Compared with each CDR in, an amino acid sequence with at least 80% identity, or an amino acid sequence with 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.
  • the HCDR1-3 or/and the LCDR1-3 are determined according to the Kabat numbering system,
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, or/and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein, the HCDR1-3 or/and the LCDR1 -3 selected from:
  • the HCDR1-3 is SEQ ID NO:7-9; or/and the LCDR1-3 is SEQ ID NO:10-12;
  • the HCDR1-3 is SEQ ID NO: 13-15; or/and the LCDR1-3 is SEQ ID NO: 16-18;
  • the HCDR1-3 is SEQ ID NO:23-25; or/and the LCDR1-3 is SEQ ID NO:26-28;
  • the HCDR1-3 is SEQ ID NO:29-31; or/and the LCDR1-3 is SEQ ID NO:32-34;
  • the HCDR1-3 is SEQ ID NO:40-42; or/and the LCDR1-3 is SEQ ID NO:43-45;
  • the HCDR1-3 is SEQ ID NO:48-50; or/and the LCDR1-3 is SEQ ID NO:51-53;
  • the HCDR1-3 is SEQ ID NO:58-60; or/and the LCDR1-3 is SEQ ID NO:61-63;
  • the HCDR1-3 is SEQ ID NO:64-66; or/and the LCDR1-3 is SEQ ID NO:67-69;
  • the HCDR1-3 is SEQ ID NO:74-76; or/and the LCDR1-3 is SEQ ID NO:77-79;
  • the HCDR1-3 is SEQ ID NO:86-88; or/and the LCDR1-3 is SEQ ID NO:89-91;
  • the HCDR1-3 is SEQ ID NO:92-94; or/and the LCDR1-3 is SEQ ID NO:95-97;
  • the HCDR1-3 is SEQ ID NO:98-100; or/and the LCDR1-3 is SEQ ID NO:101-103; or,
  • the HCDR1-3 or/and the LCDR1-3 has each CDR in the HCDR1-3 or/and the LCDR1-3 in any of the groups (1)-(12), An amino acid sequence with at least 80% identity, or at most 3 insertion, deletion or substitution mutations; preferably, the HCDR1-3 or/and the LCDR1-3 have the same amino acid sequence as any of the groups (1)-(12) A set of amino acid sequences with at least 80% identity compared to each CDR in HCDR1-3 or/and LCDR1-3; further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or/and a light chain variable region (VL), the heavy chain variable region comprising SEQ ID NOs: 1, 3 , the amino acid sequence shown in 19, 21, 37, 46, 54, 56, 71, 80, 82 or 84, or/and the light chain variable region includes SEQ ID NO: 2, 4, 20, 22, 38 , 47, 55, 57, 72, 81, 83 or 85; or, the heavy chain variable region or/and the light chain variable region respectively include the same as any of the above heavy chain variable regions or /An amino acid sequence that has at least 80% identity compared to the light chain variable region; further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100% identity.
  • the antibody or antigen-binding fragment thereof includes a heavy chain constant region sequence and/or a light chain constant region sequence selected from a complete constant region sequence or Fragments thereof, the constant region fragments include CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region The region is selected from the group consisting of human or murine kappa constant regions or lambda constant regions; optionally, the antibody or antigen-binding fragment thereof includes a complete heavy chain and a light chain, the heavy chain consisting of the VH and heavy chain constant regions, The heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 5, 39 or 73, the light chain is composed of the VL and the light chain constant region, and the light chain constant region has an amino acid sequence as SEQ ID NO: The amino acid sequence shown in 6.
  • the aforementioned ligand-drug conjugate with the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof wherein n is selected from a real number from 1 to 16, for example, n is selected from 2 to 12
  • n is selected from a real number ranging from 4 to 10
  • n is selected from a real number ranging from 5 to 9
  • n is selected from a real number ranging from 6 to 8.
  • n is selected from a real number of 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.
  • the present disclosure also provides a drug-linker compound or a pharmaceutically acceptable salt thereof, the general structural formula of which is L'-D, wherein:
  • Drug unit D is as defined in any of the preceding paragraphs;
  • L' is selected from The b-terminal is covalently connected to the drug unit D, and L 1 , L 2 , m1, m2, and m3 are as defined by any one of the above.
  • L' is selected from The b end is covalently connected to the drug unit D, m1 is selected from 5, and L 1 is selected from Gly-Gly-Phe-Gly tetrapeptide residues or Ala-Ala-Ala tripeptide residues.
  • 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 is selected from Gly-Gly-Phe-Gly tetrapeptide residues or Val-Lys dipeptide residues.
  • L' is selected from one of the following chemical structures:
  • the drug-linker compound of the present disclosure having the general formula L'-D or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • R 1 , X 1 , X, R 4 , R 5 , R 6 and R 7 are as defined above.
  • the compound represented by formula (DH) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the present disclosure provides a pharmaceutical composition, which includes the ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable salt thereof. Excipients.
  • the present disclosure provides a method for treating mammalian tumors, comprising administering a therapeutically effective amount of the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n to a mammal in need of the treatment, preferably a human. or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • the present disclosure provides the use of the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of drugs for treating tumors.
  • the present disclosure provides the use of the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in treating tumors.
  • the present disclosure provides the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating tumors.
  • the present disclosure provides a pharmaceutical composition, which includes the compound represented by the aforementioned formula (D-H) of the present disclosure or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients.
  • the present disclosure provides a method for treating mammalian tumors, which includes administering a therapeutically effective amount of a compound represented by the aforementioned formula (D-H) or a pharmaceutically acceptable salt thereof to a mammal in need of the treatment, preferably a human. or pharmaceutical compositions thereof.
  • the present disclosure provides the use of the compound represented by the aforementioned formula (D-H) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a drug for treating tumors.
  • the present disclosure provides the use of the compound represented by the aforementioned formula (D-H) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the treatment of tumors.
  • the present disclosure provides a compound represented by the aforementioned formula (D-H) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating tumors.
  • the present disclosure provides a method for preparing a ligand-drug conjugate with the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, including preparing the aforementioned ligand-drug conjugate with the general formula L'-D
  • the step of coupling the drug-linker compound to the aforementioned ligand, optionally, the ligand is an antibody or an antigen-binding fragment thereof.
  • the present disclosure provides a method for preparing a ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, which includes combining the aforementioned drug unit D of the present disclosure with the aforementioned ligand unit
  • the step of connecting Pc; optionally, connecting through the aforementioned linker unit L; optionally, the ligand is an antibody or an antigen-binding fragment thereof.
  • the present disclosure provides the use of the compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof in the preparation of the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or its pharmaceutically acceptable salt.
  • the use of accepted salts, and/or the compound represented by the aforementioned formula (DH) or its pharmaceutically acceptable salt in the preparation of the aforementioned drug-linker compound of the general formula L'-D or its pharmaceutically acceptable salt Uses in Salt.
  • the ligand-drug conjugates provided by the present disclosure have significant anti-inflammatory activity and/or reduced toxic side effects.
  • Figure 1 shows the X-ray single crystal diffraction analysis results of compound 19-P1.
  • Figure 2 shows the tumor growth curve of the OVCAR3 subcutaneous tumor model.
  • Figure 3 shows the tumor growth curve of the OVCAR3 subcutaneous tumor model.
  • Figure 4 shows the tumor growth curve of the OVCAR3 subcutaneous tumor model.
  • Figure 5 shows the mouse body weight change curve of the OVCAR3 subcutaneous tumor model.
  • Figure 6 shows the tumor growth curve of the H838 subcutaneous tumor model.
  • Figure 7 shows the body weight change curve of H838 subcutaneous tumor model mice.
  • ligand refers to a macromolecular compound that recognizes and binds to an antigen or receptor associated with a target cell.
  • the function of the ligand is to present the drug to the target cell population bound to the ligand.
  • ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells.
  • the ligand or ligand unit is represented as Pc, and the ligand can form a connection bond with the linker unit through a heteroatom on the ligand.
  • the ligand is selected from an antibody or antigen-binding fragment selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody; in some embodiments of the disclosure,
  • the antibodies are monoclonal antibodies.
  • linker or “linker unit” refers to a chemical structural segment or chemical bond that is connected to a ligand at one end and a drug at the other end.
  • drug refers to a small molecule compound that is biologically active in an organism.
  • the drug is a glucocorticoid receptor agonist or its corresponding phosphate ester molecule with anti-inflammatory function.
  • ligand-drug conjugate refers to a ligand connected to a biologically active drug through a stable linker unit.
  • the "ligand-drug conjugate” is an antibody-drug conjugate (ADC).
  • ADC refers to the combination of a monoclonal antibody or an antibody fragment with a stable linker unit. Biologically active drugs are linked.
  • DAR drug-to-antibody ratio
  • antibody is used in its broadest sense and refers to a polypeptide that contains sufficient sequence from the variable domain of an immunoglobulin heavy chain and/or sufficient sequence from the variable domain of an immunoglobulin light chain to be capable of specifically binding to an antigen or Peptide combination.
  • Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • Antibody as used herein includes alternative protein scaffolds or artificial scaffolds with grafted 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, as well as fully synthetic scaffolds, which contain, for example, biocompatible polymers.
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibodies as used herein include typical “quadruplex antibodies”, which are immunoglobulins composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain in which N It consists 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 direction from end to C end; and, when When the full-length antibody is of IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant region in the N-terminal to C-terminal direction.
  • Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.
  • Antibodies as used herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpacas.
  • Heavy-chain antibodies HCAbs
  • Ig new antigen receptor IgNAR
  • antibodies herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents and vertebrates, such as camelids, sheep Camel, guanaco, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (eg shark).
  • Antibodies as used herein include, but are 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.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for possible variants (e.g., containing naturally occurring mutations or arising during the production of the preparation), such variants are usually present in small amounts. ), each antibody comprising the population is identical and/or binds the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • the modifier "monoclonal" herein should not be construed as requiring production of the antibody or antigen-binding molecule by any particular method.
  • monoclonal antibodies can be produced by a variety of techniques, including (but not limited to) hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus. Other methods known in the art.
  • natural antibody refers to antibodies produced and paired by the immune system of a multicellular organism.
  • engineered antibodies herein refers to non-natural antibodies obtained through genetic engineering, antibody engineering and other technologies.
  • engineered antibodies include humanized antibodies, small molecule antibodies (such as scFv, etc.), bispecific antibodies, etc. Antibodies, etc.
  • the term "monospecific" is intended to mean having one or more binding sites, where each binding site binds the same epitope of the same antigen.
  • multispecific antibody refers to an antibody having at least two antigen-binding sites, each of the at least two antigen-binding sites binding to a different epitope of the same antigen or to a different epitope of a different antigen. combine.
  • terms such as “bispecific,” “trispecific,” “tetraspecific,” etc. refer to the number of different epitopes to which the antibody/antigen-binding molecule can bind.
  • valency indicates the presence of a specified number of binding sites in the antibody/antigen binding molecule.
  • monovalent bivalent
  • tetravalent hexavalent
  • Fully-length antibody “intact antibody” and “intact antibody” are used interchangeably herein and refer to having a structure that is substantially similar to the structure of a native antibody.
  • Antigen-binding fragment and “antibody fragment” herein are used interchangeably. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have Ability to bind antigen.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH and scFv.
  • Papain digestion of intact antibodies generates two identical antigen-binding fragments, termed "Fab” fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain and the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab’-SH is a Fab’ fragment in which the cysteine residues of the constant domain carry free thiol groups. Pepsin treatment produces an F(ab')2 fragment with two antigen binding sites (two Fab fragments) and part of the Fc region.
  • Fv fragment is the smallest fragment produced by IgG and IgM, containing a complete antigen-binding site. Fv fragments have the same binding properties and similar three-dimensional binding properties as Fab. The VH and VL chains of Fv fragments interact with each other non-covalently. functions combined.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • 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 useful in the present 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.
  • a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).
  • diabody in which the VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, forcing the domain to be separated from another
  • the complementary domains of one chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123(1994)).
  • chimeric antibody refers to an antibody in which part of the light chain or/and heavy chain is derived from a An antibody (which may originate from a specific species or belong to a specific antibody class or subclass), and another part of the light chain or/and heavy chain is derived from another antibody (which may originate from the same or different species or belong to same or different antibody class or subclass), but nevertheless retains 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)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody) and the heavy and light chain variable regions of the antibody are The light chain constant region is derived from a secondary antibody (eg, a human antibody).
  • a first antibody e.g., a murine antibody
  • a secondary antibody eg, a human antibody
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology with that of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, the FR and/or constant region in the variable region) comes from Human immunoglobulins (receptor antibodies).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity, the ability to enhance immune response, etc.
  • Fully human antibody refers to an antibody having variable regions in which both FRs and CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, “fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • variable region refers to the region contained in the heavy chain or light chain of an antibody that allows the antibody to bind to the antigen.
  • "Heavy chain variable region” is used interchangeably with “VH” and “HCVR”, and “light chain variable region” is used interchangeably.
  • Area” is used interchangeably with “VL” and “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007).
  • VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably in this article, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is due to its spatial structure. It can form precise complementarity with the antigen epitope, so it is also called complementarity determining region.
  • HVR hypervariable region
  • VH heavy chain variable region
  • VL light chain variable region
  • This region is due to its spatial structure. It can form precise complementarity with the antigen epitope, so it is also called complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • LCDR light chain variable region
  • frame region or "FR region” herein are used interchangeably and refer to those amino acid residues other than the CDRs in the heavy or light chain variable region of an antibody.
  • FR region usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR in this article can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but are not limited to AbRSA website (http://cao.labshare .cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign .cgi#results).
  • CDRs herein include overlaps and subsets of differently defined amino acid residues.
  • heavy chain constant region refers to the carboxyl-terminal portion of the antibody heavy chain that is not directly involved in binding of the antibody to the antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's Variable domains have more conserved amino acid sequences.
  • "Heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • "Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • 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 When an antibody is used, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxyl-terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen.
  • the light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxyl-terminal portion of the antibody resulting from papain hydrolysis of the intact antibody, which typically contains the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (according to Kabat numbering system Lys 447 of this system may be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain, and accordingly, the Fc region may or may not include Lys447.
  • identity can be calculated by aligning the sequences for optimal comparison purposes (e.g., for the purpose of determining the percent "identity" of two amino acid sequences or two nucleic acid sequences).
  • the alignment may introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the molecules are identical 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.
  • the percent identity between two sequences varies as a function of the identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm that has been integrated into the GAP program of the GCG software package (available at www.gcg.com), use the Blossum 62 matrix or The PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package uses the NWSgapdna.CMP matrix with gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • n is a real number between 1 and 16” herein means that n is any real number greater than or equal to 1 and less than or equal to 16.
  • tautomer refers to a functional group isomer resulting from the rapid movement of an atom in a molecule between two positions.
  • Compounds of the present disclosure may exhibit tautomerism.
  • Tautomeric compounds can exist in two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually yield a mixture whose physical and chemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form is dominant; in phenols, the enol form is dominant. This disclosure encompasses all tautomeric forms of the compounds.
  • stereoisomer refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.
  • the compounds of the present disclosure may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, or asymmetric double bonds, and therefore the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms.
  • Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S) )-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic or other mixtures thereof, such as enantiomers or diastereomers Enriched mixtures, all of the above isomers and mixtures thereof are within the definition of the compounds of the present disclosure. There may be additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms in substituents such as alkyl groups.
  • the compounds of the present disclosure containing asymmetric atoms can be isolated in an optically active pure form or in a racemic form.
  • the optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents. .
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence state of the specific atom is normal and the substituted compound is stable.
  • the term "optionally” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that description includes the occurrence Said event or circumstance and the non-occurrence of said event or circumstance.
  • the ethyl group is "optionally" substituted by halogen, which means that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (CH 2 CH 2 F, CH 2 CH 2 Cl, etc.), or polysubstituted. (CHFCH 2 F, CH 2 CHF 2 , CHFCH 2 Cl, CH 2 CHCl 2, etc.) or completely substituted (CF 2 CF 3 , CF 2 CCl 3 , CCl 2 CCl 3, etc.). It will be understood by those skilled in the art that any substitution or substitution pattern that is sterically impossible and/or cannot be synthesized will not be introduced for any group containing one or more substituents.
  • variable e.g, R a , R b
  • R a , R b its definition in each instance is independent. For example, if a group is replaced by 2 R b , there are separate options for each R b .
  • linking group When the number of a linking group is 0, such as -(CH 2 ) 0 -, it means that the linking group is a bond.
  • the direction of connection is arbitrary.
  • the structural unit When X in is selected from “C 1 -C 3 alkylene-O", at this time O-ring B", you can also connect ring A and ring B from right to left to form “ring AOC 1 -C 3 alkylene-ring B”.
  • C m -C n in this article refers to having an integer number of carbon atoms in the range of mn.
  • alkyl refers to a hydrocarbon group of the general formula C n H 2n+1 , which alkyl group may be straight or branched.
  • C 1 -C 6 alkyl is understood to mean a straight-chain or branched saturated hydrocarbon radical 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 base, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methyl Pentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl base, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C 1 -C 3 "Alkyl" refers to an alkyl group
  • C 1 -C 6 alkyl described herein may further include “C 1 -C 3 alkyl”.
  • alkynyl refers to a linear or branched unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one triple bond.
  • C 2 -C 6 alkynyl is understood to mean preferably a straight-chain or branched hydrocarbon radical which contains one or more triple bonds and has 2, 3, 4, 5 or 6 carbon atoms.
  • C 2 -C 6 alkynyl examples include, but are not limited to, ethynyl (-C ⁇ CH), prop-1-ynyl (1-propynyl, -C ⁇ CCH 3 ), prop-2-ynyl (-CH 2 C ⁇ CH), but-1-ynyl, but-2-ynyl or but-3-ynyl.
  • C 2 -C 6 alkynyl may include “C 2 -C 3 alkynyl", and examples of “C 2 -C 3 alkynyl” include ethynyl (-C ⁇ CH), prop-1-ynyl (1- Propargyl, -C ⁇ CCH 3 ), prop-2-ynyl (-CH 2 C ⁇ CH).
  • cycloalkyl refers to a fully saturated carbocyclic ring that exists in the form of a single ring, a branched ring, a bridged ring or a spiro ring.
  • C 3 -C 6 cycloalkyl should be understood to mean a saturated monocyclic, paracyclic, spirocyclic or bridged ring having 3 to 6 carbon atoms. Specific examples include but are not limited to cyclopropyl, cyclobutyl base, cyclopentyl, cyclohexyl, etc.
  • cycloalkenyl refers to a non-aromatic carbocyclic group that is not completely saturated and exists in the form of a single ring, a condensed ring, a bridged ring or a spiro ring. Unless otherwise indicated, the carbocyclic ring is typically 5 to 8 membered.
  • C 5 -C 7 cycloalkenyl refers to a cycloalkenyl group with 5, 6 or 7 ring atoms.
  • C 5 -C 7 cycloalkenyl may include the range “C 5 -C 6 cycloalkenyl” and the like.
  • C 5 -C 6 cycloalkenyl refers to a cycloalkenyl group with 5 or 6 ring atoms. Specific examples include but are not limited to cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadiene Key et al.
  • 4-membered heterocyclyl means a heterocyclyl with a number of ring atoms of 4, 5, 6 or 7, and its ring atoms contain 1-3 heteroatoms or heteroatom groups independently selected from the above.
  • 4-membered heterocyclyl include but are not limited to azetidinyl and oxetanyl
  • 5-membered heterocyclyl examples include, but are not limited to, tetrahydrofuryl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydroxazole or 2,5-dihydro-1H -pyrrolyl
  • 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, t
  • 5-6 membered heteroaryl refers to an aromatic ring group having 5 or 6 ring atoms and containing 1-3, preferably 1-2 heteroatoms independently selected from N, O and S.
  • the 5-6 membered heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, tris Azolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, etc.
  • halogen refers to fluorine, chlorine, bromine and iodine.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent or slow down (reduce) the progression of undesirable physiological changes or pathologies in the subject, such as cancer, autoimmune diseases, and viral infections.
  • beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Subjects in need of treatment include subjects who already have the condition or disease as well as subjects who are susceptible to the condition or disease or who are intended to prevent the condition or disease.
  • terms such as slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • an effective amount refers to an amount of a therapeutic agent that is effective when administered alone or in combination with another therapeutic agent to a cell, tissue or subject to prevent or alleviate the symptoms of a disease or the progression of that disease.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • subject refers to an organism undergoing treatment for a particular disease or condition as described in this disclosure.
  • subjects and patients include mammals such as humans, primates (eg, monkeys), or non-primate mammals undergoing treatment for a disease or condition.
  • the amount of a compound of the present disclosure that constitutes a "therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art. based on its own knowledge and the contents of this disclosure.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without multiple toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts refers to salts of pharmaceutically acceptable acids or bases, including salts of compounds with inorganic or organic acids, and salts of compounds with inorganic or organic bases.
  • composition refers to a mixture of one or more compounds of the present disclosure or salts thereof and pharmaceutically acceptable excipients.
  • the purpose of pharmaceutical compositions is to facilitate administration of the compounds of the present disclosure to an organism.
  • pharmaceutically acceptable excipients refers to those excipients that have no obvious irritating effect on the organism and do not impair the biological activity and performance 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.
  • the present disclosure also includes isotopically labeled compounds that are the same as those described herein, but in which one or more atoms are replaced by an atom having an atomic weight or mass number different from that typically found in nature.
  • isotopes that may be incorporated into the compounds of the present 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 respectively 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.
  • Certain isotopically labeled compounds of the present disclosure can be used in compound and/or substrate tissue distribution analyses. Tritiated (i.e. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly useful due to their ease of preparation and detectability. preferred. Positron-emitting isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • Isotopically labeled compounds of the present disclosure can generally be prepared by substituting an isotopically labeled reagent for a non-isotopically labeled reagent by following procedures similar to those disclosed in the Schemes and/or Examples below.
  • compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.
  • pharmaceutical compositions of the present disclosure may be in the form of sterile injectable aqueous solutions for intramuscular or subcutaneous administration.
  • the pharmaceutical compositions of the present disclosure may accept other vehicles or solvents when used, such as water, Ringer's solution or isotonic sodium chloride solution.
  • the daily dose is 0.001 mg/kg to 600 mg/kg body weight, preferably 0.05 mg/kg to 200 mg/kg body weight, more preferably 0.1 mg/kg to 100 mg/kg body weight, In single or divided dose form.
  • the compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other chemical synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, the embodiments of the present disclosure.
  • the ratios expressed for mixed solvents are volumetric mixing ratios.
  • % refers to wt%.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the units of NMR shifts are 10 -6 (ppm).
  • the solvents measured by NMR are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS);
  • TMS tetramethylsilane
  • IC 50 refers to the half inhibitory concentration, which refers to the concentration when half of the maximum inhibitory effect is achieved.
  • Concentration, "EC 50” refers to the concentration that causes half of the maximum effect concentration and 50% of the maximum effect.
  • 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 trichloride were added. Aluminum chloride (573.36 mg, 4.30 mmol) was added to the reaction solution at 0°C under nitrogen protection. Chloroacetonitrile (299.67 mg, 3.97 mmol) was added thereto, and the reaction solution was stirred at 90°C under nitrogen protection for 16 hours. LC-MS detects that the reaction is complete.
  • Step 2 (S)-15-(Chloromethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxane[2, Synthesis of 3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (intermediate 1-4)
  • Step 3 (S)-15-(azidomethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxane[2 ,3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (intermediate 1-5) synthesis
  • Step 4 (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-2,3,11,14-tetrahydro-12H-[1,4]dioxane[2, Synthesis of 3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (intermediate 1-6)
  • Step 5 (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-11H-[1,4] Dioxane[2,3-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2- Synthesis of hydroxyacetamide (compound 1)
  • reaction solution was filtered and purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m silica, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; acetonitrile Gradient ratio 10%-30%, elution time 12 minutes) to obtain the title compound (7 mg).
  • Step 1 Synthesis of 1-(2-amino-5-chloro-4-fluorophenyl)-2-chloroethane-1-one (intermediate 3-2)
  • Step 2 (S)-9-Chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4' :Synthesis of 6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (intermediate 3-3)
  • Step 3 (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4' :Synthesis of 6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(3-4)
  • Step 4 (S)-N-((9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyra Synthesis of prando[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (compound 3)
  • the crude product was purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m silica, 25 mm diameter, 100 mm length; water (containing 0.05% formic acid) and acetonitrile with decreasing polarity. The mixture was used as the eluent; acetonitrile gradient ratio 10%-40%, elution time 12 minutes) to obtain the title compound (2.20 mg).
  • Step 1 Synthesis of 1-(2-amino-5-bromo-4-fluorophenyl)-2-chloroethane-1-one (intermediate 5-2)
  • Step 2 (S)-9-Bromo-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4' :Synthesis of 6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (intermediate 5-3)
  • Step 3 (S)-11-(aminomethyl)-9-bromo-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4' :Synthesis of 6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (intermediate 5-4)
  • Step 4 (S)-N-((9-bromo-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxylidene-3,4,12,14-tetrahydro-1H- Synthesis of pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (compound 5)
  • the crude product was purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as a wash Deliquidation, acetonitrile gradient ratio 6%-36%, elution time 12 minutes) gave the title compound (2.09 mg).
  • Step 1 Synthesis of 1-(2-amino-4-chloro-5-methylphenyl)-2-chloroethane-1-one (intermediate 6-2)
  • Step 2 (S)-8-Chloro-11-(chloromethyl)-4-ethyl-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4 ':Synthesis of 6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (intermediate 6-3)
  • Step 3 (S)-11-(Aminomethyl)-8-chloro-4-ethyl-4-hydroxy-9-methyl-1,12-14H-pyrano[3',4':6 ,Synthesis of 7]indolizino[1,2-b]quinoline-3,14(4H)-dione (intermediate 6-4)
  • the reaction 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 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity was used as Eluent; acetonitrile gradient ratio 2%-32%, elution time 12 minutes) to obtain the title compound (11.0 mg).
  • Step 4 (S)-N-((8-chloro-4-ethyl-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H- Synthesis of pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (compound 6)
  • the crude product was purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as a wash Deliquidation, acetonitrile gradient ratio 10%-40%, elution time 12 minutes) gave the title compound (3.00 mg).
  • Step 1 Synthesis of 4,6-dibromo-2,3-dihydro-1H-indene-5-amine (intermediate 7-2)
  • Step 5 Synthesis of N-(4-(2-bromoacetyl)-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 7-6)
  • Step 6 Synthesis of 1-(5-amino-2,3-dihydro-1H-inden-4-yl)-2-chloroethan-1-one (intermediate 7-7)
  • Step 7 (S)-15-(chloromethyl)-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopenta[f Synthesis of ]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione (intermediate 7-8)
  • Step 8 (S)-15-(aminomethyl)-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopenta[f Synthesis of ]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione (intermediate 7-9)
  • the residue was purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; A mixture of decreasing polarity water (containing 0.225% formic acid) and acetonitrile was used as the eluent; acetonitrile gradient ratio 20%-40%, elution time 12 minutes) to obtain the title compound (22.0 mg).
  • Step 9 (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H-cyclopentadienyl Eno[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (compound 7) Synthesis
  • reaction solution was filtered and purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m silica, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; acetonitrile Gradient ratio 6%-36%, elution time 12 minutes) to obtain the title compound (3.00 mg).
  • the crude product was purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m silica, 25 mm diameter, 100 mm length; water (containing 0.225% formic acid) and a mixture of decreasing polarity of acetonitrile as eluent; acetonitrile gradient ratio 16 %-46%, elution time 12 minutes) to obtain the title compound (2.20 mg).
  • the crude product was purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5um, 25mm diameter, 100mm length; a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity was used as a wash Deliquidation; acetonitrile gradient ratio 16%-46%, elution time 12 minutes) to obtain the title compound (3.0 mg).
  • Step 1 Synthesis of 1-(2-fluoro-3,4-dimethoxyphenyl)ethane-1-one (intermediate 12-2)
  • Step 2 Synthesis of 1-(2-fluoro-3,4-dihydroxyphenyl)ethane-1-one (intermediate 12-3)
  • Step 3 Synthesis of 1-(4-fluorobenzo[d][1,3]dioxol-5-yl)ethane-1-one (intermediate 12-4)
  • Step 4 Synthesis of 1-(4-fluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)ethane-1-one (intermediate 12-5)
  • Step 5 Synthesis of 1-(6-amino-4-fluorobenzo[d][1,3]dioxol-5-yl)ethane-1-one (intermediate 12-6)
  • Step 6 Synthesis of N-(6-acetyl-7-fluorobenzo[d][1,3]dioxol-5-yl)acetamide (intermediate 12-7)
  • Step 7 N-(6-(2-bromoacetyl)-7-fluorobenzo[d][1,3]dioxol-5-yl)acetamide (intermediate 12-8) combine become
  • Dissolve intermediate 12-7 (380.0 mg) in acetic acid (3 mL), add hydrogen bromide in acetic acid solution (1.95 g, 33% content), and slowly add liquid bromine (256.42 mg) to the reaction solution. .
  • the reaction solution was stirred at 25°C for 1 h. After the reaction, the reaction solution was slowly poured into ice water and stirred for 0.5 h, filtered, the filter cake was washed with water (20 mL*2), and the filter cake was dried to obtain the title compound (400.0 mg).
  • Step 8 1-(6-amino-4-fluorobenzo[d][1,3]dioxol-5-yl)-2-chloroethane-1-one (intermediate 12-9 )Synthesis
  • Step 9 (S)-14-(Chloromethyl)-7-ethyl-15-fluoro-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[ 4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 12-10) Synthesis
  • Step 10 (S)-14-(aminomethyl)-7-ethyl-15-fluoro-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[ 4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 12-11) Synthesis
  • Step 11 (S)-(2-(((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1, 3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl Synthesis of )amino)-2-oxoethyl)carbamic acid tert-butyl ester (intermediate 12-12)
  • Step 12 (S)-2-amino-N-((7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[ 1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl) Synthesis of methyl)acetamide (compound 12)
  • Example 14-1 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl )methyl)-2-hydroxyacetamide (compound 14)
  • Step 1 Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethanone (intermediate 14-2)
  • Step 2 Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)ethanone (intermediate 14-3)
  • Step 3 Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl)acetamide (intermediate 14-4)
  • Step 4 Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl)acetamide (intermediate 14-5)
  • Step 5 Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)-2-chloroethanone (intermediate 14-6)
  • Step 6 (S)-14-(bromomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5- Synthesis of g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 14-7)
  • Step 7 (S)-14-(Aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5- Synthesis of g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 14-8)
  • Step 8 2-Cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl )-Synthesis of 2-hydroxyacetamide (compound 14)
  • reaction solution was filtered and purified by preparative high performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m, 30 mm diameter, 150 mm length; a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity was used as the eluent; Acetonitrile gradient ratio 4%-44%, elution time 9 minutes) to obtain the title compound (3 mg).
  • Step 9 Preparation of 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (intermediate 14-9-P1/P2)
  • Intermediate 14-9 is resolved to prepare isomers 14-9-P1 and 14-9-P2.
  • Intermediate 14-9 (1.3g) was prepared by supercritical fluid chromatography (DAICEL CHIRALPAK AD column, 10 ⁇ m silica, 30mm diameter, 250mm length; use ethanol (containing 0.1% ammonia) as eluent) to obtain intermediate 14 -9-P1 (600mg) and intermediate 14-9-P2 (600mg).
  • Step 10 Synthesis of 2-cyclopropyl-2-hydroxyacetic acid (intermediate 14-10-P1/P2)
  • intermediate 14-9-P1 (500 mg) was added to methanol (15 mL), wet palladium on carbon (10 mg, 10%) was added to the reaction solution, and the reaction solution was stirred at 25°C for 16 hours under a hydrogen atmosphere. After the reaction was completed, the reactant was filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P1 (273 mg).
  • intermediate 14-9-P2 (500 mg) was added to methanol (15 mL), wet palladium on carbon (10 mg, 10%) was added to the reaction solution, and the reaction solution was stirred at 25°C for 16 hours under a hydrogen atmosphere. After the reaction was completed, the reactant was filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P2 (279 mg).
  • Step 11 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl )-Synthesis of 2-hydroxyacetamide (compound 14-P1/P2)
  • reaction solution was purified by preparative high performance liquid chromatography (Boston Green ODS C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity was used as the eluent ( Acetonitrile gradient ratio 16%-46%, elution time 12 minutes), compound 14-P1 (22.00 mg) was obtained.
  • reaction solution was directly purified by preparative high performance liquid chromatography (Boston Green ODS C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; a mixture of decreasing polarity of water (containing 0.225% formic acid) and acetonitrile was used as the eluent (Gradient ratio of acetonitrile 16%-46%, elution time 12 minutes), compound 14-P2 (8.00 mg) was obtained.
  • preparative high performance liquid chromatography Boston Green ODS C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; a mixture of decreasing polarity of water (containing 0.225% formic acid) and acetonitrile was used as the eluent (Gradient ratio of acetonitrile 16%-46%, elution time 12 minutes), compound 14-P2 (8.00 mg) was obtained.
  • the two isomers were further analyzed by the following chiral supercritical fluid chromatography analysis method.
  • Example 14-2 (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydrofuran) Hydrogen-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline -14-yl)methyl)-2-hydroxyacetamide (compound 14-S)
  • Step 1 Synthesis of (S)-4-benzyl-3-(2-cyclopropylacetyl)oxazolidin-2-one (intermediate 3)
  • starting material 1 150.0g
  • 4-dimethylaminopyridine 160.15g
  • starting material 2 221.2g
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • dichloromethane (1500 mL) was added to the reaction solution to dilute, then washed twice with water (500 mL), once with 2N HCl (500 mL), once with saturated sodium bicarbonate solution (500 mL), and once with saturated saline solution (500 mL). 500 mL) and washed once, 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).
  • Step 4 Synthesis of (S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetic acid benzyl ester (intermediate 7)
  • Step 5 Synthesis of (S)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester (intermediate 8)
  • the retention time of intermediate 8 is 3.013 minutes; it is basically consistent with the retention time (2.990 minutes) of intermediate 14-9-P1 in Example 14-1 under the same chromatographic analysis conditions.
  • Intermediate 8 has the same configuration as intermediate 14-9-P1, and they are the same compound.
  • Step 6 Synthesis of (S)-2-cyclopropyl-2-hydroxyacetic acid (intermediate 9)
  • Step 7 (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H -[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14- Synthesis of (methyl)-2-hydroxyacetamide (compound 14-S)
  • 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%: 20%-50 %, 12 min) to obtain the title compound (21 mg).
  • the retention time of Compound 14-S under the above chiral supercritical fluid chromatography conditions is 3.654 minutes; it is basically consistent with the retention time (3.673 minutes) of Compound 14-P1 prepared in Example 14-1 under the same chromatographic analysis conditions. . Therefore, it is judged that the configuration of compound 14-S is the same as that of compound 14-P1 prepared in Example 14-1, and they are the same compound.
  • Step 1 Synthesis of 1-(5-amino-2-bromopyridin-4-yl)ethanone (intermediate 15-2)
  • Step 3 Synthesis of 1-(5-amino-2-bromopyridin-4-yl)-2-bromoethanone (intermediate 15-4)
  • Step 4 Synthesis of 1-(5-amino-2-bromopyridin-4-yl)-2-chloroethanone (intermediate 15-5)
  • Step 5 (S)-9-Bromo-11-(chloromethyl)-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7 Synthesis of indolozino[1,2-b][1,7]naphthyridine-3,14(4H)-dione (intermediate 15-6)
  • Step 6 (S)-11-(aminomethyl)-9-bromo-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7 Synthesis of indolozino[1,2-b][1,7]naphthyridine-3,14(4H)-dione (intermediate 15-7)
  • Step 7 (S)-N-((9-bromo-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3 Synthesis of ',4':6,7]indolizino[1,2-b][1,7]naphthyridin-11-yl)methyl)-2-hydroxyacetamide (compound 15)
  • reaction solution was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; Acetonitrile gradient ratio 8%-28%, elution time 12 minutes) to obtain the title compound (1.00 mg).
  • Step 1 Synthesis of 1-(6-amino-3-chloro-2,4-difluorophenyl)-2-chloroethane-1-one (intermediate 16-2)
  • Step 2 (S)-9-Chloro-11-(chloromethyl)-4-ethyl-8,10-difluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3' Synthesis of ,4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (intermediate 16-3)
  • Step 3 (S)-11-(aminomethyl)-9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3' Synthesis of ,4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (16-4)
  • Step 4 (S)-N-((9-chloro-4-ethyl-8,10-difluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropane-1-carboxamide (compound 16) synthesis
  • reaction solution was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity was used as the eluent; Acetonitrile gradient ratio 14%-34%, elution time 12 minutes) to obtain the title compound (3 mg).
  • reaction solution was filtered and purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient Ratio 25%-45%, elution time 12 minutes) to obtain the title compound (1.20 mg).
  • Example 19-1 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl -2,2-d 2 )methyl)-2-hydroxyacetamide (compound 19, compound 19-P1/P2)
  • Step 1 Synthesis of 1-(benzo[d][1,3]dioxol-5-yl-2,2-d 2 )ethane-1-one (intermediate 19-2)
  • Step 3 Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl-2,2-d 2 )acetamide (intermediate 19-4)
  • Step 4 N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl-2,2-d 2 )acetamide (Intermediate 19- 5) synthesis
  • Step 5 1-(6-aminobenzo[d][1,3]dioxol-5-yl-2,2-d 2 )-2-chloroethane-1-one (intermediate 19-6) synthesis
  • Step 6 (S)-14-(Chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5- g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d 2 (Intermediate 19 -7) synthesis
  • Step 7 (S)-14-(Aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5- g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d 2 (Intermediate 19 -Synthesis of 8)
  • Step 8 2-Cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3]dioxola Eno[4,5-g]pyrano[3',4':6,7]indolozino[1,2-b]quinolin-14-yl-2,2-d 2 )methyl )-Synthesis of 2-hydroxyacetamide (compound 19)
  • Step 9 2-Cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3]dioxolo[4,5-g]pyrano[3',4':6,7]indolozino[1,2-b]quinolin-14-yl-2, 2-d 2 ) Synthesis of methyl)-2-hydroxyacetamide (compound 19-P1/P2)
  • reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as Eluent; acetonitrile gradient ratio 15%-45%, elution time 12 minutes) to obtain compound 19-P1 (3.30 mg).
  • reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as The eluent (acetonitrile gradient ratio 15%-45%, elution time 12 minutes) gave compound 19-P2 (4.0 mg).
  • the two isomers were further analyzed by the following chiral supercritical fluid chromatography analysis method.
  • Single crystal culture method Weigh 10 mg of compound 19-P1 sample and place it in a 1.5 ml centrifuge tube. Add 300 ⁇ l of pyridine. After ultrasonic dissolution, seal it with a sealing film. Use a needle to poke three small holes in the sealing film. Leave it in place for 20 to 30 seconds. Volatilize slowly at °C for 48h to obtain needle-shaped crystals.
  • the obtained single crystal sample was subjected to X-ray analysis, and the test results are shown in Table 1 and Figure 1.
  • Compound 19-S was further analyzed by the following chiral supercritical fluid chromatography analysis methods.
  • the retention time of compound 19-S prepared in this example under the above chiral supercritical fluid chromatography conditions is 2.853 minutes; the retention time is the same as that of compound 19-P1 prepared in example 19-1 under the same chromatographic analysis conditions. (2.877 minutes) is basically the same. Therefore, it is judged that compound 19-S and compound 19-P1 have the same configuration and are the same compound.
  • Step 1 Synthesis of 1-(5-aminobenzofuran-4-yl)-2-chloroethanone (intermediate 20-2)
  • Step 2 (S)-15-(Chloromethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano[3',4 Synthesis of ':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (intermediate 20-3)
  • Step 3 (S)-15-(Aminomethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano[3',4 Synthesis of ':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (intermediate 20-4)
  • reaction solution 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 ⁇ m, 25 mm diameter, 100 mm length; water (containing 0.225% formic acid) and the polarity of methanol Decreasing mixture was used as eluent; methanol gradient ratio 5%-25%, elution time 12 minutes) to obtain the title compound (17 mg).
  • Step 4 (S)-N-((8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furo[3,2-f] Pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (compound 20)
  • reaction solution was filtered and purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient Ratio 20%-40%, elution time 12 minutes) to obtain the title compound (1 mg).
  • reaction solution was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity was used as the eluent; Acetonitrile gradient ratio 20%-40%, elution time 12 minutes) to obtain the title compound (1.07 mg).
  • reaction solution was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity was used as the eluent; Acetonitrile gradient ratio 41%-61%, elution time 12 minutes) to obtain the title compound (7 mg).
  • reaction solution was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient Proportion 20%-40%, elution time 12 minutes) to obtain the target The title compound (1.80 mg).
  • reaction solution was filtered and purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient Ratio 20%-40%, elution time 12 minutes) to obtain the title compound (1.60 mg).
  • Step 1 Synthesis of (3-chloro-2-hydroxy-5-nitrophenyl)methanediol (intermediate 27-2)
  • Step 2 Synthesis of 7-chloro-5-nitrobenzofuran-2-carboxylic acid ethyl ester (intermediate 27-4)
  • Step 6 Synthesis of 1-(5-amino-7-chlorobenzofuran-4-yl)-2-chloroethan-1-one (intermediate 27-8)
  • Step 7 (S)-4-chloro-15-(chloromethyl)-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano[ Synthesis of 3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (intermediate 27-9)
  • Step 8 (S)-15-(aminomethyl)-4-chloro-8-ethyl-8-hydroxy-11,14-dihydro-12H-furo[3,2-f]pyrano Synthesis of [3',4':6,7]indolizino[1,2-b]quinoline-9,12(8H)-dione (intermediate 27-10)
  • the residue was purified by preparative high performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; water (containing A mixture of decreasing polarity (0.225% formic acid) and acetonitrile was used as the eluent; acetonitrile gradient ratio 13%-43%, elution time 12 minutes) to obtain the title compound (60.0 mg).
  • Step 9 (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-8,9,12,14-tetrahydro-11H-furo[3, 2-f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide (compound 27) synthesis
  • reaction solution was filtered, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as the eluent; Acetonitrile gradient ratio 28%-48%, elution time 12 minutes) to obtain the title compound (6.0 mg).
  • Step 1 (S)-14-(Chloromethyl)-7-ethyl-7-hydroxy-15-nitro-10,13-dihydro-11H-[1,3]dioxolo [4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 29-1 )Synthesis
  • Step 2 (S)-14-(Aminomethyl)-7-ethyl-7-hydroxy-15-nitro-10,13-dihydro-11H-[1,3]dioxolo [4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 29-2 )Synthesis
  • Step 3 2-Cyclopropyl-N-((S)-7-ethyl-7-hydroxy-15-nitro-8,11-dioxo-7,8,11,13-tetrahydro- 10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolozino[1,2-b]quinoline-14 Synthesis of -methyl)-2-hydroxyacetamide (compound 29)
  • the residue is purified by preparative high performance liquid chromatography (Boston Prime C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; water (containing 0.05% formic acid) and acetonitrile) The mixture of decreasing polarity was used as the eluent; acetonitrile gradient ratio 15%-45%, elution time 12 minutes) to obtain the title compound (5.20 mg).
  • Step 2 Synthesis of 4-chlorobenzo[d][1,3]dioxole-5-carbaldehyde (intermediate 30-3)
  • Step 3 Synthesis of 1-(4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-ol (intermediate 30-4)
  • Step 4 Synthesis of 1-(4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-one (intermediate 30-5)
  • Step 5 Synthesis of 1-(4-chloro-6-nitrobenzo[d][1,3]dioxol-5-yl)ethan-1-one (intermediate 30-6)
  • Step 6 Synthesis of 1-(6-amino-4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-one (intermediate 30-7)
  • Step 7 Synthesis of N-(6-acetyl-7-chlorobenzo[d][1,3]dioxol-5-yl)acetamide (intermediate 30-8)
  • Step 8 N-(6-(2-bromoacetyl)-7-chlorobenzo[d][1,3]dioxol-5-yl)acetamide (intermediate 30-9) synthesis
  • Step 10 (S)-15-chloro-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[ 4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 30-11) Synthesis
  • Step 11 (S)-14-(Aminomethyl)-15-chloro-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo [4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (intermediate 30-12 )Synthesis
  • reaction is cooled to room temperature, concentrated to dryness under reduced pressure, and the residue is purified by preparative high performance liquid chromatography (Boston Prime C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; water (containing 0.225% formic acid) and acetonitrile A mixture of decreasing polarity was used as the eluent; acetonitrile gradient ratio 5%-25%, elution time 12 minutes) to obtain the title compound (16.0 mg).
  • preparative high performance liquid chromatography Boston Prime C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; water (containing 0.225% formic acid) and acetonitrile A mixture of decreasing polarity was used as the eluent; acetonitrile gradient ratio 5%-25%, elution time 12 minutes
  • Step 12 N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3] Dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)- Synthesis of 2-cyclopropyl-2-hydroxyacetamide (compound 30)
  • Step 3 Synthesis of 5-amino-7-chloro-2,3-dihydro-1H-inden-4-ol (intermediate 33-4)
  • Step 4 Synthesis of 5-acetamido-7-chloro-2,3-dihydro-1H-inden-4-yl acetate (intermediate 33-5)
  • Step 6 Synthesis of 5-acetamido-7-chloro-2,3-dihydro-1H-inden-4-yl trifluoromethanesulfonate (intermediate 33-7)
  • Step 7 Synthesis of N-(4-(1-butoxyvinyl)-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 33-8)
  • Step 8 Synthesis of N-(4-acetyl-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 33-9)
  • Step 9 Synthesis of N-(4-(2-bromoacetyl)-7-chloro-2,3-dihydro-1H-inden-5-yl)acetamide (intermediate 33-10)
  • Step 10 Synthesis of 1-(5-amino-7-chloro-2,3-dihydro-1H-inden-4-yl)-2-chloroethane-1-one (intermediate 33-11)
  • Step 11 (S)-4-chloro-15-(chloromethyl)-8-ethyl-1,2,3,8,11,14-hexahydro-9H,12H-cyclopenta[f Synthesis of ]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12-dione (intermediate 33-12)
  • Step 12 (S)-15-(aminomethyl)-4-chloro-8-ethyl-8-hydroxy-1,2,3,8,11,14-hexahydro-9H,12H-cyclopentadien Synthesis of enzo[f]pyrano[3',4':6,7]indolozino[1,2-b]quinoline-9,12-dione (intermediate 33-13)
  • Step 13 (S)-N-((4-chloro-8-ethyl-8-hydroxy-9,12-dioxo-2,3,8,9,12,14-hexahydro-1H,11H -Cyclopenta[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-15-yl)methyl)-2-hydroxyacetamide Synthesis of (Compound 33)
  • reaction solution is filtered and purified by preparative high performance liquid chromatography (Boston Green ODS C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; a mixture of water (containing 0.05% FA) and acetonitrile with decreasing polarity is used as the eluent ; Acetonitrile gradient ratio 32%-52%, elution time 12 minutes) to obtain the title compound (2.0 mg).
  • reaction solution is filtered and purified by preparative high performance liquid chromatography (Boston Prime C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; a mixture of water (containing 0.05% FA) and acetonitrile with decreasing polarity is used as the eluent ; Acetonitrile gradient ratio 35%-55%, elution time 12 minutes) to obtain the title compound (2.0 mg).
  • reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Boston Prime C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; water (containing 0.225% formic acid) and acetonitrile with decreasing polarity The mixture was used as the eluent (acetonitrile gradient ratio 15%-45%, elution time 12 minutes) to obtain the title compound (6.50 mg).
  • reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; water (containing 0.225% formic acid) and the polarity of acetonitrile The decreasing mixture was used as the eluent; acetonitrile gradient ratio 17%-47%, elution time 12 minutes), to obtain compound 35-P1 (2.87 mg).
  • reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column 5 ⁇ m silica, 30 mm diameter, 150 mm length; water (containing 0.225% formic acid) and the polarity of acetonitrile The decreasing mixture was used as the eluent; acetonitrile gradient ratio 17%-47%, elution time 12 minutes), to obtain compound 35-P2 (2.01 mg).
  • the two isomers were further analyzed by the following chiral supercritical fluid chromatography analysis method.
  • reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column 5 ⁇ m, 25 mm diameter, 100 mm length; a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity was used as Eluent (acetonitrile gradient ratio 18%-48%, elution time 12 minutes), the title compound (2.40 mg) was obtained.
  • the starting materials L1-14-1 (25g), lead acetate (43.79g) and pyridine (6.98g) were dissolved in a mixed solvent of tetrahydrofuran (600mL) and toluene (200mL), heated to 85°C under a nitrogen atmosphere, and stirred Reaction 18h. After the reaction was completed, it was cooled to room temperature, the reaction solution was filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (chromatography column: 330g Silica Flash Column, mobile phase gradient 0-75% ethyl acetate/petroleum ether, flow rate 100mL/min) to obtain the title compound (18g).
  • the residue is purified by column chromatography (chromatography column: 120g Silica Flash Column, mobile phase gradient 0 ⁇ 45% ethyl acetate/petroleum ether, flow rate 80mL/min), and then further purified by high performance liquid chromatography (chromatographic column: Boston Prime C18 150*30mm*5 ⁇ m; mobile phase: [A : water (0.225% formic acid), B: acetonitrile]; B%: 42%-82%, 13min) to obtain the title compound (1.4g).
  • the residue was purified by high performance liquid chromatography (chromatographic column: Boston Prime C18 150*30mm*5 ⁇ m; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 24%-64%, 13min), and then further purified by supercritical fluid chromatography (chromatographic column: DAICEL CHIRALPAK IC column, 10 ⁇ m silica, 30mm diameter, 250mm length; using isopropyl alcohol (containing 0.1% ammonia water) as the eluent), the intermediate L1-14-4-P1 (130 mg) and the intermediate L1-14-4-P2 (130 mg) were purified.
  • the two isomers were further analyzed by the following chiral high-performance liquid chromatography analysis method.
  • Step 4 Synthesis of intermediates L1-14-5-P1 and L1-14-5-P2
  • intermediate L1-14-4-P2 130 mg
  • intermediate L1-14-5-P2 350 mg
  • Step 5 Synthesis of intermediates L1-14-6-P1 and L1-14-6-P2
  • intermediate L1-14-5-P2 350 mg
  • intermediate L1-14-6-P2 220 mg
  • intermediate L1-14-7-P2 (361 mg) was prepared according to the above method.
  • Step 7 Synthesis of intermediates L1-14-8-P1 and L1-14-8-P2
  • intermediate L1-14-7-P2 (361 mg) as raw material
  • intermediate L1-14-8-P2 (260 mg) was prepared according to the above method.
  • Step 8 Synthesis of intermediates L1-14-9-P1 and L1-14-9-P2
  • intermediate L1-14-8-P2 260 mg
  • intermediate L1-14-9-P2 420 mg
  • Step 9 Synthesis of intermediates L1-14-10-P1 and L1-14-10-P2
  • intermediate L1-14-9-P2 (420 mg) as raw material
  • intermediate L1-14-10-P2 (320 mg) was prepared according to the above method.
  • Step 10 Synthesis of intermediates L1-14-12-P1 and L1-14-12-P2
  • intermediate L1-14-10-P2 (320 mg) and intermediate L1-14-11 (194.27 mg) as raw materials, intermediate L1-14-12-P2 (387 mg) was prepared according to the above method.
  • Step 11 Synthesis of intermediates L1-14-13-P1 and L1-14-13-P2
  • intermediate L1-14-12-P2 (387 mg) as raw material
  • intermediate L1-14-13-P2 (86 mg) was prepared according to the above method.
  • Step 12 Synthesis of compounds L1-14-P1 and L1-14-P2
  • reaction solution was purified by high performance liquid chromatography (chromatographic column: Boston Green ODS 150*30mm*5 ⁇ m; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 26%- 46%, 12 min), compound L1-14-P1 (12.3 mg).
  • the two isomers were further analyzed separately by the following chiral high-performance liquid chromatography method.
  • Example 38 N-((12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-15-fluoro-7-hydroxy-8,11-dioxo- 7,8,11,13-Tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolazino [1,2-b]quinoline -14-yl)-3,8,11,14,17-pentaoxo-5-oxy-2,7,10,13,16-pentaazaoctadecane-18-yl)-6-( 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (compound L1-35-P1)
  • 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 %, 10min), L1-35-P1 (2.3mg) was obtained.
  • Example 39-1 N-((12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7, 8,11,13-Tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolozino[1 ,2-b]quinolin-14-yl-2,2-d 2 )-3,8,11,14,17-pentaoxo-5-oxy-2,7,10,13,16-penta Azastedecyl-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (compound L1-19-P1), isomer Synthesis of Form L1-19-P2 and Racemate L1-19
  • 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 %, 12min) to obtain L1-19-P1 (6.0mg).
  • Compound L1-19-P2 (19.0 mg) was prepared according to the above method using intermediate L1-14-13-P2 (30.0 mg) and intermediate 19-8 (25.2 mg) as raw materials.
  • the two isomers were further analyzed by the following chiral high-performance liquid chromatography analysis method.
  • 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 the racemate compound L1-19 (15.4 mg).
  • Example 39-2 N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo- 7,8,11,13-Tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolazino [1,2-b]quinolin-14-yl-2,2-d 2 )-3,8,11,14,17-pentaoxo-5-oxy-2,7,10,13,16 -Pentaazaoctadecan-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound L1-19-S)
  • L1-14-4-S was further analyzed by the following chiral high-performance liquid chromatography analysis methods.
  • L1-14-4-S Under the above chiral supercritical fluid chromatography conditions, the retention time of L1-14-4-S is 4.385 minutes; it is basically consistent with the retention time of compound L1-14-4-P1 (4.471 minutes) under the same chromatographic analysis conditions. Therefore, L1-14-4-S and L1-14-4-P1 have the same configuration and are the same compound.
  • reaction solution 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).
  • L1-19-S was further analyzed separately by the following chiral high-performance liquid chromatography method.
  • the retention time of L1-19-S is 3.748 minutes; and the retention time of compound L1-19-P1 prepared in Example 39-1 under the same chromatographic analysis conditions (3.775 minutes ) are basically the same. Therefore, it is judged that L1-19-S and L1-19-P1 have the same configuration and belong to the same compound.
  • the sequence of the anti-human HER2 monoclonal antibody is shown in Table 2 below.
  • the nucleic acid sequence encoding the VH and VL of the antibody was recombined into the expression vector pTT5 with signal peptide and heavy chain constant region/light chain constant region sequence to obtain expression.
  • Recombinant plasmid of VH-CH1-Fc/VL-CL After verification by sequencing, the plasmid was extracted and transfected into host cells. After culturing, the culture supernatant of cells secreting antibodies is obtained.
  • Anti-human p95HER2 monoclonal antibody was generated by immunizing mice.
  • the immunogen is hu p95HER2.ECD-Fc protein.
  • Hybridoma cells are prepared from spleen lymphocytes of mice whose serum antibody titer is high and the titer tends to a plateau. Positive hybridoma cells are screened through conventional methods in this field such as ELISA and FACS. Cloning, further preparation of antibodies using serum-free cell culture methods, and purification and sequencing of antibodies. The mouse anti-human p95HER2 antibody was obtained after sequencing.
  • the DVD-Ig antibody is composed of VH1-linker-VH2-CH1-Fc and VL1-linker-VL2-CL. Among them, VH1 and VL1 target HER2, VH2 and VL2 target p95HER2, and the linker can be omitted.
  • the specific sequences of bispecific antibodies are shown in Table 4. According to the designed structure, bispecific antibody light and heavy chain expression plasmids were constructed on the pTT5 vector and expressed in HEK293 cells.
  • CDH6-Ab and CDH6-Ab-1 are shown in Table 5 below (the antibody sequences are from US20200171163A1).
  • CDH6-Ab-2 and CDH6-Ab-3 were generated by immunizing mice.
  • the immunogens are human CDH6-hFc protein and HEK293T cells overexpressing human CDH6.
  • the spleen lymphocytes of mice with high antibody titers in the serum are selected to prepare hybridoma cells. Positive hybridoma clones are screened through conventional methods in the field such as ELISA and FACS. , the antibody was further prepared using a serum-free cell culture method, and the antibody was purified and sequenced to obtain the mouse anti-human CDH6 antibody and its variable region sequence.
  • the nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5 with signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 70) and heavy chain constant region/light chain constant region sequences to obtain expression VH-CH1-Fc /VL-CL recombinant plasmid.
  • the sequence of the anti-human LIV-1 monoclonal antibody LIV1-Ab-1 is shown in Table 7 below (the source of the antibody sequence is US20200165335A).
  • the nucleic acid sequence encoding the antibody VH and VL was recombined into a structure with a signal peptide and a heavy chain constant region ( sequence)/light chain constant region sequence, a recombinant plasmid expressing LIV1-Ab-1 was obtained. After verification by sequencing, the plasmid was extracted. Add the plasmid and transfection reagent PEI (Polysciences, Cat. No.: 24765-1) to OPTI-MEM (Gibco, Cat. No.: 11058021), mix well and let it stand for 15 minutes.
  • PEI Polysciences, Cat. No.: 24765-1
  • OPTI-MEM Gabco, Cat. No.: 11058021
  • the anti-human ROR1 monoclonal antibody ROR1-Ab-1 variable region sequence is from patent WO2020198531A2
  • the ROR1-Ab-2 variable region sequence is from patent CN113521300A
  • the ROR1-Ab-3 variable region sequence is from patent WO2020074724A1 (see Table 8) .
  • the nucleic acid sequences encoding the antibody VH and VL were recombined into the pTT59 expression vector carrying CH and CL of human IgG1 to obtain recombinant plasmids expressing ROR1-Ab-1, ROR1-Ab-2 and ROR1-Ab-3 respectively.
  • Antibody purification Use ProteinA affinity chromatography to purify the above antibodies from the cell culture supernatant.
  • the Protein A affinity column is washed with 6M guanidine hydrochloride for 3-5 times the column volume, and then with pure water for 3-5 times the column volume.
  • Use a buffer system such as 1 ⁇ PBS (pH 7.4) as the balancing buffer to balance the chromatography column 3-5 times the column volume.
  • the cell supernatant is loaded and combined at a low flow rate. The flow rate is controlled so that the retention time is about 1 minute or longer.
  • the chromatography column is washed with 1 ⁇ PBS (pH7.4) for 3-5 times the column volume until the UV absorption drops back to the baseline. .
  • 0.1M acetic acid/sodium acetate (pH3.0-3.5) buffer to elute the sample. Collect the elution peaks according to UV monitoring.
  • the eluted product can be subjected to solution replacement using methods well known to those skilled in the art, such as using ultrafiltration tubes for ultrafiltration concentration and solution replacement to the required buffer system, or using size exclusion such as G-25 desalting to replace it with the required buffer system.
  • Buffer system or use a high-resolution size exclusion column such as Superdex 200 to remove aggregate components in the elution product to improve sample purity. After purification, the protein that meets the purity requirements is dialyzed and replaced with subsequent coupling and detection.
  • SEC Purity Analysis Apply SEC-HPLC method to analyze the protein sample to be tested, characterize the molecular size uniformity of the recombinant protein, and determine the purity of the recombinant protein.
  • the HPLC used in this method is Agilent 1260, the chromatographic column is TSKgel G3000SWXL (purchased from Tosoh Bioscience), the mobile phase is 200mM phosphate buffer, pH 7.0/isopropyl alcohol (v/v 9:1), and the detection temperature is 25°C.
  • the flow rate was 0.5mL/min, the detection wavelength was 280nm, the target protein loading amount was 50 ⁇ g, and the analysis time was 40min.
  • 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 minutes.
  • the instrument is Thermo Q Exactive Plus.
  • the main parameters of the mass spectrometer are spray voltage 3.8kV, capillary heating temperature 300°C, sheath gas flow rate 35arb, precursor ion scanning range 800-3000, etc.
  • the mass spectrometry data analysis software Biopharma Finder 4.1 is used, and the mass spectrometry data is passed through Respect.
  • the algorithm uses deconvolution processing to calculate the molecular weight information of the light and heavy chain mass spectrum peaks and the mass spectrum response signals of each component respectively, thereby calculating the DAR value of the ADC sample to be tested.
  • the antibodies ROR1-Ab-1, ROR1-Ab-2 and ROR1-Ab-3 were coupled to compound L1-0 respectively to prepare ADC-L1-0-8 and ADC-L1-0- 9.
  • ADC-L1-0-10 the DAR values are 6.6, 5.5, and 7.5 respectively; couple the antibody LIV1-Ab-1 with the compound L1-0 to obtain ADC-L1-0-7, the DAR values are 6.7 respectively;
  • the antibodies trastuzumab and pertuzumab were coupled to compound L1-00 respectively to obtain ADC-L1-00-2 and ADC-L1-00-11, with DAR values of 6.8 and 7.3 respectively; the antibodies trastuzumab and pertuzumab were coupled to compound L1-0 respectively.
  • ADC-L1-0-2 and ADC-L1-0-11 were obtained, both with DAR values of 7.3; the antibodies CDH6-Ab, CDH6-Ab-2, and CDH6-Ab-3 were coupled to compound L1-0 respectively to obtain ADC -L1-0-3, ADC-L1-0-5 and ADC-L1-0-6, the DAR values are 7.4, 7.4 and 7.2 respectively.
  • Example 41 Biological activity and related property testing
  • 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
  • 0.25% Trypsin-EDTA Gibco (USA)
  • bovine insulin Solarbio#I8040
  • 96-well plate (Greiner Bio-one#655098) was purchased from Corning (USA)
  • Cell-Titer Glo reagent Promega#G7568 was purchased from Promega Grid Corporation (USA).
  • HCT116 cells and SKBR3 cells were cultured in McCoy's 5a culture medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C and 5% CO2 .
  • OVCAR3 cells were cultured in McCoy's 5a culture medium containing 20% fetal bovine serum.
  • the 1640 culture medium + 2 ⁇ g/mL bovine insulin + 1% penicillin-streptomycin was cultured at 37°C and 5% CO2 . Cells in the logarithmic growth phase can be used for experiments.
  • Cell proliferation activity detection Cell-Titer Glo reagent was used to detect the inhibitory activity of compounds on the proliferation of HCT116, SKBR3 and OVCAR3 cell lines.
  • HCT116 cells (1500 cells per well), SKBR3 cells (3000 cells per well) and OVCAR3 cells (5000 cells per well) were seeded in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours.
  • the compound solution to be tested After adding the compound solution to be tested (the compound is dissolved in DMSO to make the compound concentration 1mM, then use DMSO to dilute the compound to 3 ⁇ M, dilute 3 times for a total of 9 concentrations, transfer 10 ⁇ L of the prepared compound solution to a 96-well plate, and make it The final concentration is 0-300nM), and continue culturing at 37°C and 5% CO2 .
  • HCT116 cells were cultured for 3 days, and SKBR3 cells and OVCAR3 cells were cultured for 5 days. Add Cell-Titer Glo reagent to detect cell viability.
  • a negative control group and a positive control group were set up as Bottom and Top respectively.
  • the negative control group does not add cells, only adds the same volume of culture medium, and other operations are consistent with the experimental group; the positive control group does not add the test drug, and other operations are consistent with the experimental group.
  • Signal refers to the signal value of the experimental group
  • Bottom refers to the average signal value of the negative control group
  • Top refers to the average signal value of the positive control group.
  • the disclosed compound exhibited strong proliferation inhibitory activity on HCT116 cells, SKBR3 cells and OVCAR3 cells.
  • the corresponding anti-cell proliferation activities of the disclosed compounds are detailed in Table 10.
  • This experiment uses a Biacore 8K (GE) instrument and uses multi-cycle kinetics to determine the relationship between the test antibody BsAb02-P and human p95HER2 (hu p95HER2.ECD-Fc, SEQ ID NO: 104, italics are the extracellular region of human p95HER2, and the underline is the Fc tag :MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT EPKSSDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSPG
  • the experimental running buffer is 1 ⁇ HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (Cat.#BR-1006-69, GE), the flow cell temperature is set to 25°C, and the sample The bin temperature is set to 16°C. Both were pretreated with running buffer.
  • Use Protein A biosensor chip (Cat. #29127556, GE) to affinity capture a certain amount of the antibody to be tested, and then flow a certain concentration of human p95HER2 antigen or human HER2 antigen on the surface of the chip, and use Biacore 8K instrument (GE) in real time The reaction signal is detected to obtain association and dissociation curves.
  • the antigen-antibody complex is washed and regenerated with glycine-hydrochloric acid regeneration solution (Cat. #BR-1003-54, GE) at pH 1.5.
  • glycine-hydrochloric acid regeneration solution Cat. #BR-1003-54, GE
  • the flow rate was 30 ⁇ L/min, starting from 50nM, diluted at 1:1, and setting up a series of concentration gradients; the dissociation time was long. up to 900s, and finally washed with 10mM glycine-hydrochloric acid solution (pH 1.5) at a flow rate of 30 ⁇ L/min for 30s to complete the regeneration of the chip surface.
  • Test Example 3-1 Flow cytometry experiment (FACS) to detect the association between anti-HER2 ADC and antibodies and SKBR3 tumor cells Synthetic activity
  • SKBR3 cells (derived from ATCC) were collected and counted, and then spread into a 96-well plate (Corning, 3795) at 2 ⁇ 10 5 cells/well. Add gradient dilutions of the sample to be tested and incubate 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 incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and the mean fluorescence intensity (MFI) was analyzed using a flow cytometer (BD FACSCanto TM II). Use Graphpad Prism software to perform a four-parameter curve fitting analysis on the data and obtain the EC 50 value. The results are shown in Table 13.
  • Test Example 3-2 Anti-ROR1 ADC and antibody binding activity test to stably transfected cell line MCF7-hROR1 clone 2G2
  • the nucleotide sequence encoding the human ROR1 amino acid sequence was cloned into the pLVX lentiviral vector, and viral particles were prepared in HEK293T cells.
  • DMEM containing 10% (w/w) fetal calf serum (ExCell Bio, Cat. No. FND500) containing 5 ⁇ g/ml puromycin (Gibco, Cat. No. A1113803) (Gibco, Cat. No. 11995073) culture medium for 1 week, using ROR1-Ab-1 and goat anti-mouse IgG (H+L) antibody (Jackson, Cat. No.
  • Test Example 3-3 Flow cytometry experiment (FACS) to detect the binding activity of anti-LIV-1 ADC and antibodies to OVCAR3 tumor cells
  • OVCAR3 cells (derived from ATCC) belong to a tumor model with high expression of LIV-1. OVCAR3 cells were collected and counted, and spread into a 96-well plate (Corning, 3795) at 2 ⁇ 10 5 cells/well. Add gradient dilutions of the sample to be tested and incubate at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 Goat anti-human Fc secondary antibody (JacksonImmuno, 109-605-098) was added and incubated at 4°C for 1 hour.
  • Test Example 4-1 Anti-HER2-ADC tumor cell proliferation activity test 1
  • Human breast cancer cell line SKBR3 was purchased from ATCC
  • human breast cancer cell line SKBR3-p95HER2 was constructed from Simcere Pharmaceuticals (for the construction method, please refer to patent application CN202210094806.6)
  • human ovarian cancer cell line SKOV3 was purchased from ATCC
  • bovine Serum, McCoy's 5a medium, penicillin-streptomycin and 0.25% Trypsin-EDTA were purchased from Gibco (USA, product number is the same as test example 1)
  • 96-well plates were purchased from Corning (USA, product number is the same as test example 1)
  • Cell -Titer Glo reagent was purchased from Promega (USA, product number is the same as test example 1).
  • SKBR3 cells SKBR3-p95HER2 cells and SKOV3 cells were cultured in McCoy's 5a culture medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C and 5% CO2 , and were in logarithmic growth. cells can be used for experiments.
  • Cell proliferation activity detection Cell-Titer Glo reagent was used to detect the inhibitory activity of ADC on the proliferation of three cell lines: SKBR3, SKBR3-p95HER2 and SKOV3.
  • SKBR3 cells (3000 per well), SKBR3-p95HER2 cells (3000 per well) and SKOV3 cells (600 per well) were seeded in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. .
  • ADC is diluted with the culture medium of the corresponding cells mentioned above, adjust the ADC concentration to 20nM or 200nM, then continue to perform 3-fold gradient dilution with the culture medium for a total of 8 concentrations, transfer 10 ⁇ L of the configured ADC solution to 96 wells plate, starting from a total concentration of 2 nM or 20 nM), and continue culturing at 37°C and 5% CO2 .
  • SKBR3 cells, SKBR3-p95HER2 cells and SKOV3 cells were cultured for 5 days. Add Cell-Titer Glo reagent to detect cell viability.
  • a negative control group and a positive control group were set up as Bottom and Top respectively.
  • the negative control group does not add cells, only adds the same volume of culture medium, and other operations are consistent with the experimental group; the positive control group does not add the test drug, and other operations are consistent with the experimental group.
  • Signal refers to the signal value of the experimental group
  • Bottom refers to the average signal value of the negative control group
  • Top refers to the average signal value of the positive control group.
  • 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)
  • 0.25% Trypsin-EDTA (Gibco#25200-056) were purchased from Gibco (USA)
  • 96-well plate (Greiner Bio-one#655098) was purchased from Corning Incorporated (USA)
  • Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).
  • SKBR3 cells use McCoy's 5a culture medium containing 10% fetal bovine serum + 1% penicillin-streptomycin
  • T47D cells use 1640 culture medium containing 10% fetal bovine serum + 1% penicillin-streptomycin
  • two strains Cells were cultured at 37°C and 5% CO2 , and cells in the logarithmic growth phase could be used for experiments.
  • Cell proliferation activity detection Cell-Titer Glo reagent was used to detect the inhibitory activity of ADC on the proliferation of SKBR3 and T47D cell lines.
  • SKBR3 and T47D cells were digested and blown away from the cell culture flask, resuspended in the corresponding fresh culture medium, and the cell density was adjusted.
  • T47D cells were seeded at 2000 cells/90 ⁇ L/well in a 96-well plate and cultured overnight at 37°C and 5% CO2 . Dilute the ADC concentration to 1000nM with complete culture medium, and perform 3-fold gradient dilution for a total of 8 concentration gradients.
  • SKBR3 cells were seeded at 5000 cells/90 ⁇ L/well in a 96-well plate and cultured overnight at 37°C and 5% CO2 . Dilute the ADC concentration to 1000 nM with complete culture medium, and perform 5-fold gradient dilution for a total of 9 concentration gradients. Then transfer 10 ⁇ L of the ADC dilute solution to a 96-well plate, that is, the total concentration of ADC from the beginning is 100 nM. The 96-well plate was cultured at 37°C and 5% CO2 for 3 days. Add Cell-Titer Glo reagent to detect cell viability.
  • a negative control group and a positive control group were set up as Bottom and Top respectively.
  • the negative control group does not add cells, only adds the same volume of culture medium, and other operations are consistent with the experimental group; the positive control group does not add the test drug, and other operations are consistent with the experimental group.
  • the percent inhibition (%Inhibition) was calculated and fitted to obtain the IC50 of the compound.
  • Signal refers to the signal value of the experimental group
  • Bottom refers to the average signal value of the negative control group
  • Top refers to the average signal value of the positive control group.
  • the anti-HER2-ADC of the present disclosure showed strong proliferation inhibitory activity against human breast ductal carcinoma cell line T47D and human breast cancer cell line SKBR3, as shown in Table 18.
  • OVCAR3 cells were collected and counted, and then spread into a 96-well plate (Corning, 3795) at 2 ⁇ 10 5 cells/well. Add gradient dilutions of the sample to be tested and incubate at 4°C for 1 hour. After washing twice with ice-cold PBS, Alexa Fluor-647 Goat anti-human Fc secondary antibody (JacksonImmuno, 109-605-098) was added and incubated at 4°C for 1 hour. After washing twice with ice-cold PBS, the cells were resuspended and the mean fluorescence intensity (MFI) was analyzed using a flow cytometer (BD FACSCanto TM II). Use Graphpad Prism software to perform a four-parameter curve fitting analysis on the data and obtain the EC 50 value. The results are shown in Table 19.
  • Test Example 6-1 anti-CDH6-ADC anti-tumor cell proliferation activity test
  • Human ovarian cancer cell line OVCAR3 (CDH6 high-expression cell line) was purchased from ATCC, human ovarian teratoma cell line PA-1 was purchased from ATCC, bovine serum, 1640 medium (Gibco#A10491-01), MEM Medium (Gibco #11095-080), MEM NEAA (Gibco #11140-050), Sodium Pyruvate (Gibco #11360-070), Penicillin-Streptomycin and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from The bovine insulin was purchased from Gibco, the 96-well plate was purchased from Corning (USA), and the Cell-Titer Glo reagent was purchased from Promega (USA).
  • OVCAR3 cells were cultured in 1640 culture medium containing 20% fetal bovine serum + 2 ⁇ g/mL bovine insulin + 1% penicillin-streptomycin at 37°C and 5% CO 2.
  • PA-1 cells were cultured in 1640 culture medium containing 10
  • the MEM culture medium of % fetal calf serum + 1% MEM NEAA + 1% sodium pyruvate + 1% penicillin-streptomycin was cultured at 37°C and 5% CO2 . Cells in the logarithmic growth phase can be used for experiments.
  • Cell proliferation activity detection Cell-Titer Glo reagent was used to detect the inhibitory activity of ADC on the proliferation of OVCAR3 and PA-1 cell lines. Digest and blow off OVCAR3 and PA-1 cells from the cell culture flask, resuspend them in the corresponding fresh culture medium, adjust the cell density, and inoculate OVCAR3 cells (5000 cells per well) and PA-1 cells (800 cells per well). in 96-well plate in 37°C and 5% CO2 for 24 hours.
  • ADC is diluted with the culture medium of the corresponding cells mentioned above to make the ADC concentration 100nM, then continue to perform 3-fold gradient dilution with the culture medium for a total of 8 concentrations, and transfer 10 ⁇ L of the configured ADC solution to a 96-well plate. , so that the final concentration is 0-10 nM), place it at 37°C and 5% CO 2 to continue culturing.
  • OVCAR3 cells and PA-1 cells were cultured for 5 days. Add Cell-Titer Glo reagent to detect cell viability.
  • a negative control group and a positive control group were set up as Bottom and Top respectively.
  • the negative control group does not add cells, only adds the same volume of culture medium, and other operations are consistent with the experimental group; the positive control group does not add the test drug, and other operations are consistent with the experimental group.
  • Signal refers to the signal value of the experimental group
  • Bottom refers to the average signal value of the negative control group
  • Top refers to the average signal value of the positive control group.
  • Test Example 6-2 anti-LIV-1-ADC anti-tumor cell proliferation activity test
  • 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) was purchased from Gibco (USA), bovine insulin (Solarbio#I8040) was purchased from Solarbio, 96-well plate (Greiner Bio-one#655098) was purchased from Corning (USA), Cell- Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).
  • OVCAR3 cells were cultured in 1640 culture medium containing 20% fetal bovine serum + 2 ⁇ g/mL bovine insulin + 1% penicillin-streptomycin at 37°C and 5% CO 2
  • H838 cells were cultured in 1640 culture medium containing 10% fetal bovine serum.
  • the 1640 culture medium containing serum + 1% penicillin-streptomycin was cultured at 37°C and 5% CO 2. Only cells in the logarithmic growth phase can be used for experiments.
  • Cell proliferation activity detection Cell-Titer Glo reagent was used to detect the inhibitory activity of ADC on the proliferation of OVCAR3 and H838 cell lines. Digest and blow OVCAR3 or H838 cells from the cell culture flask, resuspend them in the corresponding fresh culture medium, and adjust the cell density to 1500 cells/90 ⁇ L/well for OVCAR3 cells and 450 cells/90 ⁇ L/well for H838 cells. Inoculate into a 96-well plate and culture overnight at 37°C and 5% CO2 . Dilute the ADC concentration to 5000nM with complete culture medium, and perform 3-fold gradient dilution for a total of 8 concentration gradients.
  • a negative control group and a positive control group were set up as Bottom and Top respectively.
  • the negative control group does not add cells, only adds the same volume of culture medium, and other operations are consistent with the experimental group; the positive control group does not add the test drug, and other operations are consistent with the experimental group.
  • the percent inhibition (%Inhibition) was calculated and fitted to obtain the IC50 of the compound.
  • Signal refers to the signal value of the experimental group
  • Bottom refers to the average signal value of the negative control group
  • Top refers to the average signal value of the positive control group.
  • the anti-LIV-1-ADC of the present disclosure showed strong proliferation inhibitory activity against the human ovarian cancer cell line OVCAR3 and the human non-small cell lung cancer cell line H838. See Table 21 for details.
  • Test Example 6-3 Anti-ROR1-ADC anti-tumor cell proliferation activity test
  • the human breast cancer cell line hROR1-MCF7 was constructed from First Statement, DMEM medium (Gibco #11995-065), Penicillin-Streptomycin (Gibco #15140-122) and 0.25% Trypsin-EDTA (Gibco #25200-056) was purchased from Gibco (USA), 96-well plate (Greiner Bio-one #655098) was purchased from Corning (USA), Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA) U.S).
  • hROR1-MCF7 cells were cultured in DMEM culture medium containing 10% fetal calf serum + 1% penicillin-streptomycin at 37°C and 5% CO 2. Only cells in the logarithmic growth phase can be used for experiments.
  • Cell proliferation activity detection Cell-Titer Glo reagent was used to detect the inhibitory activity of ADC on the proliferation of hROR1-MCF7 cell line.
  • the hROR1-MCF7 cells were digested and blown away from the cell culture flask, resuspended in the corresponding fresh culture medium, and the cell density was adjusted.
  • the hROR1-MCF7 cells were 1700 cells/90 ⁇ L/well, seeded in a 96-well plate, and placed Cultivate overnight at 37°C and 5% CO2 . Dilute the ADC concentration to 500 nM with complete culture medium, and perform 4-fold gradient dilution for a total of 9 concentration gradients.

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Abstract

本公开涉及一类结构新颖的配体-药物偶联物或其药学上可接受的盐，具体而言，本公开提供了结构通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、其制备方法、含有所述偶联物的药物组合物、及其在治疗肿瘤中的用途。

Description

喜树碱类衍生物及配体-药物偶联物
相关申请的交叉引用
本申请要求于2022年5月12日提交的申请号为202210515898.0的中国专利申请、2022年8月19日提交的申请号为202210999585.7的中国专利申请以及2023年4月14日提交的申请号为202310406469.4的中国专利申请的优先权权益，这些专利申请文件通过引用整体并入本文，用于所有目的。
技术领域
本公开属于生物医药领域，涉及一类结构新颖的配体-药物偶联物、其制备方法、含有该偶联物的药物组合物以及作为抗肿瘤药物的用途。
背景技术
抗体-药物偶联物(antibody-drug conjugate，ADC)作为新型的靶向药物，通过将特异性结合肿瘤细胞表面抗原的单克隆抗体与具有生物活性的毒素分子相连，将抗体的肿瘤靶向性和毒素分子的高效杀伤作用相结合，同时又避免了前者疗效偏低和后者毒副作用过大、成药性差等缺陷。与以往传统的化疗药物相比，ADC药物能精准地靶向肿瘤细胞，并降低对正常细胞的影响，以达到更为安全有效的抗肿瘤效果。
2000年第一个抗体药物偶联物Mylotarg(gemtuzumab ozogamicin)获美国FDA批准上市，用于治疗成人急性髓细胞白血病(AML)。2011年美国FDA批准新型靶向ADC药物Adcetris(bretuximab vedotin)上市，用于治疗霍奇金淋巴瘤以及系统性间变性大细胞淋巴瘤。Mylotarg和Adcetris都是针对血液瘤的治疗药物。2013年，Kadcyla(ado-trastuzumab emtansine，T-DM1)被美国FDA批准，用于治疗HER2阳性同时对曲妥珠单抗(Trastuzumab)和紫杉醇有抗药性的晚期或者转移性乳腺癌，是第一个被批准的治疗实体瘤的ADC药物。
ADC一般包括三个部分：抗体、连接子和毒素。喜树碱类衍生物是其中一类应用于ADC开发的毒素，它通过抑制拓扑异构酶I从而达到抗肿瘤作用。第一三共公司(Daiichi Sankyo)采用喜树碱类衍生物依喜替康作为毒素，针对HER2靶点，开发了ADC药物Enhertu(Trastuzumab deruxtecan，DS-8201)，于2019年被美国FDA批准上市。临床研究表明，Enhertu对HER2阳性的乳腺癌、胃癌和非小细胞肺癌等都有较好的治疗效果。
尽管目前已有多个ADC药物上市，但依然存在一些安全性问题和耐药性问题，仍有极大的未满足的临床需求。因此，本领域亟需进一步开发更为有效和安全的ADC药物。
发明内容
本公开提供一种配体-药物偶联物或其药学上可接受的盐，其结构通式为Pc-(L-D)n，
其中，
Pc为配体单元；
L为连接子单元；
D为以下式(D-I)所示的药物单元：
其中，
X选自NH或O；
R1选自卤素、CN、C1-C6烷基、C3-C6环烷基或C2-C6炔基，所述C1-C6烷基、C3-C6环烷基或C2-C6炔基任选被一个或多个Ra1取代；
X1选自CR2或N；
R2选自H、卤素、CN，或者R1、R2与它们连接的原子共同形成5-6元杂环基，所述5-6元杂环基任选被一个或多个Ra2取代；
R4选自H、C1-C3烷基、C3-C6环烷基或4-7元杂环基，所述C1-C3烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Ra4取代；
R5选自H、卤素、CN、NH2或NO2，或者R1、R5与它们连接的原子共同形成5-6元杂环基、5-6元杂芳基或C5-C7环烯基，所述5-6元杂环基、5-6元杂芳基或C5-C7环烯基任选被一个或多个Ra5取代；
R6选自H或C1-C3烷基；
R7选自H、C1-C3烷基或者C3-C6环烷基，或者R6、R7与其连接的C原子共同形成C3-C6环烷基，所述C3-C6环烷基任选被一个或多个Ra7取代；
每一个Ra1、Ra2、Ra4、Ra5、Ra7独立地选自D、卤素、CN、＝O、OH、NH2、C1-C3烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C3烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
每一个Rb独立地选自卤素、CN、＝O、C1-C3烷基、OH、O(C1-C3烷基)、NH2、NH(C1-C3烷基)或N(C1-C3烷基)2；
条件是：i)当R1选自甲基，R2选自F时，R6、R7与其连接的C原子共同形成环丙基；
ii)当X选自NH时，R5不选自H；iii)式(D-I)所示化合物不包含
并且，n为1～16的实数。
在一些实施方案中，每一个Ra1、Ra2、Ra4、Ra5、Ra7独立地选自D、卤素、CN、＝O、OH、NH2、C1-C3烷基、C3-C6环烷基或4-7元杂环基。
在一些实施方案中，每一个Ra2和Ra7独立地选自D。
在一些实施方案中，R1选自卤素、C1-C3烷基、C3-C6环烷基或C2-C3炔基。
在一些实施方案中，R1选自Cl、Br、甲基、环丙基或乙炔基。
在一些实施方案中，R1选自Cl、Br或甲基。
在一些实施方案中，R2选自H、卤素、CN，或者R1、R2与它们连接的原子共同形成5-6元杂环基，所述5-6元杂环基含有1或2个氧原子作为环原子，所述5-6元杂环基任选被一个或多个D原子取代。
在一些实施方案中，R2选自H、卤素、CN，或者R1、R2与它们连接的原子共同形成5-6元杂环基，所述5-6元杂环基含有1或2个氧原子作为环原子。
在一些实施方案中，R2选自H、F或Cl，或者R1、R2与它们连接的原子共同形成
在一些实施方案中，R2选自H、F或Cl，或者R1、R2与它们连接的原子共同形成
在一些实施方案中，R5选自H、卤素、NH2或NO2，或者R1、R5与它们连接的原子共同形成5-6元杂芳基或C5-C6环烯基，所述5-6元杂芳基或C5-C6环烯基任选被一个或多个Ra5取代。
在一些实施方案中，R5选自H、Cl、F、NH2或NO2，或者R1、R5与它们连接的原子共同形成
在一些实施方案中，R4选自H或C1-C3烷基。
在一些实施方案中，R4选自H。
在一些实施方案中，R6选自H或甲基。
在一些实施方案中，R7选自H、C1-C3烷基或者任选被一个或多个D取代的C3-C6环烷基，或者R6、R7与其连接的C原子共同形成C3-C6环烷基。
在一些实施方案中，R7选自H、甲基、异丙基或者任选被一个或多个D取代的环丙基，或者R6、R7与其连接的C原子共同形成环丙基。
在一些实施方案中，R1、R2与其各自连接的原子共同形成R6选自H或甲基，R7选自H、甲基、异丙基或者任选被一个或多个D取代的环丙基，或者R6、R7与其连接的C原子共同形成环丙基。
在一些实施方案中，结构单元选自
在一些实施方案中，R1选自甲基，R2选自F，R6、R7与其连接的C原子共同形成环丙基。
在一些实施方案中，X选自NH，R5选自Cl、F、NH2或NO2。
在一些实施方案中，式(D-I)所示的药物单元选自式(D-Ia)所示的药物单元：
其中，R1、R2、R4、R5、R6、R7如上文定义。
在一些实施方案中，式(D-I)所示化合物选自以下所示化合物：



在一些实施方案中，本公开提供一种配体-药物偶联物或其药学上可接受的盐，其结构通式为Pc-(L-D)n，
其中，
Pc、L、n如前文定义；
D选自以下化合物之一：

在一些实施方案中，连接子单元L选自 其a端与配体单元Pc共价连接，b端与药物单元D共价连接，其中，m1、m2各自独立地选自整数2～8，m3选自整数1～16，L1、L2各自独立地选自由1至8个氨基酸构成的肽残基，所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代。
在一些实施方案中，所述L1、L2各自独立地选自由2、3或4个氨基酸构成的肽残基， 所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代。
在一些实施方案中，所述L1为Gly-Gly-Phe-Gly四肽残基或Ala-Ala-Ala三肽残基。
在一些实施方案中，所述L2为Gly-Gly-Phe-Gly四肽残基或Val-Lys二肽残基。
在一些实施方案中，m1选自5。
在一些实施方案中，m2选自2，m3选自8。
在一些实施方案中，连接子单元L选自以下化学结构：
其a端与配体单元Pc共价连接，b端与药物单元D共价连接。
在一些实施方案中，本公开通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：





其中Pc和n如前文定义。
在一些实施方案中，前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐，其中所述配体单元Pc可选自多肽、抗体或其抗原结合片段。
在一些实施方案中，配体单元Pc可特异性结合选自以下组的一种或多种抗原：HER2、p95HER2、HER3、CD3、CD16、ROR1、DLL3、CDH6、CD70、CD5、CD20、BCMA、EGFR、VEGF和LIV-1。
在一些实施方案中，所述抗体或其抗原结合片段为单特异性、双特异性、三特异性、或四特异性的。
在一些实施方案中，所述抗体选自Trastuzumab、Pertuzumab或Rituximab。
在一些实施方案中，所述Pc为特异性结合HER2、p95HER2、CDH6、ROR1或LIV-1的抗体或其抗原结合片段；所述抗体或其抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)；
在一些实施方案中，(1)所述重链可变区包括SEQ ID NO：1、3、19、21、37、46、54、56、71、80、82或84所示的VH中所含的HCDR1、HCDR2和HCDR3；或/和所述轻链可变区包括SEQ ID NO：2、4、20、22、38、47、55、57、72、81、83或85所示的VL中所含的LCDR1、LCDR2和LCDR3；或(2)所述重链可变区或/和所述轻链可变区包括与第(1)组中所述HCDR1-3或/和LCDR1-3中的每个CDR相比，具有至少80％同一性的氨基酸序列，或至多发生3个插入、缺失或替换突变的氨基酸序列；进一步的，所述至少80％同一性为85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性。可选地，所述HCDR1-3或/和所述LCDR1-3根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定。
在一些实施方案中，所述重链可变区包括HCDR1、HCDR2和HCDR3，或/和所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中，所述HCDR1-3或/和所述LCDR1-3选自以下：
(1)所述HCDR1-3为SEQ ID NO:7-9；或/和所述LCDR1-3为SEQ ID NO:10-12；
(2)所述HCDR1-3为SEQ ID NO:13-15；或/和所述LCDR1-3为SEQ ID NO:16-18；
(3)所述HCDR1-3为SEQ ID NO:23-25；或/和所述LCDR1-3为SEQ ID NO:26-28；
(4)所述HCDR1-3为SEQ ID NO:29-31；或/和所述LCDR1-3为SEQ ID NO:32-34；
(5)所述HCDR1-3为SEQ ID NO:40-42；或/和所述LCDR1-3为SEQ ID NO:43-45；
(6)所述HCDR1-3为SEQ ID NO:48-50；或/和所述LCDR1-3为SEQ ID NO:51-53；
(7)所述HCDR1-3为SEQ ID NO:58-60；或/和所述LCDR1-3为SEQ ID NO:61-63；
(8)所述HCDR1-3为SEQ ID NO:64-66；或/和所述LCDR1-3为SEQ ID NO:67-69；
(9)所述HCDR1-3为SEQ ID NO:74-76；或/和所述LCDR1-3为SEQ ID NO:77-79；
(10)所述HCDR1-3为SEQ ID NO:86-88；或/和所述LCDR1-3为SEQ ID NO:89-91；
(11)所述HCDR1-3为SEQ ID NO:92-94；或/和所述LCDR1-3为SEQ ID NO:95-97；
(12)所述HCDR1-3为SEQ ID NO:98-100；或/和所述LCDR1-3为SEQ ID NO:101-103；或，
(13)所述HCDR1-3或/和所述LCDR1-3具有与第(1)-(12)组中任一组所述HCDR1-3或/和LCDR1-3中的每个CDR相比，至少80％同一性，或至多发生3个插入、缺失或替换突变的氨基酸序列；优选地，所述HCDR1-3或/和所述LCDR1-3具有与第(1)-(12)组中任一组所述HCDR1-3或/和LCDR1-3中的每个CDR相比，至少80％同一性的氨基酸序列；进一步的，所述至少80％同一性为85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性。
在一些实施方案中，所述抗体或其抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)，所述重链可变区包括SEQ ID NO：1、3、19、21、37、46、54、56、71、80、82或84所示的氨基酸序列，或/和所述轻链可变区包括SEQ ID NO：2、4、20、22、38、47、55、57、72、81、83或85所示的氨基酸序列；或者，所述重链可变区或/和所述轻链可变区分别包括与上述任一重链可变区或/和轻链可变区相比，具有至少80％同一性的氨基酸序列；进一步的，所述至少80％同一性为85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性。
在一些实施方案中，所述抗体或其抗原结合片段包括重链恒定区序列和/或轻链恒定区序列，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区片段包括CH1，铰链区，CH2，CH3或Fc；可选地，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；可选地，所述抗体或其抗原结合片段包括完整的重链和轻链，所述重链由所述VH和重链恒定区组成，所述重链恒定区具有如SEQ ID NO:5、39或73所示的氨基酸序列，所述轻链由所述VL和轻链恒定区组成，所述轻链恒定区具有如SEQ ID NO:6所示的氨基酸序列。
在一些实施方案中，前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐，其中n选自1～16的实数，例如n选自2～12的实数，例如n选自4～10的实数，例如n选自5～9的实数，例如n选自6～8的实数。
在一些实施方案中，n选自5～9的实数，例如n为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或9.0。
本公开还提供了一种药物-连接子化合物或其药学上可接受的盐，其结构通式为L’-D，其中：
药物单元D如前文所述任一所定义；
L’选自其b端与药物单元D共价连接，L1、L2、m1、m2、m3如前文所述任一所定义。
在一些实施方案中，L’选自其b端与药物单元D共价连接，m1选自5，L1选自Gly-Gly-Phe-Gly四肽残基或Ala-Ala-Ala三肽残基。
在一些实施方案中，L’选自其b端与药物单元D共价连接，m2选自2，m3选自8，L2选自Gly-Gly-Phe-Gly四肽残基或Val-Lys二肽残基。
在一些实施方案中，L’选自以下化学结构之一：
其b端与药物单元D共价连接。
在一些实施方案中，本公开通式为L’-D的药物-连接子化合物或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：




本公开还提供了以下式(D-H)所示的化合物或其药学上可接受的盐：
其中，
R1、X1、X、R4、R5、R6、R7如前文所述任一所定义。
在一些实施方案中，式(D-H)所示的化合物或其药学上可接受的盐选自以下所示化合物或其药学上可接受的盐：



另一方面，本公开提供了一种药物组合物，其包含本公开前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐和药学上可接受的辅料。
另一方面，本公开提供了治疗哺乳动物肿瘤的方法，包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物。
另一方面，本公开提供了前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物在制备治疗肿瘤的药物中的用途。
另一方面，本公开提供了前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物在治疗肿瘤中的用途。
另一方面，本公开提供了治疗肿瘤的前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物。
另一方面，本公开提供了一种药物组合物，其包含本公开前述式(D-H)所示的化合物或其药学上可接受的盐和药学上可接受的辅料。
另一方面，本公开提供了治疗哺乳动物肿瘤的方法，包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物。
另一方面，本公开提供了前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物在制备治疗肿瘤的药物中的用途。
另一方面，本公开提供了前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物在治疗肿瘤中的用途。
另一方面，本公开提供了治疗肿瘤的前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物。
另一方面，本公开提供了通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐的制备方法，包括将本公开前述通式为L’-D的药物-连接子化合物与前述配体偶联的步骤，可选地，所述配体为抗体或其抗原结合片段。
另一方面，本公开提供了通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐的制备方法，包括将本公开前述药物单元D与前述配体单元Pc连接的步骤；可选地，通过前述连接子单元L连接；可选地，配体为抗体或其抗原结合片段。
另一方面，本公开提供了前述式(D-H)所示的化合物或其药学上可接受的盐在制备前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐中的用途，和/或前述式(D-H)所示的化合物或其药学上可接受的盐在制备前述通式为L’-D的药物-连接子化合物或其药学上可接受的盐中的用途。
本公开提供的配体-药物偶联物具有显著的抗炎活性和/或降低的毒副作用。
附图的简要说明
图1显示了化合物19-P1 X射线单晶衍射分析结果。
图2显示了OVCAR3皮下瘤模型的肿瘤生长曲线。
图3显示了OVCAR3皮下瘤模型的肿瘤生长曲线。
图4显示了OVCAR3皮下瘤模型的肿瘤生长曲线。
图5显示了OVCAR3皮下瘤模型的小鼠体重变化曲线。
图6显示了H838皮下瘤模型的肿瘤生长曲线。
图7显示了H838皮下瘤模型小鼠体重变化曲线。
术语定义和说明
除非另有说明，本公开中所用的术语具有下列含义，本公开中记载的基团和术语定义，包括其作为实例的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
术语“配体”是指能识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。在本公开的实施方案中，配体或配体单元表示为Pc，配体可通过配体上的杂原子与连接子单元形成连接键。在本公开的一些实施方案中，配体选自抗体或抗原结合片段，所述抗体选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；在本公开的一些实施方案中，抗体为单克隆抗体。
术语“连接子”或“连接子单元”是指一端与配体连接而另一端与药物相连的化学结构片段或化学键。
术语“药物”指在生物体内具有生物活性的小分子化合物。在本公开的一些实施方案中，药物为具有抗炎功能的糖皮质激素受体激动剂或其相应的磷酸酯分子。
术语“配体-药物偶联物”，指配体通过稳定的连接子单元与具有生物活性的药物相连。在本公开的一些实施方案中，“配体-药物偶联物”为抗体-药物偶联物(antibody drug conjugate，ADC)，ADC指将单克隆抗体或者抗体片段通过稳定的连接子单元与具有生物活性的药物相连。
术语“DAR”或“药物抗体比率”是指每个抗体分子连接的小分子糖皮质激素受体激动剂药物的平均数目。在本公开的抗体-药物偶联物中，DAR由变量“n”定义，n既可以是整数，也可以是小数。
术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文的“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer等人,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque等人,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文的“抗体”包括典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文的“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
本文的“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文的“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原驼、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文的“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体或全人抗体。
术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体，即，除了可能的变异体(例如含有天然存在的突变或在制剂的生产过程中产生，此类变体通常以少量存在)之外，包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。本文修饰语“单克隆”不应解释为需要通过任何特定方法产生所述抗体或抗原结合分子。举例 来说，单克隆抗体可通过多种技术制得，包括(但不限于)杂交瘤技术、重组DNA方法、噬菌体库展示技术和利用含有全部或部分人免疫球蛋白基因座的转基因动物的方法和其它本领域已知的方法。
术语“天然抗体”是指通过多细胞生物体的免疫系统产生和配对的抗体。本文的术语“工程化抗体”是指通过基因工程、抗体工程等技术获得的非天然抗体，示例性地，“工程化抗体”包括人源化抗体、小分子抗体(例如scFv等)、双特异性抗体等。
术语“单特异性”是指表示具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
术语“多特异性抗体”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此，术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文的“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文的“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2、Fv、VHH和scFv。
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此，本文的术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段以及重链的VH域和第一恒定域(CH1)的抗体片段。Fab’片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同，包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’)2片段。
“Fv片段”是由IgG和IgM产生的最小片段，包含完整的抗原结合位点，Fv片段具有与Fab相同的结合特性和相似的三维结合特性，Fv片段的VH和VL链通过非共价相互作用结合在一起。
术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过连接子(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-连接子-VH-COOH或NH2-VH-连接子-VL-COOH。合适的现有技术连接子由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的连接子，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本公开的其他连接子由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。
术语“双抗体(diabody)”，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
术语“嵌合抗体(Chimeric antibody)”是指这样的抗体，其轻链或/和重链的一部分源自一个 抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly等人；Morrison等人,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
术语“人源化抗体”是指经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区中的FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文的“全人抗体”可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文的“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
术语“可变区”是指抗体重链或轻链中所包含的使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt等人,Kuby Immunology,第6版,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文的术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本文的术语“框架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
本文的“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文的CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文的术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文的术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文的术语“Fc”是指完整抗体经木瓜蛋白酶水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据Kabat编号系 统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文的术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
本文中“n为1-16的实数”指n为大于或等于1且小于或等于16的任意实数。
本文中表示连接位点。
本文中消旋体或者对映体纯的化合物的图示法来自Maehr，J.Chem.Ed.1985，62:114-120。除非另有说明，用楔形键和虚楔键表示一个立体中心的绝对构型，用黑实键和虚键表示一个立体中心的相对构型(如脂环化合物的顺反构型)。
术语“互变异构体”是指因分子中某一原子在两个位置迅速移动而产生的官能团异构体。本公开化合物可表现出互变异构现象。互变异构的化合物可以存在两种或多种可相互转化的种类。互变异构体一般以平衡形式存在，尝试分离单一互变异构体时通常产生一种混合物，其理化性质与化合物的混合物是一致的。平衡的位置取决于分子内的化学特性。例如，在很多脂族醛和酮如乙醛中，酮型占优势；而在酚中，烯醇型占优势。本公开包含化合物的所有互变异构形式。
术语“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体，包括顺反异构体、对映异构体和非对映异构体。
本公开的化合物可以具有不对称原子如碳原子、硫原子、氮原子、磷原子或不对称双键，因此本公开的化合物可以存在特定的几何或立体异构体形式。特定的几何或立体异构体形式可以是顺式和反式异构体、E型和Z型几何异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，以及其外消旋混合物或其它混合物，例如对映异构体或非对映体富集的混合物，以上所有这些异构体以及它们的混合物都属于本公开化合物的定义范围之内。烷基等取代基中可存在另外的不对称碳原子、不对称硫原子、不对称氮原子或不对称磷原子，所有取代基中涉及到的这些异构体以及它们的混合物，也均包括在本公开化合物的定义范围之内。本公开的含有不对称原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来，光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生 所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，是指乙基可以是未被取代的(CH2CH3)、单取代的(CH2CH2F、CH2CH2Cl等)、多取代的(CHFCH2F、CH2CHF2、CHFCH2Cl、CH2CHCl2等)或完全被取代的(CF2CF3、CF2CCl3、CCl2CCl3等)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。
当任何变量(例如Ra、Rb)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，如果一个基团被2个Rb所取代，则每个Rb都有独立的选项。
当一个连接基团的数量为0时，比如-(CH2)0-，表示该连接基团为键。
当其中一个变量选自化学键或不存在时，表示其连接的两个基团直接相连，比如A-L-Z中L代表键时表示该结构实际上是A-Z。
当本文中涉及到的连接基团若没有指明其连接方向，则其连接方向是任意的。例如当结构单元中的X选自“C1-C3亚烷基-O”时，此时X既可以按照与从左到右的方向连接环A和环B构成“环A-C1-C3亚烷基-O-环B”，也可以按照从右到左的方向连接环A和环B构成“环A-O-C1-C3亚烷基-环B”。
本文中的Cm-Cn，是指具有m-n范围中的整数个碳原子。
术语“烷基”是指通式为CnH2n+1的烃基，该烷基可以是直链或支链的。术语“C1-C6烷基”应理解为表示具有1、2、3、4、5或6个碳原子的直链或支链饱和烃基。所述烷基包括但不限于甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1，2-二甲基丙基、新戊基、1，1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3，3-二甲基丁基、2，2-二甲基丁基、1，1-二甲基丁基、2，3-二甲基丁基、1，3-二甲基丁基或1，2-二甲基丁基等；术语“C1-C3烷基”指含有1至3个碳原子的烷基，例如甲基、乙基、正丙基、异丙基。
本文所述“C1-C6烷基”可以进一步包含“C1-C3烷基”。
术语“炔基”是指由碳原子和氢原子组成的直链或支链的具有至少一个三键的不饱和脂肪族烃基。例如，术语“C2-C6炔基”应理解为优选表示直链或支链的烃基，其包含一个或多个三键并且具有2、3、4、5或6个碳原子。“C2-C6炔基”的实例包括但不限于乙炔基(-C≡CH)、丙-1-炔基(1-丙炔基、-C≡CCH3)、丙-2-炔基(-CH2C≡CH)、丁-1-炔基、丁-2-炔基或丁-3-炔基。“C2-C6炔基”可以包含“C2-C3炔基”，“C2-C3炔基”实例包括乙炔基(-C≡CH)、丙-1-炔基(1-丙炔基、-C≡CCH3)、丙-2-炔基(-CH2C≡CH)。
术语“环烷基”指完全饱和的且以单环、并环、桥环或螺环等形式存在的碳环。术语“C3-C6环烷基”应理解为表示饱和的单环、并环、螺环或桥环，其具有3～6个碳原子，具体实例包括但不限于环丙基、环丁基、环戊基、环己基等。
术语“环烯基”是指不完全饱和的且以单环、稠环、桥环或螺环等形式存在的非芳香族碳环基。除非另有指示，该碳环通常为5至8元环。术语“C5-C7环烯基”是指环原子数为5、6或7的环烯基，具体实例包括但不限于环戊烯基、环戊二烯基、环己烯基、环己二烯基、环庚烯基或环庚二烯基等。术语“C5-C7环烯基”可以包含“C5-C6环烯基”等范围。术语“C5-C6环烯基”是指环原子数为5或6的环烯基，具体实例包括但不限于环戊烯基、环戊二烯基、环己烯基、环己二烯基等。
术语“杂环基”是指完全饱和的或部分饱和的单环、并环、螺环或桥环基团，其环原子中含有1-5个杂原子或杂原子团(即含有杂原子的原子团)，所述“杂原子或杂原子团”包括但不限于氮原子(N)、氧原子(O)、硫原子(S)、磷原子(P)、硼原子(B)、-S(＝O)2-、-S(＝O)-、-P(＝O)2-、-P(＝O)-、-NH-、-S(＝O)(＝NH)-、-C(＝O)NH-或-NHC(＝O)NH-等。术语“4-7元杂环基”意指环原子数目为4、5、6或7的杂环基，且其环原子中含有1-3个独立选自上文所述的杂原子或杂原子团。其中，4元杂环基的实例包括但不限于氮杂环丁烷基、氧杂环丁烷基；5元杂环基的 实例包括但不限于四氢呋喃基、二氧杂环戊烯基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基、4,5-二氢噁唑或2,5-二氢-1H-吡咯基；6元杂环基的实例包括但不限于四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基、三噻烷基、四氢吡啶基或4H-[1,3,4]噻二嗪基；7元杂环基的实例包括但不限于二氮杂环庚烷基。优选地，“4-7元杂环基”可以包含“4-7元杂环烷基”、“5-6元杂环基”、“5-6元杂环烷基”等范围。
术语“5-6元杂芳基”指具有5或6个环原子的芳族环基，且其包含1-3个，优选1-2个独立选自N、O和S的杂原子。特别地，5-6元杂芳基选自噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、三唑基、噻二唑基、吡啶基、哒嗪基、嘧啶基、吡嗪基或三嗪基等。
术语“卤”或“卤素”是指氟、氯、溴和碘。
术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症、自身免疫性疾病和病毒感染的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
术语“受试者”是指接受对如本公开所述的特定疾病或病症的治疗的生物体。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
构成“治疗有效量”的本公开化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指药学上可接受的酸或碱的盐，包括化合物与无机酸或有机酸形成的盐，以及化合物与无机碱或有机碱形成的盐。
术语“药物组合物”是指一种或多种本公开的化合物或其盐与药学上可接受的辅料组成的混合物。药物组合物的目的是有利于对有机体给予本公开的化合物。
术语“药学上可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising可理解为开放的、非排他性的意义，即“包括但不限于”。
本公开还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本公开化合物。可结合到本公开化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、31P、32P、35S、18F、123I、125I和36Cl等。
某些同位素标记的本公开化合物(例如用3H及14C标记)可用于化合物和/或底物组织分布分析中。氚化(即3H)和碳-14(即14C)同位素对于由于它们易于制备和可检测性是尤其 优选的。正电子发射同位素，诸如15O、13N、11C和18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本公开化合物。
本公开的药物组合物可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。例如，本公开的药物组合物可以是用于肌内或皮下给药的无菌注射水溶液形式。本公开的药物组合物在使用时可接受其它溶媒或溶剂，如水、林格氏液或等渗氯化钠溶液。
本文所述化合物的所有施用方法中，每天给药的剂量为0.001mg/kg到600mg/kg体重，优选为0.05mg/kg到200mg/kg体重，更优选0.1mg/kg到100mg/kg体重，以单独或分开剂量的形式。
本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其它化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开的实施例。
本公开具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本公开的化学变化及其所需的试剂和物料。为了获得本公开的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
本领域合成路线规划中的一个重要考量因素是为反应性官能团(如本公开中的氨基、羧基)选择合适的保护基，例如，可参考Greene's Protective Groups in Organic Synthesis(4th Ed).Hoboken,New Jersey:John Wiley&Sons,Inc.本公开引用的所有参考文献整体上并入本公开。
具体实施方式
下面通过实施例对本公开进行详细描述，但并不意味着对本公开任何不利限制。本文已经详细地描述了本公开，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本公开精神和范围的情况下针对本公开具体实施方式进行各种改变将是显而易见的。本公开所使用的所有试剂是市售的，无需进一步纯化即可使用。
除非另作说明，混合溶剂表示的比例是体积混合比例。
除非另作说明，否则，％是指wt％。
化合物经手工或软件命名，市售化合物采用供应商目录名称。
化合物的结构是通过核磁共振(NMR)和/或质谱(MS)来确定的。NMR位移的单位为10-6(ppm)。NMR测定的溶剂为氘代二甲基亚砜、氘代氯仿、氘代甲醇等，内标为四甲基硅烷(TMS)；“IC50”指半数抑制浓度，指达到最大抑制效果一半时的浓度,“EC50”指能半数最大效应浓度，引起50％最大效应的浓度。
实施例1、(S)-N-((8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-11H-[1,4]二噁己环并[2,3-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物1)
步骤1：1-(7-氨基-2,3-二氢苯并[b][1,4]二噁己环-6-基)-2-氯乙烷-1-酮(中间体1-2)的合成
将反应物1-1(500mg,3.31mmol)溶于1,2-二氯乙烷(3mL)中，反应液降温至0℃，向其中加入三氯化硼(1M,2.65mL)和三氯化铝(573.36mg，4.30mmol)，反应液于氮气保护下0℃向其中加入氯乙腈(299.67mg,3.97mmol)，反应液于氮气保护下90℃搅拌16h。LC-MS检测反应完毕。待反应冷却至室温，依次加入冰水(30mL)和1N HCl(10mL)，然后搅拌30min。向反应液中加入二氯甲烷(30mL*3)萃取3遍，合并有机相用饱和食盐水(30mL)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。减压浓缩至干，粗品经制备薄层色谱法(二氧化硅，石油醚：乙酸乙酯＝9:1)得到标题化合物(210mg)。
MS m/z(ESI):228.0[M+H]+。
步骤2：(S)-15-(氯甲基)-8-乙基-8-羟基-2,3,11,14-四氢-12H-[1,4]二噁己环并[2,3-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12(8H)-二酮(中间体1-4)的合成
将中间体1-2(100mg,439.28μmol)和中间体1-3(115.64mg,439.28μmol)溶于无水甲苯(3mL)中，向其中加入对甲苯磺酸吡啶盐(22.08mg,87.86μmol)，反应液于氮气保护下100℃搅拌16h。LC-MS检测反应完毕。待反应冷却至室温，反应液过滤，滤饼用乙醇(5mL*2)洗涤得到标题化合物粗品(130mg)。
MS m/z(ESI):455.1[M+H]+。
步骤3：(S)-15-(叠氮甲基)-8-乙基-8-羟基-2,3,11,14-四氢-12H-[1,4]二噁己环并[2,3-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12(8H)-二酮(中间体1-5)的合成
将中间体1-4(120mg,263.82μmol)溶于二甲亚砜(1mL)中，向其中加入叠氮化钠(25.73mg,395.73μmol)，反应液于氮气保护下25℃搅拌3h。LC-MS检测反应完毕。向其中加入冰水(2mL)搅拌0.5h，过滤得到标题化合物粗品(90mg)。
MS m/z(ESI):462.1[M+H]+。
步骤4：(S)-15-(氨基甲基)-8-乙基-8-羟基-2,3,11,14-四氢-12H-[1,4]二噁己环并[2,3-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12(8H)-二酮(中间体1-6)的合成
将中间体1-5(90mg,195.05μmol)溶于无水甲苯(1mL)中，向其中加入亚磷酸三乙酯(81.02mg,487.62μmol)，反应液于氮气保护下100℃搅拌3h。反应液降温至25℃，向其中加入盐酸甲醇溶液(0.5mL)，反应液于氮气保护下85℃搅拌16h。LC-MS检测反应完毕。待反应冷却至室温，过滤得到标题化合物(18mg)。
MS m/z(ESI):436.1[M+H]+。
步骤5：(S)-N-((8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-11H-[1,4]二噁己环并[2,3-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物1)的合成
将中间体1-6(18mg,41.34μmol)和2-羟基乙酸(15.72mg,206.69μmol)溶于无水N,N-二甲基甲酰胺(1mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)(23.58mg,62.01μmol)和N,N-二异丙基乙胺(DIEA)(16.03mg，124.02μmol)，反应液于25℃搅拌3h。LC-MS检测反应完毕。反应液过滤，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例10％-30％,洗脱时间12分钟)得到标题化合物(7mg)。
MS m/z(ESI):494.1[M+H]+。
1H 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,1H),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)。
实施例3、(S)-N-((9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺(化合物3)
步骤1：1-(2-氨基-5-氯-4-氟苯基)-2-氯乙烷-1-酮(中间体3-2)的合成
将三氯化硼(1M，13.74mL)溶于1,2-二氯乙烷(24mL)中，反应液降温至0℃，向其中加入反应物3-1(2g，13.74mmol)和氯乙腈(1.56g，20.61mmol)，反应在0℃下搅拌10min，向其中加入三氯化铝(2.38g，17.86mmol)。之后反应液在氮气保护下升至25℃搅拌10min。反应液于氮气保护下90℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，依次缓慢加入冰水(50mL)和5％HCl(10mL)于25℃下搅拌30min，再加入二氯甲烷(50mL)，有机相用水(2mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。减压浓缩至干，粗品经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例40％-60％，洗脱时间10分钟)得到标题化合物(320mg)。
MS m/z(ESI):222.0[M+H]+。
步骤2：(S)-9-氯-11-(氯甲基)-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(中间体3-3)的合成
将中间体3-2(220mg,990.80μmol)和中间体1-3(273.86mg,1.04mmol)溶于甲苯(2mL)中，向其中加入对甲基苯磺酸吡啶盐(24.90mg,99.08μmol)。反应液在100℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，加入乙醇(1mL),反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤得到标题化合物粗品(270mg)。
MS m/z(ESI):449.0[M+H]+。
步骤3：(S)-11-(氨基甲基)-9-氯-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(3-4)的合成
将中间体3-3(50mg,111.29μmol)溶于乙醇(1mL)中，向其中加入乌洛托品(23.40mg,166.94μmol)。反应液在90℃搅拌1.5h。LC-MS检测反应完毕。待反应冷却至室温，减压浓缩至干，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.225％甲酸)和甲醇的极性递减的混合物作为洗脱液；甲醇梯度比例0％-30％,洗脱时间12分钟)得到标题化合物(22mg)。
MS m/z(ESI):430.1[M+H]+。
1H 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)。
步骤4：(S)-N-((9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺(化合物3)的合成
将3-4(22mg,51.18μmol)和2-羟基乙酸(19.46mg,255.92μmol)溶于无水N,N-二甲基甲 酰胺(1mL)，向其中加入HATU(29.19mg,76.77μmol)和N,N-二异丙基乙胺(19.84mg，153.55μmol)，反应液于25℃搅拌1.5h。LC-MS检测反应完毕。反应液过滤，减压浓缩至干，粗品经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例10％-40％，洗脱时间12分钟)得到标题化合物(2.20mg)。
MS m/z(ESI):488.1[M+H]+。
1H 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＝5.7Hz,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)。
实施例5、(S)-N-((9-溴-4-乙基-8-氟-4-羟基-3,14-二氧亚基-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺(化合物5)
步骤1：1-(2-氨基-5-溴-4-氟苯基)-2-氯乙烷-1-酮(中间体5-2)的合成
将三氯化硼(1M，10.53mL)溶于1,2-二氯乙烷(24mL)中，反应液降温至0℃，向其中加入中间体5-1(2g，10.53mmol)和氯乙腈(1.19g，15.79mmol)，反应在0℃下搅拌10min，向其中加入三氯化铝(1.82g，13.68mmol)。反应液在氮气保护下，25℃搅拌10min。随后升温至90℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，依次缓慢加入冰水(50mL)和5％HCl(10mL)于25℃下搅拌30min，再加入二氯甲烷(50mL)，有机相用水(2mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例39％-49％，洗脱时间12分钟)得到标题化合物(380mg)。
MS m/z(ESI):265.9[M+H]+。
步骤2：(S)-9-溴-11-(氯甲基)-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(中间体5-3)的合成
将中间体5-2(200mg,750.48μmol)和中间体1-3(207.44mg,788.01μmol)溶于无水甲苯(4mL)中，向其中加入对甲基苯磺酸吡啶盐(22.63mg,90.06μmol)。反应液在100℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，加入乙醇(1mL)，反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤得到标题化合物粗品(200mg)。
MS m/z(ESI):493.0[M+H]+。
步骤3：(S)-11-(氨基甲基)-9-溴-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(中间体5-4)的合成
将中间体5-3(200mg,405.10μmol)溶于乙醇(4mL)中，向其中加入乌洛托品(113.58mg,810.19μmol)。反应液在90℃搅拌1.5h。LC-MS检测反应完毕。待反应冷却至室温，减压浓缩至干，粗品经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例6％-26％，洗脱时间12分钟)得到标题化合物(30mg)。
MS m/z(ESI):476.0[M+H]+。
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)。
步骤4：(S)-N-((9-溴-4-乙基-8-氟-4-羟基-3,14-二氧亚基-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺(化合物5)的合成
将中间体5-4(15mg,26.88μmol)和2-羟基乙酸(10.22mg,134.41μmol)溶于无水N,N-二甲基甲酰胺(1mL)，向其中加入HATU(15.33mg,40.32μmol)和N,N-二异丙基乙胺(10.42mg，80.65μmol)，反应液于25℃搅拌1h。LC-MS检测反应完毕。反应液过滤，减压浓缩至干，粗品经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液，乙腈梯度比例6％-36％，洗脱时间12分钟)得到标题化合物(2.09mg)。
MS m/z(ESI):532.0[M+H]+。
1H 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)。
实施例6、(S)-N-((8-氯-4-乙基-4-羟基-9-甲基-3,14-二氧亚基-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺(化合物6)
步骤1：1-(2-氨基-4-氯-5-甲基苯基)-2-氯乙烷-1-酮(中间体6-2)的合成
将三氯化硼(1M，2.82mL)溶于1,2-二氯乙烷(8mL)中，反应液降温至0℃，向其中加入反应物6-1(0.5g，3.53mmol)和氯乙腈(319.91g，4.24mmol)，反应在0℃下搅拌10min，向其中加入三氯化铝(612.09mg，4.59mmol)。反应液在氮气保护下25℃搅拌10min。之后反应液升温至90℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，依次缓慢加入冰水(25mL)和5％HCl(5mL)于25℃下搅拌30min，再加入二氯甲烷(20mL)，有机相用 水(20mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。经制备薄层色谱法(二氧化硅，石油醚：乙酸乙酯＝9:1)得到标题化合物(500mg)。
MS m/z(ESI):218.0[M+H]+
步骤2：(S)-8-氯-11-(氯甲基)-4-乙基-4-羟基-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(中间体6-3)的合成
将中间体6-2(250mg,1.15mmol)和中间体1-3(316.87mg,1.20mmol)溶于甲苯(5mL)中，向其中加入对甲基苯磺酸吡啶盐(34.57mg,137.56μmol)。反应液在100℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，加入乙醇(1mL)，反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤得到标题化合物粗品(230mg)。
MS m/z(ESI):445.1[M+H]+。
步骤3：(S)-11-(氨基甲基)-8-氯-4-乙基-4-羟基-9-甲基-1,12-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(中间体6-4)的合成
将中间体6-3(49.56mg,111.29μmol)溶于乙醇(0.5mL)中，向其中加入乌洛托品(23.40mg,166.94μmol)。反应液在90℃搅拌1.5h。LC-MS检测反应完毕。待反应冷却至室温，减压浓缩至干，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例2％-32％,洗脱时间12分钟)得到标题化合物(11.0mg)。
MS m/z(ESI):426.2[M+H]+。
步骤4：(S)-N-((8-氯-4-乙基-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺(化合物6)的合成
将中间体6-4(11mg,25.83μmol)和2-羟基乙酸(9.82mg,129.15μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(14.73mg,38.74μmol)和N,N-二异丙基乙胺(10.01mg，77.49μmol)，反应液于25℃搅拌1h。LC-MS检测反应完毕。反应液过滤，减压浓缩至干，粗品经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液，乙腈梯度比例10％-40％，洗脱时间12分钟)得到标题化合物(3.00mg)。
MS m/z(ESI):484.1[M+H]+。
1H 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)
实施例7、(S)-N-((8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-1H,11H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物7)
步骤1：4,6-二溴-2,3-二氢-1H-茚-5-胺(中间体7-2)的合成
将中间体7-1(10.0g)溶解在无水乙腈中，在0℃下分批加入N-溴代丁二酰亚胺(NBS)(27.5g)，随后在室温下搅拌过夜。将反应液过滤，滤液减压浓缩，残余物溶于200mL乙酸乙酯，并用水(100mL*2)洗涤。所得有机相用无水硫酸钠干燥，硅胶拌样，然后置于硅藻土上，用500mL石油醚淋洗，滤液浓缩得到标题化合物(17.0g)。
MS m/z(ESI):289.9[M+H]+。
步骤2：4-溴-2,3-二氢-1H-茚-5-胺(中间体7-3)的合成
将中间体7-2(15.0g)和氯化亚锡(15.0g)溶解在75mL醋酸中，并加入6N的浓盐酸140mL，在90℃下反应3h。将反应体系冷却至室温，浓缩除去醋酸，残留物溶解在乙酸乙酯100mL中，用饱和碳酸钠水溶液调节pH至8左右，过滤，滤液分去有机相，水相再用乙酸乙酯(50mL*3)萃取。合并有机相并用无水硫酸钠干燥，过滤，滤液减压浓缩，得到标题化合物(10g)。
MS m/z(ESI):212.0[M+H]+。
步骤3：N-(4-溴-2,3-二氢-1H-茚-5-基)乙酰胺(中间体7-4)的合成
将中间体7-3(10.0g)溶于无水二氯甲烷100mL，加入三乙胺(10.6g)，并在0℃下缓慢滴加乙酰氯(5.5g)，然后反应体系在室温下搅拌过夜。反应液用水(100mL*2)洗涤，有机相用无水硫酸钠干燥，过滤。滤液减压浓缩，残余物用硅胶柱层析法纯化(石油醚：乙酸乙酯＝90：10)得到标题化合物(7.1g)。
MS m/z(ESI):254.0[M+H]+。
步骤4：N-(4-乙酰基-2,3-二氢-1H-茚-5-基)乙酰胺(中间体7-5)的合成
在氮气保护下，将中间体7-4(6.7g)和三丁基-(2-乙氧基乙烯基)锡(10.4g)溶于100mL无水1,4-二氧六环中，然后加入二(三苯基膦)二氯化钯(1.8g)，然后反应体系在100℃下搅拌过夜。反应液冷却至室温，加入3N的盐酸30mL，室温下搅拌1h。用硅藻土过滤反应液，所得滤液用乙酸乙酯100mL稀释，并用水(100mL*2)洗涤。有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经硅胶柱层析法纯化(石油醚：乙酸乙酯＝85：15)得到标题化合物(4.7g)。
MS m/z(ESI):218.1[M+H]+。
步骤5：N-(4-(2-溴乙酰基)-2,3-二氢-1H-茚-5-基)乙酰胺(中间体7-6)的合成
将中间体7-5(4.7g)溶于50mL醋酸中，加入33％的氢溴酸-乙酸溶液7.3g，室温下缓慢滴加溴素(2.85g)，然后室温下继续搅拌反应3h。反应结束后，将反应液倒入冰水中， 搅拌至大量固体析出，过滤，用石油醚洗涤滤饼，所得固体干燥得到标题化合物(5.0g)。
MS m/z(ESI):296.0[M+H]+。
步骤6：1-(5-氨基-2,3-二氢-1H-茚-4-基)-2-氯乙-1-酮(中间体7-7)的合成
将中间体7-6(5.0g)溶于30mL乙醇中，加入6N浓盐酸35mL，反应体系在80℃下搅拌2h。反应体系冷却至室温，减压浓缩除去溶剂，残留物用二氯甲烷100mL溶解，用饱和碳酸氢钠水溶液调节pH至7左右，分去有机相，水相再用二氯甲烷(50mL*2)萃取。合并有机相并用无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经硅胶柱层析法纯化(石油醚：乙酸乙酯＝75：25)得到标题化合物(840mg)。
MS m/z(ESI):210.0[M+H]+。
步骤7:(S)-15-(氯甲基)-8-乙基-8-羟基-1,2,3,8,11,14-六氢-9H,12H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12-二酮(中间体7-8)的合成
将中间体7-7(100mg)和中间体1-3(125.55mg)溶于甲苯(5mL)中，向其中加入对甲基苯磺酸吡啶盐(5.99mg)。反应液在90℃搅拌18h。待反应冷却至室温，加入乙醇(1mL)，反应液于25℃搅拌0.5h。反应液过滤，滤饼用石油醚(2mL*2)洗涤得到标题化合物(180mg)。
MS m/z(ESI):437.0[M+H]+。
步骤8：(S)-15-(氨基甲基)-8-乙基-8-羟基-1,2,3,8,11,14-六氢-9H,12H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12-二酮(中间体7-9)的合成
将中间体7-8(50mg)溶于甲醇(1mL)和N,N-二甲基甲酰胺(1mL)的混合溶液中，向其中加入乌洛托品(483.13mg)。反应液在50℃搅拌4h。反应结束后，反应液冷却至室温，加入浓盐酸(0.5mL)搅拌0.5h，然后减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例20％-40％,洗脱时间12分钟)得到标题化合物(22.0mg)。
MS m/z(ESI):418.2[M+H]+。
步骤9：(S)-N-((8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-1H,11H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物7)的合成
将中间体7-9(15mg)和羟基乙酸(10.93mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(27.32mg)和二异丙基乙胺(4.64mg)，反应液于25℃搅拌1h。反应结束后，反应液过滤，滤液减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例30％-50％,洗脱时间12分钟)得到标题化合物(9.0mg)。
MS m/z(ESI):476.2[M+H]+。
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),3.09(t,J＝7.4Hz,2H),2.23-2.15(m,2H),1.95-1.80(m,2H),0.88(t,J＝7.3Hz,3H)。
实施例9、(S)-N-((4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-1-羟基环丙烷-1-甲酰胺(化合物9)
将中间体9-1(6.00mg,14.66μmol,可根据专利文献WO2020219287报道方法合成)和中间体9-2(4.49mg,43.97μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(8.36mg,21.98μmol)和N,N-二异丙基乙胺(5.68mg，43.97μmol)，反应液于25℃搅拌1h。LC-MS检测反应完毕。反应液过滤，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例6％-36％，洗脱时间12分钟)得到标题化合物(3.00mg)。
MS m/z(ESI):494.2[M+H]+。
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),5.52(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)。
实施例10、(S)-N-((8-氯-4-乙基-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-1-羟基环丙烷-1-甲酰胺(化合物10)
将中间体6-4(6.24mg,14.66μmol)和中间体9-2(4.49mg,43.97μmol)溶于N,N-二甲基甲酰胺(1mL)中，向其中加入HATU(8.36mg,21.98μmol)和DIEA(5.68mg,43.97μmol)，反应液25℃搅拌1h。LC-MS检测反应完毕。反应液过滤，减压浓缩至干。粗品经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例16％-46％，洗脱时间12分钟)得到标题化合物(2.20mg)。
MS m/z(ESI):510.1[M+H]+。
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)。
实施例11、N-(((S)-8-氯-4-乙基-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-环丙基-2-羟基乙酰胺(化合物11)
将中间体6-4(6.0mg,14.09μmol)和中间体11-1(8.18mg,70.45μmol)溶于N,N-二甲基甲酰胺(0.5mL)中，向其中加入HATU(8.04mg,21.13μmol)和DIEA(5.46mg,42.27μmol)，反应液25℃搅拌1h。LC-MS检测反应完毕。反应液过滤，减压浓缩至干，粗品经制备高效液相色谱纯化(YMC-Actus Triart C18柱5um，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例16％-46％，洗脱时间12分钟)得到标题化合物(3.0mg)。
MS m/z(ESI):524.1[M+H]+。
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)。
实施例12、(S)-2-氨基-N-((7-乙基-15-氟-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)乙酰胺(化合物12)
步骤1：1-(2-氟-3,4-二甲氧苯基)乙烷-1-酮(中间体12-2)的合成
将中间体12-1(25.0g)溶于1,2-二氯乙烷(DCE，250mL)中，反应液冷却至0℃，向其中缓慢加入三氯化铝(64.04g)，再向反应液中滴加乙酰氯(64.04g)。反应液于氮气氛围下0℃搅拌2h。反应结束后，向其中加入水(300mL)，使用乙酸乙酯(150mL*3次)萃取，合并有机相，用无水硫酸钠干燥。过滤后，有机相经减压浓缩。残留物以硅胶柱层析法纯化(120g快速硅胶柱,梯度0～50％石油醚/乙酸乙酯，流速70mL/min)，得到标题化合物(21.0g)。
MS m/z(ESI):199.0[M+H]+。
步骤2：1-(2-氟-3,4-二羟基苯基)乙烷-1-酮(中间体12-3)的合成
将中间体12-2(15.00g)溶于无水二氯甲烷(DCM，150mL)中，反应液冷却至-78℃，向缓慢向其中滴加三溴化硼(56.88g)，反应液于氮气氛围下-78℃搅拌2h，再升温至0℃反应4h。反应结束后，反应液缓慢滴入冰水中淬灭，淬灭完毕后，使用乙酸乙酯(150mL*3次)萃取，合并有机相，用无水硫酸钠干燥。过滤后，有机相经减压浓缩。残留物以硅胶柱层析法纯化(120g快速硅胶柱,梯度0～50％石油醚/乙酸乙酯，流速70mL/min)，得到标题化合物(8.50g)。
MS m/z(ESI):171.0[M+H]+。
步骤3：1-(4-氟苯并[d][1,3]二氧杂环戊烯-5-基)乙烷-1-酮(中间体12-4)的合成
将中间体12-3(4.0g)溶于无水N,N-二甲基甲酰胺(40mL)中，向其中加入碳酸铯(11.49g)和1,2-二碘甲烷(18.89g)。反应液于氮气氛围下100℃搅拌8min。反应结束后，反应液缓慢倒入水中，使用乙酸乙酯(50mL*3次)萃取，合并有机相，用无水硫酸钠干燥。过滤后，有机相经减压浓缩。残留物以硅胶柱层析法纯化(24g快速硅胶柱,梯度0～15％石油醚/乙酸乙酯，流速60mL/min)得到标题化合物(2.0g)。
MS m/z(ESI):183.0[M+H]+。
步骤4：1-(4-氟-6-硝基苯并[d][1,3]二氧杂环戊烯-5-基)乙烷-1-酮(中间体12-5)的合成
将中间体12-4(2.0g)溶于无水二氯甲烷(15mL)中，向其中加浓硫酸(5.38g,98％质量分数)，反应液降温至0℃。再向反应液缓慢滴加浓硝酸(3.46g,68％质量分数)。反应液于25℃搅拌2h。反应结束后，将反应液缓慢滴入冰水(50mL)中，再加入乙酸乙酯(50mL)，有机相用水(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩至干。残留物以硅胶柱层析法纯化(24g快速硅胶柱,梯度0～40％石油醚/乙酸乙酯，流速60mL/min)得到标题化合物(1.8g)。
1H NMR(400MHz,氘代氯仿)δ＝7.51(s,1H),6.26(s,2H),2.63(s,3H)。
步骤5：1-(6-氨基-4-氟苯并[d][1,3]二氧杂环戊烯-5-基)乙烷-1-酮(中间体12-6)的合成
将中间体12-5(1.8g)溶于无水甲醇(18mL)和水(9mL)中，向其中加入氯化铵(635.83mg)和铁粉(2.21mg)。氮气氛围下反应液于80℃搅拌2h。反应结束后，待反应冷却至室温。反应液过滤，滤液用乙酸乙酯(50mL)稀释，有机相用水(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩至干，得到标题化合物(1.5g)。
MS m/z(ESI):198.0[M+H]+。
步骤6：N-(6-乙酰基-7-氟苯并[d][1,3]二氧杂环戊烯-5-基)乙酰胺(中间体12-7)的合成
将中间体12-6(500.0mg)溶于无水二氯甲烷(5mL)中，向其中加入吡啶(601.79mg)，氮气氛围下向反应中滴加乙酰氯(398.14mg)。氮气氛围下反应液于25℃搅拌1.5h。反应结束后，有机相经减压浓缩至干。残留物以硅胶柱层析法纯化(12g快速硅胶柱,梯度0～40％石油醚/乙酸乙酯，流速60mL/min)得到标题化合物(380.0mg)。
1H NMR(400MHz,氘代氯仿)δ＝11.72(s,1H),8.18(d,J＝1.0Hz,1H),6.10(s,2H),2.64(d,J＝8.4Hz,3H),2.22(s,3H)。
步骤7：N-(6-(2-溴乙酰基)-7-氟苯并[d][1,3]二氧杂环戊烯-5-基)乙酰胺(中间体12-8)的合 成
将中间体12-7(380.0mg)溶于乙酸(3mL)中，向其中加入溴化氢的乙酸溶液(1.95g,33％含量)，再向反应液中缓慢滴加液溴(256.42mg)。反应液在25℃搅拌1h。反应结束后，反应液缓慢倒入冰水中搅拌0.5h，过滤，滤饼用水(20mL*2)洗涤，滤饼干燥，得到标题化合物(400.0mg)。
MS m/z(ESI):317.8[M+H]+。
步骤8：1-(6-氨基-4-氟苯并[d][1,3]二氧杂环戊烯-5-基)-2-氯乙烷-1-酮(中间体12-9)的合成
将中间体12-8(400.0mg)溶于无水乙醇(2mL)和浓盐酸(2mL)中，反应液于60℃搅拌3h。反应结束后，待反应冷却至室温，依次缓慢加入冰水(20mL)，用饱和碳酸氢钠调节至pH＝8，再加入乙酸乙酯(40mL),有机相用水(20mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩至干，得到标题化合物(220.0mg)。
MS m/z(ESI):232.0[M+H]+。
步骤9：(S)-14-(氯甲基)-7-乙基-15-氟-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体12-10)的合成
将中间体12-9(200.0mg)和中间体1-3(227.32mg)溶于甲苯(3mL)中，向其中加入对甲基苯磺酸吡啶盐(21.70mg)。反应液在90℃搅拌16h。反应结束后，待反应冷却至室温，加入乙醇(1mL),反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(5mL*2)洗涤，得到标题化合物(320.0mg)。
MS m/z(ESI):459.0[M+H]+。
步骤10：(S)-14-(氨基甲基)-7-乙基-15-氟-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体12-11)的合成
将中间体12-10(260.00mg)溶于无水甲醇(1mL)和无水N,N-二甲酰甲酰胺(1mL)中，向其中加入乌洛托品(238.32mg)。反应液在50℃搅拌3h。反应结束后，待反应冷却至室温，减压浓缩至干，经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例0％-30％,洗脱时间14分钟)得到标题化合物(85.0mg)。
MS m/z(ESI):440.0[M+H]+。
1H 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)。
步骤11：(S)-(2-(((7-乙基-15-氟-7-羟基-8,11-二氧代-8,10,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙基)氨基甲酸叔丁酯(中间体12-12)的合成
将中间体12-11(7mg)和2-((叔-丁氧羰基)氨基)乙酸(5.58mg)溶于无水N,N-二甲基甲酰胺(1mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(12.11mg)和二异丙基乙胺(2.06mg)，反应液于25℃搅拌1h。反应结束后，将反应液浓缩至干，得到标题化合物(8.00mg)。
MS m/z(ESI):597.3[M+H]+。
步骤12：(S)-2-氨基-N-((7-乙基-15-氟-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)乙酰胺(化合物12)的合成
将中间体12-12(6mg)溶于二氯甲烷(0.5mL)，向其中加入三氟乙酸(902.27mg)，反应液于25℃搅拌1h。反应结束后，反应液减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例2％-32％,洗脱时间12分钟)，得到标题化合物(1.3mg)。
MS m/z(ESI):497.1[M+H]+。
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)。
实施例13、2-环丙基-N-(((S)-8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-1H,11H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物13)
将中间体7-9(10mg)和中间体11-1(8.34mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(13.66mg)和二异丙基乙胺(3.10mg)，反应液于25℃搅拌1h。反应结束后，反应液过滤，滤液减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例34％-54％,洗脱时间12分钟)得到标题化合物(0.8mg)。
MS m/z(ESI):516.2[M+H]+。
1H 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)
实施例14-1、2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物14)
步骤1：1-(6-硝基苯并[d][1,3]二氧杂环戊烯-5-基)乙酮(中间体14-2)的合成
将中间体14-1(10.0g,60.92mmol)溶于硝基甲烷(100mL)中，向其中缓慢加入硝酸(35.43g,365.50mmol,65％纯度)，反应液于25℃搅拌2.5h。反应结束后，向反应液中缓慢加入饱和碳酸氢钠溶液，调节pH至7-8，再向其中加入二氯甲烷(100mL)，有机相用水 (50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。经制备薄层色谱法纯化(石油醚：乙酸乙酯＝1:2)得到标题化合物(5g)。
MS m/z(ESI):210.0[M+H]+。
步骤2：1-(6-氨基苯并[d][1,3]二氧杂环戊烯-5-基)乙酮(中间体14-3)的合成
将中间体14-2(2.37g,11.33mmol)溶于无水乙醇(25mL)中，向其中加入钯碳(0.2g,10％纯度)，反应液于氢气保护下25℃搅拌16h。反应结束后，反应液过滤，滤饼用乙酸乙酯洗涤2遍，滤液经减压浓缩至干得到标题化合物(1.6g)。
MS m/z(ESI):180.1[M+H]+。
步骤3：N-(6-乙酰基苯并[d][1,3]二氧杂环戊烯-5-基)乙酰胺(中间体14-4)的合成
将中间体14-3(1.0g,5.58mmol)溶于二氯甲烷(10mL)中，反应液冷却至0℃向其中加入N,N-二异丙基乙胺(DIEA)(1.08g,8.37mmol)和乙酰氯(569.55mg,7.26mmol)。反应液在25℃搅拌1.5h。反应结束后，反应液经减压浓缩至干得到标题化合物(1.23g)。
MS m/z(ESI):222.1[M+H]+。
步骤4：N-(6-(2-溴乙酰基)苯并[d][1,3]二氧杂环戊烯-5-基)乙酰胺(中间体14-5)的合成
将中间体14-4(1.23g,5.00mmol)溶于乙酸(12mL)中，向其中加入溴化氢的乙酸溶液(1.84g,7.51mmol,33％纯度)，再缓慢向其中加入液溴(959.69mg,6.01mmol)反应液于25℃搅拌1h。反应结束后，反应液倒入冰水中搅拌10min。过滤，滤饼用水洗涤2遍，减压浓缩至干，向残余物中加入乙酸乙酯(2mL)和石油醚(10mL)，反应液在25℃下搅拌0.5h。反应液过滤，滤饼经干燥得到标题化合物(500mg)。
MS m/z(ESI):300.0[M+H]+。
步骤5：1-(6-氨基苯并[d][1,3]二氧杂环戊烯-5-基)-2-氯乙酮(中间体14-6)的合成
将中间体14-5(0.2g,666.43μmol)溶于无水乙醇(1mL)和浓盐酸(1mL)中，反应液于60℃搅拌16h。反应结束后，待反应冷却至室温，依次缓慢加入冰水(10mL)和饱和碳酸氢钠(10mL)，再加入二氯甲烷(50mL)，有机相用水(20mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。经制备薄层色谱法(石油醚：乙酸乙酯＝6:1)得到标题化合物(160mg)。
MS m/z(ESI):214.0[M+H]+。
步骤6：(S)-14-(溴甲基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体14-7)的合成
将中间体14-6(50mg,234.06μmol)和中间体1-3(61.62mg,234.06μmol)溶于甲苯(1mL)中，向其中加入对甲基苯磺酸吡啶盐(5.88mg,23.41μmol)。反应液在90℃搅拌16h。反应结束后，待反应冷却至室温，加入乙醇(1mL)，反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤后，干燥得到标题化合物(60mg)。
MS m/z(ESI):441.1[M+H]+。
步骤7：(S)-14-(氨基甲基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体14-8)的合成
将中间体14-7(55.00mg,124.76μmol)溶于乙醇(1mL)中，向其中加入乌洛托品(52.47mg,374.29μmol)。反应液在80℃搅拌1.5h。反应结束后，待反应冷却至室温，减压浓缩至干，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和甲醇的极性递减的混合物作为洗脱液；甲醇梯度比例0％-27％,洗脱时间12分钟)得到标题化合物(10mg)。
MS m/z(ESI):422.1[M+H]+。
步骤8：2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物14)的合成
将中间体14-8(10.00mg,20.17μmol)和中间体11-1(23.42mg,201.71μmol)溶于无水 N,N-二甲基甲酰胺(0.5mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)(11.50mg,30.26μmol)和N,N-二异丙基乙胺(7.82mg，60.51μmol)，反应液于25℃搅拌1.5h。反应结束后，反应液过滤，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例4％-44％,洗脱时间9分钟)得到标题化合物(3mg)。
MS m/z(ESI):520.1[M+H]+。
1H 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)。
步骤9：2-环丙基-2-羟基乙酸苄酯(中间体14-9-P1/P2)的制备
对中间体14-9进行拆分制备得到异构体14-9-P1和14-9-P2。取中间体14-9(1.3g)经超临界流体色谱制备(DAICEL CHIRALPAK AD柱，10μm二氧化硅，30mm直径，250mm长度；使用乙醇(含有0.1％氨水)作为洗脱液)得到中间体14-9-P1(600mg)和中间体14-9-P2(600mg)。
通过以下手性超临界流体色谱条件对上述两个异构体进一步分析。
中间体14-9-P1：
在上述手性超临界流体色谱条件下，其保留时间为2.990分钟；
1H 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)。
中间体14-9-P2：
在上述手性超临界流体色谱条件下，其保留时间为2.661分钟；
1H 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)。
步骤10：2-环丙基-2-羟基乙酸(中间体14-10-P1/P2)的合成
氢气氛围下，将中间体14-9-P1(500mg)加入到甲醇(15mL)中,向反应液中加入湿钯碳(10mg,10％)，反应液在氢气氛围下25℃搅拌16小时。反应结束后，反应物经过滤，滤液经减压浓缩，得中间体14-10-P1(273mg)。
1H NMR(400MHz,METHANOL-d4)δ3.63(d,J＝7.2Hz,1H),1.21-1.09(m,1H),0.61-0.40(m,4H)。
氢气氛围下，将中间体14-9-P2(500mg)加入到甲醇(15mL)中,向反应液中加入湿钯碳(10mg,10％)，反应液在氢气氛围下25℃搅拌16小时。反应结束后，反应物经过滤，滤液经减压浓缩，得中间体14-10-P2(279mg)。
1H NMR(400MHz,METHANOL-d4)δ3.63(d,J＝7.2Hz,1H),1.19-1.08(m,1H),0.60-0.39(m,4H)。
步骤11：2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物14-P1/P2)的合成
将中间体14-8(40.00mg)和中间体14-10-P1(28.11mg)溶于无水N,N-二甲基甲酰胺(1mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)(46.02mg)和N,N-二异丙基乙胺(31.28mg)，反应液于25℃搅拌1.5h。反应结束后，反应液经制备高效液相色谱纯化(Boston Green ODS C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例16％-46％,洗脱时间12分钟)，得化合物14-P1(22.00mg)。
MS m/z(ESI):520.1[M+H]+。
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)。
将中间体14-8(10.00mg)和中间体14-10-P2(8.27mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(18.05mg)和N,N-二异丙基乙胺(6.13mg)，反应液于25℃搅拌1.5h。反应结束后，反应液直接经制备高效液相色谱纯化(Boston Green ODS C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例16％-46％,洗脱时间12分钟)，得化合物14-P2(8.00mg)。
MS m/z(ESI):520.1[M+H]+。
1H 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)。
通过以下手性超临界流体色谱分析方法分别对两个异构体进一步分析。

化合物14-P1：
在上述手性超临界流体色谱条件下，其保留时间为3.673分钟；
化合物14-P2：
在上述手性超临界流体色谱条件下，其保留时间为3.735分钟。
实施例14-2：(S)-2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物14-S)
步骤1：(S)-4-苄基-3-(2-环丙基乙酰基)噁唑烷-2-酮(中间体3)的合成
称取起始原料1(150.0g)，4-二甲氨基吡啶(160.15g)，和起始原料2(221.2g)，溶解于1500mL二氯甲烷中，并在室温下搅拌15min。称取1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI,359.0g)，分批加入反应反应液，加毕，在室温下搅拌约5h。反应结束后，向反应液中加入二氯甲烷(1500mL)稀释，然后依次用水(500mL)洗涤2次，2N HCl(500mL)洗涤一次，饱和碳酸氢钠溶液(500mL)洗涤一次，饱和食盐水溶液(500mL)洗涤一次，有机相用无水硫酸钠干燥，过滤后滤液减压浓缩至干，得标题化合物(302g)。
1H 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).
步骤2：(S)-4-苄基-3-((S)-2-环丙基-2-羟基乙酰基)噁唑烷-2-酮(中间体5)的合成
称取中间体3(200g)，溶解于2000mL无水四氢呋喃中，在氮气保护下，-78℃下搅拌15min。随后，向反应液中滴加二(三甲基硅基)氨基钠(443.5mL,2M的四氢呋喃溶液)。滴加结束，反应液在-78℃下搅拌30min。将中间体4(201.5g)溶解于700mL四氢呋喃中溶清，并缓慢滴加入反应反应液。滴加结束，-78℃下搅拌2h。随后，向反应液中加入冰醋酸220mL，淬灭反应。滴毕后逐渐升温至室温，并向反应液中加入2N HCl 600mL，室温(20-25℃)搅拌10h。随后，反应液减压浓缩，并向残留物中加入乙酸乙酯(1000mL)和水(200mL)，搅拌20min。分离水相用乙酸乙酯(500mL x2)萃取两次，合并有机相，依次用饱和NaHCO3溶液400mL，饱和Na2S2O3溶液400mL和饱和食盐水各洗涤两次，所得有机相用无水硫酸钠干燥，过滤后，滤液减压浓缩至干，残留物用硅胶柱色谱纯化(石油醚:乙酸乙酯＝1/30)得标题化合物(128.7g)。
1H 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).
步骤3：(S)-4-苄基-3-((S)-2-((叔丁基二甲基硅基)氧基)-2-环丙基乙酰基)噁唑烷-2-酮(中间体6)的合成
称取中间体5(128.7g)，溶解于1300mL二氯甲烷中，并加入咪唑(56.17g)，在冰浴下搅拌15min。随后分批向反应液中加入TBSCl(107.3g)，并在室温下搅拌3h。向反应液中加入200mL 2N HCl搅拌20min，分液。有机相依次用饱和NaHCO3溶液200mL和饱和食盐水各洗涤两次。有机相用无水硫酸钠干燥，过滤后，滤液减压浓缩至干，残留物用硅胶柱色谱纯化(石油醚:乙酸乙酯＝80：1)得标题化合物(160g)。
1H 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).
步骤4：(S)-2-((叔丁基二甲基硅基)氧基)-2-环丙基乙酸苄酯(中间体7)的合成
称取苄醇(62.18g)，溶解于500mL四氢呋喃中，并在-25℃下搅拌。量取正丁基锂(213.6mL,2.5M的四氢呋喃溶液)，缓慢滴加入反应液。滴毕，在-25℃下搅拌1h。称取中间体6(160g)，溶于320mL四氢呋喃中，并在-25℃下缓慢滴加入反应液中。滴毕，在-15℃下搅拌3h。向反应液中加入饱和NH4Cl(200mL)溶液淬灭反应。随后减压浓缩，并向反应液中加入甲基叔丁基醚400mL和水(150mL),搅拌30min，分液，水相用甲基叔丁基醚(200mL x2)萃取两次，合并有机相，饱和食盐水(250mL)洗涤一次，无水硫酸钠干燥，过滤后，滤液经减压浓缩，残留物经硅胶柱色谱纯化(石油醚:乙酸乙酯＝100:1)，得标题化合物(125g)。
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).
步骤5：(S)-2-环丙基-2-羟基乙酸苄酯(中间体8)的合成
称取中间体7(125g)，溶于1200mL四氢呋喃中，并加入冰醋酸(35.1g)，在室温下搅拌5min。随后向反应液中加入四丁基氟化铵(TBAF,585mL,1M的四氢呋喃溶液)，并将反应液置于45℃下反应4h。反应液减压浓缩除去四氢呋喃(600mL)，并向残余物中加入300mL水和400mL甲基叔丁基醚，搅拌20min，分液，水相用甲基叔丁基醚(200mL)萃取两次，合并有机相，依次用饱和NaHCO3溶液200mL和饱和食盐水各洗涤两次，有机相用无水硫酸钠干燥，过滤后，滤液减压浓缩至干，残留物硅胶柱色谱纯化(石油醚:乙酸乙酯＝60:1)，得标题化合物(68.9g)。
1H 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).
通过以下手性超临界流体色谱条件对中间体8进一步分析。
中间体8在上述手性超临界流体色谱条件下，其保留时间为3.013分钟；与实施例14-1中间体14-9-P1在相同色谱分析条件下的保留时间(2.990分钟)基本一致，中间体8与中间体14-9-P1构型相同，两者为相同的化合物。
步骤6：(S)-2-环丙基-2-羟基乙酸(中间体9)的合成
将中间体8(5g)溶于甲醇(80mL)中，向反应液中加入湿钯碳(10％质量含量,0.7g)，并在氢气氛围下，25℃搅拌16h。反应结束后，过滤反应液，滤液减压浓缩至干后，得标题化合物(2.4g)。
1H NMR(400MHz,METHANOL-d4)δ＝3.63(d,J＝7.3Hz,1H),1.20-1.09(m,1H),0.61-0.39(m,4H)。
步骤7：(S)-2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物14-S)的合成
将中间体14-8(90mg)和中间体9(49.60mg)溶于无水N,N-二甲基甲酰胺(1mL)，向其中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟膦盐(121.81mg)和N,N-二异丙基乙胺(82.81mg)，反应液于25℃搅拌16h。反应结束后，反应液经高效液相色谱法纯化(柱子:Boston Green ODS 150*30mm*5um；流动相:[A:水(0.225％甲酸)，B:乙腈]；B％:20％-50％,12min)得标题化合物(21mg)。
MS m/z(ESI):520.0[M+H]+。
1H 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)。
通过以下手性超临界流体色谱条件对化合物14-S进一步分析。
化合物14-S在上述手性超临界流体色谱条件下，其保留时间为3.654分钟；与实施例14-1制备得到的化合物14-P1在相同色谱分析条件下的保留时间(3.673分钟)基本一致。因此判断化合物14-S与实施例14-1制备得到的化合物14-P1构型相同，两者为相同的化合物。
实施例15、(S)-N-((9-溴-4-乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b][1,7]萘啶-11-基)甲基)-2-羟基乙酰胺(化合物15)
步骤1：1-(5-氨基-2-溴吡啶-4-基)乙酮(中间体15-2)的合成
将中间体15-1(500mg,3.67mmol)溶于无水四氢呋喃(90mL)中，向其中加入碳酸氢钠(617.04mg,7.34mmol)和2-吡咯烷酮三溴化氢盐(1.64g，5.03mmol)，反应液于25℃搅拌8h。反应结束后，反应液过滤，经制备薄层色谱法(二氧化硅，石油醚：乙酸乙酯＝20:1)得到标题化合物(300mg)。
MS m/z(ESI):214.9[M+H]+。
步骤2：N-(4-乙酰基-6-溴吡啶-3-基)乙酰胺(中间体15-3)的合成
将中间体15-2(150mg,697.52mmol)溶于二氯甲烷(2mL)中，反应液冷却至0℃向其中加入N,N-二异丙基乙胺(180.30mg,1.40mmol)和乙酰氯(109.51mg,1.40mmol)。反应液在25℃搅拌3h。反应结束后，反应液减压浓缩至干，经制备薄层色谱法(二氧化硅，石油醚：乙酸乙酯＝3:1)得到标题化合物(100mg)。
MS m/z(ESI):257.0[M+H]+。
步骤3：1-(5-氨基-2-溴吡啶-4-基)-2-溴乙酮(中间体15-4)的合成
将中间体15-3(95.00mg,273.45μmol)溶于乙酸(2mL)中，向其中加入溴化氢的乙酸溶液(100.57g,410.18μmol,33％纯度)，再缓慢向其中加入液溴(48.07mg,300.80μmol)反应液于25℃搅拌1h。反应结束后，反应液减压浓缩至干，经制备薄层色谱法(二氧化硅，石油醚：乙酸乙酯＝3:1)得到标题化合物(45mg)。
MS m/z(ESI):292.9[M+H]+。
步骤4：1-(5-氨基-2-溴吡啶-4-基)-2-氯乙酮(中间体15-5)的合成
将中间体15-4(50.00mg,170.10μmol)溶于浓盐酸(1mL)中，反应液于60℃搅拌16h。反应结束后，待反应冷却至室温，依次缓慢加入冰水(10mL)和饱和碳酸氢钠(10mL)，再加入二氯甲烷(30mL),有机相用水(20mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩至干得到标题化合物(25mg)。
MS m/z(ESI):248.9[M+H]+。
步骤5：(S)-9-溴-11-(氯甲基)-4-乙基-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b][1,7]萘啶-3,14(4H)-二酮(中间体15-6)的合成
将中间体15-5(25mg,100.20μmol)和中间体1-3(26.38mg,100.20μmol)溶于甲苯(0.5mL)中，向其中加入对甲基苯磺酸吡啶盐(2.52mg,10.02μmol)。反应液在90℃搅拌16h。 反应结束后，待反应冷却至室温，加入乙醇(1mL),反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤得到标题化合物(30mg)。
MS m/z(ESI):475.9[M+H]+。
步骤6：(S)-11-(氨基甲基)-9-溴-4-乙基-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b][1,7]萘啶-3,14(4H)-二酮(中间体15-7)的合成
将中间体15-6(30mg,62.93μmol)溶于乙醇(1mL)中，向其中加入乌洛托品(17.64mg,125.86μmol)。反应液在80℃搅拌2h。反应结束后，待反应冷却至室温，减压浓缩至干，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和甲醇的极性递减的混合物作为洗脱液；甲醇梯度比例0％-25％,洗脱时间12分钟)得到标题化合物(4mg)。
MS m/z(ESI):457.0[M+H]+。
步骤7：(S)-N-((9-溴-4-乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b][1,7]萘啶-11-基)甲基)-2-羟基乙酰胺(化合物15)的合成
将中间体15-7(4mg,8.75μmol)和羟基乙酸(3.33mg,43.74μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(4.99mg,13.12μmol)和N,N-二异丙基乙胺(3.39mg，26.24μmol)，反应液于25℃搅拌2h。反应结束后，反应液过滤，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例8％-28％,洗脱时间12分钟)得到标题化合物(1.00mg)。
MS m/z(ESI):515.0[M+H]+。
1H 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,1H),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)。
实施例16、(S)-N-((9-氯-4-乙基-8,10-二氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-1-羟基环丙烷-1-甲酰胺(化合物16)
步骤1：1-(6-氨基-3-氯-2,4-二氟苯基)-2-氯乙烷-1-酮(中间体16-2)的合成
将三氯化硼(1M，6.11mL)溶于1,2-二氯乙烷(12mL)中，反应液降温至0℃，向其中加入反应物16-1(1g，6.11mmol)和氯乙腈(784.73mg，10.39mmol)，反应在0℃下搅拌10min，向其中加入三氯化铝(1.06g，7.95mmol)。之后反应液在氮气保护下升至25℃搅拌10min。反应液于氮气保护下90℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，依次缓慢加入冰水(25mL)和5％盐酸(5mL)于25℃下搅拌30min，再加入二氯甲烷(50mL), 有机相用水(2mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。减压浓缩至干，粗品经制备薄层色谱法(二氧化硅，石油醚：乙酸乙酯＝9:1)得到标题化合物(340mg)。
MS m/z(ESI):240.0[M+H]+。
步骤2：(S)-9-氯-11-(氯甲基)-4-乙基-8,10-二氟-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(中间体16-3)的合成
将中间体16-2(0.2g，833.22μmol)和中间体1-3(219.34mg，833.22μmol)溶于甲苯(4mL)中，向其中加入对甲基苯磺酸吡啶盐(20.94mg，83.32μmol)。反应液在100℃搅拌18h。LC-MS检测反应完毕。待反应冷却至室温，加入乙醇(1mL)，反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤得到标题化合物粗品(190mg)。
MS m/z(ESI):467.1[M+H]+。
步骤3：(S)-11-(氨基甲基)-9-氯-4-乙基-8,10-二氟-4-羟基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(16-4)的合成
将中间体16-3(50mg，107.01μmol)溶于乙醇(1mL)中，向其中加入乌洛托品(45.00mg，321.03μmol)。反应液在80℃搅拌1.5h。LC-MS检测反应完毕。待反应冷却至室温，减压浓缩至干，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例2％-32％，洗脱时间12分钟)得到标题化合物(1.22mg)。
MS m/z(ESI):448.0[M+H]+。
1H 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)。
步骤4：(S)-N-((9-氯-4-乙基-8,10-二氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-1-羟基环丙烷-1-甲酰胺(化合物16)的合成
将化合物16-4(5.75mg,12.84μmol)和中间体9-2(3.93mg,38.52μmol)溶于N,N-二甲基甲酰胺(0.5mL)中，向其中加入HATU(7.32mg,19.26μmol)和二异丙基乙基胺(4.98mg,38.52μmol)，反应液30℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例14％-34％,洗脱时间12分钟)得到标题化合物(3mg)。
MS m/z(ESI):532.1[M+H]+。
1H 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)。
实施例17、(S)-N-((8-氯-4-乙基-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基-2-甲基丙酰胺(化合物17)
将中间体6-4(5mg,11.74μmol)和中间体17-1(2.44mg,23.48μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(8.95mg,23.48μmol)和N,N-二异丙基乙胺(1.19mg,11.74μmol)，反应液于25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱 纯化(Waters Xbridge C18柱5μm二氧化硅，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例22％-42％,洗脱时间12分钟)得到标题化合物(1mg)。
MS m/z(ESI):512.1[M+H]+。
1H 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)。
实施例18、N-(((S)-8-氯-4-乙基-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基-3-甲基丁酰胺(化合物18)
将中间体6-4(5mg,11.74μmol)和中间体18-1(2.77mg,23.48μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(8.95mg,23.48μmol)和N,N-二异丙基乙胺(1.19mg,11.74μmol)，反应液于25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例25％-45％,洗脱时间12分钟)得到标题化合物(1.20mg)。
MS m/z(ESI):526.1[M+H]+。
1H 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)。
实施例19-1、2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基-2,2-d2)甲基)-2-羟基乙酰胺(化合物19,化合物19-P1/P2)
步骤1：1-(苯并[d][1,3]二氧杂环戊烯-5-基-2,2-d2)乙烷-1-酮(中间体19-2)的合成
将中间体19-1(3g)溶于无水DMF溶液(25mL)中，加入氘代二氯甲烷(8.57g)和碳酸钾(8.18g)，加毕，升温至90℃搅拌16h。随后将反应液加入到水(100mL)中，并用乙酸乙酯(200mL*2)萃取，合并有机相用饱和食盐水(100mL)洗涤，并用无水硫酸钠干燥。过滤后，滤液减压浓缩至干，残余物通过柱层析色谱法纯化(乙酸乙酯/石油醚＝5:1)，得到标题化合物(2.4g)。
MS m/z(ESI):167.1[M+H]+。
步骤2：1-(6-硝基苯并[d][1,3]二氧杂环戊烯-5-基-2,2-d2)乙烷-1-酮(中间体19-3)的合成
将中间体19-2(2.4g)溶于无水醋酸(10mL)中，在0℃滴加浓硝酸(32.50g,70％含量)，加完0℃搅拌10min。随后升温至室温，搅拌1h。反应完毕后，将反应液滴加入到冰水(200mL)中，过滤后，滤饼干燥得到标题化合物(1.9g)。
MS m/z(ESI):212.0[M+H]+。
1H NMR(400MHz,DMSO-d6)δ7.69(s,1H),7.30(s,1H),2.49(s,3H).
步骤3：N-(6-乙酰苯并[d][1,3]二氧杂环戊烯-5-基-2,2-d2)乙酰胺(中间体19-4)的合成
将中间体19-3(1.8g)溶于醋酸(25mL)中，加入醋酸酐(1.84g)和还原铁粉(4.76g)，室温搅拌1h。反应完毕后，过滤，滤液减压浓缩至干，残余物通过柱层析色谱法纯化(乙酸乙酯/石油醚＝5:1)，得到标题化合物(1.5g)。
MS m/z(ESI):224.1[M+H]+。
步骤4：N-(6-(2-溴乙酰基)苯并[d][1,3]二氧杂环戊烯-5-基-2,2-d2)乙酰胺(中间体19-5)的合成
将HBr的乙酸溶液(2.39g,33％含量)滴加到中间体19-4(1.45g)的无水醋酸(25mL)溶液中，然后再滴加Br2(1.07g)，滴加完，室温搅拌1h。反应完毕后，反应液减压浓缩至干，残余物加入到水(50mL)中，用乙酸乙酯(50mL*2)萃取，有机相用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤后，残余物通过柱层析色谱法纯化(乙酸乙酯/石油醚＝5:1)，得到标题化合物(1.3g,)。
MS m/z(ESI):302.1[M+H]+。
步骤5：1-(6-氨基苯并[d][1,3]二氧杂环戊烯-5-基-2,2-d2)-2-氯乙烷-1-酮(中间体19-6)的合成
将中间体19-5(1.2g)和浓盐酸(144.82mg)溶于乙醇(15mL)中，反应液在60℃下搅拌16h。反应完毕后，反应液减压浓缩至干，残余物通过高效液相色谱法纯化(YMC-Actus Triart C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.05％NH4HCO3)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例40％-50％)，得到标题化合物(577mg)。
MS m/z(ESI):216.0[M+H]+。
步骤6：(S)-14-(氯甲基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮-2,2-d2(中间体19-7)的合成
将中间体19-6(100.0mg)和中间体1-3(109.87mg)溶于甲苯(1mL)和乙酸(1mL)中，向其中加入对甲基苯磺酸吡啶盐(5.24mg)。反应液在100℃搅拌16h。反应结束后，待反应冷却至室温，直接将反应液经减压浓缩至干。加入乙醇(5mL),反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(5mL*2)洗涤得到标题化合物(100.0mg)。
MS m/z(ESI):443.0[M+H]+。
步骤7：(S)-14-(氨基甲基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮-2,2-d2(中间体19-8)的合成
将中间体19-7(100.00mg)溶于无水乙醇(1.5mL)和无水N,N-二甲酰甲酰胺(1.5mL)中，向其中加入乌洛托品(94.97mg)。反应液在50℃搅拌6h。反应结束后，反应液经减压浓缩至干，残余物经高效液相色谱法纯化(柱子:Boston Green ODS 150*30mm*5μm；流动相:[A:水(甲酸)，B:乙腈]；B％:0％-30％,12min)得到标题化合物(25.0mg)。
MS m/z(ESI):424.0[M+H]+。
步骤8：2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊 烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基-2,2-d2)甲基)-2-羟基乙酰胺(化合物19)的合成
将中间体19-8(7mg)和中间体11-1(5.76mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(12.57mg)和二异丙基乙胺(4.27mg)，反应液于25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例20％-50％,洗脱时间12分钟)，得到标题化合物(2.60mg)。
MS m/z(ESI):522.1[M+H]+。
1H 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)。
步骤9：2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基-2,2-d2)甲基)-2-羟基乙酰胺(化合物19-P1/P2)的合成
将中间体19-8(7mg)和中间体14-10-P1(5.76mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(12.57mg)和二异丙基乙胺(4.27mg)，反应液于25℃搅拌1h。反应结束后，反应液减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例15％-45％,洗脱时间12分钟)，得化合物19-P1(3.30mg)。
MS m/z(ESI):522.1[M+H]+。
1H NMR(400MHz,DMSO-d6)δ＝8.62(t,J＝6.0Hz,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)。
将中间体19-8(7mg)和中间体14-10-P2(5.76mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(12.57mg)和二异丙基乙胺(4.27mg)，反应液于25℃搅拌1h。反应结束后，反应液减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例15％-45％,洗脱时间12分钟)得化合物19-P2(4.0mg)。
MS m/z(ESI):522.1[M+H]+。
1H NMR(400MHz,DMSO-d6)δ＝8.62(t,J＝6.1Hz,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)。
通过以下手性超临界流体色谱分析方法分别对两个异构体进一步分析。

化合物19-P1：
在上述手性高效液相色谱条件下，其保留时间为2.877分钟；
化合物19-P2：
在上述手性高效液相色谱条件下，其保留时间为2.690分钟。
化合物19-P1构型确证(X射线单晶衍射方法)
单晶培养方法：称取10mg化合物19-P1样品置于1.5ml离心管中，加入300μl吡啶，超声溶清后用封口膜封口，用针头在封口膜上扎三个小孔，于20～30℃下缓慢挥发48h，得针状晶体。
所得单晶样品进行X-射线分析，测试结果见表1和图1。
表1化合物19-P1的单晶样品和晶体数据
通过上述X-射线晶体衍射实验，确定化合物19-P1的化学结构为：
实施例19-2、(S)-2-环丙基-N-(((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基-2,2-d2)甲基)-2-羟基乙酰胺(化合物19-S)的合成
将中间体19-8(2.4g)和中间体9(1645.5mg)溶于无水N,N-二甲基甲酰胺(25mL)，向其中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟膦盐(3.24g)和N,N-二异丙基乙胺(1465.11mg)，反应液于25℃搅拌3h。反应结束后，反应液经减压浓缩至干，向残余物中加入乙酸乙酯(100mL)搅拌16小时。过滤后，向滤饼中加入甲醇(50mL)搅拌16小时。过滤后，得标题化合物(1.6g)。
MS m/z(ESI):522.1[M+H]+。
1H 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)。
通过以下手性超临界流体色谱分析方法分别对化合物19-S进一步分析。
本实施例制得的化合物19-S在上述手性超临界流体色谱条件下，其保留时间为2.853分钟；与实施例19-1制得的化合物19-P1在相同色谱分析条件下的保留时间(2.877分钟)基本一致。因此判断化合物19-S和化合物19-P1构型相同，为相同的化合物。
实施例20、(S)-N-((8-乙基-8-羟基-9,12-二氧代-8,9,12,14-四氢-11H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物20)
步骤1：1-(5-氨基苯并呋喃-4-基)-2-氯乙酮(中间体20-2)的合成
将三氯化硼(1M，9.61mL)溶于二氯乙烷(14mL)中，反应液降温至0℃，向其中加入中间体20-1(1.6g，12.02mmol)和氯乙腈(1.36g，18.03mmol)，反应在0℃下搅拌10min，向其中加入三氯化铝(1.92g，14.42mmol)。反应液在氮气保护下升至25℃搅拌10min。反应液于氮气保护下90℃搅拌18h。反应结束后，反应液冷却至室温，依次缓慢加入冰水(50mL)和5％HCl(10mL)于25℃下搅拌30min，再加入二氯甲烷(60mL)，有机相用水(30mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。经制备薄层色谱法(石油醚：(乙酸乙酯+乙醇＝3:1)＝9:1)得到标题化合物(300mg)。
MS m/z(ESI):210.0[M+H]+。
1H NMR(400MHz,氯仿-d)δ＝7.72(d,J＝2.1Hz,1H),7.53(d,J＝9.0Hz,1H),6.89(d,J＝1.4Hz,1H),6.66(d,J＝9.0Hz,1H),4.78(s,2H)
步骤2：(S)-15-(氯甲基)-8-乙基-8-羟基-11,14-二氢-12H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12(8H)-二酮(中间体20-3)的合成
将中间体20-2(200mg，954.07μmol)和中间体1-3(251.15mg，954.07μmol)溶于无水甲苯(4mL)中，向其中加入对甲苯磺酸吡啶盐(23.98mg，95.41μmol)，反应液于氮气保护下90℃搅拌16h。反应结束后，反应液冷却至室温，反应液过滤，滤饼用乙醇(3mL*2)洗涤，得到标题化合物(300mg)。
MS m/z(ESI):437.1[M+H]+。
步骤3：(S)-15-(氨基甲基)-8-乙基-8-羟基-11,14-二氢-12H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12(8H)-二酮(中间体20-4)的合成
将中间体20-3(70mg,160.24μmol)溶于乙醇(0.5mL)和无水N,N-二甲基甲酰胺(0.5mL)中，向其中加入乌洛托品(89.85mg,640.96μmol)。反应液在25℃搅拌3h。反应结束后，反应液冷却至室温，减压浓缩至干，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和甲醇的极性递减的混合物作为洗脱液；甲醇梯度比例5％-25％,洗脱时间12分钟)得到标题化合物(17mg)。
MS m/z(ESI):418.1[M+H]+。
步骤4：(S)-N-((8-乙基-8-羟基-9,12-二氧代-8,9,12,14-四氢-11H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物20)
将中间体20-4(5.00mg,11.98μmol)和羟基乙酸(4.55mg,59.89μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(6.83mg,17.97μmol)和N,N-二异丙基乙胺(4.64mg，35.94μmol)，反应液于25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱 纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例15％-35％,洗脱时间12分钟)得到标题化合物(3mg)。
MS m/z(ESI):476.2[M+H]+。
1H 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＝5.3Hz,2H),3.88(d,J＝5.6Hz,2H),1.93-1.82(m,2H),0.89(t,J＝7.2Hz,3H)。
实施例21、(S)-N-((9-氯-4-乙基-8,10-二氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺(化合物21)
将化合物16-4(7mg,15.63μmol)和羟基乙酸(1.78mg,25.48μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(11.89mg,31.26μmol)和二异丙基乙胺(2.02mg,15.63μmol)，反应液于25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例20％-40％,洗脱时间12分钟)得到标题化合物(1mg)。
MS m/z(ESI):506.1[M+H]+。
1H 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)。
实施例22、(S)-N-((9-氯-4-乙基-8,10-二氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基-2-甲基丙酰胺(化合物22)
将化合物16-4(20mg,44.66μmol)和中间体17-1(13.95mg,133.98μmol)溶于N,N-二甲基甲酰胺(0.5mL)中，向其中加入HATU(25.47mg,66.99μmol)和N,N-二异丙基乙胺(17.32mg,133.98μmol)，反应液25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例20％-40％,洗脱时间12分钟)得到标题化合物(1.07mg)。
MS m/z(ESI):534.2[M+H]+。
1H 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)。
实施例23、N-(((S)-9-氯-4-乙基-8,10-二氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-环丙基-2-羟基乙酰胺(化合物23)
将化合物16-4(7.0mg,15.63μmol)和中间体11-1(9.08mg,78.16μmol)溶于N,N-二甲基甲酰胺(0.5mL)中，向其中加入HATU(8.92mg,23.45μmol)和二异丙基乙胺(6.06mg,46.89μmol)，反应液25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(YMC-Actus Triart C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例41％-61％,洗脱时间12分钟)得到标题化合物(7mg)。
MS m/z(ESI):546.2[M+H]+。
化合物23(7mg)经过制备超临界流体色谱分离纯化(柱子:DAICEL CHIRALCEL OD-H(250mm*30mm,5μm)；流动相:A:二氧化碳；B:乙醇；B％:50％；流速:80毫升/分钟)，得到化合物23-1(2.1mg,RT:5.106min)和化合物23-2(2.09mg,RT:5.641min)。
化合物23-1：
1H 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)
MS m/z(ESI):546.2[M+H]+。
化合物23-2：
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)
MS m/z(ESI):546.2[M+H]+。
实施例25、(R)-N-(((S)-9-氯-4-乙基-8,10-二氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基丙酰胺(化合物25)
将化合物16-4(6mg,13.40μmol)和中间体25-1(2.41mg,26.80μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(10.09mg,26.80μmol)和二异丙基乙胺(1.73mg,13.49μmol)，反应液于25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例20％-40％,洗脱时间12分钟)得到标 题化合物(1.80mg)。
MS m/z(ESI):520.1[M+H]+。
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)。
实施例26、(S)-N-(((S)-9-氯-4-乙基-8,10-二氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)-2-羟基丙酰胺(化合物26)
将化合物16-4(6mg,13.40μmol)和中间体26-1(2.41mg,26.80μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(10.09mg,26.80μmol)和N,N-二异丙基乙胺(1.73mg,13.49μmol)，反应液于25℃搅拌1h。反应结束后，反应液过滤，经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例20％-40％,洗脱时间12分钟)得到标题化合物(1.60mg)。
MS m/z(ESI):520.1[M+H]+。
1H 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),5.51(s,2H),5.45(s,2H),4.90(s,2H),4.07-3.96(m,1H),1.93-1.81(m,2H),1.21(d,J＝6.8Hz,3H),0.87(t,J＝7.3Hz,3H)。
实施例27、(S)-N-((4-氯-8-乙基-8-羟基-9,12-二氧代-8,9,12,14-四氢-11H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物27)
步骤1：(3-氯-2-羟基-5-硝基苯基)甲二醇(中间体27-2)的合成
将中间体27-1(6g)溶于乙酸(25mL)中，反应液降温到0℃向其中缓慢加入硝酸(9.64g)。反应液在25℃搅拌4h。反应结束后，反应液缓慢低滴加到冰水中，然后用乙酸乙酯(60 mL)萃取三次，有机相用无水硫酸钠干燥，过滤后，减压浓缩至干得到标题化合物(5.88g)。
1H NMR(400MHz,METHANOL-d4)δ＝8.16(d,J＝1.4,2.4Hz,1H),8.07-8.01(m,1H),5.68(s,1H)
步骤2：7-氯-5-硝基苯并呋喃-2-甲酸乙酯(中间体27-4)的合成
将中间体27-2(5.88g)，中间体27-3(7.69g)和碳酸钾(7.41g)溶于丙酮(60mL)中，反应液在70℃搅拌6h。反应结束后，向其中加入水(50ml)，然后用乙酸乙酯(50mL)萃取三次，有机相用无水硫酸钠干燥，过滤后，减压浓缩至干得到标题化合物(4.5g)。
1H NMR(400MHz,氯仿-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)
步骤3：7-氯-5-硝基苯并呋喃-2-羧酸(中间体27-5)的合成
将中间体27-4(4.5g)溶于甲醇(50mL)中，向其中缓慢滴加氢氧化钠水溶液(2g in 25ml H2O)，反应液在25℃搅拌3h。反应结束后，向其中加入水(120ml)，然后用乙酸乙酯(50mL)萃取两次。水相用稀盐酸调节pH值到1，再用乙酸乙酯(50mL)萃取三次，有机相用无水硫酸钠干燥，过滤后，减压浓缩至干得到标题化合物(4.0g)
1H NMR(400MHz,氯仿-d)δ＝8.45(d,J＝2.1Hz,1H),8.25(d,J＝2.1Hz,1H),7.58(s,1H)
步骤4：7-氯-5-硝基苯并呋喃(中间体27-6)的合成
将中间体27-5(4g)和氧化铜(1.05g)溶于喹啉(28mL)中，反应液通入氮气并在200℃搅拌0.5h。反应结束后，降温到0℃向其中缓慢滴加入稀盐酸(80ml)，然后加水(30ml)并用乙酸乙酯(30mL)萃取三次。有机相用无水硫酸钠干燥，过滤后，减压浓缩至干得到标题化合物(2.4g)
1H NMR(400MHz,氯仿-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)
步骤5：7-氯苯并呋喃-5-胺(中间体27-7)的合成
将中间体27-6(2.4g)和铁粉(1.55g)溶于甲醇(5mL)中，向其中滴加氯化铵水溶液(148.91mg,5ml)，反应液通入氮气并在80℃搅拌0.5h。反应结束后，降温到25℃向其中加入水(10ml)，并用乙酸乙酯(10mL)萃取三次。有机相用无水硫酸钠干燥，过滤后，减压浓缩至干得到标题化合物(1.2g)。
MS m/z(ESI):167.8[M+H]+。
步骤6：1-(5-氨基-7-氯苯并呋喃-4-基)-2-氯乙-1-酮(中间体27-8)的合成
将三氯化硼(671.17mg)溶于1.2二氯乙烷(8mL)中，反应液降温至0℃，向其中加入中间体27-7(1.2g)和氯乙腈(702.75mg)，反应在0℃下搅拌10min，向其中加入三氯化铝(1.24g)。之后反应液在氮气保护下升至25℃搅拌10min。反应液于氮气保护下90℃搅拌18h。反应结束后，待反应冷却至室温，依次缓慢加入冰水(5mL)和5％HCl(1mL)于25℃下搅拌30min，再加入二氯甲烷(4mL)，有机相用水(2mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。过滤后，减压浓缩至干，残余物经制备薄层色谱法(二氧化硅，石油醚：(乙酸乙酯与乙醇的3/1混合溶剂)＝9:1)得到标题化合物(500mg)。
MS m/z(ESI):243.8[M+H]+。
步骤7:(S)-4-氯-15-(氯甲基)-8-乙基-8-羟基-11,14-二氢-12H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12(8H)-二酮(中间体27-9)的合成
将中间体27-8(450mg)和中间体1-3(480.35mg)溶于甲苯(5mL)中，向其中加入对甲基苯磺酸吡啶盐(23.17mg)。反应液在90℃搅拌18h。反应结束后，待反应冷却至室温，加入乙醇(1mL)，反应液于25℃搅拌0.5h。反应液过滤，滤饼用石油醚(2mL*2)洗涤，干燥后得到标题化合物(500mg)。
MS m/z(ESI):471.0[M+H]+。
步骤8：(S)-15-(氨基甲基)-4-氯-8-乙基-8-羟基-11,14-二氢-12H-呋喃并[3,2-f]吡喃并 [3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12(8H)-二酮(中间体27-10)的合成
将中间体27-9(450mg)溶于甲醇(1mL)和N,N-二甲基甲酰胺(1mL)的混合溶液中，向其中加入乌洛托品(267.71mg)。反应液在50℃搅拌4h。反应结束后，反应液冷却至室温，加入浓盐酸(2mL)搅拌，然后减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例13％-43％,洗脱时间12分钟)得到标题化合物(60.0mg)。
MS m/z(ESI):451.9[M+H]+。
步骤9：(S)-N-((4-氯-8-乙基-8-羟基-9,12-二氧代-8,9,12,14-四氢-11H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物27)的合成
将中间体27-10(10mg)和2-羟基乙酸(5.05mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(16.83mg)和二异丙基乙胺(2.86mg)，反应液于25℃搅拌1h。反应结束后，反应液过滤，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例28％-48％,洗脱时间12分钟)得到标题化合物(6.0mg)。
MS m/z(ESI):510.1[M+H]+。
1H 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,1H),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)。
实施例28、N-(((S)-4-氯-8-乙基-8-羟基-9,12-二氧代-8,9,12,14-四氢-11H-呋喃并[3,2-f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-环丙基-2-羟基乙酰胺(化合物28)
将中间体27-10(10mg)和中间体11-1(8.34mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(13.65mg)和二异丙基乙胺(3.10mg)，反应液于25℃搅拌1h。反应结束后，反应液过滤，滤液减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例34％-54％,洗脱时间12分钟)得到标题化合物(6.2mg)。
MS m/z(ESI):550.1[M+H]+。
1H 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)。
实施例29、2-环丙基-N-(((S)-7-乙基-7-羟基-15-硝基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物29)
步骤1：(S)-14-(氯甲基)-7-乙基-7-羟基-15-硝基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体29-1)的合成
将中间体14-7(500.0mg)溶于硫酸(15mL)中，反应液冷却至0℃。再缓慢向其中加入硝酸(357.35g，70％纯度)，反应液于25℃搅拌1.5h，反应完毕后，依次缓慢加入冰水(10mL)再加入二氯甲烷(30mL)，有机相用水40mL(20mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。过滤后，滤液经减压浓缩至干得到标题化合物粗品(280.0mg)。
MS m/z(ESI):486.0[M+H]+。
步骤2：(S)-14-(氨基甲基)-7-乙基-7-羟基-15-硝基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体29-2)的合成
将中间体29-1(270.0mg)溶于甲醇(2mL)和四氢呋喃(2mL)中，向其中加入乌洛托品(233.73mg)。反应液在60℃搅拌16h。反应完毕后，待反应冷却至室温，减压浓缩至干，残余物经制备高效液相色谱纯化(YMC-Pack CN C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％FA)和甲醇的极性递减的混合物作为洗脱液；甲醇梯度比例9％-29％,洗脱时间12分钟)得到标题化合物(15.0mg)。
MS m/z(ESI):467.1[M+H]+。
步骤3：2-环丙基-N-(((S)-7-乙基-7-羟基-15-硝基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物29)的合成
将中间体29-2(12.00mg)和中间体11-1(14.94mg)溶于无水N,N-二甲基甲酰胺(1mL)，向其中加入HATU(14.67mg)和N,N-二异丙基乙胺(9.98mg)，反应液于25℃搅拌1.5h。反应完毕后，反应液过滤，滤液减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例15％-45％,洗脱时间12分钟)得到标题化合物(5.20mg)。
MS m/z(ESI):565.2[M+H]+。
1H 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)。
实施例30、N-(((S)-15-氯-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-环丙基-2-羟基乙酰胺(化合物30)
步骤1：2-氯-3,4-二羟基苯甲醛(中间体30-2)的合成
将中间体30-1(20.0g)溶于无水二氯甲烷(100mL)中，反应液冷却至0℃，向其中缓慢加入三溴化硼(87.41g)，反应液于25℃搅拌4h。反应结束后，反应液缓慢倒入冰水中，再加入乙酸乙酯(200mL),有机相用水(100mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥，过滤后，滤液减压浓缩至干，残余物经乙酸乙酯：石油醚＝1：4溶液搅洗，过滤后得到标题化合物(14g)。
MS m/z(ESI):173.1[M+H]+。
步骤2：4-氯苯并[d][1,3]二氧杂环戊烯-5-甲醛(中间体30-3)的合成
将中间体30-2(10.0g)溶于无水N,N-二甲基甲酰胺(100mL)中，向其中加入碳酸铯(28.32g)和二碘甲烷(23.28g)。反应液于100℃搅拌1h。反应完毕后，待反应冷却至室温，依次缓慢加入水(100mL)，再加入乙酸乙酯(150mL)，有机相用水(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥，过滤后，滤液经减压浓缩至干得到标题化合物(5.2g)。
MS m/z(ESI):185.4[M+H]+。
步骤3：1-(4-氯苯并[d][1,3]二氧杂环戊烯-5-基)乙-1-醇(中间体30-4)的合成
将中间体30-3(5.0g)溶于无水四氢呋喃(100mL)中，反应液冷却至-78℃，向其中缓慢加入甲基溴化镁(4.85g,3M)。氮气保护下反应液于25℃搅拌4h。反应结束后，依次缓慢加入水(50mL)，再加入乙酸乙酯(100mL),有机相用水(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥，过滤后，滤液经减压浓缩至干，残余物经制备柱层析色谱法纯化(石油醚：乙酸乙酯＝2:1)得到标题化合物(5.3g)。
1H NMR(400MHz,氯仿-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)
步骤4：1-(4-氯苯并[d][1,3]二氧杂环戊烯-5-基)乙-1-酮(中间体30-5)的合成
将中间体30-4(5.2g)溶于无水二氯甲烷(100mL)中，反应液冷却至0℃，向其中缓慢加 入戴斯-马丁试剂(DMP)(16.49g)。氮气保护下反应液于25℃搅拌2h。反应结束后，依次缓慢加入水(50mL)，再加入乙酸乙酯(100mL)，有机相用水(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。过滤后，滤液减压浓缩至干，残余物经制备柱层析色谱法纯化(石油醚：乙酸乙酯＝3:1)得到标题化合物(3.0g)。
MS m/z(ESI):199.2[M+H]+。
步骤5：1-(4-氯-6-硝基苯并[d][1,3]二氧杂环戊烯-5-基)乙-1-酮(中间体30-6)的合成
将中间体30-5(2.50g)溶于无水二氯甲烷(20mL)中，向其中缓慢加入浓硫酸(1.23g)和硝酸(5.67g)。反应液在25℃搅拌3h。反应结束后，反应液缓慢倒入冰水中，再加入乙酸乙酯(150mL)，有机相用水(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥，过滤后，滤液减压浓缩至干，残余物经制备柱层析色谱法(石油醚：乙酸乙酯＝2:1)得到标题化合物(1.8g)。
MS m/z(ESI):244.1[M+H]+。
步骤6：1-(6-氨基-4-氯苯并[d][1,3]二氧杂环戊烯-5-基)乙-1-酮(中间体30-7)的合成
将中间体30-6(0.80g)溶于无水甲醇(6mL)中，向其中加入Raney Ni(400.0mg)。反应液在氢气氛围下25℃搅拌16h。反应结束后，反应液经过滤，滤液减压浓缩至干得到标题化合物(510.0mg)。
MS m/z(ESI):214.2[M+H]+。
步骤7：N-(6-乙酰基-7-氯苯并[d][1,3]二氧杂环戊烯-5-基)乙酰胺(中间体30-8)的合成
将中间体30-7(260.0mg)溶于无水二氯甲烷(5mL)中，反应液冷却至0℃向其中加入N,N-二异丙基乙胺(235.95mg)和氯乙酰(143.32mg)。反应液在25℃搅拌1.5h。反应结束后，反应液减压浓缩至干，残余物经制备层析色谱法纯化(石油醚：乙酸乙酯＝5:1)得到标题化合物(140.0mg)。
MS m/z(ESI):256.0[M+H]+。
步骤8：N-(6-(2-溴乙酰基)-7-氯苯并[d][1,3]二氧杂环戊烯-5-基)乙酰胺(中间体30-9)的合成
将中间体30-8(110.00mg)溶于乙酸(2mL)中，向其中加入溴化氢的乙酸溶液(158.24mg,33％含量)，再缓慢向其中加入液溴(72.20mg)反应液于25℃搅拌1h。反应结束后，反应液缓慢倒入冰水中，再加入乙酸乙酯(30mL)，有机相用水(20mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩至干得到标题化合物(110.0mg)。
MS m/z(ESI):334.0[M+H]+。
步骤9：1-(6-氨基-4-氯苯并[d][1,3]二氧杂环戊烯-5-基)-2-氯乙-1-酮(中间体30-10)的合成
将中间体30-9(110.00mg)溶于无水乙醇(1mL)和浓盐酸(1mL)中，反应液于60℃搅拌16h。反应结束后，待反应冷却至室温，依次缓慢加入冰水(10mL)和饱和碳酸氢钠(10mL)，再加入二氯甲烷(30mL),有机相用水(20mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。过滤后，滤液经减压浓缩至干，残余物经制备薄层色谱法纯化(石油醚：乙酸乙酯＝3:1)得到标题化合物(75mg)。
MS m/z(ESI):248.0[M+H]+。
步骤10：(S)-15-氯-14-(氯甲基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体30-11)的合成
将中间体30-10(75.00mg)和中间体1-3(83.57mg)溶于甲苯(3mL)中，向其中加入对甲基苯磺酸吡啶盐(PPTS)(11.4mg)。反应液在90℃搅拌16h。反应结束后，待反应冷却至室温，加入乙醇(1mL)，反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤，干燥后得到标题化合物(75.0mg)。
MS m/z(ESI):475.1[M+H]+。
步骤11：(S)-14-(氨基甲基)-15-氯-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并 [4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(中间体30-12)的合成
将中间体30-11(70.00mg)溶于无水甲醇(2mL)和无水四氢呋喃(1mL)中，向其中加入乌洛托品(61.94mg)。反应液在80℃搅拌2h。反应结束后，待反应冷却至室温，减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例5％-25％,洗脱时间12分钟)得到标题化合物(16.0mg)。
MS m/z(ESI):456.1[M+H]+。
步骤12：N-(((S)-15-氯-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-环丙基-2-羟基乙酰胺(化合物30)的合成
将中间体30-12(5.00mg)和中间体11-1(6.37mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(6.26mg)和N,N-二异丙基乙胺(4.25mg)，反应液于25℃搅拌1h。反应结束后，反应液过滤，滤液减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例31％-51％,洗脱时间12分钟)得到标题化合物(2.30mg)。
MS m/z(ESI):554.2[M+H]+。
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)。
实施例31、(S)-N-((15-氯-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物31)
将中间体30-12(5mg)和羟基乙酸(4.17mg)溶于N,N-二甲基甲酰胺(0.5mL)中，向其中加入HATU(6.26mg)和N,N-二异丙基乙胺(4.25mg)，反应液25℃搅拌1h。反应结束后，反应液过滤，滤液减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例20％-40％,洗脱时间12分钟)得到标题化合物(2.50mg)。
MS m/z(ESI):514.2[M+H]+。
1H 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)
实施例33、(S)-N-((4-氯-8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-1H,11H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物33)
步骤1：7-氯-2,3-二氢-1H-茚-4-醇(中间体33-2)的合成
将中间体33-1(500mg)溶于无水乙腈(5mL)中，向其中加入NCS(547mg)，加毕，在25℃条件下搅拌2小时。反应结束后，向反应液加入水(10mL)，用乙酸乙酯萃取(10mL*3次)，饱和食盐水(30mL)洗涤有机层，有机层用无水硫酸钠干燥。过滤后，有机相经减压浓缩除去溶剂，残余物经硅胶柱层析法纯化(石油醚:乙酸乙酯＝100:1～10:1)得到标题化合物(600mg)。
MS m/z(ESI):169.0[M+H]+.
步骤2：7-氯-5-硝基-2,3-二氢-1H-茚-4-醇(中间体33-3)的合成
将中间体33-2(10g)溶于AcOH(50mL)和H2O(10mL)中，在0℃向其中加入HNO3(8.62g,65％质量分数)。反应液于0℃下搅拌2小时。待反应完毕，缓慢将反应液加入冰水(200mL)。过滤后，经减压浓缩除去溶剂得到标题化合物(10g)。
MS m/z(ESI):214.0[M+H]+.
步骤3：5-氨基-7-氯-2,3-二氢-1H-茚-4-醇(中间体33-4)的合成
将中间体33-3(5g)溶于无水DCM(50mL)，向其中加入AcOH(14.04g)和Zn(7.61g)，反应液于25℃下搅拌12小时。待反应完毕，依次加入水(200mL)和乙酸乙酯(200mL)，有机相用饱和碳酸氢钠水溶液(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩除去溶剂得到标题化合物(4g)。
MS m/z(ESI):184.0[M+H]+.
步骤4：5-乙酰氨基-7-氯-2,3-二氢-1H-茚-4-基乙酸酯(中间体33-5)的合成
将中间体33-4(4g)溶于无水二氯甲烷(40mL)，向其中加入乙酸酐(Ac2O)(6.67g)和三乙胺(TEA)(6.61g)，反应液于25℃下搅拌12小时。待反应完毕，依次加入水(200mL)和乙酸乙酯(200mL)，有机相用饱和NaCl水溶液(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩除去溶剂，残余物经硅胶柱层析法纯化(石油醚:乙酸乙酯＝20:1～5:1)得到标题化合物(4g)。
MS m/z(ESI):268.0[M+H]+.
步骤5：N-(7-氯-4-羟基-2,3-二氢-1H-茚-5-基)乙酰胺(中间体33-6)的合成
将中间体33-5(4g)溶于无水甲醇(20mL)，向其中加入K2CO3(6.19g)，反应液于25℃下 搅拌12小时。待反应完毕，依次加入水(200mL)和乙酸乙酯(200mL)，有机相用饱和NaCl水溶液(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩除去溶剂，残余物经硅胶柱层析法纯化(石油醚:乙酸乙酯＝20:1～1:1)得到标题化合物(2.8g)。
MS m/z(ESI):226.0[M+H]+.
步骤6：5-乙酰氨基-7-氯-2,3-二氢-1H-茚-4-基三氟甲烷磺酸酯(中间体33-7)的合成
将中间体33-6(500mg)溶于二氯甲烷(5mL)，向其中加入三氟甲磺酸酐(Tf2O)(749mg)和三乙胺(TEA)(672mg)，反应液于25℃下搅拌12小时。待反应完毕，依次加入水(50mL)和乙酸乙酯(50mL)，有机相用饱和NaCl水溶液(50mL*2)洗涤，洗涤后的有机相用适量无水硫酸钠干燥。有机相经减压浓缩除去溶剂，残余物经硅胶柱层析法纯化(石油醚:乙酸乙酯＝20:1～1:1)得到标题化合物(580mg)。
MS m/z(ESI):358.0[M+H]+.
步骤7：N-(4-(1-丁氧基乙烯基)-7-氯-2,3-二氢-1H-茚-5-基)乙酰胺(中间体33-8)的合成
将中间体33-7(430mg)和乙烯基正丁醚(360.60mg)溶于二氧六环(20mL)中，加入二异丙基乙胺(DIEA)(466mg)，1,1'-双(二苯基膦)二茂铁(DPPF)(66mg)和三(二亚苄基丙酮)二钯(Pd2(dba)3)(110mg)，反应液于80℃氮气保护下搅拌反应16小时。反应结束后，反应液用水(50ml)稀释，乙酸乙酯萃取(50ml*3次)，合并有机相，用无水硫酸钠干燥。过滤后，有机相经减压浓缩除去溶剂，残余物经硅胶柱层析法纯化(石油醚:乙酸乙酯＝20:1～1:1)得到标题化合物(300mg)。
MS m/z(ESI):308.0[M+H]+.
步骤8：N-(4-乙酰基-7-氯-2,3-二氢-1H-茚-5-基)乙酰胺(中间体33-9)的合成
将中间体33-8(200mg)溶于二氧六环(5mL)中，加入1N HCl(5mL),25℃下搅拌反应2小时。反应结束后，有机相经减压浓缩除去溶剂得到标题化合物(150mg)。
MS m/z(ESI):252.0[M+H]+.
步骤9：N-(4-(2-溴乙酰基)-7-氯-2,3-二氢-1H-茚-5-基)乙酰胺(中间体33-10)的合成
将中间体33-9(300mg)溶于HBr/AcOH(4mL，33％质量分数)中，加入NBS(318.20mg)，反应液于25℃下搅拌反应2小时。待反应完毕，反应液经减压浓缩除去溶剂，得到标题化合物(390mg)。
MS m/z(ESI):330.0[M+H]+.
步骤10:1-(5-氨基-7-氯-2,3-二氢-1H-茚-4-基)-2-氯乙烷-1-酮(中间体33-11)的合成
将中间体33-10(300mg)溶于无水乙醇加入HCl(12M,8.00mL)，反应液于80℃下搅拌反应2小时。待反应完毕，反应液经减压浓缩除去溶剂，残余物经制备高效液相色谱纯化(色谱柱:Gemini NX C18 5μm*10*150mm；流动相:A:水(0.225％甲酸v/v),B:乙腈；B％:30％-70％)纯化得到标题化合物(64mg)。
MS m/z(ESI):244.0[M+H]+.
步骤11：(S)-4-氯-15-(氯甲基)-8-乙基-1,2,3,8,11,14-六氢-9H,12H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12-二酮(中间体33-12)的合成
将中间体33-11(45.00mg)和中间体1-3(48.53mg)溶于甲苯(1mL)中，向其中加入对甲基苯磺酸吡啶盐(4.63mg)。反应液在90℃搅拌16h。反应完毕后，冷却至室温，加入乙醇(1mL),反应液于25℃搅拌0.5h。反应液过滤，滤饼用乙醇(2mL*2)洗涤，干燥得到标题化合物(80.0mg)。
MS m/z(ESI):471.1[M+H]+.
步骤12：(S)-15-(氨基甲基)-4-氯-8-乙基-8-羟基-1,2,3,8,11,14-六氢-9H,12H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9,12-二酮(中间体33-13)的合成
将中间体33-12(40.00mg)溶于无水甲醇(1mL)和无水N,N-二甲基甲酰胺(0.5mL)中，向其中加入乌洛托品(35.69mg)。反应液在50℃搅拌16h。反应完毕后，冷却至室温，反 应液减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％FA)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例35％-55％,洗脱时间12分钟)得到标题化合物(15.0mg)。
MS m/z(ESI):452.1[M+H]+.
步骤13：(S)-N-((4-氯-8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-1H,11H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-羟基乙酰胺(化合物33)的合成
将中间体33-13(5.00mg)和羟基乙酸(4.21mg,55.30μmol)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(6.31mg)和N,N-二异丙基乙胺(4.29mg)，反应液于25℃搅拌1h。反应完毕，反应液过滤，经制备高效液相色谱纯化(Boston Green ODS C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.05％FA)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例32％-52％,洗脱时间12分钟)得到标题化合物(2.0mg)。
MS m/z(ESI):510.3[M+H]+.
1H 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＝5.3Hz,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).
实施例34、N-(((S)-4-氯-8-乙基-8-羟基-9,12-二氧代-2,3,8,9,12,14-六氢-1H,11H-环戊二烯并[f]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-15-基)甲基)-2-环丙基-2-羟基乙酰胺(化合物34)
将中间体33-13(5.00mg)和中间体11-1(6.42mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入HATU(6.31mg)和二异丙基乙胺(4.29mg)，反应液于25℃搅拌1h。反应完毕后，反应液过滤，经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.05％FA)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例35％-55％,洗脱时间12分钟)得到标题化合物(2.0mg)。
MS m/z(ESI):550.3[M+H]+.
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＝7.1Hz,2H),3.56(t,J＝5.8Hz,1H),3.14(t,J＝7.4Hz,2H),2.27-2.18(m,2H),1.93-1.81(m,2H),1.09-1.03(m,1H),0.88(t,J＝7.2Hz,3H),0.41-0.26(m,4H).
实施例35、2-环丙基-N-(((S)-7-乙基-15-氟-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物35)
将中间体12-11(6mg)和中间体11-1(7.93mg)溶于N,N-二甲基甲酰胺(0.5mL)中，向其中 加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(7.79mg)和N,N-二甲基二异丙胺(5.29mg)，反应液25℃搅拌1h。反应结束后，反应液减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Prime C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例15％-45％,洗脱时间12分钟)，得到标题化合物(6.50mg)。
MS m/z(ESI):538.1[M+H]+。
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)。
化合物35-P1和35-P2的合成
将中间体12-11(6mg)和中间体14-10-P1(7.93mg)溶于N,N-二甲基甲酰胺(1mL)中，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(7.79mg)和N,N-二甲基二异丙胺(5.29mg)，反应液25℃搅拌1h。反应结束后，反应液减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Green ODS C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例17％-47％,洗脱时间12分钟)，得化合物35-P1(2.87mg)。
MS m/z(ESI):538.1[M+H]+。
1H 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)。
将中间体12-11(6mg)和中间体14-10-P2(4.76mg)溶于N,N-二甲基甲酰胺(1mL)中，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(10.38mg)和N,N-二甲基二异丙胺(1.76mg)，反应液25℃搅拌1h。反应结束后，反应液减压浓缩至干，残余物经制备高效液相色谱纯化(Boston Green ODS C18柱5μm二氧化硅，30mm直径，150mm长度；用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例17％-47％,洗脱时间12分钟)，得化合物35-P2(2.01mg)。
MS m/z(ESI):538.1[M+H]+。
1H 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)。
通过以下手性超临界流体色谱分析方法分别对两个异构体进一步分析。

化合物35-P1：
在上述手性高效液相色谱条件下，其保留时间为3.519分钟；
化合物35-P2：
在上述手性高效液相色谱条件下，其保留时间为3.573分钟。
实施例36、(S)-N-((7-乙基-15-氟-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)-2-羟基乙酰胺(化合物36)
将中间体12-11(6mg)和羟基乙酸(3.21mg)溶于无水N,N-二甲基甲酰胺(0.5mL)，向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(10.38mg)和二异丙基乙胺(1.76mg)，反应液于25℃搅拌1h。反应结束后，反应液减压浓缩至干，残余物经制备高效液相色谱纯化(Waters Xbridge C18柱5μm，25mm直径，100mm长度；用水(含有0.05％甲酸)和乙腈的极性递减的混合物作为洗脱液(乙腈梯度比例18％-48％,洗脱时间12分钟)，得到标题化合物(2.40mg)。
MS m/z(ESI):498.1[M+H]+。
1H 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)。
实施例37、N-((12S)-12-苄基-4-环丙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-3,8,11,14,17-五氧代-5-氧基-2,7,10,13,16-五氮杂十八烷-18-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺(化合物L1-14)、异构体L1-14-P1和L1-14-P2的合成
步骤1：中间体L1-14-2的合成
将起始原料L1-14-1(25g)，醋酸铅(43.79g)和吡啶(6.98g)溶于四氢呋喃(600mL)和甲苯(200mL)的混合溶剂中，在氮气氛围下加热到85℃，搅拌反应18h。反应结束后冷却至室温，过滤反应液，滤液减压浓缩至干，残余物经柱层析色谱法纯化(色谱柱：330gSilica Flash Column,流动相梯度0～75％乙酸乙酯/石油醚，流速100mL/min)得到标题化合物(18g)。
1H 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)。
MS m/z(ESI):391.1[M+Na]+。
步骤2：中间体L1-14-3的合成
将中间体L1-14-2(5g),2-环丙基-2-羟基乙酸苄酯(8.40g),对甲基苯磺酸吡啶盐(PPTS,341.09mg)溶于二氯甲烷(150mL)，反应液在氮气氛围下加热到65℃，搅拌反应48h。反应结束后，冷却至室温，过滤反应液，滤液减压浓缩至干，残余物经柱层析色谱法纯化(色谱柱：120gSilica Flash Column,流动相梯度0～45％乙酸乙酯/石油醚，流速80mL/min)，随后进一步经高效液相色谱纯化(色谱柱:Boston Prime C18 150*30mm*5μm；流动相:[A：水(0.225％甲酸)，B：乙腈]；B％:42％-82％,13min)，得到标题化合物(1.4g)。
MS m/z(ESI):537.2[M+Na]+。
步骤3：中间体L1-14-4-P1和L1-14-4-P2的合成
将中间体L1-14-3(1.4g)溶于混合溶剂甲醇(15mL)和水(15mL)，加入湿钯碳(10％质量含量,0.15g)，反应液在氢气氛围下25℃搅拌反应16h。反应结束后，过滤反应液，滤液减压浓缩至干，残余物经高效液相色谱法纯化(色谱柱：Boston Prime C18 150*30mm*5μm；流动相:[A:水(0.225％甲酸)，B:乙腈]；B％:24％-64％,13min)，随后进一步经超临界流体色谱制备纯化(色谱柱：DAICEL CHIRALPAK IC柱，10μm二氧化硅，30mm直径，250mm长度；使用异丙醇(含有0.1％氨水)作为洗脱液)，纯化得到中间体L1-14-4-P1(130mg)和中间体L1-14-4-P2(130mg)。
通过以下手性高效液相色谱分析方法分别对两个异构体进一步分析。
手性高效液相色谱条件如下：
中间体L1-14-4-P1：
在上述手性高效液相色谱条件下，其保留时间为4.471分钟；
MS m/z(ESI):447.5[M+Na]+。
中间体L1-14-4-P2：
在上述手性高效液相色谱条件下，其保留时间为5.692分钟；
MS m/z(ESI):447.3[M+Na]+。
步骤4：中间体L1-14-5-P1和L1-14-5-P2的合成
将2-氯三苯甲基氯树脂(2-CTC-resin)(规格：约1.19mmol/g)(257mg)加入二氯甲烷(3mL)中,再加入中间体L1-14-4-P1(130mg)和二异丙基乙基胺(59.37mg)。反应液在氮气氛围下25℃摇床摇晃反应16h。反应结束后，树脂用甲醇(10mL)和二氯甲烷(10mL)依次洗涤，重复3次。过滤，滤饼干燥后得中间体L1-14-5-P1(330mg)。
以中间体L1-14-4-P2(130mg)为原料，根据上述方法制备得到中间体L1-14-5-P2(350mg)。
步骤5：中间体L1-14-6-P1和L1-14-6-P2的合成
将中间体L1-14-5-P1(330mg)溶于N,N-二甲基甲酰胺(5mL)中，加入哌啶(1.08g)。反应液在25℃放置于摇床摇晃1h。反应结束后，树脂用甲醇(10mL)和二氯甲烷(10mL)依次洗 涤，重复3次。过滤，滤饼干燥后得L1-14-6-P1(220mg)。
以中间体L1-14-5-P2(350mg)为原料，根据上述方法制备得到中间体L1-14-6-P2(220mg)。
步骤6：中间体L1-14-7-P1和L1-14-7-P2的合成
将中间体L1-14-6-P1(220mg)和(((9H-芴-9-基)甲氧基)羰基)-L-苯丙氨酸(230.17mg)溶于N,N-二甲基甲酰胺(5mL)中，向反应液中加入苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸盐(HBTU)(225.31mg)和二异丙基乙基胺(99.96mg)。反应液在25℃放置于摇床摇晃1h。反应结束后，树脂用甲醇(10mL)和二氯甲烷(10mL)依次洗涤，重复3次。过滤，滤饼干燥后得L1-14-7-P1(377mg)。
以中间体L1-14-6-P2(220mg)为原料，根据上述方法制备得到中间体L1-14-7-P2(361mg)。
步骤7：中间体L1-14-8-P1和L1-14-8-P2的合成
将中间体L1-14-7-P1(377mg)溶于N,N-二甲基甲酰胺(5mL)中，加入哌啶(37.22mg)。反应液在25℃放置于摇床摇晃1h。反应结束后，树脂用甲醇(10mL)和二氯甲烷(10mL)依次洗涤，重复3次。过滤，滤饼干燥后得L1-14-8-P1(270mg)。
以中间体L1-14-7-P2(361mg)为原料，根据上述方法制备得到中间体L1-14-8-P2(260mg)。
步骤8：中间体L1-14-9-P1和L1-14-9-P2的合成
将中间体L1-14-8-P1(270mg)溶于N,N-二甲基甲酰胺(5mL)中，依次加入N-((9H-芴-9-基甲氧基)羰基)甘氨酰甘氨酸(209.58mg)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸盐(224.29mg)，二异丙基乙基胺(99.51mg)。反应液在25℃放置于摇床摇晃1h。反应结束后，树脂用甲醇(10mL)和二氯甲烷(10mL)依次洗涤，重复3次。过滤，滤饼干燥后得中间体L1-14-9-P1(403mg)。
以中间体L1-14-8-P2(260mg)为原料，根据上述方法制备得到中间体L1-14-9-P2(420mg)。
步骤9：中间体L1-14-10-P1和L1-14-10-P2的合成
将中间体L1-14-9-P1(403mg)溶于N,N-二甲基甲酰胺(5mL)中，加入哌啶(1.08g)。反应液在25℃放置于摇床摇晃1h。反应结束后，树脂用甲醇(10mL)和二氯甲烷(10mL)依次洗涤，重复3次。过滤，滤饼干燥后得L1-14-10-P1(300mg)。
以中间体L1-14-9-P2(420mg)为原料，根据上述方法制备得到中间体L1-14-10-P2(320mg)。
步骤10：中间体L1-14-12-P1和L1-14-12-P2的合成
将中间体L1-14-10-P1(300mg)溶于N,N-二甲基甲酰胺(5mL)中，依次加入化合物L1-14-11(182.13mg)和二异丙基乙基胺(99.41mg)。反应液在25℃放置于摇床摇晃16h。反应结束后，树脂用甲醇(10mL)和二氯甲烷(10mL)依次洗涤，重复3次。过滤，滤饼干燥后得L1-14-12-P1(374mg)。
以中间体L1-14-10-P2(320mg)和中间体L1-14-11(194.27mg)为原料，根据上述方法制备得到中间体L1-14-12-P2(387mg)。
步骤11：中间体L1-14-13-P1和L1-14-13-P2的合成
将中间体L1-14-12-P1(374mg)加入混合溶剂二氯甲烷(8mL)和六氟异丙醇(HFIP，2mL)中，反应液在25℃下摇床摇晃反应0.5h。反应结束后，过滤反应液去除树脂，滤液减压浓缩至干，残余物用高效液相色谱法纯化(色谱柱:Boston Prime C18 150*30mm*5μm；流动相:[A:水(0.225％甲酸)，B:乙腈]；B％:15％-35％,9min)，得中间体L1-14-13-P1(74mg)。
1H NMR(400MHz,METHANOL-d4)δ＝7.33-7.20(m,5H),6.81(s,2H),4.77-4.70(m,2H),4.60-4.52(m,1H),3.96-3.70(m,6H),3.61(d,J＝7.6Hz,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).
MS m/z(ESI):679.7[M+Na]+。
以中间体L1-14-12-P2(387mg)为原料，根据上述方法制备得到中间体L1-14-13-P2(86mg)。
1H 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),1.75-1.55(m,4H),1.41-1.32(m,2H),1.16-1.06(m,1H),0.60-0.45(m,4H).
MS m/z(ESI):679.5[M+Na]+。
步骤12：化合物L1-14-P1和L1-14-P2的合成
将中间体L1-14-13-P1(31.17mg)，中间体14-8(20mg)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)(18.20mg)，吡啶(11.26mg)和1-羟基苯并三唑(12.83mg)溶解于N,N-二甲基甲酰胺(1mL)，反应液在氮气氛围下25℃搅拌反应2h。反应结束后，反应液经高效液相色谱法纯化(色谱柱:Boston Green ODS 150*30mm*5μm；流动相:[A:水(0.225％甲酸)，B:乙腈]；B％:26％-46％,12min)，化合物L1-14-P1(12.3mg)。
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,1H),2.14-2.06(m,2H),1.94-1.80(m,2H),1.52-1.42(m,4H),1.27-1.13(m,2H),1.01-0.92(m,1H),0.88(t,J＝7.3Hz,3H),0.41-0.28(m,4H)。
MS m/z(ESI):1060.3[M+H]+。
以中间体L1-14-13-P2(31.17mg)和中间体14-8(20mg)为原料，根据上述方法制备得到化合物L1-14-P2(11.4mg)。
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)
MS m/z(ESI):1060.3[M+H]+。
通过以下手性高效液相色谱法分别对两个异构体进一步分析。
手性高效液相色谱条件如下：
化合物L1-14-P1在上述手性高效液相色谱条件下，其保留时间为3.735分钟。
化合物L1-14-P2在上述手性高效液相色谱条件下，其保留时间为3.901分钟。
实施例38：N-((12S)-12-苄基-4-环丙基-1-((S)-7-乙基-15-氟-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉 -14-基)-3,8,11,14,17-五氧代-5-氧基-2,7,10,13,16-五氮杂十八烷-18-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺(化合物L1-35-P1)
将中间体L1-14-13-P1(8mg),中间体12-11(5.89mg),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(4.67mg)，吡啶(2.89mg)和1-羟基苯并三唑(3.29mg)溶解于N,N-二甲基甲酰胺(1mL)，反应液在氮气氛围下25℃搅拌反应2h。反应结束后，反应液经高效液相色谱法纯化(柱子:Boston Prime C18 150*30mm*5um；流动相:[A:水(0.225％甲酸)，B:乙腈]；B％:23％-45％,10min)，得L1-35-P1(2.3mg)。
1H 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),4.69-4.62(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)。
MS m/z(ESI):1078.2[M+H]+。
通过以下超临界流体色谱法进一步分析。
超临界流体色谱条件如下：
化合物L1-35-P1在上述超临界流体色谱条件下，其保留时间为3.732分钟。
实施例39-1：N-((12S)-12-苄基-4-环丙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基-2,2-d2)-3,8,11,14,17-五氧代-5-氧基-2,7,10,13,16-五氮杂十八烷-18-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺(化合物L1-19-P1)、异构体L1-19-P2和消旋体L1-19的合成
将中间体L1-14-13-P1(7.75mg)，中间体19-8(5.0mg)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(4.53mg)，吡啶(2.80mg)和1-羟基苯并三唑(3.19mg)溶解于N,N-二甲基甲酰胺(1mL)，反应液在氮气氛围下25℃搅拌反应2h。反应结束后，反应液经高效液相色谱法纯化(柱子:Boston Green ODS 150*30mm*5um；流动相:[A:水(0.225％甲酸)，B:乙腈]；B％:22％-52％,12min)，得L1-19-P1(6.0mg)。
MS m/z(ESI):1062.3[M+H]+。
1H NMR(400MHz,DMSO-d6)δ＝8.74-8.69(m,1H),8.59(t,J＝6.7Hz,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)。
以中间体L1-14-13-P2(30.0mg)和中间体19-8(25.2mg)为原料，根据上述方法制备得到化合物L1-19-P2(19.0mg)。
MS m/z(ESI):1062.3[M+H]+。
1H NMR(400MHz,DMSO-d6)δ＝8.73-8.63(m,1H),8.58(t,J＝6.4Hz,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)。
通过以下手性高效液相色谱分析方法分别对两个异构体进一步分析。
手性高效液相色谱条件如下：
L1-19-P1在上述手性超临界流体色谱条件下，其保留时间为3.775分钟；
L1-19-P2在上述手性超临界流体色谱条件下，其保留时间为3.973分钟。
消旋体化合物L1-19的合成
将中间体L1-14-13(30.00mg,参考L1-14-13-P1合成方法以2-环丙基-2-羟基乙酸苄酯(消旋体)为原料进行合成),中间体19-8(25.15mg),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(17.52mg),吡啶(10.84mg)和1-羟基苯并三唑(12.35mg)溶解于N,N-二甲基甲酰胺(1mL)，反应液在25℃氮气保护下，搅拌反应2h。反应结束后，反应液经高效液相色谱法纯化(柱子:Boston Green ODS 150*30mm*5um；流动相:[A:水(甲酸)，B:乙腈]；B％:33％-33％,14min)得消旋体化合物L1-19(15.4mg)。
1H 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＝7.1 Hz,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)。
MS m/z(ESI):1062.4[M+H]+。
实施例39-2：N-((4S,12S)-12-苄基-4-环丙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基-2,2-d2)-3,8,11,14,17-五氧代-5-氧基-2,7,10,13,16-五氮杂十八烷-18-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺(化合物L1-19-S)
步骤1：L1-14-3-S的合成
将中间体L1-14-2(28g)，中间体9(23.5g，由实施例14-2制得)溶于二氯甲烷(25mL)，向反应液加入三氟甲烷磺酸银(139.50mg)，反应液在氮气保护下，在25℃搅拌反应108h。反应结束后，过滤反应液，滤液减压浓缩至干，残余物经快速硅胶柱纯化(流动相梯度四氢呋喃/二氯甲醇：0～7％，流速70mL/min)，得标题化合物(10.3g)。
MS m/z(ESI):537.3[M+Na]+。
步骤2：L1-14-4-S的合成
将中间体L1-14-3-S(10g)溶于四氢呋喃(100mL)，加入湿钯碳(10％质量含量,1g)，反应液在氢气氛围下，0℃氢气氛围中搅拌反应16h。反应结束后，过滤反应液，滤液减压浓缩至干，向残余物中加入石油醚/乙酸乙酯(10mL/0.5mL)搅拌2小时，过滤后，得标题化合物(6g)。
MS m/z(ESI):447.2[M+Na]+。
通过以下手性高效液相色谱分析方法分别对L1-14-4-S进一步分析。
手性高效液相色谱条件如下：

L1-14-4-S在上述手性超临界流体色谱条件下，其保留时间为4.385分钟；与化合物L1-14-4-P1在相同色谱分析条件下的保留时间(4.471分钟)基本一致。因此L1-14-4-S与L1-14-4-P1构型相同，为同一化合物。
步骤3：L1-19-S的合成
将中间体L1-14-13-S(600mg,以L1-14-4-S为原料，参考L1-14-13-P1合成方法制备获得)，中间体19-8(502.93mg),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)(350.31mg),吡啶(216.82mg)和1-羟基苯并三唑(246.91mg)溶解于N,N-二甲基甲酰胺(2mL)，反应液在25℃氮气保护下，搅拌反应2h。反应结束后，反应液经高效液相色谱法纯化(柱子:Boston Green ODS 150*30mm*5um；流动相:[A:水(0.05％甲酸)，B:乙腈]；B％:24％-54％,12min)，得标题化合物(286mg)。
1H NMR(400MHz,DMSO-d6)δ＝8.72-8.63(m,1H),8.62-8.52(m,1H),8.34-8.26(m,1H),8.16-7.94(m,3H),7.78(s,1H),7.51(s,1H),7.26-7.13(m,6H),6.99(s,1H),6.56-6.42(m,1H),5.52-5.40(m,4H),4.85-4.60(m,3H),4.59-4.46(m,1H),4.44-4.38(m,1H),3.79-3.53(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)。
MS m/z(ESI):1062.5[M+H]+。
通过以下手性高效液相色谱法分别对L1-19-S进一步分析。
手性高效液相色谱条件如下：
L1-19-S在上述手性超临界流体色谱条件下，其保留时间为3.748分钟；与实施例39-1制得的化合物L1-19-P1在相同色谱分析条件下的保留时间(3.775分钟)基本一致。因此判断L1-19-S与L1-19-P1为同一构型，属于相同的化合物。
化合物LI-0和L1-00的合成分别参考专利文献WO2019195665A1和WO2020063676A1。
实施例40：抗体-药物偶联物的制备
40.1抗体：
40.1.1抗人HER2单克隆抗体的构建和生产
抗人HER2单克隆抗体的序列如下表2所示，将编码所述抗体VH和VL的核酸序列重组至带有信号肽和重链恒定区/轻链恒定区序列的表达载体pTT5上，得到表达VH-CH1-Fc/VL-CL的重组质粒。经测序验证后，提取质粒，并转染宿主细胞。经培养，获得分泌抗体的细胞培养上清。
表2抗人HER2抗体序列信息及其CDR分析(根据Kabat划分)

40.1.2抗人p95HER2单克隆抗体筛选
抗人p95HER2单克隆抗体通过免疫小鼠产生。免疫原为hu p95HER2.ECD-Fc蛋白，选择血清中抗体滴度高并且滴度趋于平台的小鼠的脾淋巴细胞制备杂交瘤细胞，通过ELISA、FACS等本领域常规方法筛选得到阳性杂交瘤克隆，用无血清细胞培养法进一步制备抗体，并纯化抗体和测序。经测序得到鼠源抗人p95HER2抗体。
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库和MOE(Molecular Operating Environment,分子操作环境)软件，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列，在此基础上，根据需要，进行回复突变以保证原有的亲和力和/或进行热点突变以消除分子修饰风险。最终优化的人源化抗体和对应序列如表3所示。
表3优化的人源化抗体序列信息及其CDR分析(根据Kabat划分)
40.1.3 p95HER2/HER2双特异性抗体的构建和生产
由VH1-linker-VH2-CH1-Fc和VL1-linker-VL2-CL组成DVD-Ig形式的抗体，其中，VH1和VL1靶向HER2，VH2和VL2靶向p95HER2，linker可省略。双特异性抗体具体序列参见表4。按所设计结构，分别在pTT5载体上构建双特异性抗体轻重链表达质粒，并于HEK293细胞中表达。
表4 p95HER2/HER2双特异性抗体的序列信息

40.1.4抗人CDH6抗体的构建和生产
抗人CDH6单克隆抗体CDH6-Ab和CDH6-Ab-1的序列如下表5所示(抗体序列来自US20200171163A1)。
表5抗人CDH6抗体CDH6-Ab和CDH6-Ab-1序列信息及其CDR分析(根据Kabat划分)

CDH6-Ab-2和CDH6-Ab-3通过免疫小鼠产生。免疫原为人CDH6-hFc蛋白和过表达人CDH6的HEK293T细胞，选择血清中抗体滴度高的小鼠的脾淋巴细胞制备杂交瘤细胞，通过ELISA、FACS等本领域常规方法筛选得到阳性杂交瘤克隆，用无血清细胞培养法进一步制备抗体，并纯化抗体和测序，得到鼠源抗人CDH6抗体及其可变区序列。通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库和MOE(Molecular Operating Environment)软件，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列，在此基础上，根据需要，进行回复突变以保证原有的亲和力和/或进行热点突变以消除分子修饰风险。最终优化的人源化抗体CDH6-Ab-2和CDH6-Ab-3对应的VH和VL以及根据Kabat形式划分的CDR序列如表6所示。
表6抗人CDH6抗体CDH6-Ab-2和CDH6-Ab-3序列信息及其CDR分析(根据Kabat划分)

将编码所述抗体VH和VL的核酸序列重组至带有信号肽(MGWSWILLFLLSVTAGVHS，SEQ ID NO：70)和重链恒定区/轻链恒定区序列的表达载体pTT5上，得到表达VH-CH1-Fc/VL-CL的重组质粒。将质粒和转染试剂PEI(Polysciences，货号：24765-1)加入OPTI-MEM(Gibco，货号：11058021)中混匀后静置15min，加入Expi293F细胞(厂家：Thermofisher，货号：A14527)中，放入5％CO2，120rpm，37℃摇床培养。转染第二天，加入OPM-293ProFeed(上海奥浦迈，货号：F081918-001)和6g/L葡萄糖(厂家：Sigma，货号:G7528)。转染第六天，获得分泌抗体的细胞培养上清。
40.1.5抗人LIV-1单克隆抗体的构建和生产
抗人LIV-1单克隆抗体LIV1-Ab-1的序列如下表7所示(抗体序列来源US20200165335A)，将编码所述抗体VH和VL的核酸序列重组至带有信号肽和重链恒定区(序列)/轻链恒定区序列的表达载体pTT5上，得到表达LIV1-Ab-1的重组质粒。经测序验证后，提取质粒。将质粒和转染试剂PEI(Polysciences，货号：24765-1)加入OPTI-MEM(Gibco，货号：11058021)中混匀后静置15min，加入Expi293细胞(厂家：Thermofisher，货号：A14527)中，放入5％CO2，120rpm，37℃摇床培养。转染第二天，加入OPM-293ProFeed(上海奥浦迈，货号：F081918-001)和6g/L葡萄糖(厂家：Sigma，货号:G7528)。转染第六天，收集细胞上清。
表7抗人LIV-1抗体序列信息及其CDR分析(根据Kabat划分)

40.1.6抗人ROR1单克隆抗体的构建和生产
抗人ROR1单克隆抗体ROR1-Ab-1可变区序列来自专利WO2020198531A2，ROR1-Ab-2可变区序列来自专利CN113521300A，并且ROR1-Ab-3可变区序列来自专利WO2020074724A1(见表8)。将编码所述抗体VH和VL的核酸序列重组至带有人IgG1的CH和CL的pTT59表达载体中，分别获得表达ROR1-Ab-1、ROR1-Ab-2和ROR1-Ab-3的重组质粒。
将质粒和转染试剂PEI(Polysciences，24765-1)加入OPTI-MEM(Gibco，货号：11058021)中混匀后静置15min，加入Expi293细胞(Thermofisher，A14527)中，放入5％CO2，120rpm，37℃摇床培养。转染第二天，加入OPM-293ProFeed(上海奥浦迈，F081918-001)和6g/L葡萄糖(Sigma，G7528)。转染第六天，收集细胞上清。
表8抗人ROR1抗体序列信息及其CDR分析(根据Kabat划分)

40.2抗体纯化：利用ProteinA亲和层析，从细胞培养上清中纯化上述抗体。Protein A亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外监测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于 洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。纯化后符合纯度要求的蛋白，透析换液并进行后续偶联和检测。
40.3偶联：将上述抗体加入超滤管Amicon-Ultra-30kD并浓缩换液至50mM磷酸盐，150mM NaCl，1mM EDTA，pH 6.5的缓冲液中，向抗体溶液中加入7～8倍的10mM三(2-羧乙基)膦溶液(TCEP)，将混合溶液于恒温金属摇床在25℃下还原抗体2～3h。将15～20倍(DMSO溶解)的对应药物-连接子化合物(由实施例37-39制备获得，或参照实施例37-39的方法制备获得)加入反应体系，反应液在25℃偶联2～16h。对反应产物超滤浓缩换液至磷酸盐(PBS)缓冲液，去除未反应游离小分子毒素。采用SEC和LC-MS方法对ADC产品进行纯度和DAR值分析。
40.4SEC纯度分析:应用SEC-HPLC法分析待测蛋白样品，表征重组蛋白的分子大小均一性，测定重组蛋白的纯度。本法所用的HPLC为Agilent 1260，色谱柱为TSKgel G3000SWXL(购自Tosoh Bioscience)，流动相为200mM磷酸缓冲液，pH 7.0/异丙醇(v/v 9:1)，检测温度为25℃，流速为0.5mL/min，检测波长为280nm，目标蛋白上样量为50μg，分析时间为40min。
40.5 DAR值测定：应用超高效液相色谱-质谱(UHPLC-MS)联用法测量ADC分子的DAR值。首先将待测ADC分子经过PNGase F处理，脱掉N糖修饰，再用二硫苏糖醇(Dithiothreitol，DTT)处理，37℃孵育1h，还原成轻重链，然后用Thermo Vanquish UHPLC-Q Exactive Plus质谱系统进行分析，取2μg蛋白注射到Waters ACQUITY Protein BEH分子排阻色谱柱，流动相为含0.1％甲酸、0.05％TFA、25％乙腈的水溶液，流速为0.2mL/min，分析时间30min，质谱仪为Thermo Q Exactive Plus，质谱主要参数分别为喷雾电压3.8kV，毛细管加热温度300℃，鞘气流速35arb，母离子扫描范围800-3000等，最后，应用质谱数据分析软件Biopharma Finder 4.1，通过Respect算法去卷积处理，分别计算出轻重链质谱峰的分子量信息和各组分的质谱响应信号，以此计算出待测ADC样品的DAR值。
用上述同样的方法制备同型对照抗体Ab-ISO(anti-FITC-hIgG1抗体)，并偶联Linker+Payload化合物，得到相应ADC的同型对照。
用上述同样的方法，将抗体ROR1-Ab-1、ROR1-Ab-2和ROR1-Ab-3分别与化合物L1-0偶联，制备得到ADC-L1-0-8、ADC-L1-0-9、ADC-L1-0-10，DAR值分别为6.6、5.5、7.5；将抗体LIV1-Ab-1与化合物L1-0偶联得到ADC-L1-0-7，DAR值分别为6.7；将抗体trastuzumab、pertuzumab分别与化合物L1-00偶联得到ADC-L1-00-2和ADC-L1-00-11，DAR值分别为6.8和7.3；将抗体trastuzumab、pertuzumab分别与化合物L1-0偶联得到ADC-L1-0-2和ADC-L1-0-11，DAR值均为7.3；将抗体CDH6-Ab、CDH6-Ab-2、CDH6-Ab-3分别与化合物L1-0偶联得到ADC-L1-0-3、ADC-L1-0-5和ADC-L1-0-6，DAR值分别为7.4、7.4和7.2。
表9抗体-药物偶联物、其DAR值和SEC纯度





实施例41：生物学活性及相关性质测试
以下测试例中的化合物均根据本公开上述实施例的方法制备获得。
测试例1、式(D-H)化合物抗肿瘤细胞增殖活性测试
细胞与材料：人结直肠癌细胞系HCT116购于康源博创，人乳腺癌细胞系SKBR3购于ATCC，人卵巢癌细胞系OVCAR3购于ATCC，牛血清(Gibco#10099-141C)，McCoy's 5a培养基(Gibco#16600-082)，1640培养基(Gibco#A10491-01)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，牛胰岛素(Solarbio#I8040)购于Solarbio公司，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
细胞培养：HCT116细胞和SKBR3细胞均用含10％胎牛血清+1％青霉素-链霉素的McCoy's 5a培养液于37℃、5％CO2条件下培养，OVCAR3细胞用含20％胎牛血清+2μg/mL牛胰岛素+1％青霉素-链霉素的1640培养液于37℃、5％CO2条件下培养。处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测化合物对HCT116、SKBR3和OVCAR3三个细胞株增殖的抑制活性。将HCT116细胞(每孔1500个)、SKBR3细胞(每孔3000个)和OVCAR3细胞(每孔5000个)接种于96孔板中，置于37℃、5％CO2条件下培养24小时。加入待测化合物溶液后(化合物用DMSO溶解，使化合物浓度为1mM,然后利用DMSO将化合物稀释至3μM,3倍稀释共9个浓度，转移10μL配置好的化合物溶液至96孔板中，使之终浓度为0-300nM)，置于37℃、5％CO2条件下继续培养。HCT116细胞培养3天，SKBR3细胞和OVCAR3细胞培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：计算抑制百分数(％Inhibition)并拟合化合物IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，本公开化合物对HCT116细胞、SKBR3细胞和OVCAR3细胞均展现出了较强的增殖抑制活性。本公开化合物相应的抗细胞增殖活性具体见表10。
表10式(D-H)化合物抗肿瘤细胞增殖活性

在上表中，用于指示结合活性的符号所表示含义为：
“+++”表示待测化合物对细胞的增殖抑制活性IC50范围为<10nM。
“++”表示待测化合物对细胞的增殖抑制活性IC50范围为10～100nM。
“N/A”表示未测试。
测试例2、抗p95HER2/HER2双抗BsAb02-P的Biacore测定
该实验用Biacore 8K(GE)仪器，采用多循环动力学测定待测抗体BsAb02-P与人p95HER2(hu p95HER2.ECD-Fc，SEQ ID NO:104，斜体为人p95HER2胞外区，下划线为Fc标签：MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTEPKSSDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK，自行表达制备该蛋白)和HER2(购自Acro，HE2-H5225)的亲和力。
实验运行缓冲液为1×HBS-EP+缓冲溶液(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(Cat.#BR-1006-69，GE)，流经池温度设置为25℃，样品仓温度设置为16℃。两者都用运行缓冲液预处理。用Protein A生物传感芯片(Cat.#29127556,GE)亲和捕获一定量的待测抗体，然后于芯片表面流经一定浓度的人p95HER2抗原或人HER2抗原，利用Biacore 8K仪器(GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，pH1.5的甘氨酸-盐酸再生溶液(Cat.#BR-1003-54,GE)将抗原-抗体复合物洗净再生。具体地，通过注射溶液中不同浓度的人p95HER2和人HER2抗原240s来检测其结合过程，流速为30μL/min，从50nM起始，以1:1稀释，设置一系列浓度梯度；解离时间长达900s，最后用10mM甘氨酸-盐酸溶液(pH 1.5)以30μL/min的流速洗涤30s完成芯片表面的再生。
实验得到的数据用GE Biacore 8K Evaluation version 2.0软件以(1:1)Langmuir模型进行拟合，得出结合速率(Ka)、解离速率(Kd)和亲和力数值(KD)，具体见表11-12所示。实验结果表明，本公开的p95HER2/HER2双抗能与人p95HER2和HER2以高亲和力结合。
表11 p95HER2/HER2双抗与人p95HER2蛋白的反应亲和力
表12 p95HER2/HER2双抗与人HER2蛋白的反应亲和力
测试例3-1、流式细胞实验(FACS)检测抗HER2ADC及抗体与SKBR3肿瘤细胞结 合活性
将SKBR3细胞(来源于ATCC)收集后计数，以2×105个/孔铺于96孔板(Corning，3795)。加入梯度稀释的待测样品，于4℃孵育1h。用冰PBS洗两遍后，加入Alexa Fluor-647山羊抗人Fc二抗(JacksonImmuno，109-605-098)，于4℃孵育1h。用冰PBS洗两遍后，重悬细胞，用流式细胞仪(BD FACSCantoTM II)分析平均荧光强度(MFI)。使用Graphpad Prism软件对数据进行四参数曲线拟合分析，得到EC50值，结果如表13所示。
表13.FACS检测抗HER2ADC及抗体与SKBR3肿瘤细胞的结合反应
测试例3-2、抗ROR1 ADC及抗体与稳转株细胞MCF7-hROR1 clone 2G2结合活性测试
稳转株细胞构建
编码人ROR1氨基酸序列(SEQ ID NO：105)的核苷酸序列被克隆到pLVX慢病毒载体，并在HEK293T细胞中制备病毒颗粒。对MCF7细胞系(购自中科院)进行慢病毒感染后，在含5μg/ml嘌呤霉素(Gibco，货号A1113803)的含10％(w/w)胎牛血清(ExCell Bio，货号FND500)的DMEM(Gibco,货号11995073)培养基中选择性培养1周，用ROR1-Ab-1和山羊抗鼠IgG(H+L)抗体(Jackson，货号：115605006)在流式细胞仪FACS Aria III(购自BD Biosciences)上分选不同表达水平阳性细胞群到96孔板，并置于37℃，5％(v/v)CO2培养，大约2周后选择部分细胞进行扩增选择长势较好、荧光强度较高、均一性较好的阳性细胞群继续扩大培养并液氮冻存。单克隆稳转株细胞鉴定见表14。
表14人ROR1蛋白的MCF7稳转细胞系FACS检测结果
全长人ROR1氨基酸序列(SEQ ID NO：105)：
流式细胞实验(FACS)检测抗ROR1 ADC及抗体与稳转株细胞MCF7-hROR1 clone 2G2结合活性
将上述步骤中构建的稳转株细胞MCF7-hROR1 clone 2G2在T-75细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次，加入待测ADC及抗体，起始浓度为100nM,5倍梯度稀释8个点，4°孵育1h后。用PBS洗两遍后，加入二抗：Alexa647 AffiniPure Goat Anti-Human IgG(H+L)(购自Jackson Immuno，货号：109-605-088)，于4℃孵育1h。用PBS洗两遍后，重悬细胞，经FACS(FACS CantoTM，购自BD公司)检测和分析。使用Graphpad Prism软件对数据进行四参数曲线拟合分析，得到EC50值，结果如表15所示。
表15.FACS检测抗ROR1 ADC及抗体与MCF7-hROR1 clone 2G2细胞的结合活性
测试例3-3、流式细胞实验(FACS)检测抗LIV-1 ADC及抗体与OVCAR3肿瘤细胞结合活性
OVCAR3细胞(来源于ATCC)属于LIV-1高表达的肿瘤模型，将OVCAR3细胞收集后计数，以2×105个/孔铺于96孔板(Corning，3795)。加入梯度稀释的待测样品，于4℃孵育1h。用冰PBS洗两遍后，加入Alexa Fluor-647 Goat anti-human Fc二抗(JacksonImmuno，109-605-098)，于4℃孵育1h。用冰PBS洗两遍后，重悬细胞，用流式细胞仪(BD FACSCantoTM II)分析平均荧光强度(MFI)。使用Graphpad Prism软件对数据进行四参数曲线拟合分析，得到EC50值，结果如表16所示。
表16.FACS检测抗LIV-1 ADC及抗体与OVCAR3肿瘤细胞的结合反应
测试例4-1、抗HER2-ADC肿瘤细胞增殖活性测试1
细胞与材料：人乳腺癌细胞系SKBR3购于ATCC，人乳腺癌细胞系SKBR3-p95HER2构建于先声药业(构建方法参见专利申请CN202210094806.6)，人卵巢癌细胞系SKOV3购于ATCC，牛血清，McCoy's 5a培养基，青霉素-链霉素和0.25％Trypsin-EDTA购于Gibco公司(美国，货号同测试例1)，96孔板购于康宁公司(美国，货号同测试例1)，Cell-Titer Glo试剂购于普洛麦格公司(美国，货号同测试例1)。
细胞培养：SKBR3细胞、SKBR3-p95HER2细胞和SKOV3细胞均用含10％胎牛血清+1％青霉素-链霉素的McCoy's 5a培养液于37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测ADC对SKBR3、SKBR3-p95HER2和SKOV3三个细胞株增殖的抑制活性。将SKBR3细胞(每孔3000个)、SKBR3-p95HER2细胞(每孔3000个)和SKOV3细胞(每孔600个)接种于96孔板中，置于37℃、5％CO2条件下培养24小时。加入待测ADC溶液后(ADC用上述对应细胞的培养基稀释，调整ADC浓度为20nM或200nM,然后继续以培养基进行3倍梯度稀释共8个浓度，转移10μL配置好的ADC溶液至96孔板中，使起始终浓度为2nM或20nM)，置于37℃、5％CO2条件下继续培养。SKBR3细胞、SKBR3-p95HER2细胞和SKOV3细胞培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：计算抑制百分数(％Inhibition)并拟合化合物IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：在本实验条件下，本公开ADC对SKBR3细胞、SKBR3-p95HER2细胞和SKOV3细胞均展现出了较强的增殖抑制活性，详见表17。
表17 ADC抗肿瘤细胞增殖活性
“N/A”表示未测试。
测试例4-2、抗HER2-ADC肿瘤细胞增殖活性测试2
细胞与材料：人乳腺导管癌细胞系T47D(中表达细胞系)购于ATCC，人乳腺癌细胞系SKBR3购于ATCC，McCoy's 5a培养基(Gibco#16600-082)，1640培养基(Gibco#A10491-01)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
细胞培养：SKBR3细胞用含10％胎牛血清+1％青霉素-链霉素的McCoy's 5a培养液，T47D细胞用含10％胎牛血清+1％青霉素-链霉素的1640培养液，两株细胞均在37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测ADC对SKBR3和T47D两细胞株增殖的抑制活性。将SKBR3和T47D细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度。T47D细胞为2000个细胞/90μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至1000nM,并进行3倍梯度稀释，共8个浓度梯度，然后转移10μL ADC稀释溶液至96孔板中，即ADC起始终浓度为100nM。96孔板置于37℃、5％CO2条件下培养7天。加入Cell-Titer Glo试剂，检测细胞活性。
SKBR3细胞为5000个细胞/90μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至1000nM,并进行5倍梯度稀释，共9个浓度梯度，然后转移10μL ADC稀释溶液至96孔板中，即ADC起始终浓度为100nM。96孔板置于37℃、5％CO2条件下培养3天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：
计算抑制百分数(％Inhibition)并拟合得到化合物的IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)。
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，本公开抗HER2-ADC对人乳腺导管癌细胞系T47D和人乳腺癌细胞系SKBR3展现出了较强的增殖抑制活性，见表18。
表18 ADC抗肿瘤细胞增殖活性
测试例5、抗CDH6抗体和对应ADC的细胞结合活性
将OVCAR3细胞收集后计数，以2×105个/孔铺于96孔板(Corning，3795)。加入梯度稀释的待测样品，于4℃孵育1h。用冰PBS洗两遍后，加入Alexa Fluor-647 Goat anti-human Fc二抗(JacksonImmuno，109-605-098)，于4℃孵育1h。用冰PBS洗两遍后，重悬细胞，用流式细胞仪(BD FACSCantoTM II)分析平均荧光强度(MFI)。使用Graphpad Prism软件对数据进行四参数曲线拟合分析，得到EC50值，结果如表19所示。
表19 CDH6抗体和CDH6-ADC与OVCAR3细胞的结合活性
测试例6-1、抗CDH6-ADC抗肿瘤细胞增殖活性测试
细胞与材料：人卵巢癌细胞系OVCAR3(CDH6高表达细胞株)购于ATCC，人卵巢畸胎瘤细胞系PA-1购于ATCC，牛血清，1640培养基(Gibco#A10491-01)，MEM培养基(Gibco#11095-080)，MEM NEAA(Gibco#11140-050)，丙酮酸钠(Gibco#11360-070)，青霉素-链霉素和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司，牛胰岛素购于Solarbio公司，96孔板购于康宁公司(美国)，Cell-Titer Glo试剂购于普洛麦格公司(美国)。
细胞培养：OVCAR3细胞用含20％胎牛血清+2μg/mL牛胰岛素+1％青霉素-链霉素的1640培养液于37℃、5％CO2条件下培养，PA-1细胞均用含10％胎牛血清+1％MEM NEAA+1％丙酮酸钠+1％青霉素-链霉素的MEM培养液于37℃、5％CO2条件下培养。处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测ADC对OVCAR3和PA-1两个细胞株增殖的抑制活性。将OVCAR3和PA-1细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度，将OVCAR3细胞(每孔5000个)和PA-1(每孔800个)接种于96孔板 中，置于37℃、5％CO2条件下培养24小时。加入待测ADC溶液后(ADC用上述对应细胞的培养基稀释，使ADC浓度为100nM,然后继续以培养基进行3倍梯度稀释共8个浓度，转移10μL配置好的ADC溶液至96孔板中，使之终浓度为0-10nM)，置于37℃、5％CO2条件下继续培养。OVCAR3细胞和PA-1细胞培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：计算抑制百分数(％Inhibition)并拟合化合物IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：在本实验条件下，本公开抗CDH6-ADC对人卵巢癌细胞系OVCAR3和人卵巢畸胎瘤细胞系PA-1细胞均展现出了较强的增殖抑制活性，详见表20。
表20 ADC抗肿瘤细胞增殖活性
测试例6-2、抗LIV-1-ADC抗肿瘤细胞增殖活性测试
细胞与材料：人卵巢癌细胞系OVCAR3、人非小细胞肺癌细胞系H838购于ATCC，1640培养基(Gibco#A10491-01)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，牛胰岛素(Solarbio#I8040)购于Solarbio公司，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
细胞培养：OVCAR3细胞用含20％胎牛血清+2μg/mL牛胰岛素+1％青霉素-链霉素的1640培养液于37℃、5％CO2条件下培养，H838细胞用含10％胎牛血清+1％青霉素-链霉素的1640培养液于37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测ADC对OVCAR3和H838细胞株增殖的抑制活性。将OVCAR3或H838细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度，OVCAR3细胞为1500个细胞/90μL/孔，H838细胞为450个细胞/90μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至5000nM,并进行3倍梯度稀释，共8个浓度梯度，然后转移10μL ADC稀释溶液至96孔板中，即ADC起始终浓度为500nM。96孔板置于37℃、5％CO2条件下培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：
计算抑制百分数(％Inhibition)并拟合得到化合物的IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)。
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，本公开抗LIV-1-ADC对人卵巢癌细胞系OVCAR3、人非小细胞肺癌细胞系H838展现出了较强的增殖抑制活性，详见表21。
表21 ADC抗肿瘤细胞增殖活性
测试例6-3、抗ROR1-ADC抗肿瘤细胞增殖活性测试
细胞与材料：人乳腺癌细胞系hROR1-MCF7由先声再明构建，DMEM培养基(Gibco#11995-065)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
细胞培养：hROR1-MCF7细胞用含10％胎牛血清+1％青霉素-链霉素的DMEM培养液于37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测ADC对hROR1-MCF7细胞株增殖的抑制活性。将hROR1-MCF7细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度，hROR1-MCF7细胞为1700个细胞/90μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至500nM,并进行4倍梯度稀释，共9个浓度梯度，然后转移10μL ADC稀释溶液至96孔板中，即ADC起始终浓度为50nM。96孔板置于37℃、5％CO2条件下培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物药物，其他操作与实验组一致。
数据分析：
计算抑制百分数(％Inhibition)并拟合得到化合物的IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)。
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，本公开抗ROR1-ADC对人卵巢癌细胞系hROR1-MCF7展现出了较强的增殖抑制活性，详见表22。
表22 ADC抗肿瘤细胞增殖活性

测试例7、OVCAR3皮下瘤模型药效评价-1
实验试剂：人卵巢癌OVCAR3细胞购自ATCC，RPMI-1640培养基购自Gibco(货号A104910)，胎牛血清购自Excell(货号FND500)，青霉素-链霉素购自Gibco(货号15140122)，牛胰岛素购自Yeasen(货号40107ES60)，0.25％胰酶-EDTA购自Gibco(货号25200-072)，D-PBS(无钙镁离子磷酸盐缓冲液)购自Hyclone(货号SH30256.01)，Matrigel(基质胶)购自Corning(货号356237)。
实验方法：
动物信息：Balb/c nude小鼠，雌性，5-6周，体重约14-20克，动物购自北京维通利华生物技术有限公司，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。
细胞培养：人卵巢癌OVCAR3细胞株体外培养，培养条件为RPMI-1640中加入20％胎牛血清，1％青霉素-链霉素，10μg/ml牛胰岛素，37℃、5％CO2孵箱。一周一次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞饱和度为80％-90％，数量达到要求时，收取细胞，计数。
细胞接种：将0.1ml的OVCAR3细胞悬液(含1×107个细胞，RPMI-1640：Matrigel体积比为1:1)皮下接种于每只小鼠的腋下。在接种细胞后第26天，依据肿瘤体积随机分组给药，分组当天为Day 0。
肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a x b2，a和b分别表示肿瘤的长径和短径。每周两次测量小鼠体重。
受试药物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价。TGI(％)＝[(1-(某处理组给药结束时平均瘤体积-该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]x100％。
实验结果：
在小鼠皮下移植瘤OVCAR3模型中，ADC-L1-14-P1-5在3mg/kg剂量下，单次静脉给药后均对肿瘤生长具有显著抑制作用(P<0.0001)。结果见表23和图2。
表23 OVCAR3皮下瘤模型肿瘤体积
在小鼠皮下移植瘤OVCAR3模型中，ADC-L1-19-P1-5在3mg/kg剂量下，单次静脉给药后均对肿瘤生长具有显著抑制作用(P<0.0001)。结果见表24和图3。
表24 OVCAR3皮下瘤模型肿瘤体积

测试例8、ADC血浆稳定性测试
将ADC分子(终浓度为100μg/ml)分别与人血浆(澳能生物，PB021-C)和猴血浆(仕诺达生物，SND-X0107)于37℃培养箱中孵育。将孵育当天标记为第0天，随后分别在第7天、第14天和第28天取出样品，进行游离小分子的检测。
取20μL样品，加入300μL内标工作液(乙腈配置)，涡旋混匀5分钟，离心5分钟(14000rpm)，4μL上清液LC-MS/MS(API 6500+)进样分析，结果见表25。结果表明测试的ADC分子在人和猴血浆中均较为稳定。
表25 CDH6-ADC的血浆稳定性
N/A表示未检测到游离小分子，无法计算游离小分子％。
测试例9、OVCAR3皮下瘤模型药效评价-2
实验试剂：
人卵巢癌OVCAR3细胞：ATCC
RPMI-1640培养液：Gbico；Cat No.:A104910
胎牛血清：Excell，FND500
牛胰岛素：Yeasen，40107ES60
0.25％胰酶-EDTA：Gibco,Cat No.:25200-072
D-PBS(无钙镁离子磷酸盐缓冲液)：Hyclone,Cat.No.:SH30256.01
Matrigel:Corning,Cat.No.:356237
实验方法：
动物信息：Balb/c nude小鼠，雌性，5-6周，体重约14-20克，动物购自北京维通利华生物技术有限公司，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。
细胞培养：人卵巢癌OVCAR3细胞株体外培养，培养条件为RPMI-1640(细胞培养液)中加入20％胎牛血清，1％Pen Strep，10μg/ml牛胰岛素，37℃、5％CO2孵箱。一周一次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞饱和度为80％-90％，数量达到要求时，收取细胞，计数。
细胞接种：将0.1ml/(含1×107)OVCAR3细胞悬液(RPMI-1640：Matrigel，体积比为1:1)皮下接种于每只小鼠的腋下。在接种细胞后第23天，依据肿瘤体积随机分组给药，分 组当天为Day 0。
给药：ADC-L1-19-P1-7和同型对照ADC-L1-19-P1-ISO的给药剂量均为3mg/kg，腹腔给药(Q4D)。每组6只小鼠。
肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a x b2，a和b分别表示肿瘤的长径和短径。每周两次测量小鼠体重。
化合物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价。TGI(％)＝[(1-(某处理组给药结束时平均瘤体积-该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]x100％。
实验结果：
见表26、图4和图5。
表26 OVCAR3皮下瘤模型肿瘤体积
实验结论：
在小鼠皮下移植瘤OVCAR3模型中，本公开化合物ADC-L1-19-P1-7及同型对照ADC-L1-19-P1-ISO在3mg/kg，四天一次腹腔给药对肿瘤生长具有显著抑制作用(P<0.0001)；但同型对照ADC-L1-19-P1-ISO抑瘤显著弱于ADC-L1-19-P1-7(P<0.001)。本实施例在所尝试剂量下未发现影响小鼠体重，也未引起任何小鼠死亡，小鼠可以耐受。
测试例10、NCI-H838皮下瘤模型药效评价
实验试剂：
人肺癌NCI-H838细胞：科佰
RPMI-1640培养液：Gbico；Cat No.:61870-036
胎牛血清：Gibco；Cat No.:10099-141C
0.25％胰酶-EDTA：Gibco,Cat No.:25200-072
D-PBS(无钙镁离子磷酸盐缓冲液)：Hyclone,Cat.No.:SH30256.01
Matrigel:Corning,Cat.No.:356237
实验方法：
动物信息：B-NDG小鼠，雌性，5-6周，体重约14-20克，动物购自百奥赛图，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。
细胞培养：人肺癌NCI-H838细胞株体外培养，培养条件为RPMI-1640(细胞培养液)中加入10％胎牛血清，1％Pen Strep，37℃、5％CO2孵箱。一周两次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞饱和度为80％-90％，数量达到要求时，收取细胞，计数。
细胞接种：将0.1ml/(含1×107)NCI-H838细胞悬液(RPMI-1640：Matrigel，体积比为1:1)皮下接种于每只小鼠的腋下。在接种细胞后第23天，依据肿瘤体积随机分组给药，分组当天为Day 0。
给药：ADC-L1-19-P1-7和同型对照ADC-L1-19-P1-ISO的给药剂量均为3mg/kg，均为腹腔给药(Q4D)。每组6只小鼠。
肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a x b2，a和b分别表示肿瘤的长径和短径。每周两次测量小鼠体重。
化合物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价。TGI(％)＝[(1-(某处理组给药结束时平均瘤体积-该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]x100％。
实验结果：
见表27、图6和图7。
表27 H838皮下瘤模型肿瘤体积
实验结论：
在小鼠皮下移植瘤NCI-H838模型中，本公开化合物ADC-L1-19-P1-7和同型对照ADC-L1-19-P1-ISO在3mg/kg，四天一次腹腔给药对肿瘤生长具有显著抑制作用(P<0.0001)；但同型对照ADC-L1-19-P1-ISO的抑瘤显著弱于ADC-L1-19-P1-7(P<0.001)。本实施例在所尝试剂量下未发现影响小鼠体重，也未引起任何小鼠死亡，小鼠可以耐受。
测试例11、抗LIV-1-ADC的旁观者效应测试
细胞与材料：人卵巢癌细胞系OVCAR3购于ATCC，人非小细胞肺癌细胞系NCI-H838-hLIV1-KO为自行构建，牛血清(Gibco#10099-141C)，1640培养基(Gibco#A10491-01)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，牛胰岛素(Solarbio#I8040)购于Solarbio公司，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
NCI-H838-hLIV1-KO的构建方法：针对人LIV1基因设计了六种sgRNA，然后将sgRNA克隆到pLVX慢病毒载体,并在HEK293T(购自中科院)细胞中制备病毒颗粒。对NCI-H838(购自ATCC)细胞系进行慢病毒感染后，在含1.5μg/ml嘌呤霉素(购自Gibco，货号:A1113802)的含10％(w/w)胎牛血清的RPMI 1640培养基中选择性培养2周，获得H838-LIV1 KO pool细胞株。用人抗LIV1抗体(Ladiratuzumab，自产)和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)标记，在流式细胞仪FACSAriaII(购自BD Biosciences)上分选H838-LIV1KO单克隆细胞到96孔板，并置于37℃，5％(v/v)CO2培养，大约2周后选择部分单克隆孔进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。选择长势较好、荧光强度低、单克隆细胞系继续扩大培养并液氮冻存。
细胞培养：OVCAR3细胞用含20％胎牛血清+2μg/mL牛胰岛素+1％青霉素-链霉素的1640培养液，NCI-H838-hLIV1-KO细胞用含10％胎牛血清+1％青霉素-链霉素的1640培养液，两株细胞均在37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
旁观者效应检测：通过将待测ADC与LIV-1阳性细胞OVCAR3孵育后的上清液转移至LIV-1阴性细胞NCI-H838-hLIV1-KO中继续孵育，并利用Cell-Titer Glo试剂检测该上清液对NCI-H838-hLIV1-KO细胞增殖活性的影响，进而反映ADC的旁观者效应。将OVCAR3细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度为10000个细胞/180μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至5000nM,并进行5倍梯度稀释，共8个浓度梯度，然后转移20μL ADC稀释溶液至96孔板中，即ADC起始浓度为500nM。96孔板置于37℃、5％CO2条件下培养5天。吸取150μL OVCAR3细胞培养板中的上清液，转移至提前一天接种的NCI-H838-hLIV1-KO细胞板中 (450个细胞/50μL/孔)，置于37℃、5％CO2条件下培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：
计算抑制百分数(％Inhibition)并拟合得到化合物的IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)。
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，将待测ADC与LIV-1阳性细胞OVCAR3孵育后的上清液继续与LIV-1阴性细胞NCI-H838-hLIV1-KO孵育，通过加入Cell-Titer Glo试剂检测其对阴性细胞的杀伤，结果如表28所示，表明本公开抗LIV1-ADC具有较好的旁观者效应。
表28抗LIV1-ADC旁观者效应
测试例12、抗ROR1-ADC的旁观者效应测试
细胞与材料：人乳腺癌细胞系hROR1-MCF7由先声再明构建，人乳腺癌细胞系MCF7购于ATCC，牛血清(Gibco#10099-141C)，DMEM培养基(Gibco#11995-065)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
细胞培养：hROR1-MCF7细胞和MCF7细胞用含10％胎牛血清+1％青霉素-链霉素的DMEM培养液于37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
旁观者效应检测：通过将待测ADC与ROR1阳性细胞hROR1-MCF7孵育后的上清液转移至ROR1阴性细胞MCF7中继续孵育，并利用Cell-Titer Glo试剂检测该上清液对MCF7细胞增殖活性的影响，进而反映ADC的旁观者效应。将hROR1-MCF7细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度为20000个细胞/180μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至5000nM,并进行3倍梯度稀释，共8个浓度梯度，然后转移20μL ADC稀释溶液至96孔板中，即ADC起始浓度为500nM。96孔板置于37℃、5％CO2条件下培养5天。吸取150μL hROR1-MCF7细胞培养板中的上清液，转移至提前一天接种的MCF7细胞板中(1500个细胞/50μL/孔)，置于37℃、5％CO2条件下培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：
计算抑制百分数(％Inhibition)并拟合得到化合物的IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)。
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，将待测ADC与ROR1阳性细胞hROR1-MCF7孵育后的上清液继续与ROR1阴性细胞MCF7孵育，通过加入Cell-Titer Glo试剂检测其对阴性细胞的杀伤，结果如表29所示，表明本公开抗ROR1-ADC具有较好的旁观者效应。
表29抗ROR1-ADC旁观者效应
测试例13、抗HER2-ADC的旁观者效应测试
细胞与材料：人乳腺癌细胞系SKBR3购于ATCC，人小细胞肺癌细胞系NCI-H2171购于ATCC，牛血清(Gibco#10099-141C)，McCoy's 5a培养基(Gibco#16600-082)，1640培养基(Gibco#A10491-01)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
细胞培养：SKBR3细胞用含10％胎牛血清+1％青霉素-链霉素的McCoy's 5a培养液，NCI-H2171细胞用含10％胎牛血清+1％青霉素-链霉素的1640培养液，两株细胞均在37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
旁观者效应检测：通过将ADC与HER2阳性细胞SKBR3孵育后的上清液转移至HER2阴性细胞NCI-H2171中继续孵育，并利用Cell-Titer Glo试剂检测该上清液对NCI-H2171细胞增殖活性的影响，进而反映ADC的旁观者效应。将SKBR3细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度为10000个细胞/180μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至5000nM,并进行3倍梯度稀释，共8个浓度梯度，然后转移20μL ADC稀释溶液至96孔板中，即ADC起始浓度为500nM。96孔板置于37℃、5％CO2条件下培养3天。吸取150μL SKBR3细胞培养板中的上清液，转移至提前一天接种的NCI-H2171细胞板中(6000个细胞/50μL/孔)，置于37℃、5％CO2条件下培养5天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：
计算抑制百分数(％Inhibition)并拟合得到化合物的IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)。
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，将待测ADC与HER2阳性细胞SKBR3孵育后的上清液继续与HER2阴性细胞NCI-H2171孵育，通过加入Cell-Titer Glo试剂检测其对阴性细胞的杀伤，结果如表30所示，表明本公开抗HER2-ADC具有较好的旁观者效应。
表30抗HER2-ADC旁观者效应
测试例14、抗CDH6-ADC的旁观者效应测试
细胞与材料：人卵巢癌细胞系OVCAR3购于ATCC，人卵巢癌细胞系SKOV3购于ATCC，牛血清(Gibco#10099-141C)，1640培养基(Gibco#A10491-01)，McCoy's 5a培养基(Gibco#16600-082)，青霉素-链霉素(Gibco#15140-122)和0.25％Trypsin-EDTA(Gibco#25200-056)购于Gibco公司(美国)，牛胰岛素(Solarbio#I8040)购于Solarbio公司，96孔板(Greiner Bio-one#655098)购于康宁公司(美国)，Cell-Titer Glo试剂(Promega#G7568)购于普洛麦格公司(美国)。
细胞培养：OVCAR3细胞用含20％胎牛血清+2μg/mL牛胰岛素+1％青霉素-链霉素的1640培养液于，SKOV3细胞用含10％胎牛血清+1％青霉素-链霉素的McCoy's 5a培养液，两株细胞均在37℃、5％CO2条件下培养，处于对数生长期细胞方可用于实验。
旁观者效应检测：通过将ADC与CDH6阳性细胞OVCAR3孵育后的上清液转移至CDH6阴性细胞SKOV3中继续孵育，并利用Cell-Titer Glo试剂检测该上清液对SKOV3细胞增殖活性的影响，进而反映ADC的旁观者效应。将OVCAR3细胞从细胞培养瓶内消化吹散处理，用对应的新鲜培养基重悬，调整细胞密度为20000个细胞/180μL/孔，接种于96孔板中，置于37℃、5％CO2条件下培养过夜。用完全培养基稀释ADC浓度至4500nM,并进行3倍梯度稀释，共8个浓度梯度，然后转移20μL ADC稀释溶液至96孔板中，即ADC起始浓度为450nM。96孔板置于37℃、5％CO2条件下培养5天。吸取150μL OVCAR3细胞培养板中的上清液，转移至提前一天接种的SKOV3细胞板中(700个细胞/50μL/孔)，置于37℃、5％CO2条件下培养7天。加入Cell-Titer Glo试剂，检测细胞活性。
另设阴性对照组和阳性对照组分别作为Bottom和Top。阴性对照组为不加细胞，仅加同体积的培养基，其他操作与实验组一致；阳性对照组为不加受试药物，其他操作与实验组一致。
数据分析：
计算抑制百分数(％Inhibition)并拟合得到化合物的IC50。
抑制百分数(％Inhibition)＝1-100％*(Signal-Bottom)/(Top-Bottom)。
Signal指实验组的信号值，Bottom指阴性对照组的平均信号值，Top指阳性对照组的平均信号值。
实验结果：
在本实验条件下，将待测ADC与CDH6阳性细胞OVCAR3孵育后的上清液继续与CDH6阴性细胞SKOV3孵育，通过加入Cell-Titer Glo试剂检测其对阴性细胞的杀伤，结果如表31所示，表明本公开抗CDH6-ADC具有较好的旁观者效应。
表31抗CDH6-ADC旁观者效应

Claims (34)

  1. 请求保护的权利要求为：
  2. 一种配体-药物偶联物或其药学上可接受的盐，其结构通式为Pc-(L-D)n，其中：
    Pc为配体单元；
    L为连接子单元；
    D为以下式(D-I)所示的药物单元：
    其中，
    X选自NH或O；
    R1选自卤素、CN、C1-C6烷基、C3-C6环烷基或C2-C6炔基，所述C1-C6烷基、C3-C6环烷基或C2-C6炔基任选被一个或多个Ra1取代；
    X1选自CR2或N；
    R2选自H、卤素、CN，或者R1、R2与它们连接的原子共同形成5-6元杂环基，所述5-6元杂环基任选被一个或多个Ra2取代；
    R4选自H、C1-C3烷基、C3-C6环烷基或4-7元杂环基，所述C1-C3烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Ra4取代；
    R5选自H、卤素、CN、NH2或NO2，或者R1、R5与它们连接的原子共同形成5-6元杂环基、5-6元杂芳基或C5-C7环烯基，所述5-6元杂环基、5-6元杂芳基或C5-C7环烯基任选被一个或多个Ra5取代；
    R6选自H或C1-C3烷基；
    R7选自H、C1-C3烷基或者C3-C6环烷基，或者R6、R7与其连接的C原子共同形成C3-C6环烷基，所述C3-C6环烷基任选被一个或多个Ra7取代；
    每一个Ra1、Ra2、Ra4、Ra5、Ra7独立地选自D、卤素、CN、＝O、OH、NH2、C1-C3烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C3烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
    每一个Rb独立地选自卤素、CN、＝O、C1-C3烷基、OH、O(C1-C3烷基)、NH2、NH(C1-C3烷基)或N(C1-C3烷基)2；
    条件是：i)当R1选自甲基，R2选自F时，R6、R7与其连接的C原子共同形成环丙基；ii)当X选自NH时，R5不选自H；iii)式(D-I)所示化合物不包含
    并且，n为1～16的实数。
  3. 根据权利要求1所述的配体-药物偶联物或其药学上可接受的盐，其中R1选自卤素、C1-C3烷基、C3-C6环烷基或C2-C3炔基。
  4. 根据权利要求1或2所述的配体-药物偶联物或其药学上可接受的盐，其中R2选自H、卤素、CN，或者R1、R2与它们连接的原子共同形成5-6元杂环基，所述5-6元杂环基含有1或2个氧原子作为环原子，所述5-6元杂环基任选被一个或多个D原子取代。
  5. 根据权利要求1或2所述的配体-药物偶联物或其药学上可接受的盐，其中R2选自H、F或Cl，或者R1、R2与它们连接的原子共同形成
  6. 根据权利要求1至4中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R5选自H、Cl、F、NH2或NO2，或者R1、R5与它们连接的原子共同形成
  7. 根据权利要求1至5中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R4选自H或C1-C3烷基。
  8. 根据权利要求1至6中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R7选自H、C1-C3烷基或者任选被一个或多个D取代的C3-C6环烷基，或者R6、R7与其连接的C原子共同形成C3-C6环烷基。
  9. 根据权利要求1至7中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中结构单元选自
  10. 根据权利要求1至8中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中式(D-I)所示的药物单元选自式(D-Ia)所示的药物单元：
    其中，R1、R2、R4、R5、R6、R7如权利要求1至8中任一项所定义。
  11. 根据权利要求1至9中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中式(D-I)所示化合物选自以下所示化合物：


  12. 根据权利要求1至10中任一项所述的配体-药物偶联物或其药学上可接受的盐，其 中，连接子单元L选自 其a端与配体单元Pc共价连接，b端与药物单元D共价连接，其中，m1、m2各自独立地选自整数2～8，m3选自整数1～16，L1、L2各自独立地选自由1至8个氨基酸构成的肽残基，所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代。
  13. 根据权利要求11所述的配体-药物偶联物或其药学上可接受的盐，其中，所述L1、L2各自独立地选自由2、3或4个氨基酸构成的肽残基，所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代。
  14. 根据权利要求11或12所述的配体-药物偶联物或其药学上可接受的盐，其中，所述L1为Gly-Gly-Phe-Gly四肽残基或Ala-Ala-Ala三肽残基，所述L2为Gly-Gly-Phe-Gly四肽残基或Val-Lys二肽残基。
  15. 根据权利要求11至13中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，m1选自5，m2选自2，m3选自8。
  16. 根据权利要求11至14中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，连接子单元L选自 其a端与配体单元Pc共价连接，b端与药物单元D共价连接。
  17. 根据权利要求1至15中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述配体-药物偶联物或其药学上可接受的盐选自以下配体-药物偶联物或其药学上可接受的盐：




    其中Pc和n如权利要求1定义。
  18. 根据权利要求1至16中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述配体单元Pc选自多肽、抗体或其抗原结合片段。
  19. 根据权利要求1至17中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述配体单元Pc可特异性结合选自以下组的一种或多种抗原：HER2、p95HER2、HER3、CD3、CD16、ROR1、DLL3、CDH6、CD70、CD5、CD20、BCMA、EGFR、VEGF和LIV-1。
  20. 根据权利要求17或18所述的配体-药物偶联物或其药学上可接受的盐，其中，所述 Pc为特异性结合HER2、p95HER2、CDH6、ROR1或LIV-1的抗体或其抗原结合片段；所述抗体或其抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)，可选地，其中：(1)所述重链可变区包括SEQ ID NO：1、3、19、21、37、46、54、56、71、80、82或84所示的VH中所含的HCDR1、HCDR2和HCDR3；或/和所述轻链可变区包括SEQ ID NO：2、4、20、22、38、47、55、57、72、81、83或85所示的VL中所含的LCDR1、LCDR2和LCDR3；或(2)所述重链可变区或/和所述轻链可变区包括与第(1)组中所述HCDR1-3或/和LCDR1-3中的每个CDR相比，具有至少80％同一性的氨基酸序列，或至多发生3个插入、缺失或替换突变的氨基酸序列。
  21. 根据权利要求19所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体或其抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)，所述重链可变区包括HCDR1、HCDR2和HCDR3，或/和所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中，所述HCDR1-3或/和所述LCDR1-3选自以下；
    (1)所述HCDR1-3为SEQ ID NO:7-9；或/和所述LCDR1-3为SEQ ID NO:10-12；
    (2)所述HCDR1-3为SEQ ID NO:13-15；或/和所述LCDR1-3为SEQ ID NO:16-18；
    (3)所述HCDR1-3为SEQ ID NO:23-25；或/和所述LCDR1-3为SEQ ID NO:26-28；
    (4)所述HCDR1-3为SEQ ID NO:29-31；或/和所述LCDR1-3为SEQ ID NO:32-34；
    (5)所述HCDR1-3为SEQ ID NO:40-42；或/和所述LCDR1-3为SEQ ID NO:43-45；
    (6)所述HCDR1-3为SEQ ID NO:48-50；或/和所述LCDR1-3为SEQ ID NO:51-53；
    (7)所述HCDR1-3为SEQ ID NO:58-60；或/和所述LCDR1-3为SEQ ID NO:61-63；
    (8)所述HCDR1-3为SEQ ID NO:64-66；或/和所述LCDR1-3为SEQ ID NO:67-69；
    (9)所述HCDR1-3为SEQ ID NO:74-76；或/和所述LCDR1-3为SEQ ID NO:77-79；
    (10)所述HCDR1-3为SEQ ID NO:86-88；或/和所述LCDR1-3为SEQ ID NO:89-91；
    (11)所述HCDR1-3为SEQ ID NO:92-94；或/和所述LCDR1-3为SEQ ID NO:95-97；
    (12)所述HCDR1-3为SEQ ID NO:98-100；或/和所述LCDR1-3为SEQ ID NO:101-103；或
    (13)所述HCDR1-3或/和所述LCDR1-3具有与第(1)-(12)组中任一组所述HCDR1-3或/和LCDR1-3中的每个CDR相比，至少80％同一性，或至多发生3个插入、缺失或替换突变的氨基酸序列。
  22. 根据权利要求17或18所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体或其抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)，所述重链可变区包括SEQ ID NO：1、3、19、21、37、46、54、56、71、80、82或84所示的氨基酸序列，或/和所述轻链可变区包括SEQ ID NO：2、4、20、22、38、47、55、57、72、81、83或85所示的氨基酸序列；或所述重链可变区和所述轻链可变区分别包括与上述任一重链可变区和轻链可变区相比，具有至少80％同一性的氨基酸序列。
  23. 根据权利要求17-21中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体或抗原结合片段包括重链恒定区序列和/或轻链恒定区序列，可选地，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区片段包括CH1，铰链区，CH2，CH3或Fc；可选地，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；可选地，所述抗体或抗原结合片段包括完整的重链和轻链，所述重链由所述VH和重链恒定区组成，所述重链恒定区具有如SEQ ID NO:5、39或73所示的氨基酸序列，所述轻链由所述VL和轻链恒定区组成，所述轻链恒定区具有如SEQ ID NO:6所示的氨基酸序列。
  24. 根据权利要求17-22中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体选自Trastuzumab、Pertuzumab或Rituximab。
  25. 一种药物-连接子化合物或其药学上可接受的盐，其结构通式为L’-D，其中：
    药物单元D如权利要求1-10中任一项所定义；
    连接子单元L’选自其b端与药物单元D共价连接，L1、L2、m1、m2、m3如权利要求11-15中任一项所定义。
  26. 根据权利要求24所述的药物-连接子化合物或其药学上可接受的盐，其中，L’选自其b端与药物单元D共价连接，m1选自5，并且L1选自Gly-Gly-Phe-Gly四肽残基或Ala-Ala-Ala三肽残基。
  27. 根据权利要求24所述的药物-连接子化合物或其药学上可接受的盐，其中，L’选自其b端与药物单元D共价连接，m2选自2，m3选自8，并且L2选自Gly-Gly-Phe-Gly四肽残基或Val-Lys二肽残基。
  28. 根据权利要求24至26中任一项所述的药物-连接子化合物或其药学上可接受的盐，其中，L’选自以下化学结构：
    其b端与药物单元D共价连接。
  29. 根据权利要求24至27中任一项所述的药物-连接子化合物或其药学上可接受的盐，其选自以下化合物或其药学上可接受的盐：




  30. 一种药物组合物，所述组合物包含权利要求1至23中任一项所述的配体-药物偶联物或其药学上可接受的盐，以及药学上可接受的辅料。
  31. 权利要求1至23中任一项所述的配体-药物偶联物或其药学上可接受的盐、或权利要求29所述的药物组合物在制备治疗肿瘤药物中的用途。
  32. 权利要求1至23中任一项所述的配体-药物偶联物或其药学上可接受的盐的制备方法，包括将权利要求24至28中任一项所述的药物-连接子化合物与配体偶联的步骤，可选地，所述配体为抗体或其抗原结合片段。
  33. 治疗有需要的受试者的肿瘤的方法，其包括将权利要求1至23中任一项所述的配体-药物偶联物或其药学上可接受的盐、或权利要求29所述的药物组合物施用于所述受试者。
  34. 权利要求1至23中任一项所述的配体-药物偶联物或其药学上可接受的盐、或权利要求29所述的药物组合物用于治疗受试者的肿瘤的用途。
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