WO2023061466A1 - 一种艾日布林衍生物的制备方法 - Google Patents

一种艾日布林衍生物的制备方法 Download PDF

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WO2023061466A1
WO2023061466A1 PCT/CN2022/125301 CN2022125301W WO2023061466A1 WO 2023061466 A1 WO2023061466 A1 WO 2023061466A1 CN 2022125301 W CN2022125301 W CN 2022125301W WO 2023061466 A1 WO2023061466 A1 WO 2023061466A1
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antibody
alkyl
cycloalkyl
formula
preparation
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French (fr)
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彭祥龙
陈仁杰
高大志
陈亚
张磊
郭昌山
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江苏恒瑞医药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D307/18—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/20—Oxygen atoms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the disclosure belongs to the field of medicine, and relates to a preparation method of eribulin derivatives.
  • Microtubules are powerful, filamentous cytoskeletal proteins associated with a variety of cellular functions including intracellular migration and transport, cell signaling, and maintenance of cell shape. Microtubules also play a key role in mitotic cell division by forming the mitotic spindle required for the division of chromosomes into two daughter cells.
  • the biological function of microtubules in all cells is largely regulated by their polymerization kinetics through the reversible, noncovalent addition of ⁇ and ⁇ tubulin dimers to the ends of microtubules. This kinetic behavior and the resulting control of microtubule length are integral to the proper function of the mitotic spindle.
  • Halichondrin B is a polyether macrolide compound containing only C, H, and O atoms isolated from the sponge Halichondria okadai by Japanese scientists Hirata and Uemura in 1986. Antitumor activity.
  • the molecular structure of halichondrin B is very complex, including 32 chiral centers, with more than 4 billion isomers, and the synthesis is very difficult.
  • Eribulin is a derivative of halichondrin B and is a tubulin inhibitor.
  • WO9965894 discloses the structure and synthesis method of Eribulin for the first time. On November 15, 2010, the FDA approved Eribulin mesylate (Halaven) injection for the treatment of patients with metastatic breast cancer who have received at least two chemotherapy regimens.
  • PCT/CN2021/073314 relates to a new eribulin derivative and its drug conjugate.
  • the compound and its drug conjugate have good antitumor activity.
  • the structure of the compound is as follows:
  • the purpose of the present disclosure is to provide a new preparation method of eribulin derivatives.
  • the present disclosure also provides a method for preparing the compound represented by formula (I), including the step of preparing the compound represented by formula (I-1) from the compound represented by formula (I-2), and the compound represented by formula (I-1) Show the step of compound deprotection group preparation compound shown in formula (I),
  • R 1 is selected from alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclo propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl
  • the substituent is substitute
  • R is selected from methyl
  • the methylating agent used in the step is selected from methyl iodide, dimethyl sulfate or trimethyloxonium tetrafluoroborate, preferably trimethyloxonium Onium tetrafluoroborate.
  • the molar ratio of the compound represented by the formula (I-2) to the methylating agent is 1:0.1 to 1:10, preferably 1:1 to 1:5, more preferably 1:1 ⁇ 1:3.
  • the reaction temperature is -50 to -50°C, preferably -20 to -5°C.
  • the removal of the amino-protecting group can be carried out by methods commonly used in the art.
  • the reagent for removing the amino protecting group is selected from ammonia water, methylamino alcohol solution, ethylenediamine and hydrazine, preferably methylamine ethanol solution.
  • the method further includes the step of preparing the compound represented by formula (I-2) from the compound represented by formula (I-3),
  • the solvents used in the reactions described in the present disclosure can be conventional solvents such as dimethylformamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, dioxane, toluene, One or more of dimethyl sulfoxide, diethyl ether, isopropyl ether, methyl tert-butyl ether, dichloromethane, chloroform, acetone, acetonitrile, methanol, ethanol, isopropanol, water, preferably tetrahydrofuran, ethyl acetate , dioxane, toluene, dimethyl sulfoxide, ether, isopropyl ether, dichloromethane, chloroform, acetone, acetonitrile, methanol, ethanol, isopropanol, one or more.
  • the present disclosure also provides a method for preparing a compound represented by formula (L-I), including the method for preparing a compound represented by formula (I) described in the present disclosure, wherein L is a linker,
  • the linker is extracellularly stable such that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in a cell, eg, a cancer cell.
  • the linker comprises a cleavable moiety positioned such that no portion of the linker or the antibody portion remains bound to the eribulin derivative after cleavage.
  • the cleavable moiety in the linker is a cleavable peptide moiety.
  • ADCs comprising a cleavable peptide moiety exhibit lower levels of aggregation, improved antibody:drug ratios, increased targeted killing of cancer cells, reduced non- Off-target killing of cancer cells, and/or high drug load.
  • addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker.
  • increased potency and/or cytotoxicity is in cancers expressing intermediate levels of an antigen targeted by the antibody portion of the ADC (e.g., intermediate FRA expression).
  • the cleavable peptide moiety is capable of being cleaved by an enzyme
  • the linker is one that is cleavable by an enzyme.
  • the enzyme is a cathepsin and the linker is a linker that the cathepsin is capable of cleaving.
  • an enzymatically cleavable linker eg, a cathepsin-cleavable linker
  • the linker comprises an amino acid unit, preferably comprising from 2 to 7 amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid , Peptide residues composed of amino acids of aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine -Lysine (Gly-Gly-lys), Valine-Lysine (Val-lys), Valine-Alanine (Val-Ala), Valine-Phenylalanine (Val-Phe ) or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).
  • the linker is selected from:
  • the present disclosure also provides a method for preparing an antibody-drug conjugate represented by formula (ADC-I), including the step of preparing the antibody-drug conjugate represented by formula (L-I) from a compound represented by formula (L-I) , and the step of preparing the compound shown in formula (L-I) described in the present disclosure,
  • Ab is an antibody or an antigen-binding fragment thereof
  • L is a linker that covalently connects Ab to D, said L is as defined in any one of claims 5-7; and 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 value between any two values),
  • R 1 is selected from alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclo propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl
  • the substituent is substitute
  • the antibody in the antibody-drug conjugate (ADC) of the present disclosure is selected from murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antibody or antigen-binding fragment thereof in the antibody-drug conjugate (ADC-I) is selected from anti-HER2 (ErbB2) antibodies, anti-EGFR antibodies, anti-B7-H3 antibodies, 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-CD79 antibody, anti-TROP-2 antibody, anti-HER2 (
  • the antibody in the antibody-drug conjugate (ADC-I) is a known antibody, selected from but not limited to Trastuzumab (Trastuzumab), Pertuzumab (Pertuzumab) , Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Vitin-Pinertuzumab ( Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, farletuzumab, and Glematumamab or antigen-binding fragments thereof .
  • Trastuzumab Trastuzumab
  • Pertuzumab Pertuzumab
  • Nimotuzumab Nimotuzumab
  • Enoblituzumab Enoblituzumab
  • Emibetuzumab Inotuzumab
  • the antibody-drug conjugate is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe-N-drug conjugate
  • D is Preferably Ab is farletuzumab, preferably k is selected from 2.0-2.5 or 2.5-3.5.
  • the method disclosed in the prior art can be used to prepare the compound represented by the formula (L-I) with the compound represented by the formula (I) as a reactant, or to prepare the antibody represented by the formula (ADC-I) with the compound represented by the formula (L-I) as a reactant - Drug conjugates, such as methods disclosed in PCT/CN2021/073314, which are incorporated herein in their entirety.
  • R 1 is selected from alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclo propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl
  • the substituent is substitute
  • the preparation method of the new eribulin derivatives described in this disclosure adopts a new amino protecting group, which avoids the selectivity problem in the methylation process, greatly reduces the generation of dimethylated impurities, and alleviates the The difficulty of post-reaction treatment is reduced, and the reaction yield is improved, which is beneficial to large-scale industrial production.
  • the product obtained by the existing preparation process has many dimethylated impurities, which are difficult to remove and ultimately affect the quality of the final product.
  • alkyl in the present disclosure is preferably a C 1 -C 6 alkyl.
  • alkenyl in the present disclosure is preferably a C 2 -C 6 alkenyl.
  • alkynyl in the present disclosure is preferably a C 2 -C 6 alkynyl.
  • alkylene in the present disclosure is preferably a C 1 -C 6 alkylene.
  • alkenylene group described in the present disclosure is preferably a C 2 -C 6 alkenylene group.
  • alkynylene group described in the present disclosure is preferably a C 2 -C 6 alkynylene group.
  • alkoxy in the present disclosure is preferably C 1 -C 6 alkoxy.
  • alkylsulfide group described in the present disclosure is preferably a C 1 -C 6 alkylsulfide group.
  • cycloalkyl in the present disclosure is preferably 3 to 12 membered, more preferably 3 to 6 membered cycloalkyl.
  • fused cycloalkyl group is preferably 6 to 14 membered, more preferably 7 to 10 membered fused cycloalkyl group.
  • 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.
  • the "fused heterocyclic group" described in the present disclosure is preferably 6 to 14 membered, more preferably 7 to 10 membered condensed 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 5 to 12 membered, more preferably 5 to 10 membered heteroaryl.
  • drug refers to cytotoxic drugs or immunomodulators.
  • Cytotoxic drugs can have strong chemical molecules in tumor cells that disrupt their normal growth. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, resulting in serious side effects.
  • the term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (e.g.
  • Immunomodulators are inhibitors of immune checkpoint molecules.
  • linker refers to a chemical structural fragment or bond that is connected to a ligand at one end and a drug at the other end, and can also be connected after other linkers. Then connect with the drug.
  • a joint may comprise one or more joint components.
  • exemplary linker building blocks include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenyl Alanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate ( SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl (4 -iodo-acetyl)aminobenzoate (SIAB).
  • MC 6-maleimidocaproyl
  • MP maleimidopropionyl
  • Vcit valine-citrulline
  • Linkers can include Stretch units, Spacer units, Amino Acid units and Stretcher units. It can be synthesized by methods known in the art, such as described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates release of the drug in the cell.
  • acid-labile e.g., hydrazone
  • protease-sensitive e.g., peptidase-sensitive
  • photolabile, dimethyl, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
  • stretch unit refers to a chemical structural fragment that is covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptidic chemical moiety at the other end.
  • spacer unit is a bifunctional compound structural fragment that can be used to couple amino acid units and cytotoxic drugs to form antibody-drug conjugates. This coupling method can selectively link cytotoxic drugs to amino acid units superior.
  • amino acid refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure.
  • the general formula is H 2 NCHRCOOH, R is H, substituted or unsubstituted alkyl, etc. According to the position of the amino group connected to the carbon atom in the carboxylic acid, it can be divided into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ...-amino acids.
  • amino acids that make up natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the ⁇ -carbon atom, that is, ⁇ -amino acids, including glycine (Glycine), alanine (Alanine), valine (Valine), Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid, Histidine, Asparagine, Glutamic acid, Lysine, Glutamine, Methionine, Arginine , Serine, Threonine, Cysteine, Proline, etc. Unnatural amino acids such as citrulline.
  • the extension unit is PAB
  • the structure is like a 4-iminobenzylcarbamoyl fragment, and its structure is shown in formula (VI), which is connected to D,
  • Joint components include but are not limited to:
  • MC 6-maleimidocaproyl
  • Val-Cit or "vc” valine-citrulline (an exemplary dipeptide in a protease cleavable linker)
  • PAB p-aminobenzyloxycarbonyl (exemplary of a "self-sacrificing" linker component)
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent its cleavage by cathepsin B)
  • MC(PEG) 6 -OH maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine)
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • PBS Phosphate Buffered Saline.
  • antibody-drug conjugate means that a ligand is linked to a biologically active drug through a stable linker unit.
  • antibody drug conjugate refers to linking a monoclonal antibody or antibody fragment with a toxic drug with biological activity through a stable linker unit.
  • drug loading can be expressed as the ratio of the amount of drug to the amount of antibody, that is, the average amount of drug conjugated to each antibody in the ADC.
  • the range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) linked to each antibody (Ab).
  • the drug loading is expressed as k, which may 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 any two values in between.
  • the average amount of drug per ADC molecule after conjugation can be identified using routine methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.
  • the molecular size variant determination method (CE-SDS) of the disclosed monoclonal antibody can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under reducing and non-reducing conditions, according to the molecular weight, according to the capillary electrophoresis method (2015 edition of "Chinese Pharmacopoeia” 0542), quantitatively determine the purity of recombinant monoclonal antibody products.
  • CE-SDS sodium dodecyl sulfate capillary electrophoresis
  • the loading of antibody-drug conjugates can be controlled by the following non-limiting methods, including:
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by 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 classes, or isotypes, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are mu, delta, and gamma chains, respectively , ⁇ chain, and ⁇ chain.
  • IgG can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • the antibody described in the present disclosure is preferably a specific antibody against a cell surface antigen on a target cell, and the sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly, which is a variable region (Fv region); The rest of the amino acid sequence at the end is relatively stable and is 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 antibodies, also known as complementarity determining regions (CDR).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal 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.
  • 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 this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
  • chimeric antibody is 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 that secretes a mouse-derived 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 required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying 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 the 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), as well as in Kabat, E.A. et al.
  • the humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].
  • 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 relevant technologies for 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, etc.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody.
  • 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 (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs joined by a synthetic linker.
  • CDRs complementarity
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thus making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule. chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883).
  • single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
  • Antigen-binding portions 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 an IgG antibody molecule with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which About half and the entire L chain is held together by disulfide bonds.
  • F(ab')2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin 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 prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.
  • single-chain antibody single-chain Fv or “scFv” is meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules.
  • 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 the repeated GGGGS amino acid sequence or variants thereof, for example 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. Immuno 1.31:94-106, 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.
  • One of the most commonly used definitions of the six 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 CDR1, CDR L2, and L3 of 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.
  • Amino acid sequence boundaries for CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • 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.
  • epitope refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
  • An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996)).
  • antibodies bind with an affinity (KD) of less than about 10 "7M , eg, about less than 10 "8M , 10 "9M or 10 " 10M 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 into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer 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, wherein 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 (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or can integrate into the genome of the host cell after introduction, thereby following The host genome is replicated together (eg, non-episomal mammalian vectors).
  • Antigen-binding fragments can also be prepared by conventional methods.
  • one or more human FR regions are added to the non-human CDR region by genetic engineering methods.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT) by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 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 that are readily transformed include members of the enterobacteriaceae such as strains of Escherichia coli or Salmonella; the 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.
  • Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, purify with an A or G Sepharose FF column with adjusted buffer.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
  • Amino acid sequence identity means that when the amino acid sequences are aligned and gaps are introduced as necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, the difference between a first sequence and a second sequence is The percentage of amino acid residues that are identical to each other. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various 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. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • peptide refers to a compound fragment between an amino acid and a protein. It is formed by connecting two or more amino acid molecules through peptide bonds. It is a structural and functional fragment of a protein, such as hormones, enzymes, etc. All peptides.
  • sucrose refers to a biomacromolecule composed of three elements, C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides.
  • fluorescent probe refers to a characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescence properties (excitation and emission wavelength, intensity, lifetime and polarization, etc.) can vary with the properties of the environment, such as polarity, refractive index A class of fluorescent molecules that can be sensitively changed due to changes in viscosity, viscosity, etc., which can change one or several fluorescent properties by non-covalent interaction with nucleic acid (DNA or RNA), protein or other macromolecular structures, which can be used for research Properties and behavior of macromolecular substances.
  • DNA or RNA nucleic acid
  • protein or other macromolecular structures which can be used for research Properties and behavior of macromolecular substances.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a linear 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-dimethyl Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl group, 2,3-dimethylbutyl group, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being preferably one or more of the following groups independently selected from alkyl radical, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, carboxyl or carboxylate.
  • the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloal
  • 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 containing 3 to 12 carbon atoms, more preferably containing 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Base, cyclooctyl, etc.; polycyclic cycloalkyl includes spiro ring, fused ring and bridged ring cycloalkyl. "Carbocycle” refers to the ring system in a cycloalkyl group.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 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), but excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • ring atoms Preferably it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 6 ring atoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, 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 (that is, rings sharing adjacent pairs of carbon atoms) group, preferably 6 to 10 membered, having a conjugated pi-electron system, such as benzene base and naphthyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is the aryl ring.
  • “Aromatic ring” refers to a ring system in an aryl group. Non-limiting examples of aryl groups include:
  • Aryl groups 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, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio, carboxyl or carboxylate, preferably phenyl.
  • 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, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazine group, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl.
  • heteroaryl may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring.
  • Heteroaryl refers to a ring system in a heteroaryl group.
  • Non-limiting examples of heteroaryl groups include:
  • Heteroaryl groups may 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 group is a suitable group known in the art for carboxyl protection, see the carboxyl protecting group in the literature ("Protective Groups in Organic Synthesis", 5 Th Ed. TW Greene & P. GM Wuts), as an example,
  • the carboxyl protecting group can be substituted or unsubstituted C 1-10 straight chain or branched chain alkyl, substituted or unsubstituted C 2-10 straight chain or branched alkenyl or alkynyl, substituted or unsubstituted Substituted C 3-8 cyclic alkyl, substituted or unsubstituted C 5-10 aryl or heteroaryl, or (C 1-8 alkyl or aryl) 3 silyl, etc.
  • amino-protecting group is a suitable group known in the art for amino-protection, referring to the amino-protecting group in the literature ("Protective Groups in Organic Synthesis", 5 Th.Ed.TW Greene & P.GM Wuts), preferably , the amino protecting group can be (C 1-10 alkyl or aryl) acyl, for example: formyl, acetyl, benzoyl, etc.; can be (C 1-6 alkyl or C 6-10 aryl base) sulfonyl; it can also be (C 1-6 alkoxy or C 6-10 aryloxy) carbonyl, for example: Boc or Cbz; it can also be substituted or unsubstituted alkyl, for example: trityl 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).
  • DMB trityl 2,4-dimethoxybenzyl
  • PMB
  • leaving group refers to an atom or functional group that breaks away from a larger molecule in a chemical reaction.
  • Representative leaving groups include halogen, substituted sulfonyloxy, phosphoryloxy, amino, (R j ) 3 N-, cyano, R j S-, etc., wherein R j is independently selected from hydrogen atom or C 1 -C 6 alkyl.
  • the substituted sulfonyloxy group may be C 1 -C 6 alkylsulfonyloxy, perfluoro C 1 -C 6 alkylsulfonyloxy, arylsulfonyloxy, aralkylsulfonyloxy and the like.
  • C 1 -C 6 alkylsulfonyloxy include C 1 -C 6 linear or branched alkylsulfonyloxy, such as methylsulfonyloxy, ethylsulfonyloxy, n-propyl ylsulfonyloxy, isopropylsulfonyloxy, n-butylsulfonyloxy, tert-butylsulfonyloxy, n-pentylsulfonyloxy and n-hexylsulfonyloxy.
  • perfluoro C 1 -C 6 alkylsulfonyloxy groups include C 1 -C 6 linear or branched perfluoroalkylsulfonyloxy groups, such as trifluoromethylsulfonyloxy, 1,1 ,2,2,2-pentafluoro-1-ethylsulfonyloxy, 1,1,2,2,3,3,3-heptafluoro-1-propylsulfonyloxy and 1,1,2 ,2,3,3,4,4,4-Nafluoro-1-butylsulfonyloxy.
  • arylsulfonyloxy examples include: optionally having 1 to 3 alkyl groups selected from C 1 to C 6 straight chain or branched chain, C 1 to C 6 straight chain or branched chain alkyl on the benzene ring; , phenylsulfonyloxy and naphthylsulfonyloxy substituents of the group consisting of nitro and halogen atoms.
  • phenylsulfonyloxy optionally having substituents include phenylsulfonyloxy, 4-methylphenylsulfonyloxy, 2-methylphenylsulfonyloxy, 4-nitro Phenylsulfonyloxy, 4-tolylsulfonyloxy, 2-nitrophenylsulfonyloxy, 3-chlorophenylsulfonyloxy and the like.
  • naphthylsulfonyloxy include ⁇ -naphthylsulfonyloxy, ⁇ -naphthylsulfonyloxy and the like.
  • aralkylsulfonyloxy groups include: phenyl (which optionally has 1 to 3 alkyl groups selected from C 1 to C 6 straight or branched chains, C 1 to C 6 straight chains on the benzene ring, chain or branched chain alkane, nitro and halogen atom substituents) substituted C 1 to C 6 straight chain or branched alkylsulfonyloxy; and C 1 to C 6 straight chain or branched by naphthyl Branched chain alkylsulfonyloxy.
  • alkylsulfonyloxy substituted by phenyl include benzylsulfonyloxy, 2-phenylethylsulfonyloxy, 4-phenylbutylsulfonyloxy, 4-methylbenzyl Sulfonyloxy, 2-methylbenzylsulfonyloxy, 4-nitrobenzylsulfonyloxy, 4-methylbenzylsulfonyloxy, 3-chlorobenzylsulfonyloxy and the like.
  • alkylsulfonyloxy group substituted with naphthyl examples include ⁇ -naphthylmethylsulfonyloxy, ⁇ -naphthylmethylsulfonyloxy and the like.
  • 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 does not occur.
  • a heterocyclic group optionally substituted with an alkyl group means that an alkyl group may but need not be present, and the description includes cases where the heterocycle group is substituted with an alkyl group and cases where the heterocycle group is not substituted with an alkyl group .
  • the bond configuration is not specified, i.e. if there is configurational isomerism in the chemical structure, the bond can be or or both and Two configurations.
  • the bond If the configuration is not specified, it can be the Z configuration or the E configuration, or both configurations.
  • the known starting materials of the present disclosure can be adopted or synthesized according to methods known in the art, or can be purchased from ABCR GmbH & Co.KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Darui Chemicals and other companies.
  • the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.
  • the hydrogenation reaction is usually vacuumized and filled with hydrogen, and the operation is repeated 3 times.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, which is 20°C to 30°C.
  • Suction filter concentrate the reaction solution to dryness, add 200mL methyl tert-butyl ether, filter, add 100mL 1M potassium hydrogensulfate aqueous solution to wash the mother liquor three times, and wash the aqueous phase twice with tertiary methyl ether; combine the organic phases and dry over anhydrous magnesium sulfate , concentrated and dried to obtain the crude product.
  • Column chromatography obtained 4.74 g of compound 1-1; yield: 96%, HPLC purity 98%.

Abstract

公开了一种艾日布林衍生物的制备方法。具体而言，公开了一种如式(I)所示化合物的制备方法，该制备方法收率高、反应条件温和，适合工业化生产。

Description

一种艾日布林衍生物的制备方法
本申请要求申请日为2021年10月14日的中国专利申请202111196863.7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，涉及一种艾日布林衍生物的制备方法。
背景技术
微管为与包括细胞内迁移和转运、细胞信号传导和维持细胞形状的多种细胞功能相关的有力的细丝状细胞骨架蛋白。微管也在有丝分裂细胞分裂中通过形成染色体分成两个子细胞所需的有丝分裂纺锤体而起到关键作用。所有细胞中微管的生物功能大部分由其聚合动力学调节，这通过α和β微管蛋白二聚物可逆、非共价地加在微管两端进行。这种动力学行为和所产生的对微管长度的控制为有丝分裂纺锤体的适当功能所不可缺少的。甚至微管动力学的微小改变也会牵涉轴检查点，抑制有丝分裂时细胞周期进展，且随后引起细胞死亡(Mukhtar等人(2014)Mol.Cancer Ther.13：275-84)。由于癌细胞的细胞分裂快速，所以与正常细胞相比，其一般对结合于微管蛋白且破坏其正常功能的化合物更加敏感。因此，微管蛋白抑制剂和其它靶向微管剂有望成为一类治疗癌症的药物(Dumontet和Jordan(2010)Nat.Rev.Drug Discov.9：790-803)。
软海绵素B(Halichondrin B)是1986年日本科学家Hirata和Uemura从海绵Halichondria okadai中分离出了的一种只包含C、H、O原子的聚醚大环内脂类化合物，具有极强的体外抗肿瘤活性。软海绵素B的分子结构非常复杂，包含32个手性中心，有超过40亿个异构体，合成难度非常大。艾日布林是halichondrin B的衍生物，是一种微管蛋白抑制剂，WO9965894首次公开了艾日布林的结构及合成方法。2010年11月15日，FDA批准甲磺酸艾日布林(Halaven)注射液用于治疗至少接受过两种化疗方案的转移性乳腺癌患者。
PCT/CN2021/073314涉及一种新的艾日布林衍生物及其药物偶联物，该化合物及其药物偶联物具有良好的抗肿瘤活性，化合物结构如下所示：
Figure PCTCN2022125301-appb-000001
发明内容
本公开的目的在于提供一种新的艾日布林衍生物的制备方法。
本公开还提供了一种如式(I)所示化合物的制备方法，包括式(I-2)所示化合物制备式(I-1)所示化合物的步骤，以及式(I-1)所示化合物脱保护基制备式(I)所示化合物的步骤，
Figure PCTCN2022125301-appb-000002
其中，R 1选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选C 1-6烷基或C 3-8环烷基，更优选甲基。
在某些实施方式中，R 1选自甲基，所述步骤中使用的甲基化试剂选自碘甲烷、硫酸二甲酯或三甲基氧鎓四氟硼酸盐，优选三甲基氧鎓四氟硼酸盐。
在某些实施方式中，所述的式(I-2)所示化合物与甲基化试剂的摩尔比为1:0.1～1:10，优选1:1～1:5，更优选1:1～1:3。
在某些实施方式中，反应温度-50～-50℃，优选-20～-5℃。
脱除氨基保护基可采用本领域常用的方法。在某些实施方式中，脱除氨基保护基的试剂选自氨水、甲胺醇溶液、乙二胺和肼，优选甲胺乙醇溶液。
在某些实施方式中，所述方法还包括式(I-3)所示化合物制备式(I-2)所示化合物的步骤，
Figure PCTCN2022125301-appb-000003
本公开所述的反应中使用的溶剂可以是常规溶剂，例如二甲基甲酰胺、1-甲基-2-吡咯烷酮、二甲基亚砜、四氢呋喃、乙酸乙酯、二氧六环、甲苯、二甲亚砜、乙醚、异丙醚、甲基叔丁基醚、二氯甲烷、氯仿、丙酮、乙腈、甲醇、乙醇、异丙醇、水中的一种或多种，优选四氢呋喃、乙酸乙酯、二氧六环、甲苯、二甲亚砜、乙醚、异丙醚、二氯甲烷、氯仿、丙酮、乙腈、甲醇、乙醇、异丙醇中的一种或多种。
本公开还提供了一种制备式(L-I)所示化合物的方法，包括本公开所述的制备式(I)所示化合物的方法，其中L为连接子，
Figure PCTCN2022125301-appb-000004
在某些实施方案中，连接子在细胞外是稳定的，使得ADC在存在于细胞外环境中时保持完整，但在例如癌细胞的细胞中内化时能够裂解。在一些实施方案中，当ADC进入表达对ADC的抗体部分具有特异性的抗原的细胞时，艾日布林衍生物药物部分从抗体部分裂解，且裂解释放艾日布林衍生物的未修饰形式。在一些实施方案中，连接子包含被定位成使得所述连接子或所述抗体部分中没有部分在裂解后保持结合于艾日布林衍生物的可裂解部分。
在某些实施方案中，连接子中的可裂解部分为可裂解肽部分。在一些实施方案中，相对于包含替代可裂解部分的ADC，包含可裂解肽部分的ADC显示较低的聚集水平，改善的抗体：药物比率，增加的癌细胞的靶向杀死，减少的非癌细胞的脱靶杀死，和/或较高的药物负载。在一些实施方案中，相对于不可裂解的连接子，添加可裂解部分增加细胞毒性和/或效力。在一些实施方案中，增加的效力和/或细胞毒性是在表达中等水平的 由ADC的抗体部分所靶向的抗原(例如中等FRA表达)的癌症中。在一些实施方案中，可裂解肽部分能够由酶裂解，且连接子为酶能够裂解的连接子。在一些实施方案中，酶为组织蛋白酶，且连接子为组织蛋白酶能够裂解的连接子。在某些实施方案中，与替代分裂机制相比，酶能够裂解的连接子(例如组织蛋白酶能够裂解的连接子)显示上述改善特性中的一种或多种。
在某些实施方案中，连接子包含氨基酸单元，所述氨基酸单元优选包含由2至7个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸的氨基酸构成的肽残基，更优选缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
在某些实施方案中，所述连接子选自：
Figure PCTCN2022125301-appb-000005
Figure PCTCN2022125301-appb-000006
本公开还提供了一种式(ADC-I)所示抗体-药物偶联物的制备方法，包括式(L-I)所示化合物制备式(ADC-I)所示抗体-药物偶联物的步骤，以及本公开所述的制备式(L-I)所示化合物的步骤，
Ab-(L-D) k
(ADC-I)
其中，Ab为抗体或其抗原结合片段，
L为将Ab共价连接于D的连接子，所述L如权利要求5-7任意一项所定义；且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)，
-D如下式所示：
Figure PCTCN2022125301-appb-000007
其中，R 1选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选C 1-6烷基或C 3-8环烷基，更优选甲基。
在一些实施方案中，本公开抗体-药物偶联物(ADC)中所述抗体选自鼠源抗体、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-药物偶联物(ADC-I)中所述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、抗CD79抗体、抗TROP-2抗体、抗CD79B抗体、抗Mesothelin抗体、抗叶酸受体α(FRA)抗体或其抗原结合片段。
在一些实施方案中，所述抗体-药物偶联物(ADC-I)中抗体为已知抗体，选自但不限于选自曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)、恩波妥珠单抗(Enoblituzumab)、依玛妥珠单抗(Emibetuzumab)、奥英妥珠单抗(Inotuzumab)、维汀-匹那妥珠单抗(Pinatuzumab)、维布妥昔单抗(Brentuximab)、吉妥单抗(Gemtuzumab)、比伐珠单抗(Bivatuzumab)、莫洛伐妥单抗(Lorvotuzumab)、cBR96、farletuzumab和Glematumamab或其抗原结合片段。
在某些实施方式中，所述抗体-药物偶联物为
Figure PCTCN2022125301-appb-000008
其中D为
Figure PCTCN2022125301-appb-000009
优选Ab为farletuzumab，优选k选自2.0～2.5或2.5～3.5。
可采用现有技术公开的方法以式(I)所示化合物为反应物制备式(L-I)所示化合物，或以式(L-I)所示化合物为反应物制备式(ADC-I)所示抗体-药物偶联物，例如PCT/CN2021/073314等公开的方法，在此全文引入。
本公开还提供了式(I-1)所示化合物，
Figure PCTCN2022125301-appb-000010
其中，R 1选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙 基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选C 1-6烷基或C 3-8环烷基，更优选甲基。
本公开所述的新的艾日布林衍生物的制备方法，采用了新的氨基保护基，避免了甲基化过程中的选择性问题，极大降低了双甲基化杂质的产生，减轻了反应后处理的难度，并且提高了反应收率，有利于大批量的工业化生产。现有的制备工艺得到的产品存在较多的双甲基化杂质，难以除去，最终影响终产品的质量。
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“药物”是指细胞毒性药物或免疫调节剂。细胞毒性药物能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶。免疫调节剂是免疫关卡分子的抑制剂。
术语“连接子”、“连接单元”、“接头单元”、“接头”或“连接片段”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头可以包含一种或多种接头构件。例示性的接头构件包括6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(Val-Cit或vc)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)，及那些源自与接头试剂的偶联的：N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(SMCC，在本文中也称作MCC)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。接头可以包括拉伸单元、间隔单元、氨基酸单元和延伸单元。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 Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“拉伸单元”指一端通过碳原子与抗体共价连接而另一端与氨基酸单元、二硫化物部分、磺酰胺部分或非肽化学部分相连的化学结构片段。
术语“间隔单元”是一种双功能化合结构片段，可用于偶联氨基酸单元和细胞毒性药物最终形成抗体-药物偶联物，这种偶联方式可以将细胞毒性药物选择性的连接到氨基酸单元上。
术语“氨基酸”是指分子结构中含有氨基和羧基，并且氨基和羧基都直接连接在-CH-结构上的有机化合物。通式是H 2NCHRCOOH，R为H、取代或未取代烷基等。根据氨基连结在羧酸中碳原子的位置，可分为α、β、γ、δ、ε……-氨基酸。在生物界中，构成天然蛋白质的氨基酸具有其特定的结构特点，即其氨基直接连接在α-碳原子上，即α-氨基酸，包括甘氨酸(Glycine)、丙氨酸(Alanine)、缬氨酸(Valine)、亮氨酸(Leucine)、异亮氨酸(Isoleucine)、苯丙氨酸(Phenylalanine)、色氨酸(Tryptophan)、酪氨酸(Tyrosine)、天冬氨酸(Aspartic acid)、组氨酸(Histidine)、天冬酰胺(Asparagine)、谷氨酸(Glutamic acid)、赖氨酸(Lysine)、谷氨酰胺(Glutamine)、甲硫氨酸(Methionine)、精氨酸(Arginine)、丝氨酸(Serine)、苏氨酸(Threonine)、半胱氨酸(Cysteine)、脯氨酸(Proline)等。非天然氨基酸如瓜氨酸。如本领域技术人员所公知的，非天然氨基酸并不构成天然蛋白质，因此也不参与本公开中抗体的合成。本公开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。
Figure PCTCN2022125301-appb-000011
Figure PCTCN2022125301-appb-000012
本公开中延伸单元为PAB，结构如4‐亚氨基苄基氨甲酰基片段，其结构如式(VI)所示，连接在D上，
Figure PCTCN2022125301-appb-000013
缩写
接头组件包括但不限于：
MC＝6-马来酰亚氨基己酰基，结构如下：
Figure PCTCN2022125301-appb-000014
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的例示二肽)
瓜氨酸＝2-氨基-5-脲基戊酸
PAB＝对氨基苄氧羰基(“自我牺牲”接头组件的例示)
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)
MC(PEG) 6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯
SMCC＝琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯
IT＝亚氨基硫烷
PBS＝磷酸缓冲盐溶液。
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毒性药物相连。
术语“载药量”可以表示为药物量和抗体量的比值，即ADC中每个抗体所偶联的药物的平均数量。载药量的范围可以是每个抗体(Ab)连接1-20个，优选1-10个细胞毒性药物(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。
本公开单抗分子大小变异体测定法(CE-SDS)可采用十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法，在还原和非还原条件下，依据分子量大小，按毛电泳法(2015年版《中国药典》0542)，定量测定重组单克隆抗体产品的纯度。
可以用以下非限制性方法控制抗体-药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
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。本公开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，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。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“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过肽键相互连接而成，是蛋白质的结构与功能片段，如激素、酶类等本质上都是肽。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糖等。
术语“荧光探针”是指在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命和偏振等)可随所处环境的性质，如极性、折射率、粘度等改变而灵敏地改变的一类荧光性分子，其与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变，可用于研究大分子物质的性质和行为。
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 PCTCN2022125301-appb-000015
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 PCTCN2022125301-appb-000016
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)。
术语“离去基团”指在化学反应中从一较大分子中脱离的原子或官能基。具有代表性的离去基团有卤素、取代的磺酰基氧基、磷酰基氧基、氨基、(R j) 3N-、氰基、R jS-等，其中R j独立地选自氢原子或C 1～C 6烷基。
取代的磺酰基氧基可以是C 1～C 6烷基磺酰基氧基、全氟C 1～C 6烷基磺酰基氧基、芳基磺酰基氧基、芳烷基磺酰基氧基等。
C 1～C 6烷基磺酰基氧基的具体例子包括C 1～C 6直链或支链的烷基磺酰基氧基，例如甲基磺酰基氧基、乙基磺酰基氧基、正丙基磺酰基氧基、异丙基磺酰基氧基、正丁基磺酰基氧基、叔丁基磺酰基氧基、正戊基磺酰基氧基和正己基磺酰基氧基。
全氟C 1～C 6烷基磺酰基氧基的具体例子包括C 1～C 6直链或支链的全氟烷基磺酰基氧基，例如三氟甲基磺酰基氧基、1,1,2,2,2-五氟-1-乙基磺酰基氧基、1,1,2,2,3,3,3-七氟-1-丙基磺酰基氧基和1,1,2,2,3,3,4,4,4-九氟-1-丁基磺酰基氧基。
芳基磺酰基氧基的例子包括：任选地在苯环上具有1至3个选自由C 1～C 6直链或支链的烷基、C 1～C 6直链或支链的烷、硝基和卤原子组成的组的取代基的苯基磺酰基氧基和萘基磺酰基氧基。任选地具有取代基的苯基磺酰基氧基的具体例子包括苯基磺酰基氧基、4-甲基苯基磺酰基氧基、2-甲基苯基磺酰基氧基、4-硝基苯基磺酰基氧基、4-甲苯基磺酰基氧基、2-硝基苯基磺酰基氧基、3-氯苯基磺酰基氧基等等。萘基磺酰基氧基的具体例子包括α-萘基磺酰基氧基、β-萘基磺酰基氧基等等。
芳烷基磺酰基氧基的例子包括：被苯基(其任选地在苯环上具有1至3个选自C 1～C 6直链或支链的烷基、C 1～C 6直链或支链的烷、硝基和卤原子的取代基)取代的C 1～C 6直链或支链的烷基磺酰基氧基；和被萘基取代的C 1～C 6直链或支链的烷基磺酰基氧基。被苯基取代的烷基磺酰基氧基的具体例子包括苄基磺酰基氧基、2-苯基乙基磺酰基氧基、4-苯基丁基磺酰基氧基、4-甲基苄基磺酰基氧基、2-甲基苄基磺酰基氧基、4-硝基苄基磺酰基氧基、4-甲苄基磺酰基氧基、3-氯苄基磺酰基氧基等等。被萘基取代的烷基磺酰基氧基的具体例子包括α-萘基甲基磺酰基氧基、β-萘基甲基磺酰基氧基等等。
“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 PCTCN2022125301-appb-000017
并未指定构型，即如果化学结构中存在构型异构，键
Figure PCTCN2022125301-appb-000018
可以为
Figure PCTCN2022125301-appb-000019
或
Figure PCTCN2022125301-appb-000020
或者同时包含
Figure PCTCN2022125301-appb-000021
和
Figure PCTCN2022125301-appb-000022
两种构 型。本公开所述化合物的化学结构中，键
Figure PCTCN2022125301-appb-000023
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。
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。
氢化反应通常抽真空，充入氢气，反复操作3次。
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 PCTCN2022125301-appb-000024
步骤1：化合物1-2的合成
称取碳酸氢钠(2.0g，24mmol)于反应瓶中，加入10mL纯化水，将化合物1-3(4.95g，6.00mmol)加130mL纯化水溶解，加入到反应瓶中，称取N-乙氧羰基邻苯二甲酰亚胺 (5.3g，24mmol)于上述反应液中，加毕，抽真空氩气保护，室温搅拌反应至结束。向反应液中加入DCM萃取，水相用DCM萃取，合并有机相，用饱和氯化钠洗，无水硫酸镁干燥，抽滤，浓缩，干燥，得到粗品。柱层析得到4.89g化合物1-2，收率：93％。
步骤2：化合物1-1的合成
称取:三甲基氧鎓四氟硼酸盐(13.4g，90.5mmol)以及1,8-双二甲氨基萘(19.4g，90.5mmol)于反应瓶中，加毕，抽真空氩气保护，加入100mL无水DCM，冷却至-10℃；将1-2(4.86g，5.66mmol)溶解在150mL DCM中，加入至上述反应液中，保温反应过夜。中控至原料基本反应完全，停止反应。抽滤，浓缩反应液至干，加入200mL甲基叔丁基醚，过滤，母液加入100mL 1M硫酸氢钾水溶液洗涤三次，水相用甲叔醚洗涤2次；合并有机相，无水硫酸镁干燥，浓缩，干燥，得到粗品。柱层析得到4.74g化合物1-1；收率：96％，HPLC纯度98％。
步骤3：化合物1的合成
称取化合物1-1量6.0g于反应瓶中，加入180mL甲胺的乙醇溶液，氩气保护，室温搅拌反应过夜，中控至原料反应完全，停止反应。减压浓缩至干，加入200mL DCM以及100mL纯化水萃取，有机相用100mL纯化水洗涤3次，合并水相，DCM萃取2次，合并有机相，无水硫酸镁干燥，浓缩，得到粗品。柱层析得到4.2g化合物1；总收率：86.7％。
实施例2
Figure PCTCN2022125301-appb-000025
向反应瓶中加入化合物2(17.6g，根据PCT/CN2021/073314方法制备)，126ml DMF，搅拌溶解，加入碳酸氢钠，20-25℃搅拌，将化合物1溶于63mlDMF，用恒压滴液漏斗滴加到上述反应液，滴加完毕，继续20-25℃反应2.5-3h，TLC检测反应完全，加入1134ml乙酸乙酯/异丁醇(v:v＝9:1),1134ml饮用水，110ml饱和NaCl，萃取分液，剩余水相继续用乙酸乙酯/异丁醇(v:v＝9:1)萃取两次,合并有机相用饱和NaCl洗涤，无水硫酸钠干燥，减压浓缩干得到粗品。粗品柱层析得16.9g化合物L-1，收率：74％。
对比例1
Figure PCTCN2022125301-appb-000026
室温下，将1-2’(190mg,0.179mmol)，三甲基氧四氟硼酸盐(212mg,1.433mmol)，1,8-双二甲氨基萘(306mg,1.43mmol)加入到反应瓶中；加入4.5mL的二氯甲烷，然后氮气球保护在室温下搅拌至反应结束，加20mL的MTBE稀释，过滤，用约20mL的MTBE洗滤饼；合并滤液用1N盐酸洗涤；有机相用无水硫酸钠干燥；过滤，滤液减压蒸干；经硅胶柱(PE:EtOAc＝1：1)纯化后得到产物1-1’(154mg)，收率80％，HPLC纯度92％。
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 (13)

  1. 一种如式(I)所示化合物的制备方法，包括式(I-2)所示化合物制备式(I-1)所示化合物的步骤，以及式(I-1)所示化合物脱保护基制备式(I)所示化合物的步骤，
    Figure PCTCN2022125301-appb-100001
    其中，R 1选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选C 1-6烷基或C 3-8环烷基，更优选甲基。
  2. 根据权利要求1所述的制备方法，其中R 1选自甲基，所述步骤中使用的甲基化试剂选自碘甲烷、硫酸二甲酯或三甲基氧鎓四氟硼酸盐，优选三甲基氧鎓四氟硼酸盐。
  3. 根据权利要求2所述的制备方法，其中所述的式(I-2)所示化合物与甲基化试剂的摩尔比为1:0.1～1:10，优选1:1～1:5，更优选1:1～1:3。
  4. 根据权利要求1-3任意一项所述的制备方法，其中所述制备方法还包括式(I-3)所示化合物制备式(I-2)所示化合物的步骤，
    Figure PCTCN2022125301-appb-100002
  5. 一种式(L-I)所示化合物的制备方法，包括权利要求1-4任意一项所述的如式(I)所示化合物的制备方法，其中L为连接子，
    Figure PCTCN2022125301-appb-100003
  6. 根据权利要求5所述的制备方法，其中所述连接子包含氨基酸单元，所述氨基酸单元优选包含由2至7个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸的氨基酸构成的肽残基，更优选缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
  7. 根据权利要求5所述的制备方法，其中所述连接子选自：
    Figure PCTCN2022125301-appb-100004
    Figure PCTCN2022125301-appb-100005
  8. 一种式(ADC-I)所示抗体-药物偶联物的制备方法，包括式(L-I)所示化合物制备式(ADC-I)所示抗体-药物偶联物的步骤，以及权利要求5-7任意一项所述的制备式(L-I)所示化合物的步骤，
    Ab-(L-D) k
    (ADC-I)
    其中，Ab为抗体或其抗原结合片段，
    L为将Ab共价连接于D的连接子，所述L如权利要求5-7任意一项所定义；且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)，
    -D如下式所示：
    Figure PCTCN2022125301-appb-100006
    其中，R 1选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选C 1-6烷基或C 3-8环烷基，更优选甲基。
  9. 根据权利要求8所述的制备方法，其中所述抗体选自鼠源抗体、嵌合抗体、人源化抗体和全人源抗体。
  10. 根据权利要求8或9所述的制备方法，其中所述抗体-药物偶联物(ADC-I)中所述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、抗CD79抗体、抗TROP-2抗体、抗CD79B抗体、抗Mesothelin抗体、抗叶酸受体α(FRA)抗体或其抗原结合片段。
  11. 根据权利要求8所述的制备方法，其中所述抗体-药物偶联物(ADC-I)中抗体为已知抗体，选自但不限于选自曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)、恩波妥珠单抗(Enoblituzumab)、依玛妥珠单抗(Emibetuzumab)、奥英妥珠单抗(Inotuzumab)、维汀-匹那妥珠单抗(Pinatuzumab)、维布妥昔单抗 (Brentuximab)、吉妥单抗(Gemtuzumab)、比伐珠单抗(Bivatuzumab)、莫洛伐妥单抗(Lorvotuzumab)、cBR96、farletuzumab和Glematumamab或其抗原结合片段。
  12. 根据权利要求8所述的制备方法，其中所述抗体-药物偶联物为
    Figure PCTCN2022125301-appb-100007
    其中D为
    Figure PCTCN2022125301-appb-100008
    优选Ab为farletuzumab，优选k选自2.0～2.5或2.5～3.5。
  13. 式(I-1)所示化合物，
    Figure PCTCN2022125301-appb-100009
    其中，R 1选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如 C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选C 1-6烷基或C 3-8环烷基，更优选甲基。
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US20180009825A1 (en) * 2016-07-06 2018-01-11 Apicore Us Llc Methods of making eribulin mesylate
CN108883198A (zh) * 2016-03-02 2018-11-23 卫材研究发展管理有限公司 基于艾日布林的抗体-药物偶联物和使用方法
WO2021148003A1 (zh) * 2020-01-22 2021-07-29 上海森辉医药有限公司 艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用

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US20180009825A1 (en) * 2016-07-06 2018-01-11 Apicore Us Llc Methods of making eribulin mesylate
WO2021148003A1 (zh) * 2020-01-22 2021-07-29 上海森辉医药有限公司 艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用

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