WO2021190602A1 - 一种抗体药物偶联物的制备方法 - Google Patents

一种抗体药物偶联物的制备方法 Download PDF

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WO2021190602A1
WO2021190602A1 PCT/CN2021/083014 CN2021083014W WO2021190602A1 WO 2021190602 A1 WO2021190602 A1 WO 2021190602A1 CN 2021083014 W CN2021083014 W CN 2021083014W WO 2021190602 A1 WO2021190602 A1 WO 2021190602A1
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antibody
reaction
alkyl
drug conjugate
drug
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PCT/CN2021/083014
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English (en)
French (fr)
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梁志
林文峰
石瑞君
刘洵
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to AU2021243080A priority Critical patent/AU2021243080A1/en
Priority to MX2022011769A priority patent/MX2022011769A/es
Priority to KR1020227035939A priority patent/KR20220157425A/ko
Priority to EP21774485.3A priority patent/EP4130045A4/en
Priority to JP2022557990A priority patent/JP2023521956A/ja
Priority to BR112022019042A priority patent/BR112022019042A2/pt
Priority to CA3175048A priority patent/CA3175048A1/en
Priority to CN202180014771.4A priority patent/CN115103858A/zh
Priority to US17/913,928 priority patent/US20230241242A1/en
Publication of WO2021190602A1 publication Critical patent/WO2021190602A1/zh

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    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin

Definitions

  • the present disclosure relates to a preparation method of a class of antibody drug conjugates, in particular to the synthesis steps and purification steps of the preparation method of a class of antibody drug conjugates.
  • Chemotherapy is still one of the most important anti-cancer methods including surgery, radiotherapy, and targeted therapy. Although there are many types of highly effective cytotoxic drugs, the difference between tumor cells and normal cells is very small, which limits the wide clinical application of these anti-tumor compounds due to toxic side effects. While anti-tumor monoclonal antibodies are specific to tumor cell surface antigens, antibody drugs have become the frontline drugs for anti-tumor therapy, but when antibodies are used alone as anti-tumor drugs, the efficacy is often unsatisfactory.
  • Antibody drug conjugate connects monoclonal antibodies or antibody fragments with biologically active cytotoxic drugs through a stable chemical linker compound, making full use of the specificity of antibody binding to normal cells and tumor cell surface antigens
  • ADC Antibody drug conjugate
  • the first antibody-drug conjugate Mylotarg (gemtuzumab ozogamicin (gemtuzumab ozogamicin), Wyeth Pharmaceuticals Co., Ltd.) was approved by the U.S. FDA for the treatment of acute myeloid leukemia (Drugs of the Future ( 2000) 25(7): 686; US4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).
  • Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the U.S. FDA for the treatment of HER2-positive and at the same time resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel in late stage or metastasis Patients with breast cancer (WO2005037992; US8088387).
  • Kadcyla is the first ADC drug approved by the US FDA for the treatment of solid tumors.
  • camptothecin derivatives which have anti-tumor effects by inhibiting topoisomerase I.
  • camptothecin derivative Isartecan chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H ,12H-Benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione
  • ADC conjugate
  • the present disclosure provides a method for preparing an antibody-drug conjugate, wherein the structure of the antibody-drug conjugate is as shown in the general formula (Pc-L a -YD):
  • W is selected from a C 1-8 alkyl group, a C 1-8 alkyl group-C 3-7 cycloalkyl group, or a linear heteroalkyl group of 1 to 8 atoms
  • the linear heteroalkyl group contains 1 to 3 selected Heteroatoms from N, O and S, wherein the C 1-8 alkyl, C 3-7 cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxyl, cyano, One or more substituents of amino, C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy and C 3-7 cycloalkyl Replaced by
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and chemical bond, where p 1 is an integer from 1 to 20;
  • L 3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy and C 3-7 cycloalkyl group are substituted by one or more replace;
  • the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , C 1-6 alky
  • R 1 is a halogenated C 1-6 alkyl group or a C 3-7 cycloalkyl group
  • R 2 is selected from a hydrogen atom, a halogenated C 1-6 alkyl group and a C 3-7 cycloalkyl group;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-7 cycloalkyl group
  • R 5 is selected from a hydrogen atom, a C 1-6 alkyl group, a halogenated C 1-6 alkyl group, a deuterated C 1-6 alkyl group and a hydroxy C 1-6 alkyl group;
  • R 6 and R 7 are the same or different, and are each independently selected from hydrogen atom, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl, and hydroxy C 1-6 alkyl;
  • n 0 or 1
  • n 3 to 8, n is a decimal or integer
  • Pc is an antibody or an antigen-binding fragment thereof
  • the preparation method includes the following steps:
  • W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7 and m are as defined above.
  • the present disclosure provides a method for preparing an antibody-drug conjugate, the antibody-drug conjugate having a structure shown in the following formula:
  • n is 4 to 8, and n is a decimal or integer;
  • the preparation method includes the following steps:
  • the reaction temperature condition in step (a) is about 4°C to about 30°C, preferably about 20°C to about 30°C, more preferably 25°C, a non-limiting example Including about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, and about 30°C.
  • the reaction temperature condition is 13°C to 28°C or 13°C to 25°C.
  • the reaction in step (a) is carried out at a pH of about 4.5 to about 6.5, preferably the reaction is carried out at a pH of about 5.0 to about 6.0, more Preferably, the reaction is carried out at a pH of about 5.6. In a non-limiting example, the reaction is at a pH of about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, About 5.7, about 5.8, about 5.9, or about 6.0.
  • the reaction in step (a) is carried out in a buffer; in a non-limiting embodiment, the buffer is selected from a histidine buffer, a phosphate buffer And acetate buffer.
  • the reaction in step (a) is carried out in a buffer; in a non-limiting example, the buffer is a histidine-hydrochloric acid buffer.
  • the buffer is selected from a histidine salt buffer containing EDTA and a histidine-hydrochloric acid buffer containing EDTA.
  • concentration of the histidine salt buffer is 1 mM to 100 mM, 10 mM to 50 mM, 20 mM, 30 mM or 40 mM; the concentration of EDTA is 1 mM to 10 mM, 2 mM to 5 mM, 2.5 mM, 3 mM or 4 mM.
  • the buffer contains 10 mM to 50 mM histidine salt buffer and 1 mM to 10 mM EDTA.
  • the buffer contains 20 mM histidine-hydrochloric acid buffer and 2.5 mM EDTA.
  • EDTA refers to ethylenediaminetetraacetic acid.
  • the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof, 1,4-dimercaptothreitol (DTT) and ⁇ -A suitable reducing agent such as mercaptoethanol ( ⁇ -ME), preferably TCEP or a salt thereof, more preferably tris(2-carboxyethyl)phosphine hydrochloride.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT 1,4-dimercaptothreitol
  • ⁇ -A suitable reducing agent such as mercaptoethanol ( ⁇ -ME), preferably TCEP or a salt thereof, more preferably tris(2-carboxyethyl)phosphine hydrochloride.
  • the molar ratio of the reducing agent to the antibody or its antigen-binding fragment (Pc) in step (a) is 2 to 10:1, 2.6 to 7:1, 2.9 to 3.7:1, 3.2 to 3.4 :1 or 3.3:1.
  • step (b) is carried out in an organic solvent.
  • organic solvents include dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N- Dimethylacetamide (DMAc), acetonitrile or mixtures thereof.
  • step (b) includes: dissolving the compound represented by formula (La-YD) in DMSO, and combining the product of step (a) with the compound represented by formula (La-YD) The DMSO solution is mixed.
  • the above preparation method further includes step (c), and the step (c) includes purifying the product of step (b).
  • the purification can be purified by cation column chromatography or affinity column chromatography, and the filler for the cation chromatography is selected from Capto S Impact and Poros XS, preferably Capto S Impact.
  • the Capto S Impact is Capto TM S Impact.
  • the Poros XS is Poros TM XS.
  • the drug load (n) can range from 3 to 8, 4 to 8, 5 to 7, preferably 5.3 to 6.1 per antibody or antigen binding fragment (Pc) binding. , More preferably 5.7 cytotoxic drugs.
  • n is a decimal or integer. In some embodiments, n is 5.3, 5.4, 5.5, 5.6, or 5.7.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%; preferably, the ratio of antibody heavy chains that bind 4 drugs The proportion of antibody heavy chains is 4% or less, and the proportion of antibody heavy chains that are not bound to the drug is 6% or less. In a non-limiting example, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, 3% or less, 2% or less, or 1% or less; the proportion of antibody heavy chains that do not bind drugs is 6% or less and 5%. Or less, 4% or less, 3% or less, 2% or less, or 1% or less.
  • the proportion of antibody light chains that bind a drug is 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 75% or more, 80% or more , 85% or more, 90% or more or 95% or more.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs It is less than 5%.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs It is 4% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs 5% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 65% or more.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs 4% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 70% or more.
  • the ratio of the antibody heavy chain and/or the ratio of the antibody light chain is determined by reverse chromatography.
  • the preparation method is suitable for large-scale preparation.
  • the trastuzumab antibody dosage of the preparation method is 100 mg or more, preferably 1 g or more, more preferably 10 g or more, and most preferably 100 g or more.
  • the antibody-drug conjugate has a structure as shown in the general formula (Pc-L b -YD):
  • s 1 is an integer from 2 to 8;
  • the preparation method includes the following steps:
  • the aforementioned antibody-drug conjugate has the following structure:
  • Pc and n are as defined in the general formula (Pc-La-Y-D).
  • the antibody is selected from a chimeric antibody, a humanized antibody, and a fully human antibody. In some embodiments, the antibody is a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, and anti-HER3 (ErbB3) antibody.
  • HER4 (ErbB4) antibody anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, Anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody and anti-Mesothelin antibody , Or an antigen-binding fragment thereof;
  • the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, benzene Tuximab (Brentuximab), Gemtuzumab (Gemtuzumab), Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab, or antigen binding fragments thereof.
  • the antibody conjugate has a structure shown in the following formula:
  • n is 4 to 8
  • n is a decimal or integer.
  • the present disclosure provides a method for preparing an antibody-drug conjugate, the antibody-drug conjugate having a structure shown in the following formula:
  • n is 4 to 8, and n is a decimal or integer;
  • the preparation method includes the following steps:
  • the antibody-drug conjugate has a structure shown in the following formula:
  • n is 4 to 8, and n is a decimal or integer;
  • the preparation method includes the following steps:
  • Step (c) includes subjecting the product of step (b) to purification by a cation chromatography column or an affinity chromatography column.
  • the histidine-hydrochloric acid buffer containing EDTA contains 20 mM histidine-hydrochloric acid buffer and 2.5 mM EDTA.
  • the present disclosure also provides an antibody drug conjugate or a pharmaceutically acceptable salt thereof, wherein the structure of the antibody drug conjugate is as shown in the general formula (Pc-L a -YD):
  • W is selected from a C 1-8 alkyl group, a C 1-8 alkyl group-C 3-7 cycloalkyl group, or a linear heteroalkyl group of 1 to 8 atoms
  • the linear heteroalkyl group contains 1 to 3 selected Heteroatoms from N, O and S, wherein the C 1-8 alkyl, C 3-7 cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxyl, cyano, One or more substituents of amino, C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy and C 3-7 cycloalkyl Replaced by
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and chemical bond, where p 1 is an integer from 1 to 20;
  • L 3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (Q), aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy and C 3-7 cycloalkyl group are substituted by one or more replace;
  • the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (Q), aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , C 1-6 alkyl,
  • R 1 is a halogenated C 1-6 alkyl group or a C 3-7 cycloalkyl group
  • R 2 is selected from a hydrogen atom, a halogenated C 1-6 alkyl group and a C 3-7 cycloalkyl group;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-7 cycloalkyl group
  • R 5 is selected from a hydrogen atom, a C 1-6 alkyl group, a halogenated C 1-6 alkyl group, a deuterated C 1-6 alkyl group and a hydroxy C 1-6 alkyl group;
  • R 6 and R 7 are the same or different, and are each independently selected from hydrogen atom, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl, and hydroxy C 1-6 alkyl;
  • n 0 or 1
  • n is 4 to 8, n is a decimal or integer
  • Pc is an antibody or an antigen-binding fragment thereof
  • the drug load distribution of the antibody drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%; preferably, the proportion of antibody heavy chains that bind 4 drugs is less than 4% And the proportion of the heavy chain of the antibody not bound to the drug is 6% or less.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs It is less than 5%.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs It is 4% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs 5% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 65% or more.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs 4% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 70% or more.
  • the present disclosure also provides an antibody drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody drug conjugate is prepared by the aforementioned preparation method of the antibody drug conjugate; and, the antibody drug
  • the drug load distribution of the conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%; preferably, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and unbound The proportion of the drug's antibody heavy chain is less than 6%.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs It is less than 5%.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs It is 4% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs 5% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 65% or more.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs 4% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 70% or more.
  • the present disclosure also provides an antibody drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody drug conjugate has a structure shown in the following formula:
  • n is 4 to 8, and n is a decimal or integer;
  • the antibody drug conjugate is prepared by the aforementioned preparation method of the antibody drug conjugate; and the drug load distribution of the antibody drug conjugate is: in the antibody heavy chain population, 4 drugs are combined
  • the proportion of antibody heavy chain is 4% or less; preferably, the proportion of antibody heavy chain that binds 4 drugs is 4% or less, and the proportion of antibody heavy chain that does not bind drug is 6% or less.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs It is less than 5%.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs It is 4% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 4%, and the proportion of antibody heavy chains that are not bound to drugs 5% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 65% or more.
  • the drug load distribution of the antibody-drug conjugate is: in the antibody heavy chain population, the proportion of antibody heavy chains that bind 4 drugs is less than 1%, and the proportion of antibody heavy chains that are not bound to drugs 4% or less; and in the antibody light chain population, the proportion of antibody light chains that bind one drug is 70% or more.
  • the present disclosure provides a preparation method that is more conducive to large-scale production. Specifically, the product obtained by the preparation method has a narrower drug load distribution, a lower free toxin content, and a higher yield.
  • Antibody drug conjugate connects antibodies or antibody fragments with biologically active cytotoxins or small molecule drugs with cell-killing activity through a stable chemical linker compound, making full use of antibodies to tumors. Cell specificity or high expression antigen cell binding specificity and high efficiency of cytotoxicity, to avoid toxic side effects on normal cells. Compared with traditional chemotherapy drugs in the past, antibody-drug conjugates can accurately bind tumor cells and reduce the impact on normal cells.
  • Buffering agent refers to a buffering agent that withstands changes in pH through the action of its acid-base conjugated component.
  • buffers that control the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, and fumaric acid. Salt, glycylglycine and other organic acid buffers.
  • Hetidine salt buffer is a buffer containing histidine ions.
  • histidine salt buffers include buffers such as histidine-hydrochloric acid, histidine-acetic acid, histidine-phosphate, histidine-sulfuric acid, etc., preferably histidine-hydrochloric acid buffer, histidine-acetic acid
  • the buffer is prepared from histidine and acetic acid
  • the histidine hydrochloric acid buffer is prepared from histidine and hydrochloric acid or histidine and histidine hydrochloride.
  • Phosphate buffer is a buffer that includes phosphate ions.
  • examples of the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like.
  • the preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • Acetate buffer is a buffer including acetate ions.
  • acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, calcium acetate acetate, acetic acid-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate.
  • the terms “about” and “approximately” mean that the index value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value partly depends on how it is measured or determined (ie, the limit of the measurement system). For example, in every practice in the art, “about” can mean within one or more than one standard deviation. Alternatively, “about” or “substantially comprising” can mean up to 20% of the range. Furthermore, especially for biological systems or processes, the term can mean at most an order of magnitude or at most 5 times the value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about” or “substantially comprising” should be assumed to be within the acceptable error range of the specific value.
  • immunoglobulin which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibodies described in the present disclosure are preferably specific antibodies against cell surface antigens on target cells.
  • Non-limiting examples are the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met Antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 Antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin- One or more of C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody; preferably trastuzumab (trastuzumab, trade name Hercept
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment described in the present disclosure comply with the known Kabat numbering rules (LCDR1-3, HCDR1-3).
  • the antibody light chain described in the present disclosure may further include a light chain constant region, and the light chain constant region includes human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain described in the present disclosure may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
  • murine-derived antibody in the present disclosure refers to the preparation of antibodies with mice based on knowledge and skills in the art. During preparation, a test subject is injected with a specific antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed, and convert the mouse variable region
  • the gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germlines The antibody produced in the antibody framework sequence. It can overcome the strong heterogeneous reaction induced by the chimeric antibody because it carries a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation for CDR by phage display.
  • naked antibody refers to an antibody that is not conjugated to a heterologous moiety (such as a cytotoxic moiety) or a radioactive label.
  • the "antigen-binding fragment of an antibody” mentioned in the present disclosure may refer to Fab fragments, Fab' fragments, F(ab') 2 fragments, and Fv fragments and scFv fragments that bind to antigens.
  • the Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies into a polypeptide chain, which is called single chain antibody or single chain Fv (sFv).
  • antigen-binding site in the present disclosure refers to a continuous or discontinuous three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • the “ADCC” mentioned in this disclosure that is, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors directly kill the antibody-covered cells by recognizing the Fc segment of the antibody.
  • the target cell is.
  • the ADCC effect function of the antibody can be reduced or eliminated by modifying the Fc section of IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, and F234A/L235A mutation of IgG4.
  • the “mutation” in the mutant sequence described in this disclosure includes but is not limited to "back mutation", “conservative modification” or “conservative substitution or substitution”.
  • the “conservative modification” or “conservative substitution or substitution” mentioned in this disclosure refers to the substitution of other amino acids in proteins with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.)
  • the amino acids that make it possible to make frequent changes without changing the biological activity of the protein are known that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)).
  • the substitution of amino acids with similar structure or function is unlikely to disrupt biological activity.
  • the "mutated sequence” mentioned in the present disclosure refers to the nucleotide sequence and/or amino acid sequence of the present disclosure when the nucleotide sequence and/or amino acid sequence of the present disclosure is subjected to mutation modification such as appropriate substitutions, insertions, or deletions.
  • the amino acid sequence has a nucleotide sequence and/or amino acid sequence with different percent sequence identity.
  • the sequence identity described in this disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Sequence comparison and determination of the percent identity between two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • linker unit or “linking fragment” or “linking unit” refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and a drug at the other end. It can also be connected to other linkers before being connected to the antibody or drug. Connected.
  • Linkers including extensions, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker may be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers such as hydrazone
  • protease-sensitive such as peptidase-sensitive
  • light-labile linkers dimethyl linkers
  • disulfide-containing linkers Charged et al., Cancer Research 52: 127-131 ( 1992); U.S. Patent No. 5,208,020).
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification.
  • the bound antibody was eluted by the PH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably containing 1 to 10 carbons The most preferred is an alkyl group containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-Dimethylpropyl, 2,2-Dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms More preferred are lower alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.
  • Alkyl groups may be substituted or unsubstituted.
  • substituents When substituted, substituents may be substituted at any available attachment point.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkanes Group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O, and S, where the alkyl group is as defined above.
  • alkylene refers to a saturated linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is A straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, more preferably an alkylene group containing 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 -) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -) Wait.
  • the alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently optionally selected from alkyl, alkenyl, alkynyl , Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , Cycloalkylthio, heterocycloalkylthio and oxo groups are substituted by one or more substituents.
  • alkoxy refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where the definition of alkyl or cycloalkyl is as described above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 Carbon atoms, most preferably 3 to 7 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer of 0 to 2) heteroatoms, but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • spiroheterocyclic group refers to a polycyclic heterocyclic group that shares one atom (called a spiro atom) between 5- to 20-membered monocyclic rings, in which one or more ring atoms are selected from nitrogen, oxygen or S(O ) Heteroatoms of m (where m is an integer of 0 to 2), and the remaining ring atoms are carbon. It can contain one or more double bonds, but none of the rings have a fully conjugated ⁇ -electron system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spiro heterocyclic group is classified into a single spiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a single spiro heterocyclic group and a dispiro heterocyclic group. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospiro heterocyclic group.
  • Non-limiting examples of spiroheterocyclic groups include:
  • fused heterocyclic group refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system.
  • One or more rings may contain one or more Double bond, but none of the rings have a fully conjugated ⁇ -electron system, where one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), and the rest of the ring
  • the atom is carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.
  • fused heterocyclic groups include:
  • bridged heterocyclic group refers to a 5- to 14-membered polycyclic heterocyclic group with any two rings sharing two atoms that are not directly connected. It may contain one or more double bonds, but none of the rings has a complete common A conjugated ⁇ -electron system in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer of 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • bridged heterocyclic groups include:
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, non-limiting examples of which include:
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio and oxo.
  • aryl refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) with a conjugated ⁇ -electron system, preferably 6 to 10 members, such as benzene And naphthyl, preferably phenyl.
  • the aryl ring may be fused on a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples thereof include:
  • the aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycle Alkylthio.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen.
  • Heteroaryl groups are preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrakis Azole and so on.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • Heteroaryl groups may be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio And heterocycloalkylthio.
  • amino protecting group is to keep the amino group unchanged when other parts of the molecule react, and to protect the amino group with a group that is easy to remove.
  • Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.
  • cycloalkylalkyl refers to an alkyl group substituted with one or more cycloalkyl groups, preferably with a cycloalkyl group, where alkyl is as defined above and where cycloalkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, where the alkyl group is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, where the alkyl group is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • cyano refers to -CN.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of each other, substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • drug loading refers to the average amount of cytotoxic drug loaded on each antibody or antigen-binding fragment thereof in ADC, and can also be expressed as the ratio of drug amount to antibody amount, which is Integer or decimal.
  • the drug loading amount is expressed as n, which may be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 as an example.
  • Conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC can be used to identify the average number of drugs per ADC molecule after the coupling reaction.
  • antibody drug load distribution refers to the distribution of antibodies connected to different numbers of drugs in an antibody-drug conjugate population, for example, the distribution of antibodies connected to 0, 2, 4, 6, and 8 drugs in the population. It is worth noting that the DAR of 1, 3, 5 and 7 may also be included in the mixture due to the possible generation of degradation products.
  • antibody drug load distribution can be characterized by antibody heavy chains that bind different amounts of drugs, for example: H 0 represents the heavy chain of unbound drug, H 1 represents the heavy chain that binds one drug, and H 2 represents the heavy chain that binds two drugs. For the heavy chain of a drug, H 3 represents a heavy chain that binds three drugs, and H 4 represents a heavy chain that binds four drugs.
  • Exemplary heavy chain ratio H 3 which represents a ratio of 4% in the population of heavy chain of the antibody drug conjugate, in combination with three drugs is 4%.
  • the antibody drug load distribution in the present disclosure can also be characterized by antibody light chains that bind different amounts of drugs, L 0 represents an antibody light chain that does not bind a drug, and L 1 represents an antibody light chain that binds a drug.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition containing any one of the binding compounds of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, so as to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may be ineffective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • an "effective amount” includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Replacement refers to the replacement of the solvent system that dissolves the antibody protein.
  • the high salt or hypertonic solvent system containing the antibody protein is replaced by physical manipulation using a buffer system of a stable formulation, so that the antibody protein exists in the stable formulation.
  • the so-called physical operation methods include but are not limited to ultrafiltration, dialysis or centrifugation followed by reconstitution.
  • FIG 1A Plasma stability test results of ADC-19 of the present disclosure.
  • FIG. 1B Plasma stability test results of ADC-18 of the present disclosure.
  • Figure 1C The results of the plasma stability experiment of ADC-20 of the present disclosure.
  • Figure 2 Evaluation of the efficacy of ADC-21 and ADC-24 of the present disclosure on JIMT-1 tumor-bearing mice.
  • Figure 3 Evaluation of the efficacy of ADC of the present disclosure on human breast cancer cell SK-BR-3 transplanted tumor nude mice.
  • Figure 4 The results of the plasma stability experiment of ADC-25 of the present disclosure.
  • Figure 5 The therapeutic effect of ADC of the present disclosure on human brain astroblastoma U87MG transplanted tumor in nude mice.
  • Figure 6 The efficacy of ADC of the present disclosure on human pharyngeal carcinoma pleural effusion metastasis cell Detroit 562 transplanted tumor in nude mice.
  • Figure 7 The therapeutic effect of ADC of the present disclosure on human glioma U87MG transplanted tumor in nude mice.
  • the experimental methods that do not indicate specific conditions in the embodiments of this disclosure usually follow the conventional conditions or the conditions recommended by the raw material or commodity manufacturers.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR is measured with Bruker AVANCE-400 nuclear magnetic instrument, the solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard is tetramethyl sulfoxide (DMSO-d6) Based on silane (TMS), the chemical shift is given in units of 10 -6 (ppm).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • HPLC determination uses Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150 ⁇ 4.6mm chromatographic column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 ⁇ 4.6mm chromatographic column).
  • the UV-HPLC measurement uses a Thermonanodrop2000 ultraviolet spectrophotometer.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. ⁇ 0.5mm silica gel plate.
  • the known starting materials of the present disclosure can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Companies such as Darui Chemicals.
  • the reactions are all carried out under an argon atmosphere or a nitrogen atmosphere.
  • the argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenator.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction uses the CEM Discover-S 908860 microwave reactor.
  • the solution in the reaction refers to an aqueous solution.
  • reaction temperature is room temperature.
  • Room temperature is the most suitable reaction temperature, and the temperature range is 20°C to 30°C.
  • the preparation of the PBS buffer with pH 6.5 in the examples: take KH 2 PO 4 8.5g, K 2 HPO 4 .3H 2 O 8.56g, NaCl 5.85g, and EDTA 1.5g in a bottle, dilute the volume to 2L, and ultrasonic Dissolve all of it, shake well and get it.
  • the eluent system of column chromatography and the developing solvent system of thin-layer chromatography used to purify compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and In the ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.
  • Q-TOF LC/MS uses Agilent 6530 accurate mass quadrupole-time-of-flight mass spectrometer and Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 ⁇ m, 2.1 ⁇ 75mm column).
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 2 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc), B- Acetonitrile, gradient elution, flow rate: 18 mL/min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product (2-A: 1.5 mg, 2-B: 1.5 mg).
  • reaction was quenched by adding 5 mL of water to the reaction solution, the reaction solution was extracted with ethyl acetate (10 mL ⁇ 3), the organic phases were combined, washed with saturated sodium chloride solution (5 mL ⁇ 2), and the organic phase was dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with the developing solvent system B to obtain the title product 4 (2.5 mg, yield: 80.9%).
  • the benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104mg, 0.54mmol; prepared by the method disclosed in the patent application "US2005/20645”) and 2-((((9H-fluoren-9-yl) Methoxy) carbonyl) amino) acetamido) methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in the patent application "CN105829346A”) was added to the reaction flask, 5 mL of tetrahydrofuran was added, argon replaced three times, ice The temperature of the water bath was cooled to 0-5°C, potassium tert-butoxide (61mg, 0.54mmol) was added, the ice bath was removed, and the temperature was raised to room temperature and stirred for 10 minutes.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product 8 (2mg, yield: 39.0%).
  • the obtained residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, extracted with ethyl acetate (10 mL ⁇ 3), the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with the developing solvent system C to obtain the title product 9b (48 mg, yield: 19%).
  • Dissolve 9d (19 mg, 22.6 ⁇ mol) in 2 mL of dichloromethane, add 1 mL of diethylamine, and stir at room temperature for 2 hours.
  • the reaction solution was concentrated under reduced pressure, 1 mL of toluene was added and concentrated under reduced pressure, repeated twice.
  • the solid residue was concentrated under reduced pressure and pulled dry by an oil pump to obtain the crude title product 9e (17 mg), which was directly used in the next reaction without purification.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4 mg, 9-B: 1.7 mg).
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc) B-acetonitrile, gradient elution, flow rate: 18 mL/min ), the corresponding components were collected and concentrated under reduced pressure to obtain the title product (2.7mg, 2.6mg).
  • Solid sodium chloride was added to the reaction solution to saturate the aqueous phase, extracted with ethyl acetate (8mL ⁇ 8), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title product 12b (0.45g, yield: 89.3%) .
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 12 was purified by high performance liquid chromatography (separation conditions: chromatographic column: Sharpsil-T C18 5 ⁇ m 21.2*250mm; mobile phase: A-water (10mmol NH 4 OAc), B- Acetonitrile, gradient elution, flow rate: 18 mL/min), to obtain the title product (7 mg, 15 mg).
  • Example 1-13 (reference example)
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min) to obtain the title product (2mg, 2mg).
  • Dissolve 15d (19 mg, 22.56 ⁇ mol) in 2 mL of dichloromethane, add 1 mL of diethylamine, and stir at room temperature for 1.5 hours.
  • the reaction solution was concentrated under reduced pressure at 0°C, 1 mL of toluene was added, and concentrated under reduced pressure, repeated twice; 3 mL of n-hexane was added to make a slurry, and the upper layer of n-hexane was poured out, repeated three times; concentrated under reduced pressure to obtain the crude title product 15e (13.9 mg), the product It was used directly in the next reaction without purification.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), the corresponding components were collected, and concentrated under reduced pressure to obtain the title product 15 (2.5 mg, yield: 10.3%).
  • Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250mg, 1.58mmol, supplier Alfa) was dissolved in methanol (2mL) and water (1mL), sodium hydroxide (126mg, 3.15mmol) was added, and the temperature was increased. The temperature was raised to 40°C, and the reaction was stirred for 3 hours. Cool to normal temperature, concentrate under reduced pressure to remove the organic solvent, back-extract with ether (10 mL), and collect the aqueous phase. The aqueous phase was adjusted to pH 3-4 with 6N aqueous hydrochloric acid, and concentrated under reduced pressure to obtain a solid. Add 3 mL of toluene, concentrate under reduced pressure and spin dry, repeat three times. The oil pump was pulled dry to obtain the crude title product 16b (206 mg), which was directly used in the next reaction without purification.
  • the crude product 16b (206mg, 1.58mmol) was dissolved in acetonitrile (15mL), anhydrous potassium carbonate (1.09g, 7.90mmol) and tetrabutylammonium iodide (29mg, 78.51 ⁇ mol) were added, and benzyl bromide (216mg, 1.26 mmol), stirring at room temperature overnight. After filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with a developing solvent system C to obtain the title product 16c (112 mg, yield: 32.1%).
  • the obtained residue was dissolved in 3mL 1,4-dioxane, 0.5mL water was added, sodium bicarbonate (27mg, 0.32mmol) and 9-fluorene methyl chloroformate (71mg, 0.27mmol) were added, and the mixture was stirred at room temperature for 1 Hour.
  • the obtained residue was purified by C to obtain the title product 16d (24 mg, yield: 16.7%).
  • 17f (122 mg, 0.163 mmol) was dissolved in 0.8 mL of dichloromethane, 0.4 mL of trifluoroacetic acid was added, and the reaction was stirred at room temperature for 1 hour.
  • Add 15mL of dichloromethane to dilute and concentrate under reduced pressure; add 10mL of n-hexane, concentrate under reduced pressure and repeat twice; add 10mL of toluene and concentrate under reduced pressure; beat three times with a mixed solvent of 10mL of n-hexane: ether 5:1 to reach pH It was close to 7, concentrated and pumped to dryness by an oil pump to obtain 17 g (98 mg, yield: 86.8%) of the title product.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), the corresponding components were collected, and concentrated under reduced pressure to obtain the title product 17 (6 mg, yield: 27.4%).
  • Dissolve 8b (3.1g, 8.41mmol) in tetrahydrofuran (55mL), add 18a (2.0g, 9.70mmol), cool to 0 ⁇ 5°C in an ice-water bath, add potassium tert-butoxide (1.89g, 16.84mmol), ice Stir under water bath for 10 minutes.
  • ethyl acetate (30 mL) and water (20 mL)
  • stand still and separate into layers the aqueous phase is extracted with chloroform (30 mL ⁇ 5), and the organic phases are combined.
  • reaction solution was filtered and purified by high performance liquid chromatography (Separation conditions: Column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; Mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18 mL/min), the corresponding components were collected, and concentrated under reduced pressure to obtain the title product 18 (9.5 mg, yield: 56.2%).
  • the crude product 19b (230mg, 541.8 ⁇ mol) was dissolved in 7mL of dichloromethane, and 2,4-dimethoxybenzyl alcohol (136.7mg, 812.7 ⁇ mol), 1-ethyl-(3-dimethylaminopropyl) were added in sequence.
  • Base carbodiimide hydrochloride (155 mg, 808.5 ⁇ mol) and 4-dimethylaminopyridine (6.6 mg, 53.5 ⁇ mol) were stirred at room temperature for 16 hours.
  • the reaction solution was diluted with 10 mL of dichloromethane, washed with water (10 mL ⁇ 1), saturated brine (10 mL ⁇ 2), dried with anhydrous sodium sulfate, filtered and concentrated to obtain a crude product.
  • the residue obtained was purified by thin layer chromatography with the developing solvent system B to obtain the title product 19c (159 mg, yield: 51.0%)
  • reaction solution was filtered and purified by high performance liquid chromatography (Separation conditions: Column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; Mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18 mL/min), the corresponding components were collected and concentrated under reduced pressure to obtain the title product 19 (2.1 mg, yield: 32.4%).
  • the vector can be constructed, transfected into eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019), and obtained after expression and purification.
  • Embodiment 1-22 ADC-2
  • the compound 10-longer retention time compound (2.1 mg, 2.02 ⁇ mol) was dissolved in 0.10 mL DMSO, added to the above 2.0 mL solution, placed in a water bath shaker, and reacted with shaking at 25° C. for 3 hours to stop the reaction.
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-2 of the general formula of FADC-1 Solution (4.95mg/mL, 1.1mL), stored at 4°C.
  • Dissolve compound 9-shorter retention time compound 9-A (1.0mg, 0.93 ⁇ mol) in 0.10mL DMSO, add to the above 1.3mL solution, place in a water bath shaker, shake at 25°C and react for 3 hours to stop the reaction .
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-4 of FADC-4A general formula Solution (1.72mg/mL, 2.36mL), stored at 4°C.
  • Dissolve compound 9-shorter retention time compound 9-A (0.5mg, 0.42 ⁇ mol) in 0.031mL DMSO, add to the above 0.614mL solution, place in a water bath shaker, shake at 25°C and react for 3 hours, stop reaction.
  • the reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffered aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-5 of the general formula of FADC-4A Solution (3.08mg/mL, 0.82mL), stored at 4°C.
  • Dissolve compound 9-longer retention time compound 9-B (0.68mg, 0.63 ⁇ mol) in 0.10mL DMSO, add to the above 0.75mL solution, place in a water bath shaker, shake at 25°C for 3 hours to stop the reaction .
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-6 of the general formula of FADC-4B Solution (1.78mg/mL, 1.78mL), stored at 4°C.
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-9 of FADC-9A general formula Solution (2.27mg/mL, 1.11mL), stored at 4°C.
  • Dissolve compound 14-shorter retention time compound (0.64 mg, 588 nmol) in 40 ⁇ l DMSO, add it to the above reaction solution, place it in a water bath shaker, and shake the reaction at 25° C. for 3 hours to stop the reaction.
  • the reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-10 of the general formula of FADC-10 Solution (5.48mg/mL, 1.03mL), stored at 4°C.
  • UV-Vis calculated average value: n 6.25.
  • Dissolve compound 14-longer retention time compound (0.72 mg, 662 nmol) in 40 ⁇ l DMSO, add it to the above reaction solution, place it in a water bath shaker, and shake the reaction at 25° C. for 3 hours to stop the reaction.
  • the reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffered aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-11 of the general formula of FADC-10 Solution (2.13mg/mL, 1.87mL), stored at 4°C.
  • UV-Vis calculated average value: n 7.03.
  • UV-Vis calculated average value: n 6.93.
  • UV-Vis calculated average value: n 6.53.
  • UV-Vis calculated average value: n 7.61.
  • UV-Vis calculated average: n 7.89.
  • Embodiment 1-36 ADC-16
  • UV-Vis calculated average value: n 7.43.
  • UV-Vis calculated average value: n 5.42.
  • UV-Vis calculated average value: n 7.23.
  • Dissolve compound 9-shorter retention time compound 9-A (2.0 mg, 1862nmol) in 100 ⁇ l DMSO, add it to the above reaction solution, place it in a water bath shaker, and shake the reaction at 25°C for 3 hours to stop the reaction.
  • the reaction solution was desalted and purified with Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffered aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-19 of FADC-4A general formula Solution (0.73mg/mL, 13.0mL), stored at 4°C.
  • UV-Vis calculated average value: n 6.26.
  • the compound 10-longer retention time compound (2.0 mg, 1815 nmol) was dissolved in 100 ⁇ l DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25° C. for 3 hours to stop the reaction.
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the PBS buffer of the exemplary product ADC-20 of the general formula of FADC-1 Solution (0.73mg/mL, 13.0mL), stored at 4°C.
  • UV-Vis calculated average value: n 7.43.
  • UV-Vis calculated average value: n 7.23.
  • UV-Vis calculated average value: n 6.79.
  • UV-Vis calculated average value: n 7.05.
  • UV-Vis calculated average value: n 7.07.
  • UV-Vis calculated average value: n 6.87.
  • UV-Vis calculated average value: n 6.15.
  • UV-Vis calculated average value: n 6.11.
  • UV-Vis calculated average value: n 7.46.
  • UV-Vis calculated average value: n 7.24.
  • UV-Vis calculated average value: n 6.15.
  • UV-Vis calculated average value: n 6.33.
  • ADC stock solution is a kind of antibody cross-linked drug.
  • the mechanism of its treatment of diseases is to rely on the targeting of antibodies to transport toxin molecules into cells, thereby killing the cells.
  • the load of the drug plays a decisive role in the efficacy of the drug.
  • the drug load of ADC stock solution was determined by UV method.
  • UV spectrophotometry using instrument: Thermonanodrop2000 UV spectrophotometer
  • the principle is that the total absorbance of ADC stock solution at a certain wavelength is equal to that of cytotoxic drugs and monoclonal antibodies at this wavelength.
  • the sum of the absorbance value namely:
  • a 280nm ⁇ mab-280 bC mab + ⁇ Drug-280 bC Drug
  • ⁇ Drug-280 The average molar extinction coefficient of the drug at 280nm is 5100;
  • ⁇ mab-280 Trastuzumab single antigen solution or Pertuzumab single antigen solution has an average molar extinction coefficient of 214600 at 280nm;
  • C mab concentration of trastuzumab single antigen solution or pertuzumab single antigen solution;
  • the optical path length is 1 cm.
  • a 370nm ⁇ mab-370 bC mab + ⁇ Drug-370 bC Drug
  • ⁇ Drug-370 The average molar extinction coefficient of the drug at 370nm is 19000;
  • ⁇ mab-370 The extinction coefficient of trastuzumab single antigen solution or Pertuzumab single antigen solution at 370nm is 0;
  • C mab concentration of trastuzumab single antigen solution
  • the optical path length is 1 cm.
  • the drug loading can be calculated.
  • Drug load C Drug /C mab .
  • Test Example 1-1 Inhibition test of the compound of the present disclosure on tumor cell proliferation in vitro
  • the purpose of this experiment is to detect the inhibitory activity of the disclosed pharmaceutical compounds on the in vitro proliferation of U87MG cells (Cell Bank of Chinese Academy of Sciences, Catalog # TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Item No. HTB-30) .
  • the cells were treated in vitro with different concentrations of compounds, and after 6 days of culture, CTG ( Luminescent Cell Viability Assay, Promega, article number: G7573)
  • the reagent detects the proliferation of cells, and evaluates the in vitro activity of the compound based on the IC50 value.
  • the following takes the in vitro proliferation inhibition test method of U87MG cells as an example to illustrate the method in this disclosure for testing the in vitro proliferation inhibition activity of the compound of the present disclosure on tumor cells. This method is also applicable to, but not limited to, the in vitro proliferation inhibitory activity test on other tumor cells.
  • Cell plating mix the U87MG and SK-BR-3 single cell suspensions, adjust the viable cell density to 2.75 ⁇ 10 3 cells/mL and 8.25 ⁇ 10 3 cells/mL with cell culture medium, and adjust the density
  • the cell suspension was mixed and added to a 96-well cell culture plate at 180 ⁇ L/well. Only 200 ⁇ L of culture medium was added to the peripheral wells of the 96-well plate.
  • the culture plate was cultured in an incubator for 24 hours (37°C, 5% CO 2 ).
  • the initial concentration of the small molecule compound is 500 nM, and the dispensing method is as follows.
  • the initial concentration of ADC is 10 nM or 500 nM, and the dispensing method is as follows.
  • the first column of the 96-well plate add 100 ⁇ L of different samples to be tested, with a sample concentration of 100 nM or 5 ⁇ M; add 100 ⁇ L of PBS to each well of the second column to the 11th column. Take 50 ⁇ L of the sample from the first column to 100 ⁇ L of PBS in the second column, mix well, take 50 ⁇ L to the third column, and so on, dilute it 3 times to the 10th column.
  • Sample addition operation add 20 ⁇ L of the sample to be tested with different concentrations in the culture plate, each sample has two duplicate wells.
  • the culture plate was incubated in an incubator for 6 days (37°C, 5% CO 2 ).
  • Color development operation Take out the 96-well cell culture plate, add 90 ⁇ L CTG solution to each well, and incubate at room temperature for 10 minutes.
  • Reading operation Take out the 96-well cell culture plate, place it in the microplate reader (BMG labtech, PHERAstar FS), and measure the chemiluminescence with the microplate reader.
  • the small molecule fragments in the present disclosure have obvious proliferation inhibitory activity on SK-BR-3 cells and U87 cells, and the chiral center has a certain effect on the inhibitory activity of the compound.
  • Test Example 1-2 In vitro proliferation inhibition test of the antibody-drug conjugate of the present disclosure on HER2 target tumor cells
  • the purpose of this experiment is to detect the antibody-drug conjugates of the present disclosure against the HER2 target, against SK-BR-3 (human breast cancer cells, ATCC, article number HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, article number HTB-132) in vitro proliferation inhibitory activity.
  • SK-BR-3 human breast cancer cells, ATCC, article number HTB-30
  • MDA-MB-468 human breast cancer cells, ATCC, article number HTB-132
  • the test cells are SK-BR-3 and MDA-MB-468, and the cell culture medium is McCoy's 5A medium (Gibco, article number 16600-108) containing 10% FBS, containing 10% FBS.
  • EMEM medium GE, article number SH30024.01
  • L-15 medium ThermoFisher, article number 11415-114) containing 10% FBS.
  • the three cell culture medium with cells were viable cell density adjusted to 8.33 ⁇ 10 3 cells /mL,8.33 ⁇ 10 3 cells / mL and 1.39 ⁇ 10 4 cells / mL, after the cell suspension density adjusted mix Evenly, add 180 ⁇ L/well to 96-well cell culture plate. Related compounds were tested, and the results obtained are shown in Table 2 below.
  • the antibody-drug conjugates of the present disclosure against HER2 target have obvious proliferation inhibitory activity on HER2 positive cells SK-BR-3; at the same time, they have weak proliferation inhibitory activity on HER2 negative cells MDA-MB-468; they have good selection sex.
  • Test Example 1-3 Her2-ADC plasma stability test
  • the ADC-19 sample, ADC-18 sample, ADC-20 sample, human plasma, monkey plasma (Shanghai Medicilon Biopharmaceutical Co., Ltd.), and 1% BSA (Shanghai Shenggong) were used 0.22
  • the ⁇ m filter is filtered and sterilized.
  • ADC-18 has poor stability in human and monkey plasma, and the highest release rates of free toxins are 14.5% and 8.10%, respectively. It is relatively stable in 1% BSA PBS solution, as shown in Figure 1B.
  • ADC-20 has poor stability in human plasma, monkey plasma and 1% BSA PBS solution, and the highest release rates of free toxins are 21.7%, 29.7%, and 21.7%, respectively. , And has been in a degraded state in 1% BSAPBS solution, as shown in Figure 1C.
  • Test Example 1-4 Evaluation of the efficacy of JIMT-1 tumor-bearing mice
  • ADC-21 3mg/kg
  • ADC-21 10mg/kg
  • ADC-24 3mg/kg
  • ADC-24 10mg/kg
  • JIMT-1 cells (Nanjing Kebai) (5 ⁇ 10 6 /mouse, with 50% artificial basement membrane) were subcutaneously inoculated into the right ribs of mice. The tumors grew for 8 days and grew to 203.09 ⁇ 11.94mm 3 and the animals were randomly divided into groups (d1), 8 animals/group, 6 groups in total.
  • V tumor volume
  • Relative volume (RTV) V T /V 0
  • Tumor inhibition rate (%) (C RTV -T RTV )/C RTV (%)
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • C RTV and T RTV are the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively.
  • T-DM1 10mg/kg has no inhibitory effect on tumors; ADC-21, 3mg/kg tumor inhibition rate is 46.22% (P ⁇ 0.01); ADC-21, 10mg/kg tumor inhibition rate is 56.77% (P ⁇ 0.001) ); ADC-24, 3mg/kg tumor inhibition rate 62.77% (P ⁇ 0.001); ADC-24, 10mg/kg tumor inhibition rate 76.32% (P ⁇ 0.001).
  • ADC-24's anti-tumor effect is significantly better than ADC-21.
  • Test Example 1-5 Evaluation of the efficacy of SK-BR-3 tumor-bearing mice
  • Nunu nude mice were used as test animals to evaluate the efficacy of Her2-ADC antibody ADC-21 and ADC-22 after intraperitoneal injection on human breast cancer cell SK-BR-3 transplanted tumor nude mice.
  • ADC-21 1mg/kg
  • ADC-21 6mg/kg
  • ADC-22 1mg/kg
  • ADC-22 6mg/kg
  • SK-BR-3 cells (ATCC) (5 ⁇ 10 6 /mouse, with 50% artificial basement membrane) were subcutaneously inoculated into the right ribs of mice. The tumors grew for 20 days and grew to 153.34 ⁇ 11.73 mm 3 and the animals were randomly divided into groups (d0), 8 per group, 5 groups in total.
  • V tumor volume
  • Relative volume (RTV) V T /V 0
  • Tumor inhibition rate (%) (C RTV -T RTV )/C RTV (%)
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • C RTV and T RTV are the relative tumor volumes of the blank control and the experimental group at the end of the experiment, respectively.
  • the results of the experiment are shown in Figure 3.
  • the intraperitoneal injection was administered once, and the experiment ended at the 28th day.
  • the tumor inhibition rate of ADC-21 1mg/kg was 15.01%; the tumor inhibition rate of ADC-21 6mg/kg was 77.4%, and Compared with the blank control, there is a very significant difference (P ⁇ 0.001).
  • the tumor inhibition rate of ADC-22 1mg/kg was 19.82%; the tumor inhibition rate of ADC-22 6mg/kg was 98.38% (P ⁇ 0.001).
  • the anti-tumor effect of ADC-22 is also significantly better than that of ADC-21.
  • sample ADC-25 was mixed with human plasma, monkey plasma, and 1% BSA PBS solution at a final concentration of 100 ⁇ g/mL, filtered and sterilized, and incubated in a 37°C water bath. The day of incubation was recorded as On day 0, samples were taken out on day 7, day 14 and day 21, respectively, for free toxin detection.
  • ADC-25 is quite stable in human and monkey plasma, as well as 1% BSA PBS solution, the release rate of free toxin does not exceed 2%, and it stabilizes on the 14th day.
  • Test Example 1-7 Evaluation of the therapeutic effect of ADC on human brain astroblastoma U87MG transplanted tumor in nude mice
  • BALB/cA-nude nude mice were used as test animals to evaluate the efficacy of the ADC compound disclosed in this disclosure on human brain astroblastoma U87MG transplanted tumors in nude mice.
  • BALB/cA-nude nude mice purchased from Shanghai Jiesjie Laboratory Animal Co., Ltd.
  • a and b represent length and width respectively.
  • Relative volume (RTV) V T /V 0
  • Tumor inhibition rate (%) (C RTV -T RTV )/C RTV (%)
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • C RTV and T RTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.
  • Intraperitoneal injection was administered once a week for a total of 3 times.
  • the tumor inhibition rate of ADC-27 3mg/kg reached 63.3% (P ⁇ 0.0001);
  • ADC-26 3mg/kg The inhibition rate reached 49.1%.
  • ADC-27 shows stronger anti-tumor efficacy than ADC-26.
  • the tested antibodies can effectively inhibit the growth of U87MG transplanted tumors in tumor-bearing nude mice, and showed a dose-dependent manner.
  • Test Example 1-8 Evaluation of the efficacy of ADC on Detroit 562 transplanted tumors in nude mice
  • BALB/cA-nude nude mice were used as test animals to evaluate the efficacy of the ADC compound disclosed in this disclosure on human pharyngeal carcinoma pleural fluid metastasis cells Detroit 562 nude mice transplanted tumors.
  • Negative control ADC (3mg/kg): non-B7H3 target antibody conjugated with compound 20 to form an antibody-drug conjugate.
  • BALB/cA-nude nude mice purchased from Changzhou Cavins Laboratory Animal Co., Ltd.
  • a and b represent length and width respectively.
  • Relative volume (RTV) V T /V 0
  • Tumor inhibition rate (%) (C RTV -T RTV )/C RTV (%)
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • CRTV and TRTV are the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment, respectively.
  • ADC-29 The tumor inhibition rate of 3mg/kg (3mpk) reached 72.27% (P ⁇ 0.001 ); ADC-28 3mg/kg (3mpk) tumor inhibition rate reached 56.2% (P ⁇ 0.001). ADC-29 all show stronger anti-tumor efficacy than ADC-28.
  • Test Example 1-9 Evaluation of U87-MG Tumor-bearing Mice
  • Balb/c nude mice were used as test animals to evaluate the efficacy of B7H3-antibody drug conjugate after intraperitoneal injection on its human glioma cell U87MG transplantation tumor model.
  • BALB/cA-nude nude mice purchased from Shanghai Slack Laboratory Animal Co., Ltd.

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Abstract

本发明公开了一种抗体药物偶联物的制备方法，包括其合成和纯化步骤。

Description

一种抗体药物偶联物的制备方法
本申请要求2020年3月25日提交的中国专利申请(申请号CN 202010219311.2)和2021年3月19日提交的中国专利申请(申请号CN 202110297397.5)的优先权。
技术领域
本披露涉及一类抗体药物偶联物的制备方法，尤其涉及一类抗体药物偶联物的制备方法的合成步骤和纯化步骤。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
化疗依然是包括手术、放疗、以及靶向治疗法在内的最重要的抗癌手段之一。尽管高效细胞毒性药物的种类很多，但是肿瘤细胞和正常细胞之间差别很小，限制了这些抗肿瘤化合物由于毒副作用在临床上的广泛应用。而抗肿瘤单克隆抗体对于肿瘤细胞表面抗原具有特异性，抗体药物已成为抗肿瘤治疗的前线药物，但单独使用抗体作为抗肿瘤药物时，疗效经常不尽人意。
抗体药物偶联物(antibody drug conjugate，ADC)将单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒性药物相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性药物的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响(Mullard A,(2013)Nature Reviews Drug Discovery,12:329–332；DiJoseph JF,Armellino DC,(2004)Blood,103:1807-1814)。
2000年第一个抗体药物偶联物Mylotarg(吉妥珠单抗奥唑米星(gemtuzumab ozogamicin)，惠氏制药有限公司)被美国FDA批准上市，用于治疗急性髓细胞白血病(Drugs of the Future(2000)25(7):686；US4970198；US 5079233；US 5585089；US 5606040；US 5693762；US 5739116；US 5767285；US 5773001)。
2011年8月，
Figure PCTCN2021083014-appb-000001
(brentuximab vedotin，西雅图基因遗传公司)通过美国FDA快速审评通道，用于治疗霍奇金淋巴瘤以及复发性间变性大细胞淋巴瘤(Nat.Biotechnol(2003)21(7):778-784；WO2004010957；WO2005001038；US7090843；US7659241；WO2008025020)。
Figure PCTCN2021083014-appb-000002
是一种新型的ADC药物，能使药物直接作用于淋巴瘤细胞上的靶点CD30后发生内吞作用从而诱导肿瘤细胞的凋亡。
Mylotarg和Adcetris都是针对血液肿瘤进行靶向治疗，血液肿瘤和实体肿瘤相比组织结构相对简单。2013年2月，Kadcyla(ado-trastuzumab emtansine，T-DM1)获得美国FDA批准，用于治疗HER2阳性同时对曲妥珠单抗(Tratuzumab， 商品名：Herceptin)和紫杉醇有抗药性的晚期或转移性乳腺癌患者(WO2005037992；US8088387)。Kadcyla是美国FDA批准的治疗实体肿瘤的第一个ADC药物。
用于抗体药物偶联物的具有细胞毒性的小分子有几类，其中有一类是喜树碱衍生物，它们通过抑制拓扑异构酶I而具有抗肿瘤作用。报道喜树碱衍生物依沙替康(化学名：(1S,9S)-1-氨基-9-乙基-5-氟-2,3-二氢-9-羟基-4-甲基-1H,12H-苯并[de]吡喃并[3’,4’:6,7]咪唑并[1,2-b]喹啉-10,13(9H,15H)-二酮)应用于抗体药物偶联物(ADC)的文献有WO2014057687；Clinical Cancer Research(2016)22(20)：5097-5108；Cancer Sci(2016)107:1039-1046。但仍需进一步开发疗效更好的ADC药物。
发明内容
本披露提供一种抗体药物偶联物的制备方法，其中所述抗体药物偶联物的结构如通式(Pc-L a-Y-D)所示：
Figure PCTCN2021083014-appb-000003
其中：
W选自C 1-8烷基、C 1-8烷基-C 3-7环烷基或1至8个原子的直链杂烷基，所述直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 3-7环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(Q)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
R 1为卤代C 1-6烷基或C 3-7环烷基；
R 2选自氢原子、卤代C 1-6烷基和C 3-7环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-7环烷基；
R 5选自氢原子、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和羟基C 1-6烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和羟基C 1-6烷基；
m为0或1；
n为3至8，n是小数或整数；
Pc为抗体或其抗原结合片段；
所述的制备方法包括如下的步骤：
步骤(a)：在约1℃至约36℃的反应温度条件下，抗体或其抗原结合片段与还原剂反应；
步骤(b)：步骤(a)的产物与下式(La-Y-D)所示的化合物反应；
Figure PCTCN2021083014-appb-000004
其中：W，L 2，L 3，R 1，R 2，R 5，R 6，R 7和m如上所定义。
在另一个方面，本披露提供一种抗体药物偶联物的制备方法，所述抗体药物偶联物具有如下式所示的结构：
Figure PCTCN2021083014-appb-000005
其中，n为4至8，n是小数或整数；
所述的制备方法包括如下的步骤：
步骤(a)：在约1℃至约36℃的反应温度条件下，抗体或其抗原结合片段与还原剂反应；
步骤(b)：步骤(a)的产物与下式所示的化合物反应；
Figure PCTCN2021083014-appb-000006
在可选的实施方案中，步骤(a)中的所述反应温度条件为约4℃至约30℃，优选为约20℃至约30℃，更优选为25℃，非限制性的实施例包括约20℃、约21℃、约22℃、约23℃、约24℃、约25℃、约26℃、约27℃、约28℃、约29℃、约30℃。在一些实施方案中，所述反应温度条件为13℃至28℃或13℃至25℃。
在可选的实施方案中，步骤(a)中的反应是在pH为约4.5至约6.5的条件下进行的，优选所述反应是在pH为约5.0至约6.0的条件下进行的，更优选所述反应是在pH为约5.6的条件下进行的。在非限制性的实施例中，反应是在pH为约4.5、约4.6、约4.7、约4.8、约4.9、约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、或约6.0中进行的。
在可选的实施方案中，步骤(a)中的反应是在缓冲剂中进行的；在非限制性的实施例中，所述的缓冲剂选自组氨酸盐缓冲剂、磷酸盐缓冲剂和醋酸盐缓冲剂。
在可选的实施方案中，步骤(a)中的反应是在缓冲剂中进行的；在非限制性的实施例中，所述的缓冲剂为组氨酸-盐酸缓冲剂。
在可选的实施方案中，所述的缓冲剂选自含有EDTA的组氨酸盐缓冲剂和含有EDTA的组氨酸-盐酸缓冲剂。示例性地，所述组氨酸盐缓冲剂的浓度为1mM至100mM、10mM至50mM、20mM、30mM或40mM；EDTA的浓度为1mM至10mM、2mM至5mM、2.5mM、3mM或4mM。在一些实施方案中，所述的缓冲剂含有10mM至50mM的组氨酸盐缓冲剂和1mM至10mM的EDTA。在一些实施方案中，所述的缓冲剂含有20mM的组氨酸-盐酸缓冲剂和2.5mM的EDTA。EDTA指乙二胺四乙酸。
在可选的实施方案中，步骤(a)中的所述还原剂选自三(2-羧乙基)膦(TCEP)或其盐、1,4-二巯基苏糖醇(DTT)和β-巯基乙醇(β-ME)等合适的还原剂，优选为TCEP或其盐更优选为三(2-羧乙基)膦盐酸盐。
在可选的实施方案中，步骤(a)中还原剂与抗体或其抗原结合片段(Pc)的摩尔比为2至10:1、2.6至7:1、2.9至3.7:1、3.2至3.4:1或3.3:1。
在可选的实施方案中，步骤(b)是在有机溶剂中进行，优选的有机溶剂包括二甲基亚砜(DMSO)、N,N-二甲基甲酰胺(DMF)、N,N-二甲基乙酰胺(DMAc)、乙腈或其混合物。在非限制性的实施例中，步骤(b)包括：将式(La-Y-D)所示的化合物溶于DMSO，并将步骤(a)的产物与式(La-Y-D)所示的化合物的DMSO 溶液混合。
在可选的实施方案中，上述制备方法还包括步骤(c)，所述步骤(c)包括将步骤(b)的产物进行纯化。所述纯化可以采用阳离子柱层析或亲和柱层析纯化，所述阳离子层析的填料选自Capto S Impact和Poros XS，优选为Capto S Impact。在一些实施方式中，所述的Capto S Impact是Capto TM S Impact。在一些实施方式中，所述的Poros XS是Poros TM XS。
在可选的实施方案中，药物载量(n)的范围可以是每个抗体或其抗原结合片段(Pc)结合3至8个、4至8个、5至7个，优选5.3至6.1个，更优选5.7个细胞毒性药物。n是小数或整数。在一些实施方案中，n是5.3、5.4、5.5、5.6或5.7。
在可选的实施方案中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下；优选为结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为6％以下。在非限制性的实施例中，结合4个药物的抗体重链比例为4％以下、3％以下、2％以下或1％以下；未结合药物的抗体重链比例为6％以下、5％以下、4％以下、3％以下、2％以下或1％以下。或者，在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上、66％以上、67％以上、68％以上、69％以上、70％以上、75％以上、80％以上、85％以上、90％以上或95％以上。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为70％以上。在一些实施方式中，所述的抗体重链比例和/或抗体轻链比例是通过反向色谱测定的。
在可选的实施方案中，所述制备方法是适用于大规模制备的。所述制备方法的曲妥珠抗体投料量在100mg以上，优选为1克以上，更优选为10克以上，最优选为100克以上。
在可选的实施方案中，所述的抗体药物偶联物具有如通式(Pc-L b-Y-D)所示的结构：
Figure PCTCN2021083014-appb-000007
其中：
s 1为2至8的整数；
Pc、R 1、R 2、R 5、R 6、R 7、m和n如上所定义；
所述的制备方法包括如下的步骤：
步骤(a)：在约1至约36℃的反应温度条件下，抗体或其抗原结合片段与还原剂反应；
步骤(b)：步骤(a)的产物与下式(L b-Y-D)所示的化合物反应；
Figure PCTCN2021083014-appb-000008
其中：s 1、R 1、R 2、R 5、R 6、R 7和m如上所定义。
在可选的实施方案中，前述抗体药物偶联物具有如下的结构：
Figure PCTCN2021083014-appb-000009
Figure PCTCN2021083014-appb-000010
Figure PCTCN2021083014-appb-000011
Figure PCTCN2021083014-appb-000012
其中Pc和n如通式(Pc-La-Y-D)中所定义。
在可选的实施方案中，其中所述Pc为抗体或其抗原结合片段，所述抗体选自嵌合抗体、人源化抗体和全人源抗体。在一些实施方案中，所述抗体为单克隆抗体。
在可选的实施方案中，所述的抗体或其抗原结合片段选自抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体和抗Mesothelin抗体，或其抗原结合片段；
优选地，所述的抗体或其抗原结合片段选自曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、苯妥昔单抗(Brentuximab)、吉妥珠单抗(Gemtuzumab)、Bivatuzumab、Lorvotuzumab、cBR96和Glematumamab，或其抗原结合片段。
在可选的实施方案中，所述的抗体偶联物具有如下式所示的结构：
Figure PCTCN2021083014-appb-000013
其中，n为4至8，n是小数或整数。
在另一个方面，本披露提供一种抗体药物偶联物的制备方法，所述抗体药物偶联物具有如下式所示的结构：
Figure PCTCN2021083014-appb-000014
其中，n为4至8，n是小数或整数；
所述的制备方法包括如下的步骤：
步骤(a)：在反应温度为约4℃至约30℃，和pH为约4.5至约6.5的条件下，曲妥珠单抗(Trastuzumab)与TCEP反应；
步骤(b)：步骤(a)的产物与下式所示的化合物反应；
Figure PCTCN2021083014-appb-000015
在可选的实施方案中，所述抗体药物偶联物具有如下式所示的结构：
Figure PCTCN2021083014-appb-000016
其中，n为4至8，n是小数或整数；
所述的制备方法包括如下的步骤：
步骤(a)：在反应温度为约25℃，和pH为约5.6的条件下，Trastuzumab与TCEP反应，所述反应是在含有EDTA的组氨酸-盐酸缓冲剂中进行的；
步骤(b)：步骤(a)的产物与下式所示的化合物反应；
Figure PCTCN2021083014-appb-000017
步骤(c)：包括将步骤(b)的产物进行阳离子层析柱或亲和层析柱纯化。在一些实施方案中，所述含有EDTA的组氨酸-盐酸缓冲剂中含有20mM组氨酸-盐酸缓冲剂和2.5mM EDTA。
本披露还提供一种抗体药物偶联物或其药学上可接受的盐，其中所述抗体药物偶联物的结构如通式(Pc-L a-Y-D)所示：
Figure PCTCN2021083014-appb-000018
其中：
W选自C 1-8烷基、C 1-8烷基-C 3-7环烷基或1至8个原子的直链杂烷基，所述直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 3-7环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(Q)、天冬氨酸(D)中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
R 1为卤代C 1-6烷基或C 3-7环烷基；
R 2选自氢原子、卤代C 1-6烷基和C 3-7环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-7环烷基；
R 5选自氢原子、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和羟基C 1-6烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、C 1-6烷基、卤代C 1-6 烷基、氘代C 1-6烷基和羟基C 1-6烷基；
m为0或1；
n为4至8，n是小数或整数；
Pc为抗体或其抗原结合片段；
所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下；优选为结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为6％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为70％以上。
本披露还提供一种抗体药物偶联物或其药学上可接受的盐，其中所述抗体药物偶联物是由前述的抗体药物偶联物的制备方法制备获得的；并且，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下；优选为结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为6％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为70％以上。
本披露还提供一种抗体药物偶联物或其药学上可接受的盐，其中所述抗体药物偶联物具有如下式所示的结构：
Figure PCTCN2021083014-appb-000019
其中，n为4至8，n是小数或整数；
所述抗体药物偶联物是由前述的抗体药物偶联物的制备方法制备获得的；并且，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下；优选为结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为6％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。在一些实施方式中，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为1％以下，并且未结合药物的抗体重链比例为4％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为70％以上。
发明详述
本披露提供一种更利于大规模生产的制备方法。具体地，所述制备方法所获得的产品药物载量分布更窄、游离毒素含量更低、产率更高。
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
本披露将申请PCT/CN2019/107873中的全部内容引入本申请。
“抗体药物偶联物(antibody drug conjugate，ADC)”是将抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素或具有细胞杀伤活性的小分子药物相连，充分利用了抗体对肿瘤细胞的特异性或高表达抗原细胞结合的特异性和细胞毒素的高效性，避免对正常细胞的毒副作用。与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控 制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸盐缓冲剂”是包含组氨酸根离子的缓冲剂。组氨酸盐缓冲剂的实例包括组氨酸-盐酸、组氨酸-醋酸、组氨酸-磷酸、组氨酸-硫酸等缓冲剂，优选组氨酸-盐酸缓冲剂，组氨酸-醋酸缓冲剂是组氨酸与醋酸配制而成，组氨酸盐酸缓冲剂是组氨酸与盐酸或组氨酸与组氨酸盐酸盐配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选的磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、醋酸组氨酸盐、醋酸-醋酸钾、醋酸醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂是醋酸-醋酸钠。
本文所用术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本披露所述的“抗体”指免疫球蛋白，是完整抗体由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。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。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。本披露所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4 抗体或抗Mesothelin抗体中一个或多个；优选为曲妥珠单抗(曲妥珠单抗，商品名Herceptin)、帕妥珠单抗(帕妥珠单抗，也被称作2C4，商品名Perjeta)、尼妥珠单抗(尼妥珠单抗，商品名泰欣生)、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、苯妥昔单抗、吉妥珠单抗、Bivatuzumab、Lorvotuzumab、cBR96和Glematumamab。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本披露所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
在本披露中，本披露所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本披露中，本披露所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
本披露的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本披露中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进 行最少反向突变或回复突变，以保持活性。本披露的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
术语“裸抗体”，是指未与异源模块(例如细胞毒性模块)或放射性标记物缀合的抗体。
本披露中所述的“抗体的抗原结合片段”可以指具有抗原结合活性的Fab片段、Fab’片段、F(ab’) 2片段，以及与抗原结合的Fv片段scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。
本披露的术语“抗原结合位点”指抗原上连续或不连续的，由本披露抗体或抗原结合片段识别的三维空间位点。
本披露中所述的“ADCC”，即抗体依赖的细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity)，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A；IgG2/4嵌合体，IgG4的F234A/L235A突变。
本披露中所述的突变序列中的“突变”包括但不限于“回复突变”、“保守修饰”或“保守置换或取代”。本披露中所述的“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
本披露所述的“突变序列”是指对本披露的核苷酸序列和/或氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本披露的核苷酸序列和/或氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和/或氨基酸序列。本披露中所述的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
术语“接头单元”或“连接片段”或“连接单元”是指一端与抗体或其抗原结合片段连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后 再与抗体或药物相连。
接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等，Cancer Research52:127-131(1992)；美国专利No.5,208,020)。
本披露工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其 独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基。
术语“杂烷基”指含有一个或多个选自N、O和S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至7个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共 轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性实例包括：
Figure PCTCN2021083014-appb-000020
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性实例包括：
Figure PCTCN2021083014-appb-000021
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性实例包括：
Figure PCTCN2021083014-appb-000022
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接 在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2021083014-appb-000023
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2021083014-appb-000024
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基和杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2021083014-appb-000025
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、 杂环烷氧基、环烷硫基和杂环烷硫基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“环烷基烷基”指烷基被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
术语“氰基”指-CN。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“载药量”或“药物载量”(DAR)是指ADC中每个抗体或其抗原结合片段上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，其是整数或小数。在本披露的实施方式中，载药量表示为n，示例性的可以为1、2、3、4、5、6、7、8、9、10的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验和HPLC鉴定偶联反应后每个ADC分子的药物平均数量。
术语“药物载量分布”是指抗体药物偶联物群体中，连接不同数量药物的抗体的分布情况，例如，群体中连接0、2、4、6和8个药物的抗体的分布情况。值得注意的是，由于可能产生降解产物，使得1、3、5和7的DAR也可能包含于混合物中。本披露中，抗体药物载量分布可以采用结合不同数量药物的抗体重链来表征，例如：H 0表示未结合药物的重链，H 1表示结合一个药物的重链，H 2表示结合两个药物的重链，H 3表示结合三个药物的重链，H 4表示结合四个药物的重链。示例性的，H 3比例为4％即代表在抗体药物偶联物的重链群体中，结合三个药物的重链比例为4％。相应的，本披露中抗体药物载量分布也可以采用结合不同数量药物的抗体轻链来表征，L 0表示未结合药物的抗体轻链，L 1表示结合1个药物的抗 体轻链。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本披露的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“置换”是指溶解抗体蛋白的溶剂体系的置换，例如，使用稳定制剂的缓冲体系经物理操作方式将含抗体蛋白的高盐或高渗溶剂体系置换，从而使抗体蛋白存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
附图说明
图1A：本披露ADC-19的血浆稳定性实验结果。
图1B：本披露ADC-18的血浆稳定性实验结果。
图1C：本披露ADC-20的血浆稳定性实验结果。
图2：本披露ADC-21、ADC-24对JIMT-1荷瘤小鼠药效评价。
图3：本披露ADC对人乳腺癌细胞SK-BR-3移植瘤裸小鼠的疗效评价。
图4：本披露ADC-25的血浆稳定性实验结果。
图5：本披露ADC对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的疗效。
图6：本披露ADC对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效。
图7：本披露ADC对人胶质细胞瘤U87MG裸小鼠移植瘤的疗效。
具体实施方式
以下结合实施例进一步描述解释本披露，但这些实施例并非意味着限制本发明的范围。
本披露实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
一、抗体药物偶联物的制备及其性质和生物学测试
化合物的结构是通过核磁共振(NMR)或质谱(MS)来确定的。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)，化学位移是以10 -6(ppm)作为单位给出。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo,型号：Finnigan LCQ advantage MAX)。
UPLC的测定用Waters Acquity UPLC SQD液质联用仪。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。
UV-HPLC的测定使用Thermo nanodrop2000紫外分光光度计。
增殖抑制率及IC 50值的测定用PHERA starFS酶标仪(德国BMG公司)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm硅胶板。
柱层析一般使用烟台黄海200～300目硅胶为载体。
本披露的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH & Co.KG，Acros Organnics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中如无特殊说明，反应均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中如无特殊说明，反应中的溶液是指水溶液。
实施例中如无特殊说明，反应的温度为室温。
室温为最适宜的反应温度，温度范围是20℃～30℃。
实施例中pH＝6.5的PBS缓冲液的配制：取KH 2PO 4 8.5g，K 2HPO 4.3H 2O 8.56g，NaCl 5.85g，EDTA 1.5g置于瓶中，定容至2L，超声波使其全部溶解，摇匀即得。
纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂的体系包括：A：二氯甲烷和异丙醇体系，B：二氯甲烷和甲醇体系，C：石油醚和乙酸乙酯体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和酸性或碱性试剂等进行调节。
本披露部分化合物是通过Q-TOF LC/MS来表征的。Q-TOF LC/MS使用安捷伦6530精确质量数四级杆-飞行时间质谱仪和安捷伦1290-Infinity超高效液相色谱仪(安捷伦Poroshell 300SB-C8 5μm,2.1×75mm色谱柱)。
实施例1-1
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环丙烷-1-甲酰胺1
Figure PCTCN2021083014-appb-000026
向依喜替康甲磺酸盐1b(2.0mg，3.76μmol，采用专利申请“EP0737686A1”公开的方法制备而得)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-羟基环丙基甲酸1a(1.4mg，3.7μmol，采用公知的方法“Tetrahedron Letters,25(12),1269-72；1984”制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(3.8mg，13.7μmol)，加毕，在 0-5℃搅拌反应2小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(8mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物1(1.6mg，产率：82.1％)。
MS m/z(ESI):520.2[M+1]
1H NMR(400MHz,CDCl 3):δ7.90-7.84(m,1H),7.80-7.68(m,1H),5.80-5.70(m,1H),5.62-5.54(m,2H),5.44-5.32(m,2H),5.28-5.10(m,2H),3.40-3.15(m,3H),2.44(s,3H),2.23(t,1H),2.06-1.75(m,2H),1.68-1.56(m,1H),1.22-1.18(m,2H),1.04-0.98(m,2H),0.89(t,3H)。
实施例1-2
(S)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-A
(R)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-B
Figure PCTCN2021083014-appb-000027
向1b(4mg，7.53μmol)中加入2mL乙醇和0.4mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啉，搅拌至反应液变澄清。向反应液中依次加入2-环丙基-2-羟基乙酸2a(2.3mg，19.8μmol，采用专利申请“WO2013106717”公开的方法制备而得)、1-羟基苯并三唑(3mg，22.4μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(4.3mg，22.4μmol)，加毕，在0-5℃搅拌反应1小时。撤去冰水浴，加热至30℃搅拌2小时。反应液减压浓缩，所得到的粗品化合物2用高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相：A-水(10mmol NH 4OAc)，B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(2-A：1.5mg，2-B：1.5mg)。
MS m/z(ESI):534.0[M+1]。
单一构型化合物2-B(较短保留时间)
UPLC分析：保留时间1.06分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H),3.19-3.13(m,1H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83(m,1H),1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H)。
单一构型化合物2-A(较长保留时间)
UPLC分析：保留时间1.10分钟，纯度：86％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1H),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3.20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H)。
实施例1-3
(S)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-A
(R)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-B
Figure PCTCN2021083014-appb-000028
向1b(5.0mg，9.41μmol)中添加2mL乙醇和0.4mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啡啉，搅拌至反应液变澄清。向反应液中依次加入3,3,3-三氟-2-羟基丙酸3a(4.1mg，28.4μmol，供应商Alfa)、1-羟基苯并三唑(3.8mg，28.1μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5.4mg，28.2μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，加热至30℃搅拌8小时。反应液减压浓缩，所得到的粗品化合物3用高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(1.5mg，1.5mg)。
MS m/z(ESI):561.9[M+1]。
单一构型化合物(较短保留时间)
UPLC分析：保留时间1.11分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.94(d,1H),7.80(d,1H),7.32(s,1H),7.20(d,1H),6.53(s,1H),5.61-5.55(m,1H),5.45-5.23(m,3H),5.15-5.06(m,1H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.26-2.20(m,1H),2.16-2.08(m,1H), 2.02-1.94(m,1H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H)。
单一构型化合物(较长保留时间)
UPLC分析：保留时间1.19分钟，纯度：90％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.97(d,1H),7.80(d,1H),7.31(s,1H),7.16(d,1H),6.53(s,1H),5.63-5.55(m,1H),5.45-5.20(m,3H),5.16-5.07(m,1H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.22-2.14(m,1H),2.04-1.95(m,2H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H)。
实施例1-4
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环戊烷-1-甲酰胺4
Figure PCTCN2021083014-appb-000029
向1b(3.0mg，5.64μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-羟基-环戊烷甲酸4a(2.2mg，16.9μmol，采用专利申请“WO2013106717”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物4(2.5mg，产率：80.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz,CDCl 3):δ7.73-7.62(m,2H),5.75-5.62(m,1H),5.46-5.32(m,2H),5.26-5.10(m,1H),3.30-3.10(m,1H),2.43(s,3H),2.28-2.20(m,2H),2.08-1.84 (m,8H),1.69-1.58(m,2H),1.04-1.00(m,2H),0.89(t,3H)。
实施例1-5
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-(羟甲基)环丙烷-1-甲酰胺5
Figure PCTCN2021083014-appb-000030
向1b(2.0mg，3.76μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-(羟甲基)-环戊烷甲酸5a(0.87mg，7.5μmol，采用专利申请“WO201396771”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2mg，7.24μmol)，加毕，在0-5℃搅拌反应2小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(8mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物5(1.0mg，产率：50％)。
MS m/z(ESI):533.9[M+1]。
1H NMR(400MHz,CDCl 3):δ8.07(s,1H),7.23-7.18(m,2H),6.71-6.64(m,1H),6.55-6.51(m,1H),5.36-5.27(m,2H),4.67-4.61(m,2H),3.53-3.48(m,1H),3.30-3.22(m,2H),3.18-3.13(m,1H),2.71-2.61(m,2H),2.35-2.28(m,1H),2.04-1.91(m,4H),1.53-1.40(m,3H),0.91-0.75(m,4H)。
实施例1-6
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-(羟基甲基)环丁烷-1-甲酰胺6
Figure PCTCN2021083014-appb-000031
向1b(3.0mg，5.64μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-(羟基甲基)环丁烷-1-甲酸6a(2.2mg，16.9μmol；采用文献“Journal of the American Chemical Society,2014,vol.136,#22,p.8138-8142”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物6(2.1mg，产率：67.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ7.85-7.62(m,1H),6.88(br，1H),5.87-5.48(m，2H),5.47-5.33(m，1H),5.31-5.06(m，1H),4.25-3.91(m,2H),3.25(br,1H),2.60-2.32(m,3H),2.23(t,1H),2.15-1.95(m,3H),1.70-1.56(m,2H),1.41-1.17(m,9H),1.03(s,1H),0.95-0.80(m,2H)。
实施例1-7
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环丁烷-1-甲酰胺7
Figure PCTCN2021083014-appb-000032
向1b(3.0mg，5.64μmol)中添加2mL乙醇和0.4mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啡啉，搅拌至反应液变澄清。向反应液中依次加入1-羟基环丁烷甲酸7a(2.0mg，17.22μmol，供应商药石)，1-羟基苯并三唑(2.3mg，17.0μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(3.2mg，16.7μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，常温搅拌2小时。反应液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物7(2.5mg，产率：83.1％)。
MS m/z(ESI):534.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ8.28(d,1H),7.75(d,1H),7.29(s,1H),6.51(s,1H),6.12(s,1H),5.59-5.51(m,1H),5.41(s,2H),5.20-5.01(m,2H),3.27-3.17(m,1H),3.15-3.05(m,1H),2.71-2.63(m,1H),2.37(s,3H),2.12-2.05(m,1H),2.03-1.94(m,2H),1.92-1.78(m,4H),1.50-1.42(m,1H),0.90-0.83(m,4H)。
实施例1-8
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8
Figure PCTCN2021083014-appb-000033
Figure PCTCN2021083014-appb-000034
第一步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸苄酯8c
将1-羟基环丙烷-1-羧酸苄酯8a(104mg，0.54mmol；采用专利申请“US2005/20645”公开的方法制备而得)和2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯8b(100mg，0.27mmol；采用专利申请“CN105829346A”公开的方法制备而得)加入反应瓶，加入5mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(61mg，0.54mmol)，撤去冰浴，升至室温搅拌10分钟，加入20mL冰水，用乙酸乙酯(5mL×2)和氯仿(5mL×5)萃取，合并有机相并浓缩。所得残余物溶于3mL 1,4-二氧六环中，加入0.6mL水，加入碳酸氢钠(27mg，0.32mmol)和氯甲酸-9-芴甲酯(70mg，0.27mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(8mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到标题产物8c(100mg，产率：73.6％)。
MS m/z(ESI):501.0[M+1]。
第二步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸8d
将8c(50mg，0.10mmol)溶于3mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(25mg，含量10％)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到标题产物8d(41mg，产率：100％)。
MS m/z(ESI):411.0[M+1]。
第三步
(9H-芴-9-基)甲基(2-(((1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯8e
将1b(7mg，0.013mmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入8d(7mg，0.017mmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(7mg，0.026mmol)，冰浴搅拌反应35分钟。加入10mL水，用乙酸乙酯(5mL×3)萃取，有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物8e(8.5mg，产率78.0％)。
MS m/z(ESI):828.0[M+1]。
第四步
1-((2-氨基乙酰氨基)甲氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8f
将8e(4mg，4.84μmol)溶于0.2mL二氯甲烷中，加入0.1mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次，加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物8f(2.9mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):606.0[M+1]。
第五步
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8
将粗品8f(2.9mg，4.84μmol)溶于0.5mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入(S)-2(-2-(-2-(6-(2,5-二氧代-1H-吡咯-1-基)已酰氨基)乙酰氨基)乙酰氨基)-3-苯基丙酸8g(2.7mg，5.80μmol，采用专利申请“EP2907824”公开的方法制备而得)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5- 三嗪-2-基)-4-甲基氯化吗啉盐(2.7mg，9.67μmol)，冰浴搅拌反应30分钟，撤去冰浴，升至室温搅拌15分钟。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩得到标题产物8(2mg，产率：39.0％)。
MS m/z(ESI):1060.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ9.01(d,1H),8.77(t,1H),8.21(t,1H),8.08-7.92(m,2H),7.73(d,1H),7.28(s,1H),7.24-7.07(m,4H),6.98(s,1H),6.50(s,1H),5.61(q,1H),5.40(s,2H),5.32(t,1H),5.12(q,2H),4.62(t,1H),4.52(t,1H),4.40-4.32(m,1H),3.73-3.47(m,8H),3.16-3.04(m,2H),2.89(dd,1H),2.69-2.55(m,2H),2.37-2.23(m,4H),2.12-1.93(m,4H),1.90-1.74(m,2H),1.52-1.38(m,4H),1.33-1.11(m,5H),0.91-0.81(m,4H)。
实施例1-9
N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-B
Figure PCTCN2021083014-appb-000035
Figure PCTCN2021083014-appb-000036
第一步
2-环丙基-2-羟基乙酸苄酯9a
将2a(1.3g，11.2mmol；采用专利申请“WO2013/106717”公开的方法制备而得)溶于50mL乙腈中，依次加入碳酸钾(6.18g，44.8mmol)，溴化苄(1.33mL，11.2mmol)和四丁基碘化铵(413mg，1.1mmol)。将反应液室温搅拌48小时，通过硅藻土过滤，滤饼用乙酸乙酯(10mL)淋洗，合并滤液减压浓缩，用硅胶柱色谱法以展开 剂体系C纯化所得残余物，得到标题产物9a(2g，产率：86.9％)。
第二步
10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯9b
将9a(120.9mg,0.586mmol)和8b(180mg，0.489mmol)加入反应瓶，加入4mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(109mg，0.98mmol)，撤去冰浴，升至室温搅拌40分钟，加入10mL冰水，用乙酸乙酯(20mL×2)和氯仿(10mL×5)萃取，合并有机相并浓缩。所得残余物溶于4mL二氧六环中，加入2mL水，加入碳酸氢钠(49.2mg，0.586mmol)和氯甲酸-9-芴甲酯(126mg，0.49mmol)，室温搅拌2小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物9b(48mg，产率：19％)。
MS m/z(ESI):515.0[M+1]。
第三步
10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸9c
将9b(20mg，0.038mmol)溶于4.5mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(12mg，含量10％，干型)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品标题产物9c(13mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):424.9[M+1]。
第四步
(9H-芴-9-基)甲基(2-(((1-环丙基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯9d
将1b(10mg，18.8μmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入粗品9c(13mg，30.6μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(16.9mg，61.2μmol)，冰浴搅拌反应40分钟。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物9d(19mg，产率：73.6％)。
MS m/z(ESI):842.1[M+1]。
第五步
2-((2-氨基乙酰氨基)甲氧基)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺9e
将9d(19mg，22.6μmol)溶于2mL二氯甲烷中，加入1mL二乙胺，室温搅 拌2小时。反应液减压浓缩，加入1mL甲苯并减压浓缩，重复两次。往残余物中加入3mL正己烷打浆，静置后倾倒出上层清液，保留固体。将固体残余物减压浓缩，油泵拉干得到粗品标题产物9e(17mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):638.0[M+18]。
第六步
N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-B
将粗品9e(13.9mg，22.4μmol)溶于0.6mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入8g(21.2mg，44.8μmol)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(18.5mg，67.3μmol)，冰浴搅拌反应10分钟，撤去冰浴，升至室温搅拌1小时，反应生成化合物9。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(9-A：2.4mg，9-B：1.7mg)。
MS m/z(ESI):1074.4[M+1]。
单一构型化合物9-A(较短保留时间)：
UPLC分析:保留时间1.14分钟，纯度：85％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,1H),4.54-4.40(m,2H),3.76-3.64(m，4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,1H),2.80-2.62(m,1H),2.45-2.30(m,3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H)。
单一构型化合物9-B(较长保留时间)：
UPLC分析:保留时间1.16分钟，纯度：89％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.13(m,3H),6.99(s,1H),6.55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H), 5.35-5.15(m,2H),4.78-4.68(m,1H),4.60-4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,4H),0.91-0.79(m,3H),0.53-0.34(m,4H)。
实施例1-10
N-((2S,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺10-A
N-((2R,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺10-B
Figure PCTCN2021083014-appb-000037
Figure PCTCN2021083014-appb-000038
第一步
3,3,3-三氟-2-羟基丙酸苄酯10a
将3a(1.80g，12.5mmol)溶于100mL乙腈中，依次加入碳酸钾(5.17g，37.5mmol)，溴化苄(4.48mL，37.5mmol)和四丁基碘化铵(231mg，0.63mmol)。将反 应液加热至60℃搅拌5小时。将反应液冷却至室温，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物10a(980mg，产率：33.5％)。
1H NMR(400MHz,CDCl 3):δ7.43-7.36(m,5H),5.34(s,2H),4.53(s,1H),3.44(s,1H)。
第二步
1-(9H-芴-9-基)-3,6-二氧代-10-(三氟甲基)-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯10b
将8b(63mg,0.17mmol)和10a(80mg，0.34mmol)加入反应瓶，加入3mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(38mg，0.34mmol)，撤去冰浴，升至室温搅拌20分钟，加入10mL冰水，用乙酸乙酯(20mL×2)和氯仿(10mL×5)萃取，合并有机相并浓缩，所得残余物溶于2mL二氧六环中，加入0.4mL水，加入碳酸氢钠(19mg，0.23mmol)和氯甲酸-9-芴甲酯(49mg，0.19mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物10b(51mg，产率：55.3％)。
MS m/z(ESI):559.9[M+18]。
第三步
1-(9H-芴-9-基)-3,6-二氧代-10-(三氟甲基)-2,9-二氧杂-4,7-二氮杂十一-11-酸10c
将10b(15mg，0.28mmol)溶于3mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(15mg，含量10％)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到粗品标题产物10c(13mg)。
MS m/z(ESI):452.9[M+1]。
第四步
(9H-芴-9-基)甲基(2-((((3-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,1,1-三氟-3-氧代丙-2-基)氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯10d
将1b(10mg，18.8μmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入10c(13mg，28.7μmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(11mg，39.7μmol)，冰浴搅拌反应30分钟。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相，有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物10d(16mg，产率97.8％)。
MS m/z(ESI):870.0[M+1]。
第五步
2-((2-氨基乙酰氨基)甲氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟丙酰胺10e
将10d(16mg，18.4μmol)溶于0.6mL二氯甲烷中，加入0.3mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入2mL甲苯并减压浓缩，重复两次。往残余物中加入3mL正己烷打浆，静置后倾倒出上层清液，保留固体；重复三次。将固体残余物减压浓缩，油泵拉干得到粗品标题产物10e(12mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):647.9[M+1]。
第六步
N-((2S,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺10-A
N-((2R,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺10-B
将粗品10e(12mg，18.5μmol)溶于1.0mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入8g(14mg，29.6μmol)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(15mg，54.2μmol)，冰浴搅拌反应30分钟，撤去冰浴，升至室温搅拌1小时，反应生成化合物10。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(2.7mg，2.6mg)。
MS m/z(ESI):1102.0[M+1]。
单一构型化合物(较短保留时间)：
UPLC分析:保留时间1.18分钟，纯度：91％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.97(d,1H),8.85-8.76(m,1H),8.37-8.27(m,1H),8.12-8.02(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.31(s,1H),7.26-7.10(m,4H),6.99(s,1H),6.66(br,1H),6.52(s,1H),5.65-5.54(m,1H),5.41(s,1H),5.37-5.25(m,3H),5.23-5.13(m,1H),4.81-4.68(m,2H),4.51-4.41(m,1H),3.78-3.45(m,6H),3.21-3.13(m,1H),3.02-2.93(m,1H),2.77-2.63(m,2H),2.45-2.29(m,3H),2.24-2.05(m,3H),2.04-1.93(m,5H),1.90-1.75(m,2H),1.52-1.38(m,4H),0.90-0.78(m,5H)。
单一构型化合物(较长保留时间)：
UPLC分析:保留时间1.23分钟，纯度：90％(色谱柱：ACQUITY UPLC BEHC18  1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ9.05(d,1H),8.97-8.88(m,1H),8.35-8.27(m,1H),8.11-8.03(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.34(s,1H),7.29-7.13(m,4H),6.99(s,1H),6.66(br,1H),6.54(s,1H),5.64-5.55(m,1H),5.43(s,1H),5.36-5.20(m,3H),4.92-4.85(m,1H),4.82-4.72(m,2H),4.52-4.42(m,1H),3.77-3.48(m,6H),3.21-3.14(m,1H),3.03-2.95(m,1H),2.79-2.65(m,2H),2.47-2.28(m,3H),2.25-2.05(m,3H),2.05-1.94(m,5H),1.91-1.76(m,2H),1.52-1.37(m,4H),0.92-0.77(m,5H)。
实施例1-11
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丁烷-1-甲酰胺11
Figure PCTCN2021083014-appb-000039
Figure PCTCN2021083014-appb-000040
第一步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丁烷-1-羧酸苄酯11b
将1-羟基环丁烷-羧酸苄酯11a(167mg，0.81mmol，采用文献“Journal of Medicinal Chemistry,2013,vol.56,#13,p.5541-5552”公开的方法制备而得)和8b(150mg，0.41mmol)加入反应瓶，加入5mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(92mg，0.82mmol)，撤去冰浴，升至室温搅拌10分钟，加入20mL冰水，用乙酸乙酯(5mL×2)和氯仿(5mL×5)萃取，合并有机相并浓缩，所得残余物溶于3mL二氧六环中，加入0.6mL水，加入碳酸氢钠(41mg，0.48mmol)和氯甲酸-9-芴甲酯(105mg，0.41mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(8mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物11b(37mg，产率：17.6％)。
MS m/z(ESI):514.6[M+1]。
第二步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丁烷-1-羧酸11c
将11b(37mg，71.9μmol)溶于3mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂 中，加入钯碳(15mg，含量10％)，氢气置换三次，室温搅拌反应2小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到标题产物11c(35mg，产率：82％)，直接进行下一步。
第三步
(9H-芴-9-基)甲基(2-(((1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丁氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯11d
将1b(10mg，0.018mmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入11c(13mg，0.031mmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(25mg，0.091mmol)，冰浴搅拌反应40分钟。加入8mL水，用乙酸乙酯(5mL×3)萃取，有机相用饱和氯化钠溶液(8mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系A纯化所得残余物，得到标题产物11d(19mg，产率73.9％)。
MS m/z(ESI):842.3[M+1]。
第四步
1-((2-氨基乙酰氨基)甲氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丁烷-1-甲酰胺11e
将11d(19mg，22.6μmol)溶于2mL二氯甲烷中，加入1mL二乙胺，室温搅拌1.5小时。反应液减压浓缩，加入1mL甲苯减压浓缩，重复两次，加入4mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物11e(15mg)，产品不经纯化直接用于下一步反应。
第五步
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丁烷-1-甲酰胺11
将粗品11e(2mg，3.22μmol)溶于0.5mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入8g(1.5mg，3.17μmol)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2.7mg，9.67μmol)，室温搅拌30分钟。反应液用油泵旋干，除掉DMF，残余物用DCM溶解后直接用薄层层析法纯化2次(展开剂极性：DCM/MeOH＝10/1)，得到标题产物11(1mg，产率：28.8％)。
MS m/z(ESI):1073.6[M+1]。
1H NMR(400MHz,CDCl 3):δ8.70-8.60(m,1H),8.28-8.19(m,1H),8.13-7.91(m, 3H),7.79-7.71(d,1H),7.29(s,1H),7.25-7.09(m,4H),6.98(s,1H),6.71-6.62(m,1H),6.55-6.47(m,1H),5.64-5.54(m,2H),5.40(s,1H),5.35-5.27(t,2H),5.17-5.10(m,2H),4.60-4.51(m,1H),4.51-4.35(m,2H),3.93-3.78(m,3H),3.71-3.59(m,3H),3.01-2.88(m,3H),2.70-2.64(m,2H),2.44-2.30(m,3H),2.28-2.14(m,3H),2.11-1.92(m,6H),1.90-1.76(m,3H),1.51-1.39(m,4H),0.92-0.75(m,6H)。
实施例1-12
(S)-3-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基丙酰胺12-A
(R)-3-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基丙酰胺12-B
Figure PCTCN2021083014-appb-000041
第一步
3-环丙基-2-羟基丙酸12b
将12a(0.5g，3.87mmol，供应商Adamas)溶于35mL水和乙酸(V:V＝4:1)的混合溶剂中，冰水浴降温至0-5℃，滴加亚硝酸钠(0.53g，7.74mmol)的2M水溶液，升至室温搅拌反应3小时。向反应液中加入固体氯化钠，使水相饱和，用乙酸乙酯(8mL×8)萃取，无水硫酸钠干燥，过滤，浓缩，得到标题产物12b(0.45g，产率：89.3％)。
第二步
(S)-3-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基丙酰胺12-A
(R)-3-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基丙酰胺12-B
向1b(45mg，0.085mmol)中加入1.5mL乙醇和1.5mL N,N-二甲基甲酰胺，氩气置换三次，滴加0.1mL N-甲基吗啉，搅拌至反应液变澄清。向反应液中依次加入12b(90mg，0.691mmol)，1-羟基苯并三唑(34mg，0.251mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(49mg，0.256mmol)，加毕，室温搅拌反应3小时。反应液减压浓缩，所得到的粗品化合物12用高效液相色谱法纯化(分离条件：色谱柱：Sharpsil-T C18 5μm 21.2*250mm；流动相：A-水(10mmol NH 4OAc)，B-乙腈，梯度洗脱，流速：18mL/min)，得到标题产物(7mg，15mg)。
MS m/z(ESI):547.9[M+1]。
单一构型化合物(较短保留时间)
UPLC分析：保留时间1.345分钟，纯度：72％(色谱柱：ZORBAX Ecliphase Plus C18 1.8μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.42(d,1H),7.78(d,1H),7.30(s,1H),6.51(s,1H),5.60-5.50(m,2H),5.42(s,1H),5.19(q,2H),4.02-4.00(m,1H),3.21-3.11(m,2H),2.39(s,3H),2.21-2.07(m,2H),2.05-1.95(m,1H),1.92-1.68(m,4H),1.53-1.41(m,1H),0.87(t,3H),0.48-0.34(m,2H),0.14-0.01(m,2H)。
单一构型化合物(较长保留时间)
UPLC分析：保留时间1.399分钟，纯度：88％(色谱柱：ZORBAX Ecliphase Plus C18 1.8μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.36(d,1H),7.77(d,1H),7.31(s,1H),6.51(s,1H),5.58-5.51(m,1H),5.48(d,1H),5.42(s,1H),5.20(q,2H),4.09-4.02(m,1H),3.22-3.11(m,2H),2.39(s,3H),2.27-2.06(m,2H),2.05-1.95(m,1H),1.93-1.81(m,2H),1.65-1.43(m,2H),1.32-1.21(m,1H),0.87(t,3H),0.48-0.33(m,2H),0.14-0.01(m,2H)。
实施例1-13(参照例)
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟乙酰胺
Figure PCTCN2021083014-appb-000042
标题化合物13参照专利“EP2907824A1中说明书第147页的Example 76”公开的方法制备而得。
实施例1-14
N-((2R,10S)-10-苄基-2-(环丙基甲基)-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺14-A
N-((2S,10S)-10-苄基-2-(环丙基甲基)-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺14-B
Figure PCTCN2021083014-appb-000043
Figure PCTCN2021083014-appb-000044
第一步
3-环丙基-2-羟基丙酸苄酯14a
将12b(200mg，1.54mmol)溶于20mL乙腈中，依次加入碳酸钾(1.06g，7.68mmol)，溴化苄(0.16mL，1.34mmol)和四丁基碘化铵(28mg，0.07mmol)。将反应液室温搅拌48小时，通过硅藻土过滤，滤饼用乙酸乙酯(10mL)淋洗，合并滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物14a (140mg，产率：41.3％)。
第二步
10-(环丙基甲基)-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯14b
将14a(94mg,0.427mmol)和8b(130mg，0.353mmol)加入反应瓶，加入10mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(79mg，0.704mmol)，撤去冰浴，升至室温搅拌10分钟，加入20mL冰水，用乙酸乙酯(10mL×4)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物14b(50mg，产率：26.8％)。
MS m/z(ESI):529.2[M+1]。
第三步
10-(环丙基甲基)-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸14c
将14b(27mg，0.051mmol)溶于3mL乙酸乙酯，加入钯碳(7mg，含量10％，干型)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品标题产物14c(23mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):439.1[M+1]。
第四步
(9H-芴-9-基)甲基(2-((((3-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1-氧代丙-2-基)氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯14d
将1b(22mg，42.38μmol)加入反应瓶，加入3mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加三乙胺(4.3mg，42.49μmol)，加入粗品14c(23mg，51.1μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(17.6mg，63.6μmol)，冰浴搅拌反应40分钟。加入15mL水，用乙酸乙酯(8mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(15mL)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物14d(29mg，产率：79.9％)。
MS m/z(ESI):856.1[M+1]。
第五步
2-((2-氨基乙酰氨基)甲氧基)-3-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)丙酰胺14e
将14d(29mg，33.9μmol)溶于0.8mL二氯甲烷中，加入0.4mL二乙胺，室温搅拌1.5小时。反应液减压浓缩，加入1mL甲苯并减压浓缩，重复两次。往残 余物中加入3mL正己烷打浆，静置后倾倒出上层清液，重复三次。将残余物减压浓缩，油泵拉干得到粗品标题产物14e(22mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):634.1[M+1]。
第六步
N-((2R,10S)-10-苄基-2-(环丙基甲基)-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺14-A
N-((2S,10S)-10-苄基-2-(环丙基甲基)-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺14-B
将粗品14e(22mg，33.9μmol)溶于2.5mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，依次加入8g(24mg，50.8μmol)，和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(14mg，50.6μmol)，撤去冰浴，升至室温搅拌1小时，反应生成化合物14。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，得到标题产物(2mg，2mg)。
MS m/z(ESI):1088.4[M+1]。
单一构型化合物(较短保留时间)：
UPLC分析:保留时间1.18分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
单一构型化合物(较长保留时间)：
UPLC分析:保留时间1.23分钟，纯度：96％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
实施例1-15
1-((S)-9-苄基-22-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5,8,11,14,17-五氧代-2-氧杂-4,7,10,13,16-五氮杂二十二烷基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3,4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺15
Figure PCTCN2021083014-appb-000045
第一步
1-(10-(9H-芴-9-基)-5,8-二氧代-2,9-二氧杂-4,7-二氮杂癸基)环丙烷-1-羧酸苄酯15b
将8b(500mg，1.35mmol)加入反应瓶，加入6mL四氢呋喃，将1-羟基甲基环丙烷-1-甲酸苄酯15a(233mg，1.13mmol；采用专利申请“EP2862856A1中说明书第262页的Example 22-2”公开的方法制备而得)加入瓶中，氩气置换三次，冰水浴降温至0-5℃，加入氢化钠(54mg，1.35mmol)，撤去冰浴，升至室温搅拌40分钟；降至零度加入20mL冰水，用乙酸乙酯(5mL×2)和氯仿(5mL×5)萃取，合并有机相，用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到标题产物15b(15mg，产率：2.5％)。
MS m/z(ESI):515.2[M+1]。
第二步
1-(10-(9H-芴-9-基)-5,8-二氧代-2,9-二氧杂-4,7-二氮杂癸基)环丙烷-1-羧酸15c
将15b(15mg，0.029mmol)溶于2mL乙酸乙酯中，加入钯碳(3mg，含量10％,干型)，氢气置换三次，室温搅拌反应4.5小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到标题产物15c(11mg，产率：89％)。
MS m/z(ESI):425.2[M+1]。
第三步
(9H-芴-9-基)甲基(2-((((1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙基)甲氧基)甲基)氨基)2-氧代乙基)氨基甲酸酯15d
将1b(10mg，0.021mmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入15c(11mg，0.026mmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(10.7mg，0.039mmol)，加完后室温搅拌反应60分钟。加入10mL水，用乙酸乙酯(5mL×3)萃取，有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物15d(19mg，产率87.0％)。
MS m/z(ESI):842.2[M+1]。
第四步
1-(((2-氨基乙酰氨基)甲氧基)甲基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺15e
将15d(19mg，22.56μmol)溶于2mL二氯甲烷中，加入1mL二乙胺，室温搅拌1.5小时。反应液在0℃下减压浓缩，加入1mL甲苯减压浓缩，重复两次；加入3mL正己烷打浆，倾倒出上层正己烷，重复三次；减压浓缩得到粗品标题产物15e(13.9mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):620.1[M+1]。
第五步
1-((S)-9-苄基-22-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5,8,11,14,17-五氧代-2-氧杂-4,7,10,13,16-五氮杂二十二烷基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺15
将粗品15e(13.9mg，22.4μmol)溶于1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入8g(15.8mg，33.4μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(9.3mg，33.6μmol)，升至室温搅拌反应60分钟。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm  19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩得到标题产物15(2.5mg，产率：10.3％)。
MS m/z(ESI):1074.2[M+1]。
1H NMR(400MHz,DMSO-d 6):δ8.51-8.37(m,1H),8.22(t,1H),8.14-8.02(m,2H),8.011-7.94(m,1H),7.82-7.73(m,1H),7.29(s,1H),7.26-7.10(m,3H),6.98(s,1H),6.53-6.47(m,1H),5.62-5.50(m,1H),5.45-5.36(m,1H),5.35-5.23(m,2H),5.13-5.02(m,2H),4.61-4.50(m,2H),4.42-4.28(m,2H),3.76-3.61(m,3H),3.60-3.45(m,3H),3.27-3.23(m,1H),3.20-2.81(m,7H),2.75-2.61(m,3H),241-2.28(m,3H),2.23-2.13(m,2H),2.11-2.01(m,1H),2.03-1.94(m,1H),1.90(s,1H),1.87-1.74(m,2H),1.53-1.36(m,3H),1.29-1.08(m,4H),0.90-0.68(m,4H)。
实施例1-16
1-((S)-9-苄基-22-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5,8,11,14,17-五氧代-2-氧杂-4,7,10,13,16-五氮杂二十二烷基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丁烷-1-甲酰胺16
Figure PCTCN2021083014-appb-000046
Figure PCTCN2021083014-appb-000047
第一步
1-(羟基甲基)环丁烷-1-羧酸16b
将1-(羟甲基)环丁烷羧酸乙酯16a(250mg，1.58mmol，供应商Alfa)溶于甲醇(2mL)和水(1mL)，加入氢氧化钠(126mg，3.15mmol)，升温至40℃，搅拌反应3小时。冷却至常温，减压浓缩除去有机溶剂，用乙醚(10mL)反萃，收集水相。水相用6N盐酸水溶液调至pH 3-4，减压浓缩得到固体。加入3mL甲苯，减压浓缩旋干，重复三次。油泵拉干，得到粗品标题产物16b(206mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI，NEG):129.2[M-1]。
第二步
1-(羟基甲基)环丁烷-1-羧酸苄酯16c
将粗品16b(206mg，1.58mmol)溶于乙腈(15mL)，加入无水碳酸钾(1.09g，7.90mmol)和四丁基碘化铵(29mg，78.51μmol)，加入溴化苄(216mg，1.26mmol)，室温搅拌过夜。过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物16c(112mg，产率：32.1％)。
MS m/z(ESI):221.1[M+1]。
第三步
1-(10-(9H-芴-9-基)-5,8-二氧代-2,9-二氧杂-4,7-二氮杂癸基)环丁烷-1-羧酸苄酯16d
将16c(77mg，0.35mmol)和8b(100mg，0.27mmol)加入反应瓶，加入3mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(61mg，0.54mmol)，冰浴搅拌10分钟。加入20mL冰水，用乙酸乙酯(5mL)和氯仿(5mL×5)萃取，合并有机相并浓缩。所得残余物溶于3mL 1,4-二氧六环中，加入0.5mL水，加入碳酸氢钠(27mg，0.32mmol)和氯甲酸-9-芴甲酯(71mg，0.27mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物16d(24mg，产率：16.7％)。
MS m/z(ESI):551.3[M+23]。
第四步
1-(10-(9H-芴-9-基)-5,8-二氧代-2,9-二氧杂-4,7-二氮杂癸基)环丁烷-1-羧酸16e
将16d(12mg，22.7μmol)溶于1.5mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(5mg，含量10％)，氢气置换三次，室温搅拌反应2小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液减压浓缩，得到粗品标题产物16e(10mg)，产品不经纯化直接用于下一步反应。
第五步
(9H-芴-9-基)甲基(2-((((1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丁基)甲氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯16f
将1b(7.5mg，0.014mmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入粗品16e(10mg)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(6mg，0.026mmol)，冰浴搅拌反应30分钟。加入10mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物16f(10.6mg，产率87.8％)。
MS m/z(ESI):856.2[M+1]。
第六步
1-(((2-氨基乙酰氨基)甲氧基)甲基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丁烷-1-甲酰胺16g
将16f(10.6mg，12.4μmol)溶于0.6mL二氯甲烷中，加入0.3mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次；加入3mL正己烷打浆，倾倒出上层正己烷，重复三次。减压浓缩得到粗品标题产物16g(8mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):634.1[M+1]。
第七步
1-((S)-9-苄基-22-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5,8,11,14,17-五氧代-2-氧杂-4,7,10,13,16-五氮杂二十二烷基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丁烷-1-甲酰胺16
将粗品16g(8mg)溶于1mL N,N-二甲基甲酰胺，加入8g(8.8mg，18.6μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(5.2mg，18.8μmol)，室温搅拌反应30分钟。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，得到标题产物16(1.0mg，产率：7.2％)。
MS m/z(ESI):1088.0[M+1]。
实施例1-17
(1r,4r)-N-((S)-7-苄基-1-(1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙氧基)-3,6,9,12,15-五氧代-17,20,23,26,29,32,35,38,41-九氧杂-2,5,8,11,14-五氮杂四十三-43-基)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己烷-1-甲酰胺17
Figure PCTCN2021083014-appb-000048
Figure PCTCN2021083014-appb-000049
第一步
1-苯基-2,5,8,11,14,17,20,23,26,29-十氧杂三十一-31-酸叔丁酯17b
将1-苯基-2,5,8,11,14,17,20,23,26-九氧杂二十八-28-醇17a(0.34g，0.67mmol，供应商毕得)溶于10mL二氯甲烷中，依次加入氧化银(0.24g，1.01mmol)、溴乙酸叔丁酯(0.16g，0.81mmol)和碘化钾(0.07g，0.40mmol)，室温搅拌反应3小时。过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到标题产物17b(0.42g，产率：100％)。
MS m/z(ESI):636.3[M+18]。
第二步
29-羟基-3,6,9,12,15,18,21,24,27-九氧杂二十九-1-酸叔丁酯17c
将17b(417mg，0.67mmol)溶于15mL四氢呋喃，加入钯碳(110mg，含量10％，干型)，氢气置换三次，升至60℃搅拌反应3小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到粗品标题产物17c(357mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):546.2[M+18]。
第三步
29-叠氮基-3,6,9,12,15,18,21,24,27-九氧杂二十九-1-酸叔丁酯17d
将17c(357mg,0.675mmol)溶于10mL甲苯，加入叠氮磷酸二苯酯(279mg，1.014mmol)和1,8-二氮杂二环十一碳-7-烯(206mg，1.353mmol)，氩气置换三次，室温搅拌反应2小时，然后升至105℃反应19小时。反应液冷却至室温，浓缩，加入20mL水，用乙酸乙酯(10mL×4)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到粗品标题产物17d(412mg)。
MS m/z(ESI):571.3[M+18]。
第四步
29-氨基-3,6,9,12,15,18,21,24,27-九氧杂二十九-1-酸叔丁酯17e
将17d(230mg，0.415mmol)溶于8mL四氢呋喃，加入钯碳(58mg，含量10％，干型)，氢气置换三次，室温搅拌反应2小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到粗品标题产物17e(220mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):528.2[M+1]。
第五步
1-((1r,4r)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂三十一-31-酸叔丁酯17f
将(1r,4r)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己烷-1-羧酸(98.5mg，0.415mmol)溶于10mL二氯甲烷，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(190mg，0.500mmol)和N,N-二异丙基乙胺(162mg，1.253mmol)，氩气置换三次，加入粗品17e(220mg，0.417mmol)，室温搅拌反应1小时。加入15mL水，用二氯甲烷(8mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(15mL)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到标题产物17f(122mg，产率：39.2％)。
MS m/z(ESI):747.2[M+1]。
第六步
1-((1r,4r)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂三十一-31-酸17g
将17f(122mg，0.163mmol)溶于0.8mL二氯甲烷中，加入0.4mL三氟乙酸，室温搅拌反应1小时。加入15mL二氯甲烷稀释，减压浓缩；加入10mL正己烷，减压浓缩，重复两次；再加入10mL甲苯减压浓缩；用10mL正己烷：乙醚＝5:1的混合溶剂打浆三次，至pH接近7，浓缩，油泵抽干，得到标题产物17g(98mg，产率：86.8％)。
MS m/z(ESI):691.2[M+1]。
第七步
2,4-二甲氧基苄基1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙基-1-羧酸酯17h
将8d(164mg，0.40mmol)溶于二氯甲烷(5mL)，依次加入2,4-二甲氧基苄醇(81mg，0.48mmol)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(115mg，0.60mmol)和4-二甲氨基吡啶(5mg，0.041mmol)，加毕，室温搅拌反应1小时。加入20mL水，震荡后分层，水相用二氯甲烷(8mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(20mL)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物17h(124mg，产率：55.4％)。
MS m/z(ESI):583.1[M+23]。
第八步
2,4-二甲氧基苄基(S)-1-((11-苄基-1-(9H-芴-9-基)-3,6,9,12,15-五氧代-2-氧杂-4,7,10,13,16-五氮杂十七-17-基)氧基)环丙基-1-羧酸酯17j
将17h(39mg，69.6μmol)溶于0.6mL二氯甲烷中，加入0.3mL二乙胺，室温搅拌1小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次；加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩。将所得到的粗品溶于2mL N,N-二甲基甲酰胺，加入(((9H-芴-9-基)甲氧基)羰基)甘氨酰-L-苯丙氨酸17i(35mg，69.8μmol，采用专利申请“CN108853514A中说明书第13页的实施例7-12”公开的方法制备而得)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(23mg，83.1μmol)，室温搅拌1小时。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物17j(48mg，产率：83.9％)。
MS m/z(ESI):822.0[M+1]。
第九步
(S)-1-((11-苄基-1-(9H-芴-9-基)-3,6,9,12,15-五氧代-2-氧杂-4,7,10,13,16-五氮杂十七-17-基)氧基)环丙烷-1-羧酸17k
将17j(48mg，58.4μmol)溶于1.4mL 3％(v/v)的二氯乙酸的二氯甲烷溶液，冰水浴降温至0-5℃，加入三乙基硅烷(21mg，180.6μmol)，冰浴搅拌反应3小时。冰浴下减压浓缩除去一半有机溶剂，加入5mL乙醚，自然升至室温打浆，析出白色固体，过滤，收集滤饼，油泵抽干，得到标题产物17k(33mg，产率：84.1％)。
第十步
(9H-芴-9-基)甲基((S)-7-苄基-1-(1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙氧基)-3,6,9,12-四氧代-2,5,8,11-四氮杂十三-13-基)氨基甲酸酯17l
将1b(20mg，42.4μmol)加入反应瓶，加入1mL 10％(v/v)的甲醇的二氯甲烷溶液，氩气置换三次，冰水浴冷却至0～5℃，滴加一滴三乙胺，搅拌至1b溶解。将17k(33mg，49.1μmol)溶于1mL 10％(v/v)的甲醇的二氯甲烷溶液，然后滴加入上述反应液中，再加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(17.6mg，63.6μmol)。升至室温，搅拌反应1小时。加入10mL二氯甲烷和5mL水，搅拌5分钟，静置分层，收集有机相；水相用二氯甲烷(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物17l(37mg，产率：80.2％)。
MS m/z(ESI):1090.1[M+1]。
第十一步
(1r,4r)-N-((S)-7-苄基-1-(1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙氧基)-3,6,9,12,15-五氧代-17,20,23,26,29,32,35,38,41-九氧杂-2,5,8,11,14-五氮杂四十三-43-基)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己烷-1-甲酰胺17
将17l(15.5mg，14.23μmol)溶于0.6mL二氯甲烷中，加入0.3mL二乙胺，室温搅拌1.5小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次；加入3mL正己烷打浆，倾倒出上层正己烷，重复三次。减压浓缩，然后用油泵抽干。将所得粗品溶于1mL N,N-二甲基甲酰胺，加入17g(11mg，15.92μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(6.0mg，21.68μmol)，氩气置换三次，室温搅拌反应30分钟。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩得到标题产物17(6mg，产率：27.4％)。
MS m/z(ESI):1556.4[M+18]。
1H NMR(400MHz,DMSO-d 6):δ8.98(d,1H),8.76(s,1H),8.20(br,1H),8.12-7.95(m,3H),7.93-7.76(m,2H),7.75-7.66(m,2H),7.24(s,1H),7.20-7.05(m, 6H),6.97(s,1H),6.64(br,1H),6.55(d,1H),6.47(s,1H),5.61-5.52(m,2H),5.37(s,1H),5.33-5.23(m,2H),5.18(s,1H),5.13(s,1H),5.05(s,1H),5.00(s,1H),4.65-4.55(m,2H),4.53-4.45(m,1H),4.38-4.28(m,2H),3.84(s,2H),3.67(d,3H),3.60-3.40(m,33H),3.18(d,1H),3.15-3.08(m,3H),2.28(s,3H),2.00-1.92(m,3H),1.85(s,2H),1.82-1.73(m,2H),1.68-1.52(m,4H),1.29-1.15(m,3H),0.86-0.76(m,5H)。
实施例1-18
(1r,4r)-N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3,20,23,26,29,32,35,38,41,44-十氧杂-5,8,11,14,17-五氮杂四十六-46-基)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己烷-1-甲酰胺18
Figure PCTCN2021083014-appb-000050
Figure PCTCN2021083014-appb-000051
第一步
(R)-2-环丙基-2-羟基乙酸苄酯18a
(S)-2-环丙基-2-羟基乙酸苄酯18b
将2a(7.4g，63.7mmol)溶于200mL乙腈中，依次加入碳酸钾(35g，253.6mmol)，溴化苄(9.3g，54.4mmol)和四丁基碘化铵(500mg，1.36mmol)。将反应液室温搅拌16小时，通过硅藻土过滤，滤饼用乙酸乙酯(10mL)淋洗，合并滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物4.1g，进一步手性拆分纯化，得到标题产物18a(1.1g)和18b(1.2g)。
第二步
(R)-10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯18c
将8b(3.1g，8.41mmol)溶于四氢呋喃(55mL)中，加入18a(2.0g，9.70mmol)，冰水浴冷却至0～5℃，加入叔丁醇钾(1.89g，16.84mmol)，冰水浴下搅拌10分钟。加入乙酸乙酯(30mL)和水(20mL)，静置分层，水相用氯仿(30mL×5)萃取，合并 有机相。有机相减压浓缩，所得残余物溶于1,4-二氧六环(32mL)和水(8mL)，加入碳酸钠(1.78g，16.79mmol)和氯甲酸-9-芴基甲酯(2.18g，8.42mmol)，室温搅拌2小时。反应液中加入水(30mL)，用乙酸乙酯(50mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(30mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用柱层析以展开剂体系C纯化所得残余物，得到标题产物18c(1.3g，产率：30.0％)。
MS m/z(ESI):515.2[M+1]。
第三步
(R)-10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸18d
将18c(1.29g，2.51mmol)溶于乙酸乙酯(15mL)中，加入钯碳(260mg，含量10％，干型)，氢气置换三次，室温搅拌反应5小时。反应液用硅藻土过滤，滤饼用乙酸乙酯(20mL)和甲醇(20mL)淋洗，滤液浓缩，得到粗品标题产物18d(980mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):425.1[M+1]。
第四步
2,4-二甲氧基苄基(R)-10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酯18e
将粗品18d(980mg，2.31mmol)溶于二氯甲烷(15mL)中，加入2,4-二甲氧基苄醇(777mg，4.62mmol)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(664mg，3.46mmol)和4-二甲氨基吡啶(28mg，0.23mmol)，室温搅拌一小时。减压浓缩除去有机溶剂，加入20mL水，用乙酸乙酯(50mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(30mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用柱层析以展开剂体系C纯化所得残余物，得到标题产物18e(810mg，产率：61.1％)。
MS m/z(ESI):575.0[M+1]。
第五步
2,4-二甲氧基苄基(R)-2-((2-氨基乙酰氨基)甲氧基)-2-环丙基乙酸酯18f
将18e(33mg，57.4μmol)溶于0.6mL二氯甲烷中，加入0.3mL二乙胺，室温搅拌1小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次；加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物18f(21mg)，产品不经纯化直接用于下一步反应。
第六步
2,4-二甲氧基苄基(11S,19R)-11-苄基-19-环丙基-1-(9H-芴-9-基)-3,6,9,12,15-五氧代-2,18-二氧杂-4,7,10,13,16-五氮杂二十-20-酸酯18g
将粗品18f(21mg，57.4μmol)溶于3mL N,N-二甲基甲酰胺，加入17i(29mg，57.8μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(19mg，68.7 μmol)，室温搅拌1小时。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物18g(37mg，产率：77.1％)。
MS m/z(ESI):853.0[M+18]。
第七步
(11S,19R)-11-苄基-19-环丙基-1-(9H-芴-9-基)-3,6,9,12,15-五氧代-2,18-二氧杂-4,7,10,13,16-五氮杂二十-20-酸18h
将18g(37mg，44.3μmol)溶于1.4mL 3％(v/v)的二氯乙酸的二氯甲烷溶液，冰水浴降温至0-5℃，加入三乙基硅烷(15.4mg，132.4μmol)，冰浴搅拌反应3小时。冰浴下减压浓缩除去一半有机溶剂，加入5mL乙醚，自然升至室温打浆，析出白色固体，过滤，收集滤饼，油泵抽干，得到标题产物18h(24mg，产率：79.1％)。
MS m/z(ESI):708.2[M+23]。
第八步
(9H-芴-9-基)甲基((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)氨基甲酸酯18i
将1b(30mg，63.6μmol)加入反应瓶，加入1mL 10％(v/v)的甲醇的二氯甲烷溶液，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，搅拌至1b溶解。将18h(65mg，94.8μmol)溶于1mL 10％(v/v)的甲醇的二氯甲烷溶液，然后滴加入上述反应液中，再加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(27mg，97.6μmol)。升至室温，搅拌反应1小时。加入10mL二氯甲烷和5mL水，搅拌5分钟，静置分层，收集有机相；水相用二氯甲烷(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物18i(25mg，产率：35.6％)。
MS m/z(ESI):1104.4[M+1]。
第九步
(S)-2-(2-(2-氨基乙酰氨基)乙酰氨基)-N-(2-((((R)-1-环丙基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙氧基)-3-苯基丙酰胺18j
将18i(12mg，10.9μmol)溶于0.6mL二氯甲烷中，加入0.3mL二乙胺，室温搅拌1.5小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次，加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物18j(10mg)， 产品不经纯化直接用于下一步反应。
MS m/z(ESI):881.0[M+1]。
第十步
(1r,4r)-N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3,20,23,26,29,32,35,38,41,44-十氧杂-5,8,11,14,17-五氮杂四十六-46-基)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己烷-1-甲酰胺18
将粗品18j(10mg)溶于1mL N,N-二甲基甲酰胺，加入17g(8.5mg，12.3μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.6mg，16.6μmol)，室温搅拌30分钟。反应液过滤，进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物18(9.5mg，产率：56.2％)。
MS m/z(ESI):1570.2[M+18]。
1H NMR(400MHz,DMSO-d 6):δ8.77(d,1H),8.59-8.55(m,1H),8.42(d,1H),8.37-8.28(m,1H),8.25-8.06(m,2H),7.96-7.86(m,1H),7.86-7.70(m,2H),7.32-7.28(m,1H),7.25-7.14(m,3H),6.67(m,1H),5.96(s,1H),5.80-5.72(m,1H),5.62-5.52(m,2H),5.43-5.30(m,3H),5.28-5.17(m,2H),5.12-5.08(m,1H),4.72-4.35(m,8H),3.95-3.70(m,13H),3.35-3.22(m,14H),2.42-2.32(m,3H),2.05-1.98(m,4H),1.88-1.82(m,12H),1.47-1.39(m,3H),1.32-1.18(m,11H),0.90-0.80(m,4H),0.52-0.37(m,3H),0.32-0.18(m,2H)。
实施例1-19
(1r,4r)-N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3,20,23,26,29,32,35,38,41,44-十氧杂-5,8,11,14,17-五氮杂四十六-46-基)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己烷-1-甲酰胺19
Figure PCTCN2021083014-appb-000052
Figure PCTCN2021083014-appb-000053
第一步
(S)-10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯19a
将18b(252mg,1.22mmol)加入反应瓶，加入4mL二氯甲烷，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇锂(98mg,1.22mmol)，冰水浴下搅拌反应15分钟，变澄清，加入8b(300mg,814.3μmol)，冰水浴下搅拌2.5小时。加水(10mL)，分液，水相用二氯甲烷(8mL×2)萃取，合并有机相后用水(10mL×1)洗，饱和食盐水(10mL×2)洗，无水硫酸钠干燥，过滤浓缩得粗品。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物19a(282mg，产率：67.2％)。
第二步
(S)-10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸19b
将19a(280mg，0.554mmol)溶于8mL乙酸乙酯中，加入钯碳(84mg，含量10％，干型)，氢气置换三次，室温搅拌反应3小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品标题产物19b(230mg)，产品不经纯化直接 进行下一步反应。
第三步
2,4-二甲氧基苄基(S)-10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸酯19c
将粗品19b(230mg，541.8μmol)溶于7mL二氯甲烷中，依次加入2,4-二甲氧基苯甲醇(136.7mg,812.7μmol)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(155mg,808.5μmol)和4-二甲氨基吡啶(6.6mg,53.5μmol)，室温搅拌16小时。反应液用10mL二氯甲烷稀释后，用水(10mL×1)洗，饱和食盐水(10mL×2)洗，无水硫酸钠干燥，过滤浓缩得粗品。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物19c(159mg，产率：51.0％)
第四步
2,4-二甲氧基苄基(S)-2-((2-氨基乙酰氨基)甲氧基)-2-环丙基乙酸酯19d
将19c(60mg，104.4μmol)溶于1mL二氯甲烷中，加入0.5mL二乙胺，室温搅拌1小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次；加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物19d(21mg)，产品不经纯化直接用于下一步反应。
第五步
2,4-二甲氧基苄基(11S,19S)-11-苄基-19-环丙基-1-(9H-芴-9-基)-3,6,9,12,15-五氧代-2,18-二氧杂-4,7,10,13,16-五氮杂二十-20-酸酯19e
将粗品19d(36mg，102.2μmol)溶于4mL N,N-二甲基甲酰胺，加入17i(52mg，103.6μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(34.6mg，125.0μmol)，室温搅拌1小时。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物19e(70mg，产率：80.2％)。
第六步
(11S,19S)-11-苄基-19-环丙基-1-(9H-芴-9-基)-3,6,9,12,15-五氧代-2,18-二氧杂-4,7,10,13,16-五氮杂二十-20-酸19f
将19e(70mg，83.7μmol)溶于2.5mL 3％(v/v)的二氯乙酸的二氯甲烷溶液，冰水浴降温至0-5℃，加入三乙基硅烷(29mg，249.4μmol)，冰浴搅拌反应3小时。冰浴下减压浓缩除去一半有机溶剂，加入5mL乙醚，自然升至室温打浆，析出白色固体，过滤，收集滤饼，油泵抽干，得到标题产物19f(57mg，产率：99.2％)。
第七步
(9H-芴-9-基)甲基((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)氨基 甲酸酯19g
将1b(30mg，63.6μmol)加入反应瓶，加入1mL 10％(v/v)的甲醇的二氯甲烷溶液，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，搅拌至1b溶解。将19f(57mg，83.1μmol)溶于1mL 10％(v/v)的甲醇的二氯甲烷溶液，然后滴加入上述反应液中，再加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(26mg，93.9μmol)。升至室温，搅拌反应1小时。加入10mL二氯甲烷和5mL水，搅拌5分钟，静置分层，收集有机相；水相用二氯甲烷(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物19g(56mg，产率：79.8％)。
MS m/z(ESI):1103.1[M+1]。
第八步
(S)-2-(2-(2-氨基乙酰氨基)乙酰氨基)-N-(2-((((S)-1-环丙基-2-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)-3-苯基丙酰胺19h
将19g(4.6mg，4.16μmol)溶于1.5mL二氯甲烷中，加入0.75mL二乙胺，室温搅拌1.6小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次，加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物19h(4.0mg)，产品不经纯化直接用于下一步反应。
第九步
(1r,4r)-N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3,20,23,26,29,32,35,38,41,44-十氧杂-5,8,11,14,17-五氮杂四十六-46-基)-4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)环己烷-1-甲酰胺19
将粗品19h(4.0mg)溶于1mL N,N-二甲基甲酰胺，加入17g(2.9mg，4.2μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(1.5mg，5.4μmol)，室温搅拌40分钟。反应液过滤，进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物19(2.1mg，产率：32.4％)。
1H NMR(400MHz,DMSO-d 6):δ8.71-8.62(m,1H),8.59-8.51(m,1H),8.34-8.26(m,1H),8.14-8.02(m,2H),7.95-7.86(m,1H),7.83-7.69(m,2H),7.35-7.31(m,1H),7.29-7.11(m,3H),7.01(s,1H),6.72-6.50(m,3H),5.59-5.50(m,2H),5.42(s,2H),5.38-5.18(m,3H),4.79-4.69(m,2H),4.61-4.42(m,3H),3.91(s,2H),3.79-3.65(m,4H),3.63-3.44(m,13H),3.41-3.30(m,2H),3.26-3.09(m,5H),3.08-2.84 (m,4H),2.81-2.64(m,3H),2.42-2.28(m,3H),2.24-2.12(m,2H),2.05-1.93(m,4H),1.89-1.77(m,2H),1.72-1.56(m,3H),1.53-1.38(m,3H),1.34-1.10(m,11H),0.94-0.78(m,5H),0.52-0.35(m,3H)。
实施例1-20(参照例)
Figure PCTCN2021083014-appb-000054
标题化合物20参照专利“CN104755494A”中说明书第163页的实施例58”提供的方法合成。
以下抗体按抗体常规方法进行制备，例如可进行载体构建后，转染真核细胞如HEK293细胞(Life Technologies Cat.No.11625019)，表达纯化后获得。
以下为Trastuzumab的序列：
轻链
Figure PCTCN2021083014-appb-000055
重链
Figure PCTCN2021083014-appb-000056
以下为Pertuzumab的序列：
轻链
Figure PCTCN2021083014-appb-000057
重链
Figure PCTCN2021083014-appb-000058
以下为B7H3抗体1F9DS的序列：
轻链
Figure PCTCN2021083014-appb-000059
重链
Figure PCTCN2021083014-appb-000060
实施例1-21 ADC-1
Figure PCTCN2021083014-appb-000061
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；2.5mL，9.96mg/mL，0.168μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.082mL，0.82μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至5.0mg/mL，并取出2.0mL溶液往下反应。
将化合物10-较短保留时间化合物(2.1mg，2.02μmol)溶解于0.10mL DMSO中，加入到上述2.0mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-1通式的示例性产物ADC-1的PBS缓冲液(5.0mg/mL，1.1mL)，于4℃储存。
UV-HPLC计算平均值：n＝5.09。
实施例1-22 ADC-2
Figure PCTCN2021083014-appb-000062
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；2.5mL，9.96mg/mL，0.168μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.082mL，0.82μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至5.0mg/mL，并取出2.0mL溶液往下反应。
将化合物10-较长保留时间化合物(2.1mg，2.02μmol)溶解于0.10mL DMSO中，加入到上述2.0mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-1通式的示例性产物ADC-2的PBS缓冲液(4.95mg/mL，1.1mL)，于4℃储存。
UV-HPLC计算平均值：n＝7.39。
实施例1-23 ADC-3
Figure PCTCN2021083014-appb-000063
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；2.5mL，9.96mg/mL，0.168μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.082mL，0.82μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至5.0mg/mL，并取出2.0mL溶液往下反应。
将化合物8(2.1mg，2.02μmol)溶解于0.10mL DMSO中，加入到上述2.0mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-3通式的示例性产物ADC-3的PBS缓冲液(5.24mg/mL，1.1mL)，于4℃储存。
UV-HPLC计算平均值：n＝7.36。
实施例1-24 ADC-4
Figure PCTCN2021083014-appb-000064
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；3.74mL，13.38mg/mL，0.338μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.173mL，1.73μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至6.7mg/mL，并取出1.3mL溶液往下反应。
将化合物9-较短保留时间化合物9-A(1.0mg，0.93μmol)溶解于0.10mL DMSO中，加入到上述1.3mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止 反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-4A通式的示例性产物ADC-4的PBS缓冲液(1.72mg/mL，2.36mL)，于4℃储存。
UV-HPLC计算平均值：n＝7.39。
实施例1-25 ADC-5
Figure PCTCN2021083014-appb-000065
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；3.0mL，6.70mg/mL，0.136μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.067mL，0.67μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；将反应液用水浴降温至25℃，并取出0.614mL溶液往下反应。
将化合物9-较短保留时间化合物9-A(0.5mg，0.42μmol)溶解于0.031mL DMSO中，加入到上述0.614mL溶液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-4A通式的示例性产物ADC-5的PBS缓冲液(3.08mg/mL，0.82mL)，于4℃储存。
UV-HPLC计算平均值：n＝3.16。
实施例1-26 ADC-6
Figure PCTCN2021083014-appb-000066
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；3.74mL，13.38mg/mL，0.338μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.173mL，1.73μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至6.7mg/mL， 并取出0.75mL溶液往下反应。
将化合物9-较长保留时间化合物9-B(0.68mg，0.63μmol)溶解于0.10mL DMSO中，加入到上述0.75mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-4B通式的示例性产物ADC-6的PBS缓冲液(1.78mg/mL，1.78mL)，于4℃储存。
UV-HPLC计算平均值：n＝3.94。
实施例1-27 ADC-7
Figure PCTCN2021083014-appb-000067
在37℃条件下，向抗体帕妥珠单抗(Pertuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；5.0mL，10mg/mL，0.338μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.173mL，1.73μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至5.0mg/mL，并取出1.0mL溶液往下反应。
将化合物8(0.65mg，0.6μmol)溶解于0.1mL DMSO中，加入到上述1.0mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-7通式的示例性产物ADC-7的PBS缓冲液(1.42mg/mL，2.15mL)，于4℃储存。
UV-HPLC计算平均值：n＝6.91。
实施例1-28 ADC-8
Figure PCTCN2021083014-appb-000068
在37℃条件下，向抗体帕妥珠单抗(Pertuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；5.0mL，10mg/mL，0.338μmol)加入配置好的三 (2-羧乙基)膦的水溶液(10mM，0.173mL，1.73μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至5.0mg/mL，并取出1.6mL溶液往下反应。
将化合物10-较短保留时间化合物(1.04mg，1.0μmol)溶解于0.1mL DMSO中，加入到上述1.6mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-8通式的示例性产物ADC-8的PBS缓冲液(2.14mg/mL，2.31mL)，于4℃储存。
UV-HPLC计算平均值：n＝6.58。
实施例1-29 ADC-9
Figure PCTCN2021083014-appb-000069
在37℃条件下，向抗体帕妥珠单抗(Pertuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；5.0mL，10mg/mL，0.338μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.173mL，1.73μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至5.0mg/mL，并取出0.8mL溶液往下反应。
将化合物9-较短保留时间化合物9-A(0.55mg，0.5μmol)溶解于0.1mL DMSO中，加入到上述0.8mL溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-9A通式的示例性产物ADC-9的PBS缓冲液(2.27mg/mL，1.11mL)，于4℃储存。
UV-HPLC计算平均值：n＝3.16。
实施例1-30 ADC-10
Figure PCTCN2021083014-appb-000070
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.574mL，38.78nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，19.76μL，197.6nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物14-较短保留时间化合物(0.64mg，588nmol)溶解于40μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-10通式的示例性产物ADC-10的PBS缓冲液(5.48mg/mL，1.03mL)，于4℃储存。
UV-Vis计算平均值：n＝6.25。
实施例1-31 ADC-11
Figure PCTCN2021083014-appb-000071
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.646mL，43.64nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，22.24μL，222.4nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物14-较长保留时间化合物(0.72mg，662nmol)溶解于40μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-10通式的示例性产物ADC-11的PBS缓冲液(2.13mg/mL，1.87mL)，于4℃储存。
UV-Vis计算平均值：n＝7.03。
实施例1-32 ADC-12
Figure PCTCN2021083014-appb-000072
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.726mL，49.05nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，25.0μL，250.0nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物15(0.81mg，754nmol)溶解于40μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-12通式的示例性产物ADC-12的PBS缓冲液(3.34mg/mL，1.45mL)，于4℃储存。
UV-Vis计算平均值：n＝6.93。
实施例1-33 ADC-13
Figure PCTCN2021083014-appb-000073
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.287mL，19.39nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，9.88μL，98.8nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物16(0.32mg，294nmol)溶解于20μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-13通式的示例性产物ADC-13的PBS缓冲液(2.37mg/mL，0.88mL)，于4℃储存。
UV-Vis计算平均值：n＝6.53。
实施例1-34 ADC-14
Figure PCTCN2021083014-appb-000074
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.592mL，40.0nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，20.38μL，203.8nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物17(0.92mg，598nmol)溶解于40μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-14通式的示例性产物ADC-14的PBS缓冲液(0.30mg/mL，12.0mL)，于4℃储存。
UV-Vis计算平均值：n＝7.61。
实施例1-35 ADC-15
Figure PCTCN2021083014-appb-000075
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.592mL，40.0nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，20.38μL，203.8nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物18(0.93mg，599nmol)溶解于40μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-15通式的示例性产物ADC-15的PBS缓冲液(0.32mg/mL，11.8mL)，于4℃储存。
UV-Vis计算平均值：n＝7.89。
实施例1-36 ADC-16
Figure PCTCN2021083014-appb-000076
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.53mL，35.8nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，18.25μL，182.5nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物19(0.83mg，534nmol)溶解于35μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-16通式的示例性产物ADC-16的PBS缓冲液(0.32mg/mL，12.0mL)，于4℃储存。
UV-Vis计算平均值：n＝7.43。
实施例1-37 ADC-17
Figure PCTCN2021083014-appb-000077
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，2.0mL，135.12nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，43.2μL，432nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-较短保留时间化合物9-A(2.22mg，2067nmol)溶解于175μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-4A通式的示例性产物ADC-17的PBS缓冲液(1.32mg/mL，12.0mL)，于4℃储存。
UV-Vis计算平均值：n＝5.42。
实施例1-38 ADC-18(参照例)
Figure PCTCN2021083014-appb-000078
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.5mL，101.3nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，51.7μL，517nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物20(2.0mg，1934nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-18通式的示例性产物ADC-18的PBS缓冲液(0.79mg/mL，13.0mL)，于4℃储存。
UV-Vis计算平均值：n＝7.23。
实施例1-39 ADC-19
Figure PCTCN2021083014-appb-000079
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.36mL，91.9nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，46.9μL，469nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-较短保留时间化合物9-A(2.0mg，1862nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-4A通式的示例性产物ADC-19的PBS缓冲液(0.73mg/mL，13.0mL)，于4℃储存。
UV-Vis计算平均值：n＝6.26。
实施例1-40 ADC-20
Figure PCTCN2021083014-appb-000080
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.5mL，101.3nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，51.7μL，517nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物10-较长保留时间化合物(2.0mg，1815nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-1通式的示例性产物ADC-20的PBS缓冲液(0.73mg/mL，13.0mL)，于4℃储存。
UV-Vis计算平均值：n＝7.43。
实施例1-41 ADC-21(参照例)
Figure PCTCN2021083014-appb-000081
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.86mL，125.4nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，63.9μL，639nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物20(2.07mg，2001nmol)溶解于150μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-18通式的示例性产物ADC-21的PBS缓冲液(2.91mg/mL， 4.44mL)，于4℃储存。
UV-Vis计算平均值：n＝7.23。
实施例1-42 ADC-22
Figure PCTCN2021083014-appb-000082
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.88mL，127.2nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，64.9μL，649nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-较短保留时间化合物9-A(2.1mg，1955nmol)溶解于150μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-4A通式的示例性产物ADC-22的PBS缓冲液(3.56mg/mL，3.98mL)，于4℃储存。
UV-Vis计算平均值：n＝6.79。
实施例1-43 ADC-23(参照例)
Figure PCTCN2021083014-appb-000083
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，345mL，23.31μmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，11.89mL，118.9μmol)，置于水浴振荡器，于37℃振荡反应3.5小时，停止反应。将反应液用水浴降温至25℃。
将化合物20(362mg，350μmol)溶解于7.12mL MeCN和3.56mL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将 反应液通过超滤膜包先后用含有2％(v/v)MeCN和1％(v/v)DMSO的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液)、琥珀酸缓冲水溶液(pH＝5.3的0.01M的琥珀酸缓冲水溶液)脱盐纯化，之后加入蔗糖至60mg/mL、吐温20至0.2mg/mL，装瓶冻干后得到FADC-18通式的示例性产物ADC-23的冻干粉样品，于4℃储存。
UV-Vis计算平均值：n＝7.05。
实施例1-44 ADC-24
Figure PCTCN2021083014-appb-000084
在37℃条件下，向抗体曲妥珠单抗(Trastuzumab)的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，332mL，22.43μmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，11.44mL，114.4μmol)，置于水浴振荡器，于37℃振荡反应3.5小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-较短保留时间化合物9-A(241mg，224μmol)溶解于13.76mL MeCN和6.88mL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液通过超滤膜包先后用含有4％(v/v)MeCN和2％(v/v)DMSO的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液)、琥珀酸缓冲水溶液(pH＝5.3的0.01M的琥珀酸缓冲水溶液)脱盐纯化，之后加入蔗糖至60mg/mL、吐温20至0.2mg/mL，装瓶冻干后得到FADC-4A通式的示例性产物ADC-24的冻干粉样品，于4℃储存。
UV-Vis计算平均值：n＝7.07。
实施例1-45 ADC-25
Figure PCTCN2021083014-appb-000085
在37℃条件下，向抗体B7H3抗体1F9DS的PBS缓冲水溶液(pH＝6.5的0.05 M的PBS缓冲水溶液；10.0mg/mL，2.14mL，144.60nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，73.7μL，740nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-较短保留时间化合物9-A(3.0mg，2793nmol)溶解于150μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-25通式的示例性产物ADC-25的PBS缓冲液(1.28mg/mL，13.0mL)，于4℃储存。
UV-Vis计算平均值：n＝6.87。
实施例1-46 ADC-26(参照例)
Figure PCTCN2021083014-appb-000086
在37℃条件下，向抗体B7H3抗体1F9DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.89mL，60.14nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，30.1μL，300nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物20(1.0mg，967nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-26通式的示例性产物ADC-26的PBS缓冲液(1.61mg/mL，4.0mL)，于4℃储存。
UV-Vis计算平均值：n＝6.15。
实施例1-47 ADC-27
Figure PCTCN2021083014-appb-000087
在37℃条件下，向抗体B7H3抗体1F9DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.89mL，60.14nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，30.1μL，300nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-较短保留时间化合物9-A(1.02mg，950nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-25通式的示例性产物ADC-27的PBS缓冲液(1.94mg/mL，3.5mL)，于4℃储存。
UV-Vis计算平均值：n＝6.11。
实施例1-48 ADC-28(参照例)
Figure PCTCN2021083014-appb-000088
在37℃条件下，向抗体B7H3抗体1F9DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，2.36mL，159.47nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，81.3μL，810nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物20(3.0mg，2901nmol)溶解于150μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-26通式的示例性产物ADC-28的PBS缓冲液(1.29mg/mL，13.0mL)，于4℃储存。
UV-Vis计算平均值：n＝7.46。
实施例1-49 ADC-29
Figure PCTCN2021083014-appb-000089
在37℃条件下，向抗体B7H3抗体1F9DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.80mL，50.06nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，28.6μL，290nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-较短保留时间化合物9-A(1.29mg，1201nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-25通式的示例性产物ADC-29的PBS缓冲液(2.63mg/mL，2.4mL)，于4℃储存。
UV-Vis计算平均值：n＝7.24。
实施例1-50 ADC-30(参照例)
Figure PCTCN2021083014-appb-000090
在37℃条件下，向抗体B7H3抗体1F9DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.86mL，58.4nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，29.1μL，290nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物20(1.0mg，967nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-26通式的示例性产物ADC-30的PBS缓冲液(1.61mg/mL，4.0mL)，于4℃储存。
UV-Vis计算平均值：n＝6.15。
实施例1-51 ADC-31
Figure PCTCN2021083014-appb-000091
在37℃条件下，向抗体B7H3抗体1F9DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.89mL，60.14nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，30.1μL，300nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物8(1.0mg，943nmol)溶解于100μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-31通式的示例性产物ADC-31的PBS缓冲液(1.47mg/mL，4.5mL)，于4℃储存。
UV-Vis计算平均值：n＝6.33。
ADC原液药物载量分析
实验目的及原理
ADC原液是一种抗体交联物类药物，其治疗疾病的机理是依赖抗体的靶向性将毒素分子运送到细胞中，进而将细胞杀死。药物的载量对药效起着决定性的作用。使用紫外法对ADC原液的药物载量进行了测定。
实验方法
将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后，再将装有供试品溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
结果计算：采用紫外分光光度法(使用仪器：Thermo nanodrop2000紫外分光光度计)测定ADC原液载量，其原理是在某波长下ADC原液的总吸光值等于细胞毒药物与单克隆抗体在该波长下吸光值的加和，即：
(1)A 280nm＝ε mab-280bC mab+ε Drug-280bC Drug
ε Drug-280：药物在280nm平均摩尔消光系数5100；
C Drug：药物的浓度；
ε mab-280：曲妥珠单抗原液或帕妥珠单抗原液在280nm平均摩尔消光系数214600；
C mab：曲妥珠单抗原液或帕妥珠单抗原液的浓度；
b：光程长度为1cm。
同理可以得到样品在370nm下的总吸光值方程：
(2)A 370nm＝ε mab-370bC mab+ε Drug-370bC Drug
ε Drug-370：药物在370nm平均摩尔消光系数19000；
C Drug：药物的浓度；
ε mab-370：曲妥珠单抗原液或帕妥珠单抗原液在370nm消光系数为0；
C mab：曲妥珠单抗原液的浓度；
b：光程长度为1cm。
由(1)和(2)两种方程结合单克隆抗体和药物在两个检测波长下的消光系数和浓度数据可以计算出药物的载量。
药物载量＝C Drug/C mab。
生物学评价
测试例1-1：本披露化合物对肿瘤细胞体外增殖抑制测试
一、测试目的
本实验的目的是为了检测本披露药物化合物，对U87MG细胞(中科院细胞库，Catalog # TCHu138)和SK-BR-3肿瘤细胞(人乳腺癌细胞，ATCC，货号HTB-30)体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG(
Figure PCTCN2021083014-appb-000092
Luminescent Cell Viability Assay，Promega，货号：G7573)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
二、实验方法
下面以对U87MG细胞体外增殖抑制测试方法为例，用于举例说明本披露中测试本披露化合物对肿瘤细胞进行体外增殖抑制活性测试的方法。本方法同样适用于，但不限于对其它肿瘤细胞进行体外增殖抑制活性测试。
1、细胞培养：U87MG和SK-BR-3细胞分别用10％FBS的EMEM培养基(GE，货号SH30024.01)和含10％FBS的McCoy's 5A培养基(Gibco，货号16600-108)培养。
2、细胞准备：取对数生长期的U87MG和SK-BR-3细胞，用PBS(磷酸盐缓冲液，上海源培生物科技股份有限公司)洗涤1次之后，加入2-3mL胰蛋白酶(0.25％Trypsin-EDTA(1x)，Gibico,Life Technologies公司)消化2-3min，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，接着加入10-20mL细胞培养液将细胞重悬，制成单细胞悬液。
3、细胞铺板：将U87MG和SK-BR-3单细胞悬液混匀，用细胞培养液分别调整活细胞密度至2.75×10 3cells/mL和8.25×10 3cells/mL，将密度调整过后的细胞悬液混匀，以180μL/孔加入96孔细胞培养板。96孔板外周孔只加入200μL培养 基。将培养板在培养箱培养24小时(37℃，5％CO 2)。
4、化合物准备：用DMSO(二甲基亚砜，上海泰坦科技股份有限公司)溶解化合物，配制成初始浓度为10mM的存储液。
小分子化合物的起始浓度为500nM，配药方法如下。
在96孔U型底配药板第一列中分别加入30μL不同待测样品，样品浓度为100μM；第2列至第11列每孔中加入20μL DMSO。取第一列样品10μL至第二列20μL DMSO中，混匀，取10μL至第三列中，以此类推至第10列。将配药板中的药每孔取5μL至95μL EMEM培养基中，混匀，待用。
ADC的起始浓度为10nM或500nM，配药方法如下。
在96孔板第一列中分别加入100μL不同待测样品，样品浓度为100nM或5μM；第2列至第11列每孔中加入100μL PBS。取第一列样品50μL至第二列100μL PBS中，混匀，取50μL至第三列中，以此类推3倍稀释至第10列。
5、加样操作：向培养板中加入20μL配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO 2)。
6、显色操作：取出96孔细胞培养板，向每孔加入90μL CTG溶液，室温孵育10分钟。
7、读板操作：取出96孔细胞培养板，置于酶标仪(BMG labtech，PHERAstar FS)中，用酶标仪测定化学发光。
三、数据分析
用Microsoft Excel，Graphpad Prism 5对数据进行处理分析。实验结果参见下表1。
表1.本披露中的小分子片段对SK-BR-3细胞和U87细胞体外增殖抑制的IC 50值
Figure PCTCN2021083014-appb-000093
结论：本披露中的小分子片段对SK-BR-3细胞和U87细胞具有明显的增殖抑 制活性，手性中心对化合物的抑制活性有一定影响。
测试例1-2：本披露抗体药物偶联物对HER2靶标的肿瘤细胞的体外增殖抑制测试
本实验的目的是为了检测本披露针对HER2靶标的抗体药物偶联物，对SK-BR-3(人乳腺癌细胞，ATCC，货号HTB-30)和MDA-MB-468(人乳腺癌细胞，ATCC，货号HTB-132)体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG试剂对细胞的增值进行检测，根据IC 50值评价该化合物的体外活性。
按照测试例1的测试方法，测试细胞为SK-BR-3和MDA-MB-468，细胞培养液分别为含10％FBS的McCoy's 5A培养基(Gibco，货号16600-108)，含10％FBS的EMEM培养基(GE，货号SH30024.01)，和含10％FBS的L-15培养基(ThermoFisher，货号11415-114)。用细胞培养液将三株细胞分别调整活细胞密度至8.33×10 3个细胞/mL、8.33×10 3个细胞/mL和1.39×10 4个细胞/mL，将密度调整过后的细胞悬液混匀，以180μL/孔加入96孔细胞培养板。对相关化合物进行测试，得到结果见下表2。
表2.本披露抗体药物偶联物对HER2靶标的肿瘤细胞的体外增殖抑制的IC 50值
Figure PCTCN2021083014-appb-000094
结论：本披露针对HER2靶标的抗体药物偶联物对HER2阳性细胞SK-BR-3具有明显的增殖抑制活性；同时，它们对HER2阴性细胞MDA-MB-468增殖抑制活性弱；具有良好的选择性。
测试例1-3：Her2-ADC血浆稳定性实验
将ADC-19样品、ADC-18样品、ADC-20样品、人血浆、猴血浆(上海美迪西生物医药股份有限公司)、和1％BSA(Sigma)PBS溶液(上海生工)分别用0.22μm的过滤器过滤除菌。将ADC-19、ADC-18、ADC-20分别以终浓度200μg/mL加入上述无菌血浆或1％BSA PBS溶液中，置于37℃细胞培养箱中孵育，将孵育当天记为第0天，随后分别在第7天、14天和21天取出样品，进行游离毒素的检测。
取25μL样品至96孔板中；加入50μL内标工作液(100ng/mL喜树碱乙腈溶液)及150μL乙腈；涡旋混合5分钟，离心10分钟(4000rpm)，5μL进行LC/MS/MS(美国应用生物系统公司)分析。
结果显示：ADC-19在人和猴血浆，以及1％BSA PBS溶液中都相当稳定，游离毒素的释放率最高不超过2.1％，且在第14天趋于稳定，见图1A。
ADC-18在人和猴血浆中稳定性差，游离毒素的释放率最高分别为14.5％和8.10％。在1％BSA PBS溶液中比较稳定，见图1B。
ADC-20在人血浆、猴血浆和1％BSA PBS溶液中稳定性均比较差，游离毒素的释放率最高分别为21.7％、29.7％和21.7％。，且在1％BSAPBS溶液中一直处于降解状态，见图1C。
测试例1-4：JIMT-1荷瘤小鼠药效评价
1、试验目的
以nunu裸鼠为受试动物，评价Her2-ADC抗体T-DM1、ADC-21、ADC-24腹腔注射给药后，对人乳腺癌细胞曲妥珠单抗(Trastuzumab)耐药株(赫赛汀)JIMT-1移植瘤裸小鼠的疗效。
2、受试药物及材料
2-1、受试药物
T-DM1(参考US20050169933制备)
ADC-21：3mg/kg
ADC-21：10mg/kg
ADC-24：3mg/kg
ADC-24：10mg/kg
空白对照(Blank)：PBS
2-2、配制方法：均用PBS稀释配制。
2-3、试验动物
nunu裸鼠，购自北京维通利华。
3、试验方法
在小鼠右肋部皮下接种JIMT-1细胞(南京科佰)(5×10 6/只，具有50％人工基底膜)，肿瘤生长8天，长至203.09±11.94mm 3后将动物随机分组(d1)，8只/组， 共6组。
采用腹腔注射给药，共给药2次。每周测量2次瘤体积和体重，记录数据。
数据统计使用Excel 2003统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的空白对照组(Vehicle，PBS)及实验组的相对肿瘤体积。
4、试验结果
实验结果如图2显示，腹腔注射给药2次，观察至第34天时结束实验。T-DM1(10mg/kg)对肿瘤无抑制作用；ADC-21，3mg/kg的抑瘤率46.22％(P＜0.01)；ADC-21，10mg/kg的抑瘤率56.77％(P＜0.001)；ADC-24，3mg/kg的抑瘤率62.77％(P＜0.001)；ADC-24，10mg/kg的抑瘤率76.32％(P＜0.001)。在同等剂量情况下，ADC-24的抑瘤效果明显好于ADC-21。
测试例1-5：SK-BR-3荷瘤小鼠药效评价
1、试验目的
以nunu裸鼠为受试动物，评价Her2-ADC抗体ADC-21、ADC-22腹腔注射给药后对人乳腺癌细胞SK-BR-3移植瘤裸小鼠的疗效。
2、受试药物及材料
2-1、受试药物
ADC-21：1mg/kg
ADC-21：6mg/kg
ADC-22：1mg/kg
ADC-22：6mg/kg
空白对照(Blank)：PBS。
2-2、配制方法：均用PBS稀释配制。
2-3、试验动物
nunu裸鼠，购自北京维通利华。
3、试验方法
在小鼠右肋部皮下接种SK-BR-3细胞(ATCC)(5×10 6/只，具有50％人工基底膜)，肿瘤生长20天，长至153.34±11.73mm 3后将动物随机分组(d0)，8只/组，共5组。
采用腹腔注射给药1次。每周测量2次瘤体积和体重，记录数据。
数据统计使用Excel 2003统计软件：平均值以avg计算；SD值以STDEV计 算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的空白对照及实验组的相对肿瘤体积。
4、试验结果
实验结果如图3显示，腹腔注射给药1次，观察至第28天时结束实验，ADC-21 1mg/kg的抑瘤率15.01％；ADC-21 6mg/kg的抑瘤率77.4％，且和空白对照相比有非常显著差异(P＜0.001)。ADC-22 1mg/kg的抑瘤率19.82％；ADC-22 6mg/kg的抑瘤率98.38％(P＜0.001)。同为6mg/kg剂量情况下，ADC-22的抑瘤效果也明显好于ADC-21。
测试例1-6：血浆稳定性
将样品ADC-25，以100μg/mL的终浓度，分别与人血浆、猴血浆、和1％BSA PBS溶液混合均匀后，过滤除菌后置于37℃水浴锅内孵育，将孵育当天记为第0天，随后分别在第7天、14天和21天取出样品，进行游离毒素的检测。
不同时间点的样品取出后放至室温，涡旋混匀；取25μL样品至96孔板中；加入50μL内标工作液(100ng/mL喜树碱乙腈溶液)及150μL乙腈；涡旋混合5分钟，离心10分钟(4000rpm)，取上清液5μL进行LC/MS/MS分析。
结果如图4显示，ADC-25在人和猴血浆，以及1％BSA PBS溶液中都相当稳定，游离毒素的释放率最高不超过2％，且在第14天趋于稳定。
测试例1-7：ADC对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的疗效评价
1、试验目的
本实验以BALB/cA-nude裸小鼠为受试动物，评价本披露ADC化合物对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的疗效。
2、受试药物及材料
2-1、受试药物
ADC-27(3mg/kg)
ADC-26(3mg/kg)
空白对照(Blank)：pH7.4的PBS缓冲液。
2-2、配制方法：pH7.4的PBS缓冲液。
2-3、试验动物
BALB/cA-nude裸小鼠：购自上海杰思捷实验动物有限责任公司。
3、试验方法
实验用BALB/cA-nude裸小鼠，雌性，6-7周，皮下接种人脑星形胶质母细胞瘤U87MG细胞(人脑星形胶质母细胞瘤，中科院细胞库，Catalog#TCHu138)。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b 2
其中：a、b分别表示长、宽。
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的对照组(空白)及实验组的相对肿瘤体积。
4、试验结果
腹腔注射(i.p.)给药每周1次，共给药3次，观察至第22天时，ADC-27 3mg/kg的抑瘤率达到63.3％(P<0.0001)；ADC-26 3mg/kg的抑制率达到49.1％。ADC-27显示出比ADC-26更强的抗肿瘤疗效。
给药过程中各组动物体重正常，提示ADC无明显毒副作用。检测结果如表3和图5所示。所检测抗体能够有效抑制荷瘤裸鼠中U87MG移植瘤的生长，并且呈现出剂量依赖性。
表3.给药抗体对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的疗效(D22)
Figure PCTCN2021083014-appb-000095
***表示P<0.001
测试例1-8：ADC对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效评价
1、试验目的
本实验以BALB/cA-nude裸小鼠为受试动物，评价本披露ADC化合物对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效。
2、受试药物及材料
2-1、受试药物
ADC-29(3mg/kg)
ADC-28(3mg/kg)
阴性对照ADC(3mg/kg)：非B7H3靶点抗体与化合物20偶联形成的抗体药物偶联物。
2-2、配制方法：均用PBS稀释配制。
2-3、试验动物
BALB/cA-nude裸小鼠：购自常州卡文斯实验动物有限责任公司。
3、试验方法
实验用BALB/cA-nude裸小鼠，雌性，6-7周，皮下接种人咽头癌胸水转移细胞Detroit 562细胞(ATCC，Catalog
Figure PCTCN2021083014-appb-000096
CCL-138 TM)。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b 2
其中：a、b分别表示长、宽。
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的对照组(阴性对照)及实验组的相对肿瘤体积。
4、试验结果
腹腔注射给药每周1次，共给药3次，观察至第28天时，受试ADC抑瘤率分别是：ADC-29 3mg/kg(3mpk)的抑瘤率达到72.27％(P<0.001)；ADC-28 3mg/kg(3mpk)的抑瘤率达到56.2％(P<0.001)。ADC-29均显示出比ADC-28更强的抗肿瘤疗效。
给药过程中各组动物体重正常，提示ADC无明显毒副作用。检测结果如表4和图6所示。所检测抗体能够有效抑制荷瘤裸鼠中Detroit 562移植瘤的生长，并且呈现出剂量依赖性。
表4.给药抗体对荷瘤裸鼠Detroit 562移植瘤的疗效(D28)
Figure PCTCN2021083014-appb-000097
***表示P<0.001
测试例1-9：U87-MG荷瘤小鼠药效评价
1、试验目的
以Balb/c裸鼠为受试动物，在其人胶质瘤细胞U87MG移植瘤模型上评价B7H3-抗体药物偶连物腹腔注射后的疗效。
2、受试药物及材料
2-1、受试药物
ADC-30 1mg/kg
ADC-30 3mg/kg
ADC-31 1mg/kg
ADC-31 3mg/kg
空白对照(Blank)：PBS
2-2、配制方法：均用PBS稀释配制。
2-3、试验动物
BALB/cA-nude裸小鼠：购自上海斯莱克实验动物有限责任公司。
3、试验方法
在小鼠右肋部皮下接种U87MG细胞(人脑星形胶质母细胞瘤，中科院细胞库，Catalog#TCHu138)(2.5×10 6/只)，肿瘤生长14天，长至167.49mm 3后将动物随机分组(d1)，8只/组，共5组。
采用腹腔注射给药1次/周，共给药3次。每周测量2次瘤体积和体重，记录数据。
数据统计使用Excel 2003统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的空白对照组(Vehicle)及实验组的相对肿瘤体积。
4、试验结果
实验结果如图7显示，腹腔注射给药1次/周，共给药3次，观察至第18天时，受试ADC抑瘤率分别为：ADC-30 1mg/kg的抑瘤率为0.31％；ADC-30 3mg/kg的抑瘤率达到45.23％(P<0.0001)；ADC-31 1mg/kg的抑瘤率达到39.22％(P<0.01)；ADC-31 3mg/kg的抑瘤率达到80.24％(P<0.0001)。在同等剂量情况下，ADC-31的抑瘤效果明显好于ADC-30。
二、制备工艺优化
以下实施例的示例性产物具有如下式所示的结构：
Figure PCTCN2021083014-appb-000098
实施例2-1
在0℃条件下，在含有2.5mM EDTA的20mM组氨酸-盐酸缓冲液(pH 5.6)中，含61.71mg曲妥珠的抗体原液(0.0004251mmol，用20mM组氨酸-盐酸缓冲液稀释曲妥珠抗体至抗体终浓度15mg/mL)与0.7311mg三(2-羧乙基)膦盐酸盐(Sigma，0.002550mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(3.196mg，0.002975mmol)溶解于0.2062mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.2052mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-1。
反相色谱-质谱法计算平均值：n＝5.45。
实施例2-2
在13℃条件下，在含有2.5mM EDTA的20mM组氨酸-盐酸缓冲液(pH 5.6)中，含61.71mg曲妥珠的抗体原液(0.0004251mmol，用20mM组氨酸-盐酸缓冲液稀释曲妥珠抗体至抗体终浓度15mg/mL)与0.5118mg三(2-羧乙基)膦盐酸盐(Sigma，0.001785mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(3.196mg，0.002975mmol)溶解于0.2062mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.2052mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-2。
反相色谱-质谱法计算平均值：n＝5.49。
实施例2-3
在25℃条件下，在含有2.5mM EDTA的20mM组氨酸-盐酸缓冲液(pH 5.6)中，含61.71mg曲妥珠的抗体原液(0.0004251mmol，用20mM组氨酸-盐酸缓冲液 稀释曲妥珠抗体至抗体终浓度15mg/mL)与0.4021mg三(2-羧乙基)膦盐酸盐(Sigma，0.001403mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(3.196mg，0.002975mmol)溶解于0.2062mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.2052mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-3。
反相色谱-质谱法计算平均值：n＝5.39。
实施例2-4
在28℃条件下，在含有2.5mM EDTA的20mM组氨酸-盐酸缓冲液(pH 5.6)中，含61.71mg曲妥珠的抗体原液(0.0004251mmol，用20mM组氨酸-盐酸缓冲液稀释曲妥珠抗体至抗体终浓度15mg/mL)与0.3899mg三(2-羧乙基)膦盐酸盐(Sigma，0.001360mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(3.196mg，0.002975mmol)溶解于0.2062mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.2052mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-4。
反相色谱-质谱法计算平均值：n＝5.44。
实施例2-5
在37℃条件下，在含有2.5mM EDTA的20mM组氨酸-盐酸缓冲液(pH 5.6)中，含61.71mg曲妥珠的抗体原液(0.0004251mmol，用20mM组氨酸-盐酸缓冲液稀释曲妥珠抗体至抗体终浓度15mg/mL)与0.3778mg三(2-羧乙基)膦盐酸盐(Sigma，0.001318mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(3.196mg，0.002975mmol)溶解于0.2062mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.2052mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-5。
反相色谱-质谱法计算平均值：n＝5.46。
实施例2-6
在13℃条件下，在含有2.5mM EDTA的50mM PBS缓冲液(pH 6.5)中，含55.02mg曲妥珠的抗体原液(0.0003790mmol，用50mM PBS缓冲液稀释曲妥珠抗 体至抗体终浓度15mg/mL)与0.4563mg三(2-羧乙基)膦盐酸盐(Sigma，0.001592mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(2.850mg，0.002653mmol)溶解于0.1839mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.1829mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-6。
反相色谱-质谱法计算平均值：n＝5.39。
实施例2-7
在25℃条件下，在含有2.5mM EDTA的50mM PBS缓冲液(pH 6.5)中，含55.02mg曲妥珠的抗体原液(0.0003790mmol，用50mM PBS缓冲液稀释曲妥珠抗体至抗体终浓度15mg/mL)与0.3477mg三(2-羧乙基)膦盐酸盐(Sigma，0.001213mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(2.850mg，0.002653mmol)溶解于0.1839mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.1829mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-7。
反相色谱-质谱法计算平均值：n＝5.50。
实施例2-8
在37℃条件下，在含有2.5mM EDTA的50mM PBS缓冲液(pH 6.5)中，含55.02mg曲妥珠的抗体原液(0.0003790mmol，用50mM PBS缓冲液稀释曲妥珠抗体至抗体终浓度15mg/mL)与0.3259mg三(2-羧乙基)膦盐酸盐(Sigma，0.001137mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(2.850mg，0.002653mmol)溶解于0.1839mL DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.1829mL DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，加入过量半胱氨酸淬灭反应。得到FADC-4A通式的示例性产物ADC-2-8。
反相色谱-质谱法计算平均值：n＝5.47。
实施例2-9
在25℃条件下，在含有2.5mM EDTA的20mM组氨酸-盐酸缓冲液(pH 5.6)中，含127.4g曲妥珠的抗体原液(0.88mmol，用20mM组氨酸-盐酸缓冲液稀释曲 妥珠至抗体终浓度15mg/mL)与0.83g三(2-羧乙基)膦盐酸盐(Sigma，2.90mmol)在恒温水浴中搅拌反应3小时，生成中间体I溶液。
将化合物9较短保留时间化合物9-A(6.6g，6.14mmol)溶解于0.43L DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.43L DMSO，再将上述化合物9-A的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴25℃搅拌反应1小时，停止反应。
将上述反应液用Capto S Impact(GE)阳离子层析柱纯化，分别用不少于9个柱体积的含有10％(v/v)DMSO的0.05M醋酸缓冲液(pH＝5.5)和6个柱体积0.05M醋酸缓冲液(pH＝5.5)洗涤，再用0.05M醋酸缓冲液(pH 5.5，含0.39M氯化钠)进行洗脱，去除反应液中游离毒素和残留溶剂。在25℃，将阳离子洗脱液进行7倍体积等体积超滤(30kd超滤膜包)换液至0.01M琥珀酸缓冲液(pH 5.0)，得到FADC-4A通式的示例性产物ADC-A1。采用上述方法制备4个批次的样品。
反相色谱-质谱法测定得上述4个批次样品的载药量均为5.7。四个批次收率分别为100.8％，、98.9％，、97.4％和99.0％。
实施例2-10
在37℃条件下，向曲妥珠单抗的PBS缓冲液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，164mL，11.08μmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，3.55mL，35.5μmol)，置于水浴振荡器，于37℃振荡反应3.5小时，停止反应。将反应液用水浴降温至25℃。
将化合物9较短保留时间化合物9-A(185mg，172μmol)溶解于3.88mL乙腈和1.94mL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液通过超滤膜包先后用含有2％(v/v)乙腈和1％(v/v)DMSO的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液)、琥珀酸缓冲水溶液(pH＝5.3的0.01M的琥珀酸缓冲水溶液)脱盐纯化，除去小分子，得到FADC-4A通式的示例性产物ADC-B14样品，于4℃储存。
反相色谱-质谱法计算平均值：n＝5.3。
测试例2-1 药物载量分布测试
1.RP-DAR测定方法
1.1.在以下测量条件下进行RP-DAR分析：
UPLC系统：Waters H-Class超高效液相色谱仪UPLC系统
检测器：TUV检测器(测量波长：280nm)
色谱柱：Waters ACQUITY UPLC Protein BEH C4(2.1mm×150mm,1.7μm)
柱温：80℃
流速：0.3mL/min
样品室温度：20℃
运行时间：25min
流动相A：0.1％二氟乙酸(DFA)水溶液
流动相B：0.1％DFA乙腈溶液
梯度程序：27.0％B-44.0％B(0.00min-12.00min)，44.0％B-100％B(12.00min-13.00min)，100％B-100％B(13.00min-20.00min)，100％B-27.0％B(20.00min-20.04min)，27.0％B-27.0％B(20.04min-25min)
注入样品量：1.0μL
1.2.数据分析
当与药物不结合的抗体的轻链(L 0)和重链(H 0)相比时，在药物结合的轻链(一个药物结合的轻链：L 1)和药物结合的重链(一个药物结合的重链：H 1，两个药物结合的重链：H 2，三个药物结合的重链：H 3，四个药物结合的重链：H 4)的情况下，疏水性与结合药物的数目成比例的增加，并且保留时间延长。因此，以L 0、L 1、H 0、H 1、H 2、H 3和H 4的顺序进行洗脱。
由于药物接头吸收UV，根据以下表达式，使用轻链、重链和药物接头的摩尔吸光系数，根据结合的药物的数目，对所得的峰面积进行校正。计算公式如下：
轻链(εLC-280)/(εLC-280+连接药物数×ε药物-280)
重链(εHC-280)/(εHC-280+连接药物数×ε药物-280)
备注：εLC-280：轻链在280nm的摩尔消光系数；
εHC-28：重链在280nm的摩尔消光系数；
ε药物-280：毒素在280nm的摩尔消光系数。
表5.反相色谱载药量计算表
名称 连接药物数 校正峰面积百分比
L 0 0 100×L 0校正峰面积/LC校正峰面积总和
L 1 1 100×L 1校正峰面积/LC校正峰面积总和
H 0 0 100×H 0校正峰面积/HC校正峰面积总和
H 1 1 100×H 1校正峰面积/HC校正峰面积总和
H 2 2 100×H 2校正峰面积/HC校正峰面积总和
H 3 3 100×H 3校正峰面积/HC校正峰面积总和
H 4 4 100×H 4校正峰面积/HC校正峰面积总和
备注：LC校正峰面积总和＝L 0校正峰面积+L 1校正峰面积
HC校正峰面积总和＝H 0校正峰面积+H 1校正峰面积+H 2校正峰面积+H 3校正峰面积+H 4校正峰面积
载药量n＝2×Σ(连接药物数×校正峰面积百分比)/100
2.测定结果
表6.药物载量分布测定
Figure PCTCN2021083014-appb-000099
注：ADC-2-1至ADC-2-8中，H 4的含量低于检测限(<<1％)
结果显示，对于采用相同缓冲体系，不同还原反应温度制备的样品，随着还原反应温度的降低，样品药物载量分布均一程度显著提高；对于采用相同还原反应温度，不同缓冲体系制备的样品，采用组氨酸-盐酸缓冲体系的样品药物载量分布更为均一。
测试例2-2：游离毒素测试
1.游离毒素测定方法
1.1.在以下测量条件下进行HPLC分析：
HPLC系统：Waters H-Class超高效液相色谱仪UPLC系统
检测器：TUV检测器(测量波长：370nm)
色谱柱：Waters ACQUITY UPLC Petide BEH C18(
Figure PCTCN2021083014-appb-000100
2.1mm×150mm,1.7μm)
柱温：40℃
流速：0.3mL/min
样品室温度：10℃
流动相A：0.1％三氟乙酸(TFA)水溶液
流动相B：0.1％TFA乙腈溶液
梯度程序：25.0％B-25.0％B(0.00min-1.00min)，25.0％B-55.0％B(1.00min-17.00min)，55.0％B-25.0％B(17.00min-17.10min)，25.0％B-25.0％B(17.10min-20.00min)
注入样品量：5.0μL
1.2.数据分析
毒素的LOD限度的计算公式如下：
毒素限度(ppm)＝0.1×4×1000/C
注：
a)0.1为毒素LOD溶液浓度(μg/mL)，4为样品前处理时的稀释倍数，1000为单位换算系数，C为测定样品的蛋白浓度(mg/mlmL)。
b)供试品中的游离毒素峰面积小于LOD溶液的峰面积，则判定为小于限度或未检测出。
2.测定结果
对ADC-A1(批次1-4)和ADC-B14进行游离毒素检测，结果(见表7)表明采用阳离子柱层析不仅适用于大规模制备，也显著减少了游离毒素。
表7.游离毒素测定
样品 药物载量 游离毒素峰面积
ADC-A1(批次1) 5.7 760
ADC-A1(批次2) 5.7 123
ADC-A1(批次3) 5.7 70
ADC-A1(批次4) 5.7 37
ADC-B14 5.3 5423

Claims (15)

  1. 一种抗体药物偶联物的制备方法，其中所述抗体药物偶联物的结构如通式(Pc-L a-Y-D)所示：
    Figure PCTCN2021083014-appb-100001
    其中：
    W选自C 1-8烷基、C 1-8烷基-C 3-7环烷基或1至8个原子的直链杂烷基，所述直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 3-7环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
    R 1为卤代C 1-6烷基或C 3-7环烷基；
    R 2选自氢原子、卤代C 1-6烷基和C 3-7环烷基；
    或者，R 1和R 2与其相连接的碳原子一起形成C 3-7环烷基；
    R 5选自氢原子、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和羟基C 1-6烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和羟基C 1-6烷基；
    m为0或1；
    n为3至8，n是小数或整数；
    Pc为抗体或其抗原结合片段；
    所述的制备方法包括如下的步骤：
    步骤(a)：在约1℃至约36℃的反应温度条件下，抗体或其抗原结合片段与还原剂反应；
    步骤(b)：步骤(a)的产物与下式(La-Y-D)所示的化合物反应；
    Figure PCTCN2021083014-appb-100002
    其中：W，L 2，L 3，R 1，R 2，R 5，R 6，R 7和m如上所定义。
  2. 根据权利要求1所述的抗体药物偶联物的制备方法，其中步骤(a)中的所述反应温度条件为约4℃至约30℃，优选为约20℃至约30℃，更优选为约25℃。
  3. 根据权利要求1或2所述的抗体药物偶联物的制备方法，其中步骤(a)中的反应是在pH为约4.5至约6.5的条件下进行的；优选地，所述反应是在pH为约5.0至约6.0的条件下进行的；更优选地，所述反应是在pH为约5.6的条件下进行的。
  4. 根据权利要求1至3任一项所述的抗体药物偶联物的制备方法，其中步骤(a)中的反应是在缓冲剂中进行的；优选地，所述的缓冲剂选自组氨酸盐缓冲剂、磷酸盐缓冲剂和醋酸盐缓冲剂；更优选地，所述的缓冲剂为含有EDTA的组氨酸盐缓冲剂。
  5. 根据权利要求1至4任一项所述的抗体药物偶联物的制备方法，其中步骤(a)中的所述还原剂选自三(2-羧乙基)膦或其盐、1,4-二巯基苏糖醇和β-巯基乙醇，优选为三(2-羧乙基)膦盐酸盐。
  6. 根据权利要求1至5任一项所述的抗体药物偶联物的制备方法，其中所述的制备方法还包括步骤(c)，所述步骤(c)包括将步骤(b)的产物进行阳离子柱层析或亲和柱层析纯化；优选地，所述步骤(c)包括将步骤(b)的产物进行阳离子柱层析，所述阳离子层析的填料选自Capto S Impact和Poros XS，优选为Capto S Impact。
  7. 根据权利要求1至6任一项所述的抗体药物偶联物的制备方法，其中所述的抗体或其抗原结合片段选自抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、 抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体和抗Mesothelin抗体，或其抗原结合片段；
    优选地，所述的抗体或其抗原结合片段选自Trastuzumab、Pertuzumab、Nimotuzumab、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96和Glematumamab，或其抗原结合片段。
  8. 根据权利要求1至7任一项所述的抗体药物偶联物的制备方法，其中所述的抗体药物偶联物具有如下式所示的结构：
    Figure PCTCN2021083014-appb-100003
    其中，n为4至8，n是小数或整数。
  9. 根据权利要求1至8任一项所述的抗体药物偶联物的制备方法，其中n为4至8的小数或整数，优选为5至7的小数或整数，更优选为5.3至6.1的小数或整数。
  10. 根据权利要求1至9任一项所述的抗体药物偶联物的制备方法，其中所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；
    优选地，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。
  11. 一种抗体药物偶联物的制备方法，其中所述抗体药物偶联物具有如下式所示的结构：
    Figure PCTCN2021083014-appb-100004
    其中，n为4至8，n是小数或整数；
    所述的制备方法包括如下的步骤：
    步骤(a)：在反应温度为约4℃至约30℃，和pH为约5.0至约6.0的条件下，Trastuzumab与TCEP反应；
    步骤(b)：步骤(a)的产物与下式所示的化合物反应；
    Figure PCTCN2021083014-appb-100005
  12. 一种抗体药物偶联物的制备方法，其中所述抗体药物偶联物具有如下式所示的结构：
    Figure PCTCN2021083014-appb-100006
    其中，n为4至8，n是小数或整数；
    所述的制备方法包括如下的步骤：
    步骤(a)：在反应温度为约25℃，和pH为约5.6的条件下，Trastuzumab与TCEP反应，所述反应是在含有EDTA的组氨酸-盐酸缓冲剂中进行的；
    步骤(b)：步骤(a)的产物与下式所示的化合物反应；
    Figure PCTCN2021083014-appb-100007
    步骤(c)：包括将步骤(b)的产物进行阳离子层析柱纯化。
  13. 一种抗体药物偶联物或其药学上可接受的盐，其中所述抗体药物偶联物的结构如通式(Pc-L a-Y-D)所示：
    Figure PCTCN2021083014-appb-100008
    其中：
    W选自C 1-8烷基、C 1-8烷基-C 3-7环烷基或1至8个原子的直链杂烷基，所述直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 3-7环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-7环烷基中的一个或多个取代基所取代；
    R 1为卤代C 1-6烷基或C 3-7环烷基；
    R 2选自氢原子、卤代C 1-6烷基和C 3-7环烷基；
    或者，R 1和R 2与其相连接的碳原子一起形成C 3-7环烷基；
    R 5选自氢原子、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和羟基C 1-6烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、C 1-6烷基、卤代C 1-6 烷基、氘代C 1-6烷基和羟基C 1-6烷基；
    m为0或1；
    n为4至8，n是小数或整数；
    Pc为抗体或其抗原结合片段；
    并且，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；
    优选地，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。
  14. 一种抗体药物偶联物或其药学上可接受的盐，其中所述抗体药物偶联物是由权利要求1至9任一项所述的抗体药物偶联物的制备方法制备获得的；并且，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；
    优选地，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。
  15. 一种抗体药物偶联物或其药学上可接受的盐，其中所述抗体药物偶联物具有如下式所示的结构：
    Figure PCTCN2021083014-appb-100009
    其中，n为4至8，n是小数或整数；
    所述抗体药物偶联物是由权利要求11或12所述的抗体药物偶联物的制备方法制备获得的；并且，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；
    优选地，所述抗体药物偶联物的药物载量分布为：在抗体重链群体中，结合4个药物的抗体重链比例为4％以下，并且未结合药物的抗体重链比例为5％以下；以及在抗体轻链群体中，结合1个药物的抗体轻链比例为65％以上。
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