WO2022194257A1 - 一种喜树碱衍生物的制备方法 - Google Patents

一种喜树碱衍生物的制备方法 Download PDF

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WO2022194257A1
WO2022194257A1 PCT/CN2022/081542 CN2022081542W WO2022194257A1 WO 2022194257 A1 WO2022194257 A1 WO 2022194257A1 CN 2022081542 W CN2022081542 W CN 2022081542W WO 2022194257 A1 WO2022194257 A1 WO 2022194257A1
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antibody
group
alkyl
preparation
formula
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PCT/CN2022/081542
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French (fr)
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刘洋
尤凌峰
冯君
贺峰
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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 CN202280021634.8A priority Critical patent/CN117015549A/zh
Publication of WO2022194257A1 publication Critical patent/WO2022194257A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • 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
    • A61K47/54—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 organic compound
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links

Definitions

  • the present disclosure belongs to the field of medicine, and relates to a preparation method of camptothecin derivatives.
  • Antibody drug conjugates link monoclonal antibodies or antibody fragments with biologically active cytotoxins through stable chemical linker compounds, making full use of the specificity of antibodies binding to normal cells and tumor cell surface antigens and cytotoxicity, while avoiding the defects of low efficacy of the former and excessive toxic and side effects of the latter.
  • ADCs Antibody drug conjugates
  • camptothecin derivatives which have antitumor effects by inhibiting topoisomerase I.
  • camptothecin derivative ixitecan (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) for antibody coupling
  • ADC combination drug
  • ixitecan is used in a large amount as a toxin and is expensive.
  • WO2020063676, WO2020063673, CN105829346A, CN111689980A, CN112125915A and the like disclose a series of ixitecan analogs and preparation methods thereof.
  • the purpose of the present disclosure is to provide a new preparation method of camptothecin derivatives.
  • One aspect of the present disclosure provides a method for preparing a compound represented by formula (II), comprising the steps of reacting a compound represented by formula (III) with a compound represented by formula (IV),
  • R is selected from a hydrogen atom or a carboxyl protecting group
  • R 1 and R 2 are each independently selected from a hydrogen atom, a C 1-6 alkyl group, a 3-6-membered cycloalkyl group, a 6-10-membered aryl group or a 5-10-membered heteroaryl group, wherein the alkyl group, Cycloalkyl, aryl, heteroaryl optionally selected from one or more C 1 -C 6 alkyl, halogen, hydroxy, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl or C 1 -C 6 alkoxy substituents,
  • R 1 and R 2 are taken together to form the carbon atom to which they are attached, which is optionally substituted by one or more selected from C 1 -C 6 alkyl, halogen, hydroxy, amino, oxo or C 1 -C 6 alkoxy 3-6 membered cycloalkyl substituted by substituent;
  • R 3 , R 4 , and R 5 are each independently selected from hydrogen atoms or C 1-6 alkyl groups
  • n is an integer from 2 to 8;
  • n is an integer from 0 to 4.
  • the carboxyl protecting group is selected from methyl, substituted methyls, ethyl, 2-substituted ethyls, allyl, tert-butyl, alkoxyalkyls, alkanes Oxyalkoxyalkyls, 2,6-dialkylphenyls, benzyls, substituted benzyls, silyls or stannyls;
  • the substituted methyls are selected from 9-fluorenylmethyl, triisopropylsilylmethyl, cyclopropylmethyl, diphenylmethyl or triphenylmethyl;
  • the 2-substituted ethyls are selected from 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(p-toluenesulfonyl)ethyl or 2-cyanoethyl base;
  • alkoxyalkyls are selected from methoxymethyl, benzyloxymethyl or triisopropylsiloxymethyl;
  • alkoxyalkoxyalkyls are selected from methoxyethoxymethyl
  • the 2,6-dialkylphenyls are selected from 2,6-dimethylphenyl, 2,6-diisopropylphenyl or 2,6-di-tert-butyl-4-methoxybenzene base;
  • the substituted benzyls are selected from p-methylbenzyl, 2,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, p-nitrobenzyl or o-nitrobenzyl;
  • the silyl group is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl or phenyldimethylsilyl;
  • the stannyls are selected from trimethylstannyl
  • Described reaction is condensation reaction, and the condensing agent that forms amide bond includes but is not limited to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexyl Carbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1- Hydroxybenzotriazole, 1-Hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7 -Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-1-yloxytris(dimethylamino)phosphonium hexa Fluoro
  • the base is selected from organic bases or inorganic bases, and the organic bases are preferably triethylamine, diethylamine, N,N-diisopropylethylamine (diisopropylethylamine). ethylamine), pyridine, sodium hexamethyldisilazide, n-butyllithium, potassium tert-butoxide or tetrabutylammonium bromide, the inorganic base is selected from lithium hydroxide, sodium hydroxide, hydroxide Potassium, sodium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate or cesium carbonate.
  • reaction can also be carried out by an acid halide method or the like in which an acid halide is previously prepared from an acid halide such as thionyl chloride and oxalyl chloride and a carboxylic acid in the presence of a base.
  • an acid halide such as thionyl chloride and oxalyl chloride and a carboxylic acid in the presence of a base.
  • the solvent used in the reaction can be a conventional solvent, such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide, One or more of diethyl ether, isopropyl ether, methyl tert-butyl ether, acetonitrile, propionitrile, C 1 -C 6 alkyl alcohol, acetone, ethyl acetate, dichloromethane, and chloroform.
  • a conventional solvent such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide, One or more of diethyl ether, isopropyl ether, methyl tert-butyl ether, ace
  • R 1 is selected from a hydrogen atom, a C 1-6 alkyl group, a 3-6 membered cycloalkyl group, a 6-10 membered aryl group, or a 5-10 membered heteroaryl group, wherein said alkyl group , cycloalkyl, aryl, heteroaryl optionally by one or more selected from C 1 -C 6 alkyl, halogen, hydroxyl, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl substituted by substituents of C 1 -C 6 alkoxy groups,
  • R 2 is selected from hydrogen atoms or C 1 -C 6 alkyl optionally substituted by one or more substituents selected from halogen, hydroxy, amino, oxo,
  • R 1 and R 2 are taken together to form the carbon atom to which they are attached, which is optionally substituted by one or more selected from C 1 -C 6 alkyl, halogen, hydroxy, amino, oxo or C 1 -C 6 alkoxy A 3- to 6-membered cycloalkyl substituted by a substituent.
  • R 1 is selected from C 1-6 alkyl, C 1-6 haloalkyl, 3-6 membered cycloalkyl substituted C 1-6 alkyl, 6-10 membered aryl substituted C 1-6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl
  • R2 is a hydrogen atom, or R1 and R2 together with the carbon atom to which they are attached form 3 -6-membered cycloalkyl; preferably R 1 is selected from C 1-6 alkyl, C 1-6 haloalkyl, 3-6 membered cycloalkyl, R 2 is a hydrogen atom, or R 1 and R 2 are attached thereto The carbon atoms are taken together to form a 3-6 membered cycloalkyl group.
  • R 3 , R 4 , and R 5 are all hydrogen atoms.
  • R is a carboxyl protecting group
  • the method further comprises the following step of deprotecting the protecting group
  • the method includes
  • R is selected from methyl, allyl, tert-butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, preferably 2,4-Dimethoxybenzyl.
  • the method further includes
  • the present disclosure also provides a method for preparing a compound represented by formula (II-1), comprising the step of decarboxylation protecting group of the compound represented by formula (II),
  • R is a carboxyl protecting group
  • R 1 , R 2 , R 3 , R 4 , R 5 , m and n are as described above.
  • the step of decarboxylating protecting groups is performed in the presence of an acid, including common inorganic and organic acids, including but not limited to hydrochloric, acetic, sulfuric, nitric, phosphoric, formic, oxalic, lemon acid, benzenesulfonic acid, substituted benzenesulfonic acid, benzoic acid, substituted benzoic acid, maleic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, dichloroacetic acid, difluoroacetic acid, and the like.
  • an acid including common inorganic and organic acids, including but not limited to hydrochloric, acetic, sulfuric, nitric, phosphoric, formic, oxalic, lemon acid, benzenesulfonic acid, substituted benzenesulfonic acid, benzoic acid, substituted benzoic acid, maleic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, dich
  • Substituted benzenesulfonic acid or benzoic acid means that the benzenesulfonic acid or benzoic acid is selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, halogen, cyano, One or more substituents in hydroxyl and amino are substituted.
  • the molar ratio of the compound represented by the formula (II) to the acid can be 1:1-1:100, preferably 1:1-1:30, more preferably 1:1-1:10 .
  • the solvent used in the reaction can be a conventional solvent, such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide, One or more of diethyl ether, isopropyl ether, methyl tert-butyl ether, acetonitrile, propionitrile, C 1 -C 6 alkyl alcohol, acetone, ethyl acetate, dichloromethane, and chloroform.
  • a conventional solvent such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide, One or more of diethyl ether, isopropyl ether, methyl tert-butyl ether, ace
  • the present disclosure also provides a method for preparing a camptothecin derivative, comprising the steps of reacting a compound represented by formula (II-1) with a compound represented by formula (V) to prepare a compound represented by formula (I), and further comprising formula ( II) the step of preparing the compound shown in formula ((II-1) by deprotecting the compound shown,
  • R 6 is selected from hydrogen atom, deuterium atom, C 1-6 alkyl group, 6-10-membered aryl group or 5-10-membered heteroaryl group, wherein the alkyl group, aryl group and heteroaryl group are optionally One or more substituents selected from C 1 -C 6 alkyl, halogen, hydroxyl, amino and oxo are substituted, preferably R 6 is selected from hydrogen atom, C 1-6 alkyl, halogenated C 1-6 alkane group or hydroxy C 1-6 alkyl group, more preferably a hydrogen atom;
  • R is a carboxyl protecting group, and R 1 , R 2 , R 3 , R 4 , R 5 , m and n are as described above.
  • the reaction of the compound represented by the formula (II-1) with the compound represented by the formula (V) is a condensation reaction, and the condensing agent is as described above.
  • the solvent used in the reaction can be a conventional solvent, such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide, One or more of ether, isopropyl ether, methyl tert-butyl ether, acetonitrile, propionitrile, C 1 -C 6 alkyl alcohol, acetone, ethyl acetate, dichloromethane, chloroform, preferably dimethyl Formamide, 1-methyl-2-pyrrolidone, methanol, ethanol, dichloromethane, chloroform, dichloromethane/methanol, dichloromethane/ethanol, etc.
  • reaction can also be carried out by an acid halide method or the like in which an acid halide is previously prepared from an acid halide such as thionyl chloride and oxalyl chloride and a carboxylic acid in the presence of a base.
  • an acid halide such as thionyl chloride and oxalyl chloride and a carboxylic acid in the presence of a base.
  • the method further includes the method of preparing the compound of formula (II) described in this disclosure.
  • the method includes
  • R is selected from methyl, allyl, tert-butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, preferably 2,4-Dimethoxybenzyl.
  • R, R 1 , R 2 , R 3 , R 4 , R 5 , m, and n are as described above.
  • the present disclosure also provides a compound represented by formula (IIb),
  • the present disclosure also provides a compound represented by formula (IIa-1),
  • the present disclosure also provides a method for preparing an antibody-drug conjugate, comprising: the step of preparing the compound represented by formula (I) described in the present disclosure, and after the reduction of Ab, a coupling reaction with the compound represented by formula (I) , the step of obtaining the antibody-drug conjugate represented by formula (X),
  • Ab is an antibody or an antigen-binding fragment
  • k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any number in between)
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , m, and n are as described above.
  • the reducing agent is preferably TCEP, in particular, it is preferred to reduce disulfide bonds on the antibody.
  • the antibody is selected from a chimeric antibody, a humanized antibody, or a fully human antibody; preferably 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, 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-C Antibody, Anti-SLC44A4 Antibody, Anti-CD20
  • the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, or an antigen-binding fragment thereof .
  • k is 2 to 8, preferably 5 to 9.
  • Non-limiting examples include 3, 4, 5, 6, 7.2, 7.5, 8, 8.5, 9.
  • the method includes
  • the antibody-drug conjugates described in the present disclosure can be prepared with reference to known methods.
  • WO2020063676 discloses a series of preparation methods for antibody-drug conjugates, which are incorporated herein by reference in their entirety.
  • the preparation method of the camptothecin derivative described in the present disclosure has high reaction yield and lower cost, and is more suitable for industrial production.
  • the side chain reaction conditions are mild, effectively avoiding impurities generated by the decomposition of the side chain.
  • the synthesis method of the prior art is likely to cause the side chain to be broken and decomposed during the preparation process to generate impurities (eg, impurity 1, etc.), which affects the quality of the product.
  • Alkyl as used in the present disclosure is preferably a C1 - C6 alkyl group.
  • Alkenyl as used in this disclosure is preferably a C2 - C6 alkenyl.
  • alkynyl group referred to in the present disclosure is preferably a C2 - C6 alkynyl group.
  • alkylene group referred to in the present disclosure is preferably a C 1 -C 6 alkylene group.
  • alkenylene group referred to in the present disclosure is preferably a C 2 -C 6 alkenylene group.
  • alkynylene group referred to in the present disclosure is preferably a C 2 -C 6 alkynylene group.
  • alkoxy group referred to in the present disclosure is preferably a C 1 -C 6 alkoxy group.
  • alkyl sulfide group described in the present disclosure is preferably a C 1 -C 6 alkyl sulfide group.
  • Cycloalkyl as used in the present disclosure is preferably 3 to 12 membered, more preferably 3 to 6 membered cycloalkyl.
  • fused cycloalkyl is preferably 6 to 14 membered, more preferably 7 to 10 membered fused cycloalkyl.
  • heterocyclic group described in the present disclosure is preferably a 3- to 12-membered heterocyclic group, more preferably a 3- to 6-membered heterocyclic group.
  • fused heterocyclic group is preferably a 6- to 14-membered, more preferably 7- to 10-membered fused heterocyclic group.
  • aryl group described in the present disclosure is preferably a 6- to 14-membered aryl group, more preferably a 6- to 10-membered aryl group.
  • heteroaryl in the present disclosure is preferably a 5- to 12-membered heteroaryl group, more preferably a 5- to 10-membered heteroaryl group.
  • antibody-drug conjugate refers to a ligand linked to a biologically active drug through a stable linking unit.
  • antibody-drug conjugate (antibody drug conjugate, ADC) refers to linking a monoclonal antibody or antibody fragment with a biologically active glucocorticoid through a stable linking unit. Wherein the antibody or antibody fragment can be combined with the glucocorticoid molecule containing the linker through specific groups therein (eg, interchain disulfide bonds).
  • drug loading refers to the average amount of drug carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the drug loading may range from 1-20, preferably 1-10 glucocorticoids (D) linked per antibody (Ab).
  • the drug load is expressed as k, and the exemplary can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the mean of the values in between.
  • the average amount of drug per ADC molecule after conjugation can be characterized by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains, and epsilon chains.
  • IgG can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists 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 three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • murine antibody in the present disclosure refers to the use of murine antibodies in accordance with the knowledge and skill in the art. In preparation, test subjects are injected with a specific antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To build a chimeric antibody first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the mouse variable region gene from the mouse hybridoma cell. After connecting with human constant region gene into chimeric gene, it is inserted into expression vector, and finally chimeric antibody molecule is expressed in eukaryotic system or prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody, refers to the grafting of murine CDR sequences into a human antibody variable region framework, i.e. a different type of human germline antibody antibodies produced in framework sequences.
  • the heterologous reaction induced by chimeric antibodies can be overcome because they carry a large amount of murine 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 and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al.
  • humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation of the CDRs by phage display. Documents further describing methods involved in humanization using mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the present disclosure is a fully human monoclonal antibody.
  • the technologies related to the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising A bivalent fragment of two Fab fragments linked by a disulfide bridge; (iii) an Fd fragment composed of VH and CH1 domains; (iv) an Fv fragment composed of the one-armed VH and VL domains of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs)
  • single-chain Fv single-chain Fv
  • scFv single-chain Fv
  • Such single chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody.
  • Antigen binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at the 224th position of the H chain), wherein the N-terminal side of the H chain is an antibody fragment. About half and the entire L chain is held together by disulfide bonds.
  • F(ab')2 is an antibody with a molecular weight of about 100,000 obtained by digesting the lower part of two disulfide bonds in the hinge region of IgG with the enzyme pepsin and having antigen-binding activity and comprising two Fab regions linked at the hinge position Fragment.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.
  • single chain antibody is intended to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker molecule.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeats of the GGGGS amino acid sequence or variants thereof, eg using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of CDRs applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to the heavy chain variable domain CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3).
  • CDR1, HCDR2, HCDR3 there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); light
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acid residue numbers in VH are approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3)
  • CDR amino acid residues in VL are approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
  • antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation (see, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).
  • antibodies bind with an affinity (KD) of less than about 10-7 M, eg, about less than 10-8 M, 10-9 M, or 10-10 M or less.
  • KD affinity
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or may integrate into the host cell's genome after introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the present invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions.
  • Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software, from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351 get.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer.
  • Non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
  • the obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • Amino acid sequence identity refers to the alignment of amino acid sequences and, where necessary, the introduction of gaps to achieve a maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity, in the first sequence to the second sequence.
  • the percentage of amino acid residues that are identical For purposes of determining percent amino acid sequence identity, alignment can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • 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.
  • 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-methylp
  • lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl base, 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-dimethylpropyl butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl base, 2,3-dimethylbutyl, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy group, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, carboxyl or carboxylate.
  • the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalk
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups. "Carbocycle” refers to the ring system in a cycloalkyl group.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2) heteroatoms, excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • ring atoms excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 6 ring atoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidine group, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and pyrrolidinyl.
  • Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls. "Heterocycle" refers to a ring system in a heterocyclyl group.
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, preferably 6 to 10 membered, such as benzene base and naphthyl.
  • the aryl ring can be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring.
  • “Aromatic ring” refers to the ring system in an aryl group.
  • Non-limiting examples of aryl groups include:
  • Aryl may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle
  • heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • Heteroaryl is preferably 5 to 12 membered, eg imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazine base, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl.
  • heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring.
  • Heteroaromatic ring refers to the ring system in a heteroaryl group.
  • Non-limiting examples of heteroaryl groups include:
  • Heteroaryl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.
  • Carboxyl protecting groups are suitable groups known in the art for protecting carboxyl groups, see carboxyl protecting groups in the literature ("Protective Groups in Organic Synthesis", 5 Th Ed. TW Greene & P. GMWuts), as examples, Described carboxyl protecting group can be substituted or unsubstituted C 1-10 straight-chain or branched alkyl, substituted or unsubstituted C 2-10 straight-chain or branched alkenyl or alkynyl, substituted or unsubstituted C 2-10 straight-chain or branched A substituted C 3-8 cyclic alkyl group, a substituted or unsubstituted C 5-10 aryl or heteroaryl group, or a (C 1-8 alkyl or aryl) 3 silyl group, and the like.
  • amino protecting groups are suitable groups known in the art for protecting amino groups, see the literature ("Protective Groups in Organic Synthesis", 5 Th . Ed. TW Greene & P. GMWuts) for amino protecting groups, preferably , the amino protecting group can be (C 1-10 alkyl or aryl) acyl, such as: formyl, acetyl, benzoyl, etc.; can be (C 1-6 alkyl or C 6-10 aryl) can also be (C 1-6 alkoxy or C 6-10 aryloxy) carbonyl, such as: Boc or Cbz; can also be substituted or unsubstituted alkyl, such as: trityl group (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).
  • Tr trityl group
  • DMB 2,4-dimethoxybenzyl
  • PMB p-methoxybenzyl
  • Optional or “optionally” means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or instances where it does not.
  • a heterocyclic group optionally substituted with an alkyl group means that an alkyl group may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group .
  • the bond configuration is not specified, i.e. if configurational isomerism exists in the chemical structure, the bond can be or both Two configurations.
  • the bond The configuration is not specified, i.e. it can be either the Z configuration or the E configuration, or both.
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • MS was measured with a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • HPLC High performance liquid chromatography
  • Chiral HPLC analysis was determined using an Agilent 1260DAD high performance liquid chromatograph.
  • HPLC preparations used Waters 2767, Waters 2767-SQ Detector2, Shimadzu LC-20AP and Gilson-281 preparative chromatographs.
  • the CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).
  • the HPLC detection conditions of impurity 1 are: the detection chromatographic column is Sunfire C18 3.5 ⁇ m 4.6 mm*75 mm, mobile phase: TFA/MeCN/H 2 O, detection wavelength: 214 nm. Retention time of impurity 1: 6.96 min.
  • 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 TLC separation and purification products is 0.4mm ⁇ 0.5mm.
  • Silica gel column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.
  • the average inhibition rate and IC 50 value of kinases were measured with NovoStar microplate reader (BMG, Germany).
  • the known starting materials of the present disclosure can be synthesized using 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, Darui chemical companies.
  • Argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.
  • Hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1 L.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenation apparatus and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenation apparatus.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction used a CEM Discover-S 908860 microwave reactor.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, which is 20°C to 30°C.
  • the obtained residue was subjected to column chromatography (The silica gel was preliminarily basified with 0.2% TEA in DCM solution, loaded by wet method, the mobile phase was methanol and 0.05% TEA in DCM solution, 0-100:1), concentrated, and the residue was added with ether (10 mL) and shaken well. After standing, the supernatant was decanted; repeated twice, concentrated to dryness under reduced pressure to obtain compound IIb (875 mg, yield 77.9%, HPLC purity 98%).
  • Ixinotecan hydrochloride (1.45g, 3.07mmol) and DMF (44mL) were added to the reaction flask, the reaction was cooled to 0-5°C in an ice bath, then triethylamine (466mg, 4.61mmol, 642 ⁇ L) was added, and the reaction was Stir under ice bath protection, dissolve compound IIa-1 (2.82 g, 4.29 mmol) in DMF (12 mL), add it to the above reaction solution, then add DMTMM (1.27 g, 4.59 mmol), naturally rise to room temperature and stir until The reaction ends.

Abstract

式(II)所示化合物及其制备方法，以及由式(II)所示化合物制备喜树碱衍生物、抗体-药物偶联物的方法。式(II)所示化合物的制备方法包括式(III)所示化合物与式(IV)所示化合物反应的步骤，该方法收率高、反应条件温和，适合工业化生产。

Description

一种喜树碱衍生物的制备方法
本申请要求申请日为2021/3/17的中国专利申请202110286025.2的优先权。本申请引用上述中国专利申请的全文。
技术领域
本公开属于医药领域，涉及一种喜树碱衍生物的制备方法。
背景技术
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素相连，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素的高效性，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)。
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喜替康(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)应用于抗体偶联药物(ADC)的文献有WO2014057687等。其中，依喜替康作为毒素部分用量较大，且价格昂贵。
WO2020063676、WO2020063673、CN105829346A、CN111689980A、CN112125915A等公开了一系列依喜替康类似物及其制备方法。
发明内容
本公开的目的在于提供一种新的喜树碱衍生物的制备方法。
本公开一方面提供了一种如式(II)所示化合物的制备方法，包括式(III)所示化合物与式(IV)所示化合物反应的步骤，
Figure PCTCN2022081542-appb-000001
其中，
R选自氢原子或羧基保护基；
R 1、R 2各自独立地选自氢原子、C 1-6烷基、3-6元环烷基、6-10元芳基或5-10元杂芳基，其中所述的烷基、环烷基、芳基、杂芳基任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代、3-6元环烷基、6-10元芳基或C 1-C 6烷氧基的取代基所取代，
或者，R 1和R 2与其相连接的碳原子一起形成任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代或C 1-C 6烷氧基的取代基所取代的3-6元环烷基；
R 3、R 4、R 5各自独立地选自氢原子或C 1-6烷基；
n为2至8的整数；
m为0至4的整数。
在某些实施方式中，所述羧基保护基选自甲基、取代的甲基类、乙基、2-取代的乙基类、烯丙基、叔丁基、烷氧基烷基类、烷氧基烷氧基烷基类、2,6-二烷基苯基类、苄基、取代的苄基类、硅烷基类或锡烷基类；
所述取代的甲基类选自9-芴基甲基、三异丙基硅甲基、环丙基甲基、二苯基甲基或三苯基甲基；
所述2-取代的乙基类选自2,2,2-三氯乙基、2-(三甲基硅烷基)乙基、2-(对甲苯磺酰基)乙基或2-氰基乙基；
所述烷氧基烷基类选自甲氧基甲基、苄氧基甲基或三异丙基硅氧基甲基；
所述烷氧基烷氧基烷基类选自甲氧基乙氧基甲基；
所述2,6-二烷基苯基类选自2,6-二甲基苯基、2,6-二异丙基苯基或2,6-二叔丁基-4-甲氧基苯基；
所述取代的苄基类选自对甲基苄基、2,4-二甲氧基苄基、2,6-二甲氧基苄基、对硝基苄基或邻硝基苄基；
所述硅烷基类选自三甲基甲硅烷基、三乙基甲硅烷基、三异丙基甲硅烷基或苯基二甲基甲硅烷基；
所述锡烷基类选自三甲基甲锡烷基；
优选甲基、烯丙基、叔丁基、苄基、2,4-二甲氧基苄基、对甲基苄基、五氟代苯基或甲氧乙氧甲基，更优选2,4-二甲氧基苄基。
所述的反应为缩合反应，形成酰胺键的缩合剂包括但不限于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N,N'-二环己基碳化二亚胺、N,N'-二异丙基碳二酰亚胺、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲脲六氟磷酸酯、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、4-(4,6-二甲氧基三嗪)-4-甲基吗啉。
上述缩合反应优选在碱性条件下进行，所述碱选自有机碱或无机碱，所述有机碱类优选三乙胺、二乙胺、N,N-二异丙基乙胺(二异丙基乙基胺)、吡啶、六甲基二硅基氨基钠，正丁基锂、叔丁醇钾或四丁基溴化铵，所述无机碱选自氢氧化锂、氢氧化钠、氢氧化钾、氢化钠、碳酸钠、碳酸氢钠、碳酸 钾、碳酸氢钾或碳酸铯。
另外，也可利用通过在碱存在下用亚硫酰氯、草酰氯等酰卤与羧酸先制备酰卤的酰卤法等进行反应。
所述反应使用的溶剂可以是常规溶剂，例如水、二甲基甲酰胺、1-甲基-2-吡咯烷酮、四氢呋喃、甲基四氢呋喃、二氧六环、甲苯、二甲苯、二甲亚砜、乙醚、异丙醚、甲基叔丁基醚、乙腈、丙腈、C 1-C 6烷基醇、丙酮、乙酸乙酯、二氯甲烷、氯仿中的一种或多种。
在某些实施方式中，R 1选自氢原子、C 1-6烷基、3-6元环烷基、6-10元芳基或5-10元杂芳基，其中所述的烷基、环烷基、芳基、杂芳基任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代、3-6元环烷基、6-10元芳基或C 1-C 6烷氧基的取代基所取代，
R 2选自氢原子或任选被一个或多个选自卤素、羟基、氨基、氧代的取代基所取代的C 1-C 6烷基，
或者，R 1和R 2与其相连接的碳原子一起形成任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代或C 1-C 6烷氧基的取代基所取代的3-6元环烷基。
在某些实施方式中，R 1选自C 1-6烷基、C 1-6卤代烷基、3-6元环烷基取代的C 1-6烷基、6-10元芳基取代的C 1-6烷基、3-6元环烷基、6-10元芳基或5-10元杂芳基，R 2为氢原子，或者R 1和R 2与其相连接的碳原子一起形成3-6元环烷基；优选R 1选自C 1-6烷基、C 1-6卤代烷基、3-6元环烷基，R 2为氢原子，或者R 1和R 2与其相连接的碳原子一起形成3-6元环烷基。
在某些实施方式中，R 3、R 4、R 5均为氢原子。
在某些实施方式中，R为羧基保护基，所述方法还包括以下脱保护基的步骤，
Figure PCTCN2022081542-appb-000002
在某些实施方式中，所述方法包括
Figure PCTCN2022081542-appb-000003
其中，R选自甲基、烯丙基、叔丁基、苄基、2,4-二甲氧基苄基、对甲基苄基、五氟代苯基或甲氧乙氧甲基，优选2,4-二甲氧基苄基。
在某些实施方式中，所述方法还包括
Figure PCTCN2022081542-appb-000004
本公开还提供了一种如式(II-1)所示化合物的制备方法，包括式(II)所示化合物脱羧基保护基的步骤，
Figure PCTCN2022081542-appb-000005
其中，R为羧基保护基，R 1、R 2、R 3、R 4、R 5、m、n如前所述。
在某些实施方式中，脱羧基保护基的步骤在酸存在下进行，所述酸包括常见的无机酸和有机酸，包括但不限于盐酸、醋酸、硫酸、硝酸、磷酸、甲酸、草酸、柠檬酸、苯磺酸、取代的苯磺酸、苯甲酸、取代的苯甲酸、马来酸、三氟乙酸、三氟甲磺酸、二氯乙酸、二氟乙酸等。取代的苯磺酸或苯甲酸是指苯磺酸或苯甲酸被选自C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷硫基、卤素、氰基、羟基、氨基中的一个或多个取代基所取代。
在某些实施方式中，所述的式(II)所示化合物与酸的摩尔比可以是1:1- 1:100，优选1:1-1:30，更优选1:1-1:10。
所述反应使用的溶剂可以是常规溶剂，例如水、二甲基甲酰胺、1-甲基-2-吡咯烷酮、四氢呋喃、甲基四氢呋喃、二氧六环、甲苯、二甲苯、二甲亚砜、乙醚、异丙醚、甲基叔丁基醚、乙腈、丙腈、C 1-C 6烷基醇、丙酮、乙酸乙酯、二氯甲烷、氯仿中的一种或多种。
本公开还提供了一种喜树碱衍生物的制备方法，包括式(II-1)所示化合物与式(V)所示化合物反应制备式(I)所示化合物的步骤，还包括式(II)所示化合物脱保护基制备式((II-1)所示化合物的步骤，
Figure PCTCN2022081542-appb-000006
其中，R 6选自氢原子、氘原子、C 1-6烷基、6-10元芳基或5-10元杂芳基，其中所述的烷基、芳基、杂芳基任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基和氧代的取代基所取代，优选R 6选自氢原子、C 1-6烷基，卤代C 1- 6烷基或羟基C 1-6烷基，更优选氢原子；
R为羧基保护基，R 1、R 2、R 3、R 4、R 5、m、n如前所述。
式(II-1)所示化合物与式(V)所示化合物反应为缩合反应，缩合剂如前所述。所述反应使用的溶剂可以是常规溶剂，例如水、二甲基甲酰胺、1-甲基-2-吡咯烷酮、四氢呋喃、甲基四氢呋喃、二氧六环、甲苯、二甲苯、二甲亚砜、乙醚、异丙醚、甲基叔丁基醚、乙腈、丙腈、C 1-C 6烷基醇、丙酮、乙酸乙酯、二氯甲烷、氯仿中的一种或多种，优选二甲基甲酰胺、1-甲基-2-吡咯烷酮、甲醇、乙醇、二氯甲烷、氯仿、二氯甲烷/甲醇、二氯甲烷/乙醇等。另外，也可利用通过在碱存在下用亚硫酰氯、草酰氯等酰卤与羧酸先制备酰卤的酰卤法等进行反应。
在某些实施方式中，所述方法还包括本公开所述的制备式(II)所示化合物的方法。
在某些实施方式中，所述方法包括
Figure PCTCN2022081542-appb-000007
其中，R选自甲基、烯丙基、叔丁基、苄基、2,4-二甲氧基苄基、对甲基苄基、五氟代苯基或甲氧乙氧甲基，优选2,4-二甲氧基苄基。
本公开还提供了一种式(II)所示化合物：
Figure PCTCN2022081542-appb-000008
其中，R、R 1、R 2、R 3、R 4、R 5、m、n如前所述。
本公开还提供了一种式(IIb)所示化合物，
Figure PCTCN2022081542-appb-000009
本公开还提供了一种式(IIa-1)所示化合物，
Figure PCTCN2022081542-appb-000010
本公开还提供一种制备抗体-药物偶联物的方法，包括：本公开所述的制备式(I)所示化合物的步骤，以及Ab还原后，与式(I)所示化合物偶联反应，得到式(X)所示的抗体-药物偶联物的步骤，
Figure PCTCN2022081542-appb-000011
其中Ab为抗体或抗原结合片段，k为1至20(包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间任意数值)，
R 1、R 2、R 3、R 4、R 5、R 6、m、n如前所述。
还原剂优选TCEP，特别地，优选还原抗体上的二硫键。
在某些实施方式中，所述抗体选自嵌合抗体、人源化抗体或全人源抗体；优选为单克隆抗体。
在某些实施方式中，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抗体或抗Mesothelin抗体或其抗原结合片段。
在某些实施方式中，其中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、Glematumamab，或其抗原结合片段。
在某些实施方式中，k为2至8，优选为5至9。非限制性实施例包括3、4、5、6、7.2、7.5、8、8.5、9。
在某些实施方式中，所述方法包括
Figure PCTCN2022081542-appb-000012
本公开所述的抗体-药物偶联物可参考已知的方法制备，例如，WO2020063676公开了一系列抗体-药物偶联物的制备方法，在此全文引用。
本公开所述的喜树碱衍生物的制备方法，反应收率高，且成本更低，更加适合工业化生产。侧链反应条件温和，有效避免了侧链分解而产生的杂质。现有工艺的合成方法容易导致侧链在制备过程中断裂分解而产生杂质(例如杂质1等)，影响产品质量。
本公开所述的“烷基”优选C 1-C 6烷基。
本公开所述的“烯基”优选C 2-C 6烯基。
本公开所述的“炔基”优选C 2-C 6炔基。
本公开所述的“亚烷基”优选C 1-C 6亚烷基。
本公开所述的“亚链烯基”优选C 2-C 6亚链烯基。
本公开所述的“亚链炔基”优选C 2-C 6亚链炔基。
本公开所述的“烷氧基”优选C 1-C 6烷氧基。
本公开所述的“烷硫醚基”优选C 1-C 6烷硫醚基。
本公开所述的“环烷基”优选3至12元，更优选3至6元环烷基。
本公开所述的“稠环烷基”优选为6至14元，更优选为7至10元稠环烷基。
本公开所述的“杂环基”优选3至12元，更优选3至6元杂环基。
本公开所述的“稠杂环基”优选6至14元，更优选为7至10元稠杂环基。
本公开所述的“芳基”优选为6至14元，更优选为6至10元芳基。
本公开所述的“杂芳基”优选为5至12元，更优选为5至10元杂芳基。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“抗体-药物偶联物”，指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“抗体-药物偶联物”(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的糖皮质激素相连。其中抗体或抗体片段可通过其中的特定基团(例如链间二硫键)与包含接头的糖皮质激素分子相结合。
术语“载药量”是指抗体-药物偶联物群体中，每个抗体-药物偶联物分子载有的药物平均数量，也可以表示为药物量和抗体量的比值。载药量的范围可以是每个抗体(Ab)连接1-20个，优选1-10个糖皮质激素(D)。在本公开的实施方式中，载药量表示为k，示例性的可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间数值的均值。优选1-10，更优选1-8，或2-8，或2-7，或3-8，或3-7，或3-6，或4-7，或4-6，或4-5的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验、单抗分子大小变异体测定法(CE-SDS)和HPLC特征鉴定偶 联反应后每个ADC分子的药物平均数量。
术语“抗体”指免疫球蛋白，是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。
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。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。
术语“鼠源抗体”在本公开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，将鼠可变区基 因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核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框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。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(在因特网www.mrccpe.com.ac.uk/vbase可获得)，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。本公开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。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.，Natl.Acad.Sci.USA，88，2869，1991和Winter及其同事的方法[Jones等，Nature，321，522(1986)，Riechmann，等，Nature，332，323-327(1988)，Verhoeyen，等，Science，239，1534(1988)]。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本公开为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结 合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载 体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(参见Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨 基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸(参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996))。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如：大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个 实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开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。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
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。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、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中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、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基。
术语“卤素”指氟、氯、溴或碘。
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6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。“碳环”指的是环烷基中的环系。
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；更优选包含3至6个环原子。单环杂环基的非限制性实例包括吡咯烷基、咪唑烷基、四氢呋喃基、四氢噻吩基、二氢咪唑基、二氢呋喃基、二氢吡唑基、二氢吡咯基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等，优选哌啶基、吡咯烷基。多环杂环基包括螺环、稠环和桥环的杂环基。“杂环”指的是杂环基中的环系。
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。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。“芳环”指的是芳基中的环系。芳基非限制性实例包括：
Figure PCTCN2022081542-appb-000013
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、杂环烷硫基、羧基或羧酸酯基，优选苯基。
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12元，例如咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基等，优选为咪唑基、吡唑基、嘧啶基或噻唑基；更优选为吡唑基或噻唑基。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。“杂芳环”指的是杂芳基中的环系。杂芳基非限制性实例包括：
Figure PCTCN2022081542-appb-000014
Figure PCTCN2022081542-appb-000015
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、杂环烷硫基、羧基或羧酸酯基。
“羧基保护基”是本领域已知的适当的用于羧基保护的基团，参见文献(“Protective Groups in Organic Synthesis”，5 Th Ed.T.W.Greene&P.G.M.Wuts)中的羧基保护基团，作为示例，所述的羧基保护基可以是取代或非取代的C 1-10的直链或支链烷基、取代或非取代的C 2-10的直链或支链烯基或炔基、取代或非取代的C 3-8的环状烷基、取代或非取代的C 5-10的芳基或杂芳基、或(C 1-8烷基或芳基) 3硅烷基等。
“氨基保护基”是本领域已知的适当的用于氨基保护的基团，参见文献(“Protective Groups in Organic Synthesis”，5 Th.Ed.T.W.Greene&P.G.M.Wuts)中的氨基保护基团，优选地，所述的氨基保护基可以是(C 1-10烷基或芳香基)酰基，例如：甲酰基，乙酰基，苯甲酰基等；可以是(C 1-6烷基或C 6-10芳基)磺酰基；也可以是(C 1-6烷氧基或C 6-10芳基氧基)羰基，例如：Boc或Cbz；还可以是取代或非取代的烷基，例如：三苯甲基(Tr)、2,4-二甲氧基苄基(DMB)、对甲氧基苄基(PMB)或苄基(Bn)。
“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地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
本公开所述化合物的化学结构中，键
Figure PCTCN2022081542-appb-000016
并未指定构型，即如果化学结构中存在构型异构，键
Figure PCTCN2022081542-appb-000017
可以为
Figure PCTCN2022081542-appb-000018
或者同时包含
Figure PCTCN2022081542-appb-000019
Figure PCTCN2022081542-appb-000020
两种构型。本公开所述化合物的化学结构中，键
Figure PCTCN2022081542-appb-000021
并未指定构 型，即可以为Z构型或E构型，或者同时包含两种构型。
具体实施方式
以下将结合具体实例详细地解释本公开，使得本专业技术人员更全面地理解本公开具体实例仅用于说明本公开的技术方案，并不以任何方式限定本公开。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的单位给出。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-d 6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo，型号：Finnigan LCQ advantage MAX)。
高效液相色谱法(HPLC)分析使用Agilent HPLC 1200DAD、Agilent HPLC 1200VWD和Waters HPLC e2695-2489高压液相色谱仪。
手性HPLC分析测定使用Agilent 1260DAD高效液相色谱仪。
高效液相制备使用Waters 2767、Waters 2767-SQ Detecor2、Shimadzu LC-20AP和Gilson-281制备型色谱仪。
手性制备使用Shimadzu LC-20AP制备型色谱仪。
CombiFlash快速制备仪使用Combiflash Rf200(TELEDYNE ISCO)。
杂质1的HPLC检测条件为：检测色谱柱为Sunfire C18 3.5μm4.6mm*75mm，流动相：TFA/MeCN/H 2O，检测波长：214nm。杂质1的保留时间：6.96min。
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。
硅胶柱色谱法一般使用烟台黄海硅胶200～300目硅胶为载体。
激酶平均抑制率及IC 50值的测定用NovoStar酶标仪(德国BMG公司)。
本公开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 Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够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。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例1
Figure PCTCN2022081542-appb-000022
将化合物IVc(800mg，1.39mmol，根据WO2020063676公开的方法制备)溶于二氯甲烷(4mL)，加入二乙胺(2mL)，在氩气保护下室温搅拌至反应结束；10℃下，减压浓缩除去有机溶剂，残余物中加入正己烷(10mL)，振荡后静置，倒去上层清液，重复三次，最后减压浓缩至干，得到化合物IVb粗品。将上述粗品溶于DMF(10mL)，加入IIIa(658mg，1.39mmol，根据WO2020063676公开的方法制备)，DMTMM(462mg，1.67mmol)，室温搅拌至反应结束，在反应液中加入水(60mL)淬灭，乙酸乙酯萃取，合并有机相，依次用水(30mL×2)，饱和氯化钠溶液洗涤(30mL×2)，无水硫酸钠干燥，过滤，减压浓缩，所得残余物柱层析(硅胶预先用含0.2％TEA的DCM溶液碱化，湿法上样，流动相为甲醇和含0.05％TEA的DCM溶液，0～100:1)，浓缩，残余物加入乙醚(10mL)，充分振荡后静置，倒去上层清液；重复两次，减压浓缩至干，得到化合物IIb(875mg，收率77.9％，HPLC纯度98％)。
1H NMR(500MHz,DMSO-d 6)δ8.53(t,J＝6.8Hz,1H),8.28(d,J＝6.0Hz,1H),8.11(d,J＝8.0Hz,1H),8.08(d,J＝6.0Hz,1H),8.01(d,J＝5.9Hz,1H),7.25(m,5H),7.19(d,J＝6.8Hz,1H),6.99(s,2H),6.58(s,1H),6.51(d,J＝8.3Hz,1H),5.10(d,J＝12.0Hz,1H),5.01(d,J＝12.0Hz,1H),4.63–4.48(m,3H),3.78(s,3H),3.76(s,3H),3.70(m,5H),3.59(m,1H),3.37(t,J＝7.2Hz,2H),3.06(m,1H),2.80(m,1H),2.11(t,J＝7.5Hz,2H),1.47(m,4H),1.26–1.14(m,3H),1.01(m,1H),0.45(d,J＝8.4Hz,2H),0.38–0.31(m,2H).MS:m/z 829.3[M+Na] +.
实施例2
Figure PCTCN2022081542-appb-000023
在反应瓶中加入化合物IIb(3.46g，4.29mmol)，反应在冰浴下降温至0～5℃，然后加入三乙基硅烷(1.49g，12.81mmol，2mL)和140mL二氯乙酸的DCM溶液(2％，v/v)，保温搅拌至反应结束。在反应液中缓慢滴加乙醚(420mL)，加毕，混合物在室温下打浆搅拌30分钟，过滤；滤饼用乙醚(120mL)在室温继续打浆30分钟，过滤；滤饼继续用乙醚(120mL)室温打浆30分钟，过滤，减压干燥，得到目标化合物IIa-1(2.84g，收率100％，HPLC纯度92.05％，其中杂质1为0.98％)。
1H NMR(500MHz,DMSO-d 6)δ12.56(s,1H),8.51(t,J＝6.9Hz,1H),8.28(t,J＝6.0Hz,1H),8.11(d,J＝8.1Hz,1H),8.09–8.04(m,1H),8.00(t,J＝5.9Hz,1H),7.23(m,4H),7.19(d,J＝6.8Hz,1H),7.00(s,2H),4.62–4.49(m,3H),3.79–3.66(m,4H),3.60(m,2H),3.45(d,J＝7.6Hz,1H),3.41–3.38(m,1H),3.08(m,1H),2.80(dd,J＝13.8,9.8Hz,1H),2.11(t,J＝7.4Hz,2H),1.48(m,4H),1.26–1.15(m,3H),1.03(q,J＝6.5Hz,1H),0.46(m,2H),0.36(m,2H).MS:m/z 679.0[M+Na] +.
实施例3
Figure PCTCN2022081542-appb-000024
在反应瓶中加入盐酸依喜替康(1.45g，3.07mmol)和DMF(44mL)，反应在冰浴下降至0～5℃，然后加入三乙胺(466mg，4.61mmol，642μL)，反应在冰浴保护下搅拌，将化合物IIa-1(2.82g，4.29mmol)溶于DMF(12mL)后，加入至上述反应液中，再加入DMTMM(1.27g，4.59mmol)，自然升至室温搅拌至反应结束。在反应液中加入DCM-MeOH溶液(DCM:MeOH＝10：1，200mL)，水(180mL)，混合物在室温搅拌，静置分层；水相用DCM-MeOH溶液(DCM:MeOH＝10：1)萃取，合并有机相，有机相用水洗，无水硫酸钠干燥，过滤，减压浓缩，所得残余物柱层析(combinflash，展开剂：DCM:MeOH＝10:1)，得到较纯的目标产物化合物Ia(1.08g，收率33％，纯度97.02％)和部分粗品，粗品再次柱层析得到较纯的目标产物化合物Ia(813mg，收率25％，纯度94.62％)。
由于已根据其特殊的实施方案描述了本公开，某些修饰和等价变化对于精通此领域的技术人员是显而易见的且包括在本公开的范围内。

Claims (23)

  1. 一种如式(II)所示化合物的制备方法，包括式(III)所示化合物与式(IV)所示化合物反应的步骤，
    Figure PCTCN2022081542-appb-100001
    其中，
    R选自氢原子或羧基保护基；
    R 1、R 2各自独立地选自氢原子、C 1-6烷基、3-6元环烷基、6-10元芳基或5-10元杂芳基，其中所述的烷基、环烷基、芳基、杂芳基任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代、3-6元环烷基、6-10元芳基或C 1-C 6烷氧基的取代基所取代，
    或者，R 1和R 2与其相连接的碳原子一起形成任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代或C 1-C 6烷氧基的取代基所取代的3-6元环烷基；
    R 3、R 4、R 5各自独立地选自氢原子或C 1-6烷基；
    n为2至8的整数；
    m为0至4的整数。
  2. 根据权利要求1所述的制备方法，其中所述羧基保护基选自甲基、取代的甲基类、乙基、2-取代的乙基类、烯丙基、叔丁基、烷氧基烷基类、烷氧基烷氧基烷基类、2,6-二烷基苯基类、苄基、取代的苄基类、硅烷基类或锡 烷基类，优选甲基、烯丙基、叔丁基、苄基、2,4-二甲氧基苄基、对甲基苄基、五氟代苯基或甲氧乙氧甲基，更优选2,4-二甲氧基苄基。
  3. 根据权利要求1或2所述的制备方法，其中反应采用的缩合剂选自1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N,N'-二环己基碳化二亚胺、N,N'-二异丙基碳二酰亚胺、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲脲六氟磷酸酯、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、4-(4,6-二甲氧基三嗪)-4-甲基吗啉中的一种或多种。
  4. 根据权利要求1-3任意一项所述的制备方法，其中所述反应在碱性条件下进行，所述碱选自有机碱或无机碱，所述有机碱类优选三乙胺、二乙胺、N,N-二异丙基乙胺(二异丙基乙基胺)、吡啶、六甲基二硅基氨基钠，正丁基锂、叔丁醇钾或四丁基溴化铵，所述无机碱选自氢氧化锂、氢氧化钠、氢氧化钾、氢化钠、碳酸钠、碳酸氢钠、碳酸钾、碳酸氢钾或碳酸铯。
  5. 根据权利要求1-4任意一项所述的制备方法，其中，
    R 1选自氢原子、C 1-6烷基、3-6元环烷基、6-10元芳基或5-10元杂芳基，其中所述的烷基、环烷基、芳基、杂芳基任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代、3-6元环烷基、6-10元芳基或C 1-C 6烷氧基的取代基所取代，
    R 2选自氢原子或任选被一个或多个选自卤素、羟基、氨基、氧代的取代基所取代的C 1-C 6烷基，
    或者，R 1和R 2与其相连接的碳原子一起形成任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基、氧代或C 1-C 6烷氧基的取代基所取代的3-6元环烷基；
    优选R 1选自C 1-6烷基、C 1-6卤代烷基、3-6元环烷基取代的C 1-6烷基、6-10元芳基取代的C 1-6烷基、3-6元环烷基、6-10元芳基或5-10元杂芳基，R 2 为氢原子，或者R 1和R 2与其相连接的碳原子一起形成3-6元环烷基；
    更优选R 1选自C 1-6烷基、C 1-6卤代烷基、3-6元环烷基，R 2为氢原子，或者R 1和R 2与其相连接的碳原子一起形成3-6元环烷基。
  6. 根据权利要求1-5任意一项所述的制备方法，其中R 3、R 4、R 5均为氢原子。
  7. 根据权利要求1-6任意一项所述的制备方法，其中R为羧基保护基，所述方法还包括脱保护基制备式(II-1)所示化合物的步骤，
    Figure PCTCN2022081542-appb-100002
  8. 根据权利要求1-7任意一项所述的制备方法，其中所述方法包括
    Figure PCTCN2022081542-appb-100003
    其中，R选自甲基、烯丙基、叔丁基、苄基、2,4-二甲氧基苄基、对甲基苄基、五氟代苯基或甲氧乙氧甲基，优选2,4-二甲氧基苄基。
  9. 根据权利要求8所述的制备方法，其中所述方法还包括
    Figure PCTCN2022081542-appb-100004
  10. 一种喜树碱衍生物的制备方法，包括式(II-1)所示化合物与式(V)所示化合物反应制备式(I)所示化合物的步骤，还包括式(II)所示化合物脱保护基制备式((II-1)所示化合物的步骤，其中，
    R 6选自氢原子、氘原子、C 1-6烷基、6-10元芳基或5-10元杂芳基，其中所述的烷基、芳基、杂芳基任选被一个或多个选自C 1-C 6烷基、卤素、羟基、氨基和氧代的取代基所取代，优选R 6选自氢原子、C 1-6烷基，卤代C 1-6烷基或羟基C 1-6烷基，更优选氢原子；
    R为羧基保护基，R 1、R 2、R 3、R 4、R 5、m、n如权利要求1所述，
    Figure PCTCN2022081542-appb-100005
  11. 根据权利要求10所述的制备方法，所述方法还包括权利要求1-9任意一项所述的如式(II)所示化合物的制备方法。
  12. 根据权利要求10或11所述的制备方法，所述方法包括
    Figure PCTCN2022081542-appb-100006
    其中，R选自甲基、烯丙基、叔丁基、苄基、2,4-二甲氧基苄基、对甲基苄基、五氟代苯基或甲氧乙氧甲基，优选2,4-二甲氧基苄基。
  13. 一种制备抗体-药物偶联物的方法，包括权利要求10-12任意一项所述的喜树碱衍生物的制备方法的步骤，以及Ab还原后，与式(I)所示化合物偶联反应，得到式(X)所示的抗体-药物偶联物的步骤，
    Figure PCTCN2022081542-appb-100007
    其中Ab为抗体或抗原结合片段，k为1至20，R 1、R 2、R 3、R 4、R 5、R 6、m、n如权利要求1所述。
  14. 根据权利要求13所述的制备方法，其中所述抗体选自嵌合抗体、人源化抗体或全人源抗体；优选为单克隆抗体。
  15. 根据权利要求13或14所述的制备方法，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抗体或抗Mesothelin抗体或其抗原结合片段。
  16. 根据权利要求13-15任意一项所述的制备方法，其中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、Glematumamab，或其抗原结合片段。
  17. 根据权利要求13-16任意一项所述的制备方法，其中k为2至8，优选为5至9。
  18. 根据权利要求13-17任意一项所述的制备方法，其中所述方法包括
    Figure PCTCN2022081542-appb-100008
  19. 一种如式(II-1)所示化合物的制备方法，包括式(II)所示化合物脱羧基保护基的步骤，
    Figure PCTCN2022081542-appb-100009
    其中，R为羧基保护基，R 1、R 2、R 3、R 4、R 5、m、n如权利要求1所述。
  20. 根据权利要求19所述的制备方法，其中脱羧基保护基的步骤在酸存在下进行，所述酸选自盐酸、醋酸、硫酸、硝酸、磷酸、甲酸、草酸、柠檬酸、苯磺酸、取代的苯磺酸、苯甲酸、取代的苯甲酸、马来酸、三氟乙酸、三氟甲磺酸、二氯乙酸、二氟乙酸中的一种或多种。
  21. 式(II)所示化合物：
    Figure PCTCN2022081542-appb-100010
    其中，R、R 1、R 2、R 3、R 4、R 5、m、n如权利要求1所述。
  22. 根据权利要求21所述的化合物，其为式(IIb)所示化合物，
    Figure PCTCN2022081542-appb-100011
  23. 根据权利要求21所述的化合物，其为式(IIa-1)所示化合物，
    Figure PCTCN2022081542-appb-100012
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